BrJAC 2017 V4 N17 by BrJAC

Analitica Latin America Conference & Expo discussed Important Issues in Analytical Chemistry October â&#x20AC;&#x201C; December 2017 Volume 4 Number 17

BrMASS - 8 to 12 December Windsor Hotel Barra da Tijuca 7th IBERO-AMERICAN CONFERENCE ON MASS SPECTROMETRY Five days of Conferences. More than 20 Plenary Talks. 50 Invited Guests. Your chance to strengthen your networks and deepen your understanding of Mass Spectrometry - all of this in one of Brazil´s most beautiful landscapes.

SPEAKERS MASS 2018 will feature over 48 speakers from all over the world, exposing their newest and brightest ideas on Mass Spectometry.

PROGRAM A CONFERENCE YOU WON´T SOON FORGET Five days of events, full with meetings & greetings, poster presentations, oral presentations, public performances and much more.

LATEST NEWS KEEP UP TO DATE WITH MASS SPECTROMETRY Get ready for BrMASS 2018! Here you will ﬁnd the latest news about the topics and researches featured in our Congress

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About Br. J. Anal. Chem. The Brazilian Journal of Analytical Chemistry (BrJAC) is a peer-reviewed scientiﬁc journal intended for professionals and institutions acting mainly in all branches of analytical chemistry. BrJAC is an open access journal which does not charge authors an article processing fee. Scope BrJAC is dedicated to professionals involved in science, technology and innovation projects in the area of analytical chemistry at universities, research centers and in industry. BrJAC publishes original, unpublished scientiﬁc articles and technical notes that are peer reviewed in the double-blind way. In addition, it publishes reviews, interviews, points of view, letters, sponsor reports, and features related to analytical chemistry. Manuscripts submitted for publication in BrJAC, either from universities, research centers, industry or any other public or private institution, cannot have been previously published or be currently submitted for publication in another journal. For manuscript preparation and submission, please see the Guidelines for the Authors section at the end of this edition. When submitting their manuscript for publication, the authors agree that the copyright will become the property of the Brazilian Journal of Analytical Chemistry, if and when accepted for publication. Published by Visão Fokka Communication Agency Publisher Lilian Freitas MTB: 0076693/ SP lilian.freitas@visaofokka.com.br Advertisement Luciene Campos luciene.campos@visaofokka.com.br ISSN 2179-3425 printed www.brjac.com.br

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Editorial Board Editor-in-Chief Lauro Tatsuo Kubota Full Professor / Institute of Chemistry - University of Campinas - Campinas, SP, BR Associate Editors Cristina Maria Schuch Analytical Department Manager / Solvay Research & Innovation Center - Paris, FR Elcio Cruz de Oliveira Technical Consultant / Technol. Mngmt. - Petrobras Transporte S.A. and Aggregate Professor / Post-graduate Program in Metrology - Pontiﬁcal Catholic University, Rio de Janeiro, RJ, BR Fernando Vitorino da Silva Chemistry Laboratory Manager - Nestle Quality Assurance Center - São Paulo, SP, BR Marco Aurélio Zezzi Arruda Full Professor / Institute of Chemistry - University of Campinas - Campinas, SP, BR Pedro Vitoriano Oliveira Full Professor / Institute of Chemistry - University of São Paulo - São Paulo, SP, BR Renato Zanella Full Professor / Dept. of Chemistry - Federal University of Santa Maria - RS, BR Advisory Board Adriano Otávio Maldaner Criminal Expert / Forensic Chemistry Service - National Institute of Criminalistics - Brazilian Federal Police – Brasília, DF, BR Auro Atsushi Tanaka Full Professor / Dept. of Chemistry - Federal University of Maranhão, São Luís, MA, BR Carlos Roberto dos Santos President of CETESB - Environmental Company of São Paulo State, São Paulo, SP, BR Gisela de Aragão Umbuzeiro Professor / Technology School - University of Campinas - Campinas, SP, BR Isabel Cristina Sales Fontes Jardim Full Professor / Institute of Chemistry, University of Campinas, Campinas, SP, BR Janusz Pawliszyn Professor / Department of Chemistry - University of Waterloo, Ontario, Canada Joaquim de Araújo Nóbrega Full Professor / Dept. of Chemistry - Federal University of São Carlos - São Carlos, SP, BR José Anchieta Gomes Neto Associate Professor / São Paulo State University (UNESP), Institute of Chemistry, Araraquara, SP, BR José Dos Santos Malta Junior Pre-formulation Lab. Manager / EMS / NC Group – Hortolandia, SP, BR Luiz Rogerio M. Silva Quality Assurance Associate Director / EISAI Lab. – São Paulo, SP, BR Márcio das Virgens Rebouças Process & Technology Manager – GranBio Research Center - Campinas, SP, BR Marcos Nogueira Eberlin Full Professor / Institute of Chemistry - University of Campinas - Campinas, SP, BR Maria das Graças Andrade Korn Full Professor / Institute of Chemistry - Federal University of Bahia - Salvador, BA, BR Ricardo Erthal Santelli Full Professor / Institute of Chemistry - Federal University of Rio de Janeiro, RJ, BR

Contents Editorial 5th Analitica Congress – An event for sharing Knowledge and Innovation Interview Professor Fernando Lanças, who received awards such as the COLACRO and Janusz Pawliszyn medals, recently spoke with BrJAC about his work and career

1-1

2-7

Letters 5th Analitica Congress – Learning and Sharing Experiences

8-8

Technology, Innovation and Lots of Analytic Chemistry

9-9

Articles Comparison of a New Total Fat Quantiﬁcation Method in Cheese using Microwave Assisted Extraction (MAE) and Soxhlet

10-15

Study of Geographical Variation in Phyllanthus amarus Schum & Thonn using DART-TOF-MS combined with PCA

16-23

Cloud Point Extraction Spectrophotometric Method for Determination of Three Types of Cephalosporin via Diazotization Reactions with Different Reagents

24-36

Feature Analitica Latin America Conference and Expo discussed important issues for the Development of Science and Technology in Analytical Chemistry

37-44

International Institute of Chromatography offers Specialization Courses in several areas of Chromatography

45-47

Sponsor Reports US EPA 3546

48-52

Routine Quantitative Method of Analysis for Pesticides using GC Orbitrap Mass Spectrometry in accordance with SANTE/11945/2015 Guidelines

53-65

Fast Routine Analysis of Polar Pesticides in Foods by Suppressed Ion Chromatography and Mass Spectrometry 66-78 Releases Advanced Microwave Extraction System for GC and HPLC Analysis

79-79

Exactive GC Orbitrap GC-MS System - The Frontier of Routine GC-MS

81-81

TSQ Altis Triple-Stage Quadrupole Mass Spectrometer - Conﬁdent Quantitation Sensitivity and Robustness without compromise

83-83

Notices of Books

85-86

Periodicals & Websites

87-87

Events

88-89

Acknowledgement

90-92

Author's Guidelines

93-96

Br. J. Anal. Chem., 2017, 4 (17), pp 1-1

Editorial

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5 Analitica Congress – An event for sharing Knowledge and Innovation Pedro Vitoriano Oliveira Full Professor at the Department of Fundamental Chemistry, Institute of Chemistry, University of São Paulo, SP, BR pvolivei@iq.usp.br rd

The chemical industry is the 3 most important industrial sector in the Brazilian economy and is classified in the top 10 in the world. Certainly, it could be higher in the world if research and development (R&D) was the central theme of science and technology policies in Brazil. Universities and companies have long sought ways to improve the relationship, aiming to share experiences and seek pathways for scientific and technological development in Brazil. Among the several pathways being pursued, one is through the organization of events that bring together academics and professional from industries for scientific discussions. This is the proposal of the Analitica Congress. The 5th Analitica Congress was organized during the 14th Analitica Latin America Exhibition and held on September 26-28, 2017, at São Paulo Expo Center, in São Paulo. Both events were organized by the team of NürnbergMesse Brasil with complete success and a large audience. The theme “knowledge and innovation” has been explored throughout the latest Analitica Congresses to promote the exchange of knowledge between Brazilian researchers from universities and industries, aiming to overcome the new challenges in Brazilian society. Based on this idea, several aspects of Analytical Chemistry were discussed and it was possible to join specialists and show how the “knowledge and innovation” is linked to the environment, biological sciences, food chemistry, pharmacology, technological products, instrumentation, agribusiness, and petrochemistry. Topics about the distance of flight mass spectrometry and solid state ion detectors, laser ablation-based techniques for direct chemical analysis of solids, laser induced breakdown spectroscopy, X-ray fluorescence, microwave-assisted sample preparation, separation techniques, microfluidic device, and lab-on-a-chip were discussed. th The 5 Analitica Congress reached an audience of around 300 participants, including students, researchers and professionals from industries, providing a great opportunity to exchange experiences. The organizing committee, made up of representatives from universities and industry, took care to prepare an attractive program that could benefit undergraduate and graduate students, researchers, and professionals from analytical laboratories. The scientific program involved 18 plenary lectures with professors from Brazil and the USA, the last as a result of an agreement with Pittcon, 3 symposia and 111 contributions in oral and poster sessions. The work “Determination of phosphorus in fertilizers by LIBS using an electrical discharge induced by laser for improvement of sensitivity” by the graduate student Alan Lima Vieira, supervised by Prof. Dr. José Anchieta Gomes Neto, from the Institute of Chemistry, UNESP, Araraquara, was awarded the best contribution and was fully supported for presentation at Pittcon 2018. Additionally, th in the 14 Analitica Latin America Exhibition, attendees were exposed to advances in analytical instrumentation and general supplies from analytical laboratories. The Circuit of Knowledge and Innovation was organized with different activities such as the LiveLab, where attendees had the opportunity to participate in live demonstrations with analytical instruments, such as inductively coupled plasma optical emission spectrometer, mass spectrometers, and gas and liquid chromatographs, and were also offered access to the Q-Lounge and the NanoSolutions Space to increase the exchange of information between presenters and attendees. We would like to thank all of the authors of this special issue for their contributions and we have a special acknowledgment to all reviewers of the submitted manuscripts, who spent their time and shared their expertise to contribute to the quality of this issue. Additionally, we would like to thank all speakers and participants, as well as the organizing and scientific committee. Enjoy reading. 1

Br. J. Anal. Chem., 2017, 4 (17), pp 2-7

Interview

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Professor Fernando Lanças, who received awards such as the COLACRO and Janusz Pawliszyn medals, recently spoke with BrJAC about his work and career Fernando Mauro Lanças Full Professor at the Institute of Chemistry of São Carlos, University of São Paulo, São Carlos, SP, BR flancas@iqsc.usp.br Prof. Fernando Lanças is a graduate in Chemistry, Physics and Biological Sciences, after a graduation course (not completed) in Social Sciences, with emphasis on Philosophy. Lanças, who has a Masters and PhD from the Institute of Chemistry of the University of Campinas (Unicamp), is the founder of the Latin American Congress of Chromatography (COLACRO), the Brazilian Symposium on Chromatography (SIMCRO) and the International Institute of Chromatography(IIC), of which he is President. Among other awards, he received the COLACRO medal from the Argentine Chemical Association, Buenos Aires (1988) for the dissemination of Chromatography in Latin America, and the Dr. Janusz Pawliszyn Medal, in Portugal, for his expressive contribution to the development of sample preparation techniques in Latin America (2016). In addition, he has been a member of the Editorial Board of several international journals, such as the Journal of High Resolution Chromatography, Journal of Microcolumn Separations, Energy Sources, Fuel Science and Technology, Journal of Capillary Electrophoresis, Journal of Chromatographic Science, Journal of Separation Science, among others. Since 1977, Lanças has been Professor of the Institute of Physics and Chemistry of São Carlos (current Institute of Chemistry of São Carlos - IQSC) at the University of São Paulo (USP). He was Head of the Department of Fundamental Chemistry from 1997 to 2001 (two terms) and Vice-Director of the Institute of Chemistry (IQSC-USP). Lanças is also a member of the American Chemical Society (USA), the Brazilian Society of Chemistry (Brazil), the Brazilian Society for the Progress of Science (Brazil), the New York Academy of Sciences (USA), the Mediterranean Center for Separation Science (Messina, Italy), and Titular Member of the Academy of Sciences of the State of São Paulo (ACIESP, Brazil). th He founded the free access journal Scientia Chromatographica, already in its 10 volume, the only Latin American periodical devoted exclusively to separation sciences and related techniques, of which he is the editor-in-chief as well. The Professor Lanças founded the Laboratory of Chromatography (CROMA), which acts in the development of new equipment and methodologies for the preparation of samples, separation (HPLC, HRGC, SFC, CE and others) and detection, especially Mass Spectrometry coupled with High Resolution Chromatographic Techniques (LC- MS/MS, GC-MS, SFC-MS etc.). He has published more than 300 scientific papers in national and international journals, and is the author of the books: “Cromatografia Líquida Moderna”, “Cromatografia em Fase Gasosa”, “Extração em Fase Sólida”, “Fundamentos da Cromatografia Gasosa” and “Validação de Métodos Cromatográficos”. 2

Interview He has presented more than 500 papers in national and international scientific congresses, guided more than 120 postgraduate theses to date, and presented dozens of lectures in more than 20 countries. Currently, prof. Lanças continues as full Professor at IQSC-USP and is a researcher classified as 1A by the National Council for Scientific and Technological Development (CNPq). When was your first contact with Chemistry? Did you have any influencers, such as a teacher? I remember having a teacher at the elementary school, named Vilarino, who would take us to the laboratory to follow experiments. No doubt the interest in chemistry began there. I also had a professor of Organic Chemistry in high school called Idelmar, who aroused my interest in the applications of chemistry. Finally, at graduation, a professor of excellent teaching (Munif) and unparalleled enthusiasm for chemistry aroused my curiosity by the clear, steady, and enthusiastic way he presented the most complex subjects during class. My interest in teaching chemistry certainly began in this period. When did you decide to go into the area of chromatography? What motivated you for this choice? How was the beginning of your career? During my doctorate, at IQ-Unicamp, I used some principles of classical liquid chromatography, in open glass column, to separate some radioactive compounds. At this stage of my training, the focus was on radiochemistry and the use of chromatography - rather rudely - aimed only to obtain information on radiolabeled compounds. Luckily, during the period of my thesis defense, an American researcher named Luckily, during the period of my thesis defense, Harold McNair, a specialist in chromatography, was an American researcher named Harold McNair, visiting IQ-Unicamp. I looked for state-of-the-art a specialist in chromatography, was visiting IQinformation on the technique, and I became very Unicamp. After conversing with Prof. McNair, I interested in it. After conversing with Prof. McNair, I made my way to the Virginia Polytechnic and made my way to the Virginia Polytechnic and State State University, USA. University (Virginia Tech), USA, for a Pos Doc program supervised by Prof. McNair. I defended my thesis in April and in December of the same year I was already working in the laboratory of Prof. McNair, fully dedicated to chromatography, a technique that I would embrace for the rest of my career. Do you keep informed about the progress of chemistry research? What is your opinion about the current progress of research in chemistry in Brazil? What are the latest advances and challenges in scientific research in Brazil? I have always enjoyed reading and following the progress, not only of science in my specific area of practice, but also of research involving any aspect of the evolution of the human being and of our planet as a whole. Research in chemistry in Brazil has evolved a lot in the last 20 years. By the time I finished my doctorate there were a very small number of researchers in the area, all over the country. Today this number has increased (and continues to increase) and we have highly qualified research niches in virtually every area of chemistry. In my opinion, the most important is the continuity and expansion of research funding - both involving scholarships and resources for equipment acquisition, maintenance and supplies - as well as the existence of long-term programs with clear management and well-defined criteria. Also important is the support for innovative programs and projects, and not only of projects induced to attend to specific sectors of interest. What have been the most important achievements in the world of analytical research for you recently? What were the landmarks? In my opinion we did not have revolutionary innovations, but improvements in some sectors. As an example, I would like to mention the "return" of the basic and applied biological sciences, which were less prominent. In the last 20 years, with the development of genomics, proteomics has arisen and from there 3

Interview In my view, and without diminishing any analytical technique since all are important, the great advance in the last two decades was the improvement of separation techniques - especially the chromatographic techniques and their coupling to mass spectrometry.

on to metabolomics, lipidomics, foodomics, and others. In fact they do not approach completely new topics, nevertheless old subjects with a different, much more integrated vision. In this way, the area of chromatography and its association with other techniques such as mass spectrometry (GC-MS, LC-MS, SFC-MS, etc.) collaborated a lot to obtain results, which allowed for the expansion of understanding in complex areas. For example, the use of capillary liquid chromatography associated with mass spectrometry enabled - and still allows, - the achievement of very important results regarding,

for example, the proteins of a given organism. This has led to a better understanding of the function of proteins in different systems and enabled the development of specific drugs that can fight certain diseases. It is the association of tools:chromatographic instrumentation-mass spectrometry with proteomics and pharmacology. All this for the benefit of the human being. Another important example of this association has been the development of new non-invasive ways of diagnosing various diseases such as cancer (types and even stages of evolution). Today it is possible to be done by blood examination using biomarkers and liquid chromatography associated with mass spectrometry. In my view, and without diminishing any analytical technique since all are important, the great advance in the last two decades was the improvement of separation techniques - especially the chromatographic techniques - and their coupling to mass spectrometry, allowing an out of the ordinary advance in the applied analytical chemistry to areas of social relevance such as food, environment, public health, fuels among others. There are several meetings held in Brazil and in the world on Chromatography. To you, how important are these meetings for the area? How do you see the development of these meetings? Scientific events serve multiple purposes, not "just" for the presentation of papers. Researchers are looking to present the latest research findings to an interested audience, often motivating graduate students to become increasingly interested in research. In addition, researchers from similar areas have the opportunity to get to know the work of colleagues from other institutions (often from other countries) and often come to work together later through collaborations. It is also very common for manufacturers of equipment and accessories, along with their trade representatives, to participate actively in the events, bringing not only equipment but also factory specialists to countries where the majority of the public would not have access to them. This allows an immense increase of the knowledge in the host country of the event, at a very low cost to participants, with very high return. In the case of COLACRO and SIMCRO, we have brought relevant names to Latin American such as Giddings, Huber, Horvath, Sandra, Cramers, McLafferty, Marriott, Pawliszyn, Lee, Jinno, Kaiser, Schomburg, Mondello, Armstrong, Novotny and many others (more than 200 to the present). During the events, postgraduate students, professors, researchers in companies and others, come in contact with these researchers from abroad and then go to develop projects with them in their institutions abroad. You are the founder of the Brazilian Symposium on Chromatography (SIMCRO) and also the Latin American Congress of Chromatography (COLACRO). What is the importance of these events for the international and Brazilian scientific community? COLACRO was founded with the intention of serving as a forum for discussion of recent advances in the field of chromatography throughout Latin America. Its model served to inspire several other similar events in the country, in related areas. In addition to having been held several times in Brazil, the event was also held in Argentina, Chile, Mexico and Colombia, more than once, and once in Venezuela. This allowed for two complementary actions: on the one hand to strengthen the relationship between researchers from different countries, and on the other, to allow the discussion of problems common to researchers from the same country. Having always received the generous support of the manufacturers and representatives of the equipment, accessories and supplies companies, it was possible for a large number of researchers to congregate in the area (in one of the last editions in Brazil we had more than 4

Interview 1,200 registered participants). The geographical extension of COLACRO to Europe occurred in 2016 with the holding of the event in Portugal, with the collaboration of the Portuguese Society of Chemistry. The experience proved to be highly positive since it also had the participation of several researchers from Spain. Thus, with more than 30 years of existence as being one of the longest chromatography events in the world, COLACRO is already part of the permanent international calendar of events in the area. SIMCRO has the same structure as COLACRO, but with the objective of bringing together Brazilian researchers and students. As a way to ensure the wide participation of different sectors interested in chromatography and related techniques, SIMCRO brings together several workshops and specific symposiums in parallel, in order to serve the different interests of various sectors. It is the national counterpart of COLACRO, having a very similar structure, but adapted to the objectives of SIMCRO. You have received some awards. What is it like to receive this kind of recognition? What is the importance of these awards in the development of science and new technologies? The eventual awards of a researcher cannot be understood as a personal thing. In fact, it is recognition of the work developed, which always occurs with the support of many people who unfortunately end up not being recognized in the awards. Thus, the recipient of an award has to understand that he is the representative of a group of people who contributed to the result of the work to prove its relevance and thus be recognized and highlighted from the others through the awards. Research usually arises through the improvement of results previously obtained by other researchers, for whose The eventual awards of a researcher improvement we make our contribution. A good example in cannot be understood as a personal our area of action was the Nobel Prize in Chemistry in 2002 thing. In fact, it is recognition of the for John Fenn and Koichi Tanaka for the "development" work developed, which always respectively of Electrospray and MALDI. The Nobel Prize occurs with the support of many people. Committee attributed the same to Fenn and Tanaka, “for their development of soft desorption ionization methods for mass spectrometric analysis of biological macromolecules". In fact, these researchers did not develop these techniques, but rather improved them for a specific application, which brought much controversy in the scientific environment regarding the indication, because the researchers who developed the techniques were not included in the awards. This, however, does not detract from the merit of the work of these researchers, but it is important for the understanding and recognition that Science usually develops from earlier facts, often studied by researchers who will remain unknown to the general public, but whose work was fundamental to the advancement of Science. Certainly, the recognition of a work (or even the work of a researcher as a whole) brings more light to the subject and contributes to the faster development of that area. Thus both Electrospray and MALDI have undergone an impressive evolution (and still develop) from the awarding of the Nobel Prize to two researchers in the area, resulting in great contributions in several areas, notably for the large area of biosciences and their applications. For you what is the importance of the support of funding agencies such as the São Paulo Research Foundation (FAPESP), Coordination for the Improvement of Higher Education Personnel (CAPES), among others, for the scientific development of the country? In any country, even those in which the contribution of resources from outside the government sector is relevant, the role of development and funding agencies are very important. In the United States, a country often cited as an example of applying large sums of resources to research, the role of various government agencies in fostering research is critical. In addition to the National Science Foundation (NSF), several government agencies provide research resources in specific niches, such as the FDA, EPA, NASA, and others, as well as large non-profit Research Institutions such as the EPRI (Electric Power Research Institute) that have mixed resources for the development of their projects. In Brazil, as well as in countries with smaller resources for research applications, and with a population with less access to resources that allow them to enter the research system, the role of institutions such as those mentioned, is of fundamental relevance. Each one within its niche (teaching, research, extension) has made great contributions to the 5

Interview maintenance of the research system in the country. Unfortunately, the contribution of private companies to research, unlike countries in which scientific research is in a more advanced stage, is still very limited in the country, both for internal research in the company and for development of projects in collaboration with universities. How is a career in chromatography? What advice would you give to a newcomer in this area? A career in the field of chromatography is gratifying; involving aspects of teaching, research and extension, because, being a technique eminently applied can solve fundamental problems of Brazilian society. For those who want to ingress in this field, I can say that in the next 30 years, the chance of an analytical A career in the field of chromatography is gratifying; involving aspects of teaching, separation technique obscuring chromatography is unlikely. research and extension, because, being So the choice is right. In both the academic and nona technique eminently applied can solve academic sectors, the demand for good professionals in the fundamental problems of Brazilian field is great. Thus, I suggest that those interested in society. progressing in it should always seek to understand the principles behind the technique, as well as the fundamentals of the instrumentation involved, to become a chromatographer and not just an equipment operator. With the current scenario, how is it for a student to start work on chromatography in Brazil? What differs from working abroad? In the chromatography area, as in many others, today Brazil has research groups of excellent quality and very well equipped. In addition, it develops cutting-edge research in instrumentation, materials and automation, coupling with other techniques, and applications in virtually all major research areas of interest. Our group, for example, maintains collaboration with various research groups from abroad and often when our doctoral students spend a period in one of these laboratories, on their return will say that their knowledge is as good as any. I once received a curious request from a Professor at the University of São Paulo (USP), who said that after careful research he concluded that it would be more interesting for him to do a post-doctorate with us than abroad. There was agreement from USP and the project was successfully developed in São Carlos. Recently I received a Full Professor and Head of Department of an American university for a Pos Doc with resources from his country of origin. He had synthesized some polymers and wanted to study the possibility of using them in separation techniques and in order to do so, he had to associate himself with a group with experience in preparing, evaluating and using liquid chromatography micro-columns. With his coming, the work in collaboration with the research group that I coordinate produced very positive results for both parties. Thus, in certain areas (and I repeat, in Brazil, there are a large number of groups of excellence in both chromatography and other analytical techniques) and types of application, choosing the most suitable laboratory to work on is often a matter of prestige or interest in knowing a culture other than scientific. Obviously, in certain areas of application the country does not have specialists, because it does not have the problem that requires the specialty and often does not develop expertise in the area. Some examples I once received a curious request from a are bomb detection at airports, lunar rock analysis, the Professor at the University of São Paulo, detection of radon in homes, and the like, which are who said that after careful research he common in other countries. However, analytical concluded that it would be more interesting chemistry in areas such as natural products, environment, for him to do a post-doctorate with us than food, pharmaceutical analysis, fuels and, more recently, abroad. "omics" in general, is very well developed in several Brazilian laboratories, which have conditions similar to those of called "first world". One striking difference that favors the development of leading-edge research in developed countries in relation to Brazil is the ease with which the interaction between the public and private sectors occurs. This interaction is facilitated by simple and clear legal frameworks, which establish rules of interaction in a transparent way. The intrinsic characteristics of scientific research require specific government actions that address this area in a special way. Thus, in many situations obtaining three budgets for the acquisition of simple materials can in many 6

Interview cases cause a delay in the process since not all companies consulted return with the same agility. Therefore, the practical differences are due to other aspects of the country's infrastructure, not to the research groups involved. For example, while abroad an analytical standard takes about two days after purchase to arrive in the laboratory, in Brazil rarely takes less than two months. Constant power line voltage fluctuations frequently damage equipment. While parts of overseas equipment are repaired in a few days (sometimes hours) in Brazil, they must be sent to the manufacturer's representative, who will send it to the plant and will often take months for the equipment to operate again as before, and at a generally astronomical cost. These are some of the examples of the difficulties that colleagues from abroad do not know, and that in daily life greatly influence the speed with which results are produced. Thus, I think that an improvement in the country's infrastructure, in general, could put Brazilian researchers in conditions closer to those existing at the disposal of their colleagues' abroad. Aside from these practical and infrastructural and non-scientific problems, I am quite optimistic about Brazilian science. I reaffirm that in Brazil there are several groups of excellence in various specialties of Analytical Chemistry. These are equivalent to the best similar laboratories located in other countries, and are recognized by them.

Br. J. Anal. Chem., 2017, 4 (17), pp 8-8

Letter

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5 Analitica Congress Learning and Sharing Experiences

Joaquim de Araújo Nóbrega Full Professor / Department of Chemistry, Federal University of São Carlos, SP, BR djan@ufscar.br How do you know you have participated in an outstanding scientific conference? You may pick one reason: (1) you have met your collaborators from others institutions; (2) you have met students and felt good about the future perspectives of your research area; (3) you have met staff of companies and their representatives and have learned about new technologies to test your ideas; (4) you have learned about new results and trends; (5) you have had the opportunity to discuss ongoing studies of your research group; (6) and so on… I agree with all these points and I would like to add one that I consider really special: you have participated in a flow of ideas and discussions that brought you original routes for developing new studies. The 5th Analitica Congress had given to us all these. We attended attractive and extremely wellorganized and well-presented conferences that exposed us to the state-of-the-art in multiple relevant topics. To make these conference sessions even better, they were presented for professors from different generations and exposed all attendees to amazing developments coming from efforts of several Brazilian research groups. At the same time, the partnership with Pittcon had given us the opportunity to learn from professors from major American universities, which also became a great platform for starting new collaborations. Topics about separation techniques, microfluidic device, lab-on-a-chip, distance of flight mass spectrometry and solid state ion detectors, laser ablation-based techniques for direct chemical analysis of solids, laser induced breakdown spectroscopy, X-ray fluorescence and microwave-assisted sample preparation were presented. The participants also had the opportunity to present their researches in oral and poster sessions with live discussions about their achievements. Finally, during the afternoon sessions we had the chance to participate in the 1st Symposium on Analytical Innovation together with the Brazilian Academy of Pharmaceutical Sciences, discussions about nanosciences and the Q-Lounge with several presentations about instrumentation and solutions for technological demands. Reminding about the 5th Analitica Congress we certainly have good memories concerning contacts, learning experiences, current developments and new possibilities. I do not know which one reason you have picked up for evaluating your participation, but I guess you are pretty pleased with all you have seen and discussed. Be ready for the coming 6th Analitica Congress in 2019. I do not know how, but I am sure it will be even better! Why? Because I have seen an increasing participation of professional chemists, graduate students, new and experienced professors, and staff from companies. This is an essential dialogue that will bring us remarkable prospects for further collaborations that will speed up new developments with relevant economic and social impact. This is a unique flavor of Analitica Congress and gradually this arena will become the place where science meets technology and innovations bring evolution. Let´s keep this virtuous circle progressing!

Br. J. Anal. Chem., 2017, 4 (17), pp 9-9

Letter

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Technology, Innovation and Lots of Analytic Chemistry Friedel Nimax Business Manager NürnbergMesse Brasil friedel.nimax@nm-brasil.com.br th

The 5 Analitica Congress of 2017 was a success! The fully overhauled event was held in conjunction with the show, gathering over 300 congress-goers during its three days. We welcomed researchers, teachers and professionals, who had first-hand access to content and found future perspectives in relation to their projects and applications. The Congress had the important mission of bringing together the industry and academia to foster development of the sector. This joining of expertise with experience was highly beneficial and achieved the goals set. We also took the chance to express our profound thanks to congress's partners and the members of the organizing committee, who helped us make this event a success. During the morning, plenary sessions were held, with general topics presented; these were followed by scientific and technical talks and in the afternoon, the space was reserved for demos, or rather, the concept could be seen in practice and become more palpable. Finally, the Association Symposiums were held with independent areas focused on: life science, soil science, forensic science, food technology, metrology, biofuels, genetics, geochemistry, and more. On the last day, the committee also named a winner, who was guaranteed the chance to present their project at the next Analitica Congress, being held in 2019, and who also won an all-expenses-paid trip to the USA to present their work at the PITTCON Conference & Expo 2018. The event in the USA is organized annually by the Spectroscopy Society of Pittsburgh, with people from over 90 countries participating. Even during a tough year for the analytical chemistry industry, with historic cuts to public and private laboratory budgets, the 14th edition of Analitica Latin America overcome challenges, showed its strength and ended the year with a surprising result. Consolidated as the largest business platform and source of industry content, the show brought together 7,500 professionals from September 26 to 28 at São Paulo Expo, 10% more than at the previous edition in 2015. With satisfied attendees, congress-goers and exhibitors, we at NürnbergMesse Brasil are beginning to prepare the 2019 event with great optimism. Analitica Latin America is an extremely important product for us, since we know how much it helps to integrate various sectors. The analytical chemistry sector has suffered with the crisis that continues to affect Brazil, which is why the show has become an even more important platform for business and communication. We are always looking for ways to reinvent ourselves th and continue to prosper. And so I would like to invite everyone to the 6 Analitica Congress next year, to gain the latest information and find insights on the future of analytical chemistry in Brazil!

Br. J. Anal. Chem., 2017, 4 (17), pp 10-15

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Comparison of a New Total Fat Quantification Method in Cheese using Microwave Assisted Extraction (MAE) and Soxhlet Sofia Pombal1*, João Gonçalo Lourenço2, Pedro Rocha2, Daniel Ettlin3, Jesus M. Rodilla1 1 Departamento de Química, Universidade da Beira Interior, FibEnTech-Materiais Fibrosos e Tecnologias Ambientais, Rua Marquês d'Ávila e Bolama, 6200-001, Covilhã, Portugal. 2 Universidade da Beira Interior, Departamento de Química, Rua Marquês d'Ávila e Bolama, 6200-001, Covilhã, Portugal. 3 Unicam Sistemas Analíticos, Alameda António Sérgio, 1495-132, Miraflores, Algés, Portugal. In this work, a simple and rapid method for fast quantitation of total fat in cheese samples is presented. A new method based on an innovative microwave-assisted extraction (MAE) technique that allows the determination of the total fat in cheese samples was evaluated. MAE method was compared against a Soxhlet extraction based reference method in order to verify results equivalence amongst them. The data showed that MAE method is statistically equivalent to the other method, showing very good performance indicators (LOQ = 0.248%, LOD = 0.087%, U = 2.65%). In addition, it was showed that MAE method allows the determination of total fat in 12 cheese samples simultaneously in 100 minutes, having much better productivity when compared to Soxhlet based method. As well MAE greatly simplifies sample handling, but also reduces analysis time and minimizes sample loss during sample preparation, makes this method an excellent alternative to determine total fat in cheese samples. Keywords: Microwave assisted extraction, Soxhlet, total fat, cheese. INTRODUCTION Fat has high importance in human health, as it affects the texture and taste of food. In recent times, food trends have followed a dangerous course. Excessive calories, unbalanced eating, increased dependence on processed foods, the widespread trade in fast food have led to an increase in obesity and metabolic changes in our body thus causing serious health problems. Thus, more attention should be paid to the overall quality of meals. For that purpose, the use of nutrition labels, which enable consumers to choose healthier food habits, need to be considered. The Regulation EU No. 1169/2011 of the European Parliament approved new rules of food labeling, becoming clearer and more readable allowing consumers an easier choice of the product they wish to purchase. This Regulation was binding in its entirety and directly applicable in all Member States (in Portugal since December 2016). The meaning of total fat, according to this Regulation is “total lipids, including phospholipids” [1]. Consumers recognize the advantages of nutritional labeling and view them important when choosing foods. There is a key parameter on the labels to assess the quantity of fat in cheese described as “Total Fat”. Beyond the accurate quantification of fatty acids in food, this parameter is important for routine assessments in industry settings for quality assurance purposes and monitoring of the food supply. Conventional lipid extraction techniques still more commonly used are, for example, Bligh and Dyer [2], Soxhlet [3] and Folch method [4]. These techniques have been used for many years, but are techniques with many procedural steps, thus leading to a very high time for results and a high consumption of solvents. These techniques can bring about problems such as lipid degradation during extraction at high temperatures, which are applied, for example, in the Soxhlet method [5]. The Official Methods of Analysis of AOAC International, which utilizes acid digestion and extraction with diethyl ether and petroleum ether in a Mojonnier flask, tends to be the dominant technique in industry environments, although the International Organization for Standardization (ISO) method that uses a Soxhlet apparatus is also used to determine total fat composition of food samples for nutritional labelling purposes, and is a reference method for different matrixes [6]. 10

*soﬁa.pombal@gmail.com ORCID: 0000-0002-6425-6641

Comparison of a New Total Fat Quantiﬁcation Method in Cheese using Microwave Assisted Extraction (MAE) and Soxhlet

Article

Thus, new extraction methods such as supercritical fluid extraction [7], pressurized liquid extraction [7] and microwave-assisted Soxhlet extraction [8] have been perfected in order to create sustainable alternatives for the extraction of total lipids. When considering cheese samples, and classic total fat determinations, the methodologies involve a sequential two steps sample treatment process. The first one is an acid hydrolysis, employed to solubilize casein and digest other compounds to liberate the fat before extraction, followed by a Soxhlet extraction with a non-polar solvent. The objective of this study was the comparison between the Soxhlet method and the microwave assisted extraction (MAE) method, in order to allow the validation of the MAE method. Soxhlet Method The Soxhlet extraction technique was invented in 1879 by Franz von Soxhlet [3] to determine fat content in milk. A sample is placed in a cartridge which is filled gradually by a fresh condensate extractor (solvent used in extraction) of a distillation flask. The liquid reaches the level of overflow and the siphon causes the liquid to return to the distillation flask, thus entraining the analytes present in the cartridge. This process is repeated several times until extraction is complete. Extraction in Soxhlet is a discrete and continuous technique [9]. This method was generalized for extraction in agriculture before being made the most used tool in the extraction of solids and liquids in certain fields such as foodstuffs and pharmaceuticals. Currently, the Soxhlet apparatus is still commonplace in laboratories and has been the standard and reference method for solid–liquid extraction [10]. Conventional Soxhlet extraction has some attractive advantages: the main appointed is that the sample is repeatedly brought into contact with fresh portions of extractant, which facilitates displacement of the transfer equilibrium. In addition, the system remains at a relatively high temperature, caused by the heat applied in the distillation flask, which somehow reaches the extraction cavity. In addition, if proper cups are used no filtration is required after leaching. There is also the possibility that sample throughput can be increased by performing several simultaneous extractions in parallel, which is facilitated by the low cost of the basic equipment [9]. Moreover, Soxhlet extraction is a very simple methodology that requires little training, and it can be designed to extract more sample mass than other comparative techniques seen as alternatives (e.g. supercritical fluid extraction, etc.). As a wide spread technique, and known by chemist for many years, there is a quite large variety of official methods involving a sample preparation step based on Soxhlet. Compared with extractions based on supercritical fluid extraction, and particularly on analytes strongly bounded to their matrix, Soxhlet may show practically no matrix effects [11]. Nevertheless, there are some appointed major disadvantages of the Soxhlet method compared to other solid sample preparation techniques. Amongst them, the high extraction time is usually a restriction. As the extraction time is matrix and target compound dependent, it is necessary to verify and make a proper alidation of this parameter, in order to be sure how long the sample must be submitted to the process. Other disadvantage is the use of a large amount of solvent, which is the source of environmental problems, and potentially an additional expense to the laboratory as they need to eliminate it properly according regulations. On a Soxhlet extraction, sample is submitted to mix concurrently with the vapour and liquid of an adequate solvent for a long period of time. For that purpose, the solvent is maintained at the boiling point by constant heating. This can cause thermal decomposition of the thermo-labile target species. As a conventional Soxhlet apparatus does not provide any agitation, the appropriate mixing of sample and solvent can be inadequate sometimes, as proper mixing can help to expedite the process. In addition, the large amount of solvent used leads to an evaporation step after extraction. Finally, this technique is limited by the extractor and difficult to automate [9].

Pombal, S.; Lourenço, J. G.; Rocha, P.; Ettlin, D.; Rodilla, J. M.

Article Microwave Method The microwave assisted extraction (MAE) is a relatively recent method for extracting products soluble into a fluid from a wide range of materials using microwave energy [12]. MAE offers a range of benefits over other solvent extraction methods, since MAE is faster and more effective, has lower consumption of energy and solvents, and, above all, uses less toxic solvents [13]. Liquid-phase MAE process is based upon the ability of a matrix to absorb microwave energy. This varies with the chemical nature of the species being exposed to the microwave irradiations; the chemical substances absorb microwave energy at different levels [12]. The MAE process performs two steps in only a single step, i.e., it performs hydrolysis and extraction simultaneously, therefore saving several steps compared with the Soxhlet conventional chemical extraction processes. The aqueous phase, as it is acidified, promotes the absorption of microwave energy, thus allowing a rapid increase in the mean temperature. The heating of the solvents inside a closed vessel promotes the rise of pressure, and therefore the capability of extraction in temperatures above the boiling points of both media. The application of microwave energy as a heat source causes as well selective heating of the matrix over the extractant. The high temperature and the increased pressure allow a selective migration of the compounds into the solvent at a much faster rate compared to conventional extraction methods [13]. The final result is the promotion of a faster extraction speed with excellent recovery rates over other conventional methods with solvent extraction. MATERIAL AND METHODS Samples Eight cheeses collected in Beira Interior of Portugal from the certified producers were tested. They were cheeses consisting of cow, goat and sheep's milk, manufactured from a single or a mixture of these different types of milk. Reagents Cyclohexane ≥99.5% (Reag. Ph. Eur), for analysis, AC, ISO, Panreac. CAS number 110-82-7 Molecular Weight 84.16, C6H12; Sulfuric Acid 95-97% (Reag. Ph. Eur) for analytical reagent, Riedel – de Haën, CAS number 7664-93-9 Molecular Weight 98.08, H2SO4. Soxhlet method procedure The methodology used is the Reference Portuguese Normative (Norma Portuguesa 1613-1979), which is based on an AOAC Official Methods of Analysis (AOAC 1995). The procedure involves a first step of acid hydrolysis, sample drying, and sequentially, the extraction itself on the Soxhlet device. Weigh two grams of sample in an Erlenmeyer and mix with 50 mL of sulfuric acid (25% V/V). Cover the Erlenmeyer with a watch glass and place on heating until boiling. Let it boil for an hour, shaking. After 1 hour boiling, add 150 mL of boiling water. Filter the product on a filter paper and also wash the watch glass. Wash the filter paper until the pH no longer changes. Place the filter paper on a glass and allow to dry for 1 hour in oven (T = 103 °C). Remove from the oven and prepare the filter paper in an extraction cartridge. Remove all traces of gum using a cotton cloth, previously washed with chloroform and dried. Place the filter paper and cotton on the cartridge. Weigh a dry extraction flask with the boiling regulators; add 200 mL of cyclohexane and place to extract for 8 hours. After extraction, evaporate the solvent and allow the flask to dry in the oven for 1 hour; after that time allow it to cool in the air. Check the weight until it does not change by more than 0.1%. Microwave assisted extraction method (MAE) The method is based on an acid hydrolysis simultaneous to a solvent extraction on a closed Teflon vessel. MAE was carried out using an ETHOS-X advanced microwave system with a 12 samples rotor, (Milestone, Bergamo, Italy), each vessel withstanding up to 30 bar of pressure. The temperature of the extraction is measured and controlled on one reference vessel by fiber optic temperature control, and the microwave heating power is adjusted automatically by the system, in order to follow the developed temperature program. 12

Comparison of a New Total Fat Quantiﬁcation Method in Cheese using Microwave Assisted Extraction (MAE) and Soxhlet

Article

The sample (2.0 g) was weighed in a Teflon vessel and mixed with sulfuric acid 25% (10.0 mL). Cyclohexane (25.0 mL) was added to the Teflon vessel, and accurately weighed. A Teflon coated stirring bar was placed inside the vessel and all vessels were closed and positioned inside the rotor of the microwave unit. The MAE was run with a temperature program with a ramp designed to reach 125 ºC in 4 minutes and then holding that temperature for another 40 minutes. During the whole program, magnetic stirring (120 rpm) was applied, in order to enhance extraction and mixing. The maximum microwave power applied was 1200 W, automatically adjusted in order to follow the so mentioned temperature program. After irradiation, the closed Teflon vessels were allowed to be automatically cooled to ambient temperature by forced air flow inside the microwave oven for 30 min. After this, the vessels were carefully opened. Aliquots of 10 mL of the top layer (organic phase) in the vessels were carefully removed and transferred to aluminum cups and accurately weighed. The 12 cups were positioned inside an evaporation rotor (Rar-400 rotor) with Weflon® supports, where heating and vacuum conditions apply for each cup. The ® Weflon supports help to heat, whilst the system is under vacuum, in order to facilitate the fast and temperature controlled solvent evaporation, on a designed heating program of 30 minutes at 105 ºC. The cups were removed from the rotor, allowed to cool, and weighed in order to get gravimetrically the total fat results. RESULTS AND DISCUSSION Eight different identified and labeled cheese samples were submitted to MAE and Acid HydrolysisSoxhlet extraction methods. Ten replicates were made for each sample. The results of the total fat content obtained after MAE and Soxhlet procedures are shown in Table I. Table I. Results of total fat content provided by MAE and Soxhlet methods (n = 10)

Sample Ref.

MAE

Soxhlet

30.21 ± 0.80

29.40 ± 0.36

24.92 ± 1.38

26.21 ± 2.61

24.95 ± 1.02

25.47 ± 0.37

25.89 ± 2.05

27.22 ± 2.48

28.52 ± 1.03

28.00 ± 1.53

27.96 ± 1.35

28.80 ± 1.69

28.62 ± 1.74

31.25 ± 0.15

29.23 ± 1.29

29.72 ± 1.17

Total Fat in g/100 g ± SD

Statistical analysis The experimental design and statistical analyses were carried out using the Nordtest approach [14]. Over the obtained results for the different methods MAE and Soxhlet (AOAC based method), the following was observed: Method Validation A one-tailed t-test was used to compare the means of related (paired) samples in order to evaluate if the proposed method, MAE, yields results at the 95% confidence level, similar to the Soxhlet reference extraction method. The observed differences between MAE and Soxhlet methods were not significant (see Table II). The calculated t-value was compared with the theoretical value at alpha = 0.05 and 7 degrees of freedom, i.e. 2.365. As the calculated value (i.e. 1.869) is smaller than the theoretical value (2.365). Therefore, the excellent agreement found between the sets of results testifies the applicability of the proposed method. 13

Pombal, S.; Lourenço, J. G.; Rocha, P.; Ettlin, D.; Rodilla, J. M.

Article Table II. Validation test: Soxhlet method vs MAE method

Sample Ref.

XaN-XbN

-0.81

1.29

0.52

1.33

-0.52

0.84

2.63

0.49

Degrees of freedom

SD (XaN-XbN)

0.010910023

Texp.

Tcrit.

1.869

2.365

Total Fat in g/100 g

LOD and LOQ Determination As the methodology proposed is essentially a gravimetric method, we determined the limit of detection (LOD) and limit of quantiﬁcation (LOQ) through successive weighing of the cups after solvent evaporation. The mean of the variation of recorded masses (0.0015 g), the associated standard deviation (0.0047) and the mass of the cup (19.4750 g), allowed the calculation of the LOD and LOQ as follows: LOD (g) = δ + 3.3*s = 0.0169 gandLOD (%) = [LOD(g)/m(cup)]*100 = 0.087% LOQ (g) = δ + 10*s = 0.048 gandLOQ (%) = [LOQ(g)/m(cup)]*100 = 0.248% The LOD for total fat determination, calculated as the mass which gives a signal that is 3σ of the mean blank signal (where σ is the standard deviation of the blank signal), was 0.087%. The LOQ, calculated as the mass which gives a signal 10σ above the mean blank signal, was 2.248%. The blank was prepared with cyclohexane instead the samples, with previous weighting of the cups, evaporating the solvent and posterior weighting of the cups. Uncertainty Determination The determination of the uncertainty (expanded uncertainty) was based on the determination of the uncertainties associated with veracity (ubias) and precision (uprec). For the ubias estimation, a Reference material (MR) acquired from MUVA KEPTEN (muva-HA-1511 Hard Cheese) with total fat values of 24.40 g/100 g and expanded uncertainty (U) of ± 0.19 g/100 g was used. The NORDTEST approach was used and 6 trials were performed against the RM, obtaining a ﬁnal ubias of 0.49564%. For the estimation of uncertainty associated with precision, a comparison was made between the duplicate assay values (uprec = 0.16%) and control values against the control standard (stabilized cheese sample), with uprec = 1.23%, using Equation 1 for calculated value. The worst estimate was calculated for the combined uncertainty (uc) calculation, giving a value of 1.32% and the Expanded Uncertainty (U) value of U = 2.65%, using Equation 2 for calculated value.

CONCLUSIONS With the results obtained, we can conclude that the two methodologies are equivalent, that is, the statistically correspondence between the two methods is veriﬁed. The MAE method compared to the Soxhlet method represents an optimal alternative for the determination of the total fat in cheese, because the MAE is a procedure that has a much easier and faster handling than Soxhlet. Additionally, the MAE method showed good performance indices with low LOQ, LOD and U values. 14

Comparison of a New Total Fat Quantiﬁcation Method in Cheese using Microwave Assisted Extraction (MAE) and Soxhlet

Article

There are some clear advantages of using this MAE procedure, in comparison with the Soxhlet methods. The MAE process has much better handling and therefore prone to fewer errors. There are also smaller amounts of solvent involved, which is excellent for environment purposes. The methodology is also faster, not only because of its extraction times, but also because two steps - hydrolysis and extraction - can be combined at the same time.

Article

Manuscript received July 28, 2017; revised version received Jan. 15, 2018; accepted Jan. 18, 2018.

REFERENCES 1. Regulation (EU) Nº 1169/2011 of the European Parliament and of the Council. 25 October 2011. 2. Bligh, E. G.; Dyer, W. J. Can. J. Physiol. Pharm, 1959, 37, pp 911–917. 3. Soxhlet, F. V. Dingler’s Polytech. J., 1879, 232, pp 461–465. 4. Folch, J.; Lees, M; Stanley, S. J. Biophys. Chem., 1957, 226, pp 497–509. 5. Costa, D. d. S. V.; Bragagnolo, N. Eur. J. Lipid Sci. Technol., 2017, 119: n/a, 1600108. doi:10.1002/ejlt.201600108. 6. AOAC International. AOAC Official Method 996.06. Fat (Total, Saturated, and Unsaturated) in Foods - Hydrolytic Extraction Gas Chromatographic Method. Official Methods of Analysis of AOAC International, 18th ed. 2005. Gaithersburg, Maryland, USA. 7. Servaes, K.; Maesen, M.; Prandi, B.; Sforza, S.; Elst, K. J. Agric. Food Chem., 2015, 63, pp 3931– 3941. 8. Priego-Capote, F.; Luque de Castro, M. D. Talanta, 2005, 65, pp 98–103. 9. Castro, M. D. L; Priego-Capote, F. J Chromatogr A., 2010, 1217, pp 2383-2389. 10. Shin, J. M; Hwang Y. O.; Tu O. J.; Jo H. B.; Kim J. H.; Chae, Y. Z.; Rhu, K. H.; Park S. K. Food Chem., 2013, 13, pp 703-709. 11. Luque de Castro, M. D.; Valcárcel, M.; Tena, M. T. Analytical Supercritical Fluid Extraction. SpringerVerlag, Berlin, Heidelberg, 1994. 12. ElKhori, S.; Paré, J. R. J.; Bélanger, J. M. R.; Pérez E. J Food Eng, 2007, 79, pp 1110–1114. 13. Paré, J. R. J.; Bélanger, J. M. R. Microwave-assisted process (MAP™) principles and applications. In: Paré, J. R. J. & Bélanger, J. M. R. (Eds). Instrumental Methods in Food Analysis, v. 18 of Techniques and Instrumentation in Analytical Chemistry Series. Elsevier Science, Amsterdam, 1997, Chapter 10, pp. 395–420. 14. Magnusson, B.; Naykki, T.; Hovind, H.; Krysell, M. Nordtest, NT Tech Report TR 537, 2003, 52p. NT Project Nº 1589-02.

Br. J. Anal. Chem., 2017, 4 (17), pp 16-23

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Study of Geographical Variation in Phyllanthus amarus Schum & Thonn using DART-TOF-MS combined with PCA Sunil Kumar1, Vikas Bajpai1,2, Mukesh Srivastava2, Brijesh Kumar1,2* Sophisticated Analytical Instrument Facility, CSIR-Central Drug Research Institute, Lucknow-226031, Uttar Pradesh, India 2 Academy of Scientiﬁc and Innovative Research (AcSIR), New Delhi-110025, India

A rapid and versatile direct analysis in real time-time of flight-mass spectrometry (DART-TOF-MS) method was developed for chemical fingerprinting of P. amarus. Developed DART-TOF-MS method was applied to analyze nine samples collected from Uttar Pradesh (UP), West Bengal (WB) and Madhya Pradesh (MP). DART-TOF-MS fingerprinting data was analyzed using principal component analysis (PCA). Ten chemical markers from nine samples were obtained which were able to discriminate among the samples. The maximum variation was observed in the UP samples followed by WB and MP. Total 16 compounds included alkaloids and lignans were tentatively identified based on their exact mass measurement, molecular formula and literature reports. Distribution of these identified phytochemicals was also studied amongst nine samples. The developed DART-TOF-MS method and chemical markers may be used for authentication and quality control in future. Keywords: Chemical fingerprinting; Phytochemicals (Alkaloids/Lignans); Chemical Markers INTRODUCTION Phyllanthus amarus Schum & Thonn is a well known medicinal plant belonging to the family Euphorbiaceae. It is widely distributed throughout tropical and subtropical countries all over the world [1]. It is reported in Indian System of Medicine and Chinese Traditional Medicine since ancient times for the treatment of stomach problems, genitourinary system, liver, kidney and spleen [2]. P. amarus has been reported to exhibit hepatoprotective, antidiabetes, antiinflammatory, anticancer, diuretic, antioxidant, antiviral, larvicidal, antimicrobial, antibacterial, antihypoglycemic, antihypercholesterolemic and anti-HIV activities [1-3]. It is rich source of lignans, tannins (ellagitannins), triterpenes, sterols and alkaloids [4-6]. These phytochemicals especially lignans may be responsible for the therapeutic potential of the plant [7, 8]. There is no reliable and high throughput chemical fingerprinting method available to assess phytochemical variations. High performance thin layer chromatography (HPTLC) [9-14], high performance liquid chromatography (HPLC) [10, 15], gas chromatography mass spectrometry (GC-MS) [16], liquid chromatography mass spectrometry (LC-MS) [17-25] and nuclear magnetic resonance (NMR) [26] have been used previously for phytochemical analysis. These methods require chromatographic separations, sample preparation, extraction and isolation [27, 28]. So, the reported analytical methods are tedious, time-consuming and costly. Direct analysis in real time-mass spectrometry (DART) is an established ambient ionization technique for the rapid and direct analysis of samples with minimal or no sample preparation without chromatographic separation [29]. Recently, DART-MS followed by multivariate analysis were used for discrimination of plant part/species [30-35], cultivars [34-37], and detection of adulteration [40]. DART-TOF-MS combined with principal component analysis (PCA) is an efficient and high throughput method that allows studying variation in samples. Principal component analysis (PCA) is one of the most widely used multivariate techniques useful in dealing with large data sets for discrimination. PCA can also be used to construct a loadings plot that indicates variables responsible for discrimination among the samples. Scores and loadings plots are complementary and superimposable [41, 42]. 16

*brijesh_kumar@cdri.res.in; gbrikum@yahoo.com ORCID: 0000-0001-7148-1137

Study of Geographical Variation in Phyllanthus amarus Schum & Thonn using DART-TOF-MS combined with PCA

Article

The aim of this study was to develop a DART-TOF-MS method for rapid chemical fingerprinting of P. amarus samples collected from three different locations (states) of India namely UP, WB and MP. Chemical fingerprints combined with PCA were used to identify chemical markers for geographical variation. MATERIALS AND METHODS Plant material The aerial parts of P. amarus were collected from three different locations during years 2009-2011 from the districts Kolkata (WB), Lucknow (UP) and Jabalpur (MP) in India. All the specimens were deposited in the Medicinal Plant Herbarium of CSIR-CDRI, Lucknow (India) (Supplementary data - Table S1). The plant material was washed with tap water followed by shade dried at room temperature (26–28 ºC) according to Maity et. al. [19]. Dried whole plants were crushed into powder using grinding machine (Decibel, Lab Willey Griender, Model No. DB 5581-4, New Delhi, India). Powder was stored at room temperature (2628 ºC) in tight air containers until analysis. DART-MS conditions JMS-100 TLC (AccuTof ) (Jeol, Tokyo, Japan) mass spectrometer was used at atmospheric pressure ionization with a DART ion source. The mass spectrometer was operated in positive-ion mode with a resolving power of 6000 (full-width at half-maximum). The orifice 1 potential was set to 28 V, resulting in minimal fragmentation. The ring lens and orifice 2 potentials were set to 13 and 5 V, respectively. Orifice 1 was set at 100 °C. The RF ion guide potential was 300 V. The DART ion source was operated with helium gas flowing at approximately 4.0 L/min. The gas heater was set to 300 °C. The potential on the discharge needle electrode of the DART source was set to 3000 V; electrode 1 was 100 V and the grid was at 250 V. Powdered plant samples positioned in the gap between the DART source and mass spectrometer with the help of a glass capillary for fifteen time measurements of each sample. Data acquisition was from m/z 50-1000. Exact mass calibration was accomplished by including a mass spectrum of neat polyethylene glycol (PEG) (1:1 mixture PEG 200 and PEG 600) in the data file. The mass calibration was accurate to within ±0.002 u. Using the Mass Center software, the elemental composition could be determined on selected peaks. Principal component analysis PCA was performed with the STATISTICA software, Windows version 7.0 (Stat Soft, Inc., USA). Data for PCA were extracted from the fifteen repeats of nine samples of chemical fingerprint with the relative abundance ≥5%. RESULTS AND DISCUSSION Chemical fingerprints The representative chemical fingerprint spectra of P. amarus samples UP, WB and MP are shown in Figure 1 (A-I). Chemical fingerprints of all samples were approximately similar with difference in their relative abundance. Sixteen compounds were tentatively identified from chemical fingerprints on the basis of their exact mass measurement, calculated molecular formula and literature reports are shown in Table I and Figure 2. Peaks 1, 2, 3 and 4 identified as phenazine (m/z 181), norsecurinine (m/z 204), securinine (m/z 118) and dihydrosecurinine (m/z 220), respectively were alkaloids. Peaks 5-16 were identified as lignans. Peak 14 and 15 could be phyllanthin (m/z 419) and hypophyllanthin (m/z 431), respectively. The m/z 261, 387 and 436 were abundant peaks but could not be identified due to insufficient data and literature reports. All the three peaks were observed in all the samples. Similarly phyllanthin and hypophyllanthin were detected in all the samples of WB. The distribution of compounds is shown in Table I.

Kumar, S.; Bajpai, V.; Srivastava, M.; Kumar, B.

Article

Figure 1 (A-C). Comparative DART-MS chemical ďŹ ngerprint (spectra) of P. amarus samples (UP) in positive ionization mode.

Figure 1 (D-F). Comparative DART-MS chemical ďŹ ngerprint (spectra) of P. amarus samples (WB) in positive ionization mode.

Study of Geographical Variation in Phyllanthus amarus Schum & Thonn using DART-TOF-MS combined with PCA

Article

Figure 1 (G-I). Comparative DART-MS chemical fingerprint (spectra) of P. amarus samples (MP) in positive ionization mode. Table I. List of identified compounds from chemical fingerprint of P. amarus using DART-MS in positive ionization mode Calculated + m/z [M+H]

Observed m/z + [M+H]

Error (ppm)

Molecular Formula

Compound Name

181.0766

181.0762

2.20

C12H8N2

204.1019

204.1014

2.45

218.1176

218.1179

220.1338

S.Nº

Phenazine

C12H13NO2

Norsecurinine

1.37

C13H16NO2

Securinine

220.1340

-0.90

C13H17NO2

Dihydrosecurinine

341.1025

341.1018

2.05

C19H16O6

Virgatyne

355.1176

355.1171

1.40

C20H18O6

Hinokinin

359.1489

359.1482

1.95

C20H22O6

Pinoresinol

363.1802

363.1807

-1.37

C20H26O6

Secoisolariciresinol

371.1495

371.1484

2.96

C21H22O6

Dextrobursehernin

385.1651

385.1659

-2.08

C22H24O6

Urinatetralin

395.1125

395.1122

0.76

C22H18O7

Phyllamyricin E

395.1131

395.1128

0.76

C22H18O7

Justicidin A

401.1964

401.1955

2.24

C23H28O6

Lintetralin

419.2428

419.2439

-2.62

C24H34O6

Phyllanthin

431.2070

431.2065

1.16

C24H30O7

Hypophyllanthin

433.2226

433.2222

0.92

C24H32O7

Niranthin

(+) Detected; (-) Not detected; 1: 2009; 2: 2010; 3: 2011; MP: Madhya Pradesh; UP: Uttar Pradesh; WB: West Bengal

Kumar, S.; Bajpai, V.; Srivastava, M.; Kumar, B.

Article

Figure 2. Chemical structures of identiﬁed compounds from DART-TOF-MS chemical ﬁngerprint of P. amarus.

Principal component analysis: Discrimination PCA was applied on DART-MS data obtained from 15 repeats of each sample to develop a PCA model for geographical variation. Twenty five peaks (m/z 126, 130, 135, 247, 261, 262, 277, 279, 293, 296, 355, 367, 369, 387, 397, 401, 418, 419, 430, 436, 437, 450, 506, 544, and 854) were selected from chemical ingerprint to study discrimination among the samples. Finally, the peaks (m/z 126, 130, 247, 261, 262, 277, 296, 355, 387, 397, 418, 419, 430, 506, and 544) with low contributions were dropped during PCA score calculation. Hence only 10 peaks (m/z 135, 279, 293, 367, 369, 401, 436, 437, 450 and 854) showed considerable contribution for possible discrimination. PCA score is shown in Figure 3A and 3B. On the basis of these marker peaks first two principal components PC1 and PC2 hold 38.63% and 27.36% variability, respectively. Others PC3-PC10 showed variability, ranged from 12.55-0.66%. The peak at m/z 135 (38.62%) having higher cumulative effect followed by m/z 279 (27.35%) and m/z 293 (12.55%). Peaks at m/z 387, 401, 436, 437, 450 and 854 were contributed for PC1 score while the remaining peaks m/z 135, 279, 293, and 367 contributed for PC2 score. The samples from MP were close and placed in the same quadrant. The samples obtained from WB in year 2011 and 2009 were placed in same quadrant whereas samples 2010 was much apart. The maximum variation was observed in the samples of UP during all the three years 2009, 2010 and 2011. The variation observed in UP-2009 samples was due to peaks at m/z 279, 387 and 854 whereas peaks m/z 401, 436, 437 and 450 were responsible for variation in samples UP-2010. Similarly, peaks at m/z 279 and 436 influenced the samples UP-2011. 20

Study of Geographical Variation in Phyllanthus amarus Schum & Thonn using DART-TOF-MS combined with PCA

Article

Figure 3. (A) Loadings; (B) Scores 10 peaks from DART-MS ﬁngerprint of P. amarus.

Biosynthesis of secondary metabolites depends on different causes such as attack of herbivores and environmental conditions [45-49]. There are several reports on medicinal plants show geographical variations due to climatic conditions [50], seasons [51-53] or altitude and latitude [54-55]. Khan et al., 2010 [56] have also reported biosynthesis of phyllanthin in P. amarus growing at different altitudes. Therefore the variation of phytochemicals in samples may be due to environmental changes only in UP during the assessment years 2009-2011. CONCLUSION Rapid and simple DART-MS method was developed for chemical fingerprinting of P. amarus. Ten chemical markers were identified by PCA which were able for discriminated P. amarus samples. Sixteen phytochemicals included alkaloids and lignans were tentatively identified. All chemical markets may be used for quality control of P. amarus derived herbal drugs. Manuscript received Sept. 30, 2017; 1st corrected version received Jan. 24, 2018; 2nd corrected version received Feb. 26, 2018; accepted Feb. 27, 2018. CDRI communication number is 9673.

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Study of Geographical Variation in Phyllanthus amarus Schum & Thonn using DART-TOF-MS combined with PCA

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42. Bajpai, V.; Singh, A.; Arya, K. R.; Srivastava, M.; Kumar, B. Food Addit. Contam. Part A, 2015, 32, pp 799-807. 43. Vidal, R.; Ma, Y.; Sastry, S. S. Principal Component Analysis. In: Generalized Principal Component Analysis, Springer, New York, 2016, pp 25-62. 44. Li, J.; Linear, R. R. Principal component analysis. Springer Series in Statistics, New York, 2014, Chapter 9, pp 163-183. 45. Bennett, R. N.; Wallsgrove, R. M. New Phytol. 1994, 127, pp 617-633. 46. Figueiredo, A. C.; Barroso, J. G.; Pedro, L. G.; Scheffer, J. J. Flavour Fragr. J. 2008, 23, pp 213-226. 47. Neilan, B. A.; Pearson, L. A.; Muenchhoff, J.; Mofﬁtt, M. C.; Dittmann, E. Environ. Microbiol. 2013, 15, pp 1239-1253. 48. Treutter, D. Environ. Chem. Lett. 2006, 4, pp 147–157. 49. Treutter, D. Plant Biology 2005, 7, pp 581-591. 50. Kumar, S.; Yadav, A.; Yadav, M.; Yadav, J.P. BMC Research Notes 2017, 10, pp 1-12. 51. Han, W.; Chen, X.; Yu, H.; Chen, L.; Shen, M. Food Chem. 2018, 251, pp 110-114. 52. Paciﬁco, S.; Galasso, S.; Piccolella, S.; Kretschmer, N.; Pan, S. P.; Marciano, S.; Bauer, R.; Monaco, P. Food Res. Int. 2015, 69, pp 121-132. 53. Bajpai, V.; Kumar, S.; Singh, A.; Singh, J.; Negi, M. P. S.; Bag, S. K.; Kumar, N.; Konwar, R.; Kumar, B. Phytochem. Anal. 2017, 28, pp 277-288. 54. Chauhan, R. S.; Nautiyal, M. C.; Cecotti, R.; Mella, M.; Tava, A. Ind. Crops Prod. 2016, 94, pp 401404. 55. Wang, X.; Liu, P.; Wang, F.; Fu, B.; He, F.; Zhao, M. Emir. J. Food. Agric. 2017, 29, pp 359-366. 56. Khan, S.; Al-Qurainy, F.; Ram, M.; Ahmad, S.; Abdin, M.Z. J. Med. Plants Res. 2010, 4, pp 041-048. Supplementary data Table S1. Plant materials of P. amarus used in this study

Collection Date Month-Year 08-2009

08-2010

08-2011

07-2009

07-2010

07-2011

12-2009

12-2010

12-2011

S. Nº

State name (Location)

Specimens Nº

Latitude

Longitude

UP (Lucknow)

24513

26º52'24.92" N

80º52'26.74" E

WB (Kolkata)

24515

22º262'1.14" N

88º24'09.21" E

MP (Jabalpur)

24519

23º11'33.71" N

79º55'52.63" E

Br. J. Anal. Chem., 2017, 4 (17), pp 24-36

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Cloud Point Extraction Spectrophotometric Method for Determination of Three Types of Cephalosporin via Diazotization Reactions with Different Reagents Mohammed Jasim M. Hassan* and Marwah Sabbar Falih Al-Rubaiawi Department of Chemistry, College of Science, University of Al-Mustansiriyah, Baghdad, Iraq A sensitive spectrophotometric approach was developed for the determination of three types of cephalosporins, which are ceftriaxone (CFT), cefotaxime (CFX), and cefepime (CFM). This includes several steps; the ﬁrst step involves the formation of azo dye through the reaction of CFT, CFX, and CFM diazonium salt with β-naphthol, phenol, and resorcinol in alkaline medium, where measurements are collected at λmax = 545, 500, and 515 nm respectively. Beer's law is obeyed over the concentration range of 2.5 to 62.5 mg L-1 and the limits of detection and molar absorptivities for CFT, CFX and CFM are 0.194, -1 4 5 4 -1 -1 0.211, 0.198 mg L , and 0.98x10 , 0.1x10 , and 0.12x10 L mol cm respectively. The second step was the extraction of CFT and CFX via cloud point with 1.0 mL of Triton X-114 10% (v/v). Cloud point extraction allows the drugs to be accurately estimated under the optimized experimental conditions. The concentration was range between 0.25 and 6.0 mg L-1, limit of detection and molar absorptivity for CFT, -1 5 6 -1 -1 CFX at 0.053, 0.025 mg L and 6.0x10 and 0.1x10 L mol cm , while the respective preconcentration factors were 25.0, 25.0, the respective enrichment factors were 6.0, 11.0, and the respective distribution ratios were 78.0, and 198.0. The proposed method was validated and applied to estimate the CFT, CFX and CFM in pharmaceutical formulations. Keywords: ceftriaxone, cefotaxime, cefepime, visible spectrophotometry, cloud point extraction. INTRODUCTION Cephalosporins consist of a fused β-lactam-dihydrothiazine two-ring system known as 7-amino cephalosporanic acid (7-ACA) and vary in their side chain substitute at C3 (R2), and C7 (acylamido, R1) [1]. Cephalosporins have a β-lactam ring structure (Figure 1) that interferes with the synthesis of the bacterial cell wall. Cephalosporins of ceftriaxone CFT, cefotaxime CFX and cefepime CFM are used for the treatment of infections caused by Gram (+) and Gram (−) bacteria [2,3]. The chemical formula and calculated molecular weight for CFT is C18H18N8O7S3 and 554.58 g mol-1; (6R,7R)-7-((E)-2-(2-aminothiazol4-yl)-2-(methoxyimino)acetamido)-3-(((2-methyl-5,6-dioxo-1,2,5,6-tetrahydro-1,2,4-triazin-3-yl)thio)methyl) -8-oxo-5-thia-1-azabicyclo[4,2,0]oct-2-ene-2-carboxylic acid is a third-generation cephalosporin antibiotic. Like other third-generation cephalosporins, it has broad-spectrum activity against Gram-negative and Gram-positive bacteria [4,5]. CFX is a third-generation cephalosporin with a broad antibacterial spectrum and it is resistant to β-lactamases [6]. CFX is 7-[2-2-amino-4-thiazolyl)glyoxylamido]-3-(hydroxymethyl)-8oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate-72(Z)-(o-methyloxime) acetic acid [7]. CFM is (6R,7R,Z)-7-(2-(2-aminothiazol-4-yl)-2-(methoxyimino)acetamido)-3-((1methylpyrrolidinium-1-yl)methyl)-8oxo-5-thia-1-aza-bicyclo[4.2.0]oct-2-ene-2carboxylate [8]. It is a fourth-generation cephalosporin antibiotic that has widely been used as a broad spectrum antibiotic, and is effective against both Gram-positive and Gram-negative bacteria [9]. Watanabe and Tanaka ﬁrst introduced cloud point extraction (CPE) in 1978 as a somewhat green extraction technique that, instead of organic solvents, uses surfactants as the extractant [10]. Nowadays, CPE uses a non-ionic surfactant, which has attracted considerable attention as an alternative to conventional extraction techniques for separation and preconcentration [11,12]. CPE has been used to *dr.moh2004@uomustansiriyah.edu.iq ORCID: 0000-0002-9919-185X

Cloud Point Extraction Spectrophotometric Method for Determination of Three Types of Cephalosporin via Diazotization Reactions with Different Reagents

Article

extract some drugs like mefenamic acid, oxazepam, and paracetamol from human urine, and then determination by spectrophotometry [13]. Several methods have been reported for the determination of cephalosporin. The methods involve spectrophotometry [14,15], ﬂuorimetry [16], capillary electrophoresis [17], ﬂow analysis [18], electrochemical methods [19,20], and high-performance liquid chromatography [2]. In this regard, new simple and sensitive methods for the determination of CFT, CFX, or CFM, in pure solutions or in pharmaceutical formulations, are proposed. They are based on reactions of the amine groups in CFT, CFX, and CFM with β-naphtol, phenol, and resorcinol, respectively, in alkaline medium, followed by absorbance measurements at 545, 500, and 515 nm, respectively.

Figure 1. Chemical structure of cephalosporin and the three types of cephalosporin antibiotics.

MATERIAL AND METHODS Equipment The absorbance measurements were carried out in a model UV-1800 Shimadzu spectrophotometer equipped with a 1.0 cm quartz cell. Thermostated water bath, shaker, a model HI 83141 WTW pH meter (Germany), and Electronic Balance Mettle AE 200 (Germany) were used. Reagents All chemicals were of analytical grade and were purchased from Merck KGaA (Darmstadt, Germany). -1 They were used as received. Stock solutions (1000 mg L CFT, CFX, or CFM) were prepared by dissolving 0.1 g of drug in 100 mL of double distilled water. HCl, β-naphthol, phenol, and resorcinol, 50% w/v KOH, 1% w/v NaNO2, 4% w/v urea, 10% v/v Triton X-114, hexadecyltrimethylammonium bromide (HTAB), and 5% w/v Na2SO4 solutions were prepared in double distilled water. General procedure of diazotization reaction for drugs ceftriaxone, cefotaxime and cefepime -1 -1 An aliquot of a sample solution containing CFT, CFX, and CFM (2.5 − 62.5 mg L ) from 1000 mg L was transferred into a series of 20.0 mL calibrated ﬂasks and then cooled in an ice bath maintained at -4.0 ºC. -1 To this solution, 0.6 mol L of HCl followed by 1.0 mL for CFT, and 0.75 mL for CFX and CFM, from NaNO2 1.0% (w/v), were added to the mixture and left to stand for 20, 30, and 10 min for CFT, CFX, and CFM 25

Hassan, M. J. M.; Al-Rubaiawi, M. S. F.

Article respectively. Then, 1.0 mL of urea 4% (w/v) was added under shaking. The solution was further left to stand for a few minutes. Then, 1.0, 1.5, 1.5 mL of β-naphthol, phenol, and resorcinol were added to CFT, CFX, and CFM, while 0.7 mol L-1 of KOH solution was added sequentially to the mixture, and diluted to the mark with double distilled water. The azo dye that formed was monitored at λmax 545, 500, and 515 nm respectively. General procedure of cloud point extraction for drugs ceftriaxone and cefotaxime -1 Aliquots of the standard CFT and CFX solutions containing 0.25 − 6.0 mg L were prepared from 1000 mg L-1, placed into calibrated centrifuge tubes and prepared using the previous procedure. Then, 1.0 mL of Triton X−114 10% v/v, 2.0 mL of HTAB, and 2.0 mL of Na2SO4 5% w/v were added, After that, the tubes were manually shaken for 3 min and then placed in a water bath at 60 °C for 50 minutes. After the separation was complete, the tubes were transported to the ice bath for 10 min to increase the viscosity of the cloudy layer of the drug, while the decanted aqueous phase and the still cloud layer remained in the bottom of the tubes. The solutions were diluted using 0.5 mL of absolute ethanol. The obtained solutions were then directly measured using a UV-Vis spectrophotometer at λmax 545, 500 nm against a reagent blank that was prepared in the same manner. RESULTS AND DISCUSSION Absorption spectra Pink-violet-, orange-, and red-colored chromophores were formed through the coupling of diazotized CFT, CFX, and CFM with β−naphthol, phenol, and resorcinol in an alkaline medium respectively. These azo dyes were obtained with a maximum absorbance under optimized conditions, and were recorded at the wavelengths of 545, 500, and 515 nm, respectively, against the reagent blank solution, which has all the same additions as the samples except the drugs. The spectra are shown in Figures 2a, 2b and 2c.

Figure 2a. Absorption spectrum for 50 mg L-1 CFT with the reagent against the reagent blank under optimum conditions.

Cloud Point Extraction Spectrophotometric Method for Determination of Three Types of Cephalosporin via Diazotization Reactions with Different Reagents

Article

Figure 2b. Absorption spectrum for 50 mg L-1 CFX with the reagent against the reagent blank under optimum conditions.

Figure 2c. Absorption spectrum for 50 mg L-1 CFM with the reagent against the reagent blank under optimum conditions

Study of the optimum reaction conditions for the Batch method The effect of various parameters on the absorption intensity was studies for azo dye. The criteria for selecting the best analytical parameters were determined with the best absorbance for the product. The effect of acids Different concentrated acids, H2SO4, HCl, HNO3, and CH3COOH, were tested for diazotization reactions with concentrations of 0.92, 0.59, 0.82, and 0.82 mol L-1 for each respective acid in 20 mL. Among these, HCl was found to be the best because of its highest absorbance values and stability regarding diazotization. Table I shows the results. Table I. Effect of the acid VS Absorbance with 50 mg L-1 of CFT, CFX, or CFM. Type of acid

Absorbance CFT

Absorbance CFX

Absorbance CFM

HCl

0.523

0.755

0.642

H2SO4

0.401

0.456

0.525

CH3COOH

0.231

0.572

0.346

HNO3

0.393

0.622

0.507 27

Hassan, M. J. M.; Al-Rubaiawi, M. S. F.

Article Effect of the hydrochloric acid concentration The effect of acidity on the color development was studied using various concentrations of HCl that ranged from 0.148 – 0.892 mol L-1, and ﬁnal concentrations that ranged from 0.14 – 1.19 mol L-1. These -1 investigations showed that 0.6 mol L of HCl yielded absorption increases as the acid concentration increased, but the absorptivity decreased suddenly because the primary amine became inactive.

Effect of the sodium nitrite volume The effect of increasing the volume of the NaNO2 solution was studied by standardizing the absorbance of the color products at 545, 500, and 515 nm and in the volume range of 0.25 − 2.0 mL. 1.0 mL for CFT, and 0.75 mL for CFX and CFM were used. The increase caused the high proportion of contaminants that affected the formation of the salt diazonium. Effect of the reaction time after the addition of sodium nitrite The effect of the time reaction was tested by using different times that ranged from 10 – 60 min. 20, 30, and 10 min for CFT, CFX, and CFM, respectively, were long enough to obtain maximum absorption values. Effect of the urea volume The excess of nitrous acid was removed using urea. The effect of urea was tested by using different volumes (0 – 4.0 mL) of a urea solution (4% w/v). It was found that 1.0 mL of urea (4% w/v) give the highest absorbance value, and is enough to remove the excess of nitrite.

The effect of bases The effect of different bases, 50% w/v KOH, NaOH, Na2CO3, and NH4OH, on the color intensity of azo dye has been tested. The ﬁnal concentrations of each basic solution of 20 mL are respectively, 0.66, 0.09, 0.35, 1.07 mol L-1. The results, as shown in Table II, reveal that the colored azo dyes need a strong basic medium, and that the KOH solution gave the highest intensity among all the azo dyes. Table II. Effect of the base VS Absorbance with 50 mg L-1 of CFT, CFX, and CFM; HCl (0.6 mol L-1); 1.0 and 0.75 mL of NaNO2 (1% w/v); 1.0 mL of urea (4% w/v). Type of base

Absorbance CFT

Absorbance CFX

Absorbance CFM

KOH

0.589

0.839

0.735

NaOH

0.511

0.798

0.715

Na2CO3

0.451

0.621

0.541

NH3

0.393

0.523

0.623

Effect of the potassium hydroxide concentration The effect of different KOH concentrations on the absorbance were studied for the concentration range -1 -1 of 0.111 – 0.778 mol L . The ﬁnal concentrations ranged from 0.11 – 0.89 mol L of 20 mL. The investigations showed that 1.5 mL of potassium hydroxide gave a maximum absorbance; therefore, 0.7 mol L-1 was chosen for the method. It is clear that a further increase in the alkalinity will lead to subsequent deprotonation to form diazotates that are non-duplicating. 28

Cloud Point Extraction Spectrophotometric Method for Determination of Three Types of Cephalosporin via Diazotization Reactions with Different Reagents

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Effect of the reagent concentration The effect of the reagent concentrations for Î˛-Naphthol, phenol, and resorcinol were tested by using different concentrations that ranged from 3x10-5 â&#x20AC;&#x201C; 2x10-3 mol L-1. The investigations showed that 6x10-4 mol L-1 obtained the highest color intensity for the azo dye as shown in Figure 3. Above this concentration, the absorbance remained unchanged.

Figure 3. Effect of the reagent concentration VS Absorbance with 50 mg L-1 of CFT, CFX, and CFM; HCl 0.6 mol L-1; 1.0 and 0.75 mL of NaNO2 (1% w/v); 1.0 mL of urea (4% w/v); 0.7 mol L-1 KOH.

The possible reaction path may be written as follows:

Scheme 1. The proposed reaction for the formation of azo dye from CFT, CFX, and CFM.

Hassan, M. J. M.; Al-Rubaiawi, M. S. F.

Article Analytical curves obtained using the Batch method for ceftriaxone, cefotaxime, and cefepime Under the optimized experimental conditions, analytical curves were obtained through a series of standard solutions (2.5 − 62.5 mg L-1) for CFT, CFX, and CFM as shown in Figure 4.

Figure 4. Analytical curves obtained using the Batch method with 50 mg L-1 of CFT, CFX, and CFM; HCl 0.6 mol L-1; 1.0 and 0.75 mL of NaNO2 (1% w/v); 1.0 mL of urea (4% w/v); 0.7 mol L-1 KOH.

Table III. The analytical parameters obtained using the Batch method with 50 mg L-1 of CFT, CFX, and CFM; HCl 0.6 mol L-1; 1.0 and 0.75 mL of NaNO2 (1% w/v); 1.0 mL of urea (4% w/v); 0.7 mol L-1 KOH. Parameter

CFT

CFX

CFM

λmax (nm)

545

500

515

2.5- 50

2.5- 62.5

2.5- 60

-1

Linearity range (mg L ) -1

-1

Molar absorptivity (? Ɛ L mol cm )

0.1x10

0.12x10

Y=0.0134x+0.0073

Y=0.0189x+0.0099

Y=0.0172x+0.0166

0.073

0.056

0.500

0.9996

0.9994

0.9988

0.640

0.698

0.652

0.194

0.211

1.198

0.0134±0.0008

0.0189±0.00036

0.0172±0.001

0.0073±0.02

0.0099±0.0118

0.0166±0.346

Repeatability (RSD, n = 5) %

0.65

0.42

0.33

Recovery (n = 5) %

100.2

100.5

101.2

Regression equation -2

Sandell sensitivity (S μg cm ) 2

Correlation coefficient (R ) -1

Limit of quantification (mg L ) -1

Limit of detection (mg L ) C.L. for the slope (b±tsb) at 95% C.L. for the intercept (a±tsa) at 95%

0.98x10

Cloud Point Extraction Spectrophotometric Method for Determination of Three Types of Cephalosporin via Diazotization Reactions with Different Reagents

Article

Optimization of cloud point extraction for the determination of ceftriaxone and cefotaxime The effect of various parameters on the separation and extraction of CFT- and CFX-based drugs and their absorbance intensity were investigated, such as through the pH, incubation time (Et), heating temperature (T), and the amount and type of salt using a volume of 10% v/v Triton X−114 and HTAB. The criteria for selecting the best analytical extraction parameters were determined by the best absorbance for the product that gave the optimum drug concentration in the organic phase. Because the extraction technique for CFM, at cloud point, did not yield any results, the cloud layer did not appear due to the chemical structure of the drug's zwitter ion. The effect of pH Cloud point extraction depends on the pH at which the azo dye forms. The extraction of CFT and CFX using the CPE method involves extracting CFT and CFX in small volumes (in the bottom of the tube). The pH plays a main role in azo dye formation and its subsequent extraction, and it proved to be a main parameter in the CPE method. The extraction method used pH values that ranged from 4 to 14. This showed that a pH of 12 was the best value. The effect of incubation time The heating time at the optimum temperature was very necessary for the cloud point extraction of CFT and CFX; the experiment was performed with heating times that ranged from 10 – 60 min. This study showed that a time of 50 min was the best value. The effect of temperature To ensure the efﬁcient separation and preconcentration of CFT and CFM, an optimal equilibration temperature is a very crucial parameter for achieving complete separation while keeping other parameters at optimum conditions. The temperature was changed from 10 to 70 ºC, and it was observed that 60 ºC was the optimal temperature. The effect of the type of electrolyte salt The effects of the type of electrolyte salt were examined under optimized other conditions. The type of electrolyte salt was varied, such as: NaCl, CaCl2, CH3COONa, NaNO3, and Na2SO4 (all at 5% w/v), in search of the optimum value. The results are shown in Table IV, and reveal that Na2SO4 (5% w/v) yielded the maximum value. Table IV. Absorbance VS salt type on the CPE of CFT and CFX (5.0 mg L-1); pH = 12 (aqueous solution); CPE time = 50 min; T = 60 ºC. Salt (5% w/v)

Absorbance CFT

Absorbance CFX

NaCl

0.211

0.331

CaCl2

0.123

CH3COONa

0.345

0.785

NaNO3

0.332

0.775

Na2SO4

0.384

0.883

Effect of the Na2SO4 volume Different volumes of Na2SO4 (5% w/v) were investigated, and 2.0 mL of this solution showed the optimum amount of CFT and CFX due to their complete separation and extraction, and maximal absorbance. The salt increases the dryness of the surface-rich phase. It works on the growth of the micelles, reduces the temperature of the cloud point and increases the efﬁciency of the cloud point extraction.

Hassan, M. J. M.; Al-Rubaiawi, M. S. F.

Article Effect of the TritonX−114 volume A successful cloud point extraction procedure should maximize the extraction efﬁciency by minimizing the extracted layer using different amounts of non-ionic surfactant. Triton X−114 (10% v/v) was investigated with a volume range from 0.5 mL to 3.0 mL. The investigation showed that 1.0 mL was the optimal amount due to providing the best separation and extraction of CFT and CFX, and it also give the maximum absorbance. It was found that the volume of 1.0 mL yielded the maximum extraction efﬁciency by reducing the size of the cloud point layer and then by improving the preconcentration factor. Effect of the hexadecyltrimethylammonium bromide volume -1

Volumes of HTAB (0.01 mol L ) ranged from 0.5 to 3.0 mL. The results showed that 2.0 mL was the optimum volume due to providing the best separation and extraction of CFT and CFX, and it also gave the maximum absorbance. It was found that the volume of 2.0 mL gave the maximum extraction efﬁciency by reducing the size of the cloud point layer and then by improving the preconcentration factor. Analytical curve for the cloud point extraction Under the optimized experimental conditions, analytical curves were obtained for CFT and CFX drug -1 -1 concentrations ranging from 0.25 to 6.0 mg L and from 0.5 to 6.0 mg L respectively. The analytical curves are presented in Figure 5, and the linearity, regression equation, coefﬁcient of determination r2, and other analytical parameters are presented in Table V.

Figure 5. Analytical curve for CFT and CFX (5.0 mg L-1); pH = 12 (aqueous solution); CPE time = 50 min; T = 60 ºC; 2.0 mL of Na2SO4 (5% w/v); 2 mL of TritonX−114 (10% v/v); 2.0 mL of HTAB.

Cloud Point Extraction Spectrophotometric Method for Determination of Three Types of Cephalosporin via Diazotization Reactions with Different Reagents

Article

Table V. Analytical parameters of the CPE for CFT and CFX (5.0 mg L-1); pH = 12 (aqueous solution); CPE time = 50 min; T = 60 ºC; 2.0 mL of Na2SO4 (5% w/v); 2 mL of TritonX−114 (10% v/v); 2.0 mL of HTAB. Parameter

CFT

CFX

545

500

λmax (nm) -1

Linearity range (mg L )

0.25- 6.0 -1

-1

0.5- 6.0

Molar absorptivity (?Ɛ L mol cm )

6x10

0.1x10

Y=0.0823x-0.0239

Y=0.1955x-0.059

0.010

0.0054

0.9992

0.9988

Analytical sensitivity (mg L )

0.500

0.575

-1

0.176

0.084

Limit of detection (mg L )

0.053

0.025

Preconcentration factor PF

25.0

25.00

Enrichment factor EF

6.0

11.400

Distribution ratio D

78.0

198.00

C.L. for the slope (b±tsb) at 95%

0.0823±0.0016

C.L. for the intercept (ab±tsa) at 95%

0.0239±0.0053

0.46

0.70

101.44

101.22

Regression equation -2

Sandell sensitivity (S μg cm ) 2

Coefficient of determination (r ) -1

Limit of quantification (mg L ) -1

Repeatability (RSD, n = 5) % Recovery (n = 5) %

Accuracy and precision of the proposed methods The accuracy and precision of the proposed methods were examined by analyzing ﬁve replicates of three different concentrations. The accuracy was estimated by determining the relative error, and the precision was determined by the relative standard deviation (RSD%). These speciﬁc results indicate an accuracy and sensible precision for the suggest method as shown in Table VI and Table VII. There is no interference between these three types of drug because each drug was studied independently. Table VI. Accuracy and precision of the Batch method with 50 mg L-1 of CFT, CFX, and CFM; HCl 0.6 mol L-1; 1.0 and 0.75 mL of NaNO2 (1% w/v); 1.0 mL of urea (4% w/v); 0.7 mol L-1 KOH. Cephalosporin

Ceftriaxone

Cefotaxime

Cefepime

Amount of Drug mg L Taken Found

-1

Relative Error %

Recovery %

RSD%*

2.5

2.54

1.97

101.97

0.33

17.91

-0.48

99.52

0.047

49.89

-0.2

99.78

0.017

2.5

2.59

3.898

103.89

3.2

20.1

0.53

100.5

0.425

62.5

62.43

-0.097

99.9

0.134

2.5

2.45

-1.4

98.59

3.7

20.23

1.162

101.16

0.326

60.25

0.4

100.4

0.15

RSD%**

0.13

1.253

1.392

* The average of 5 determinations. ** The average of 15 determinations. 33

Hassan, M. J. M.; Al-Rubaiawi, M. S. F.

Article Table VII. Accuracy and precision of the CPE for CFT and CFX (5.0 mg L-1); pH = 12 (aqueous solution); CPE time = 50 min; T = 60 ºC; 2.0 mL of Na2SO4 (5% w/v); 2.0 mL of TritonX−114 (10% v/v); 2.0 mL of HTAB Cephalosporin

Amount of Drug mg L

Ceftriaxone

Cefotaxime

-1

Relative Error %

Recovery %

RSD%*

Taken

Found

0.25

0.248

-0.48

99.52

3.31

3.04

1.44

101.44

0.35

5.95

-0.71

99.29

0.14

0.5

0.524

4.8

104.8

1.598

3.036

1.225

101.225

5.967

-0.51

RSD%**

1.6

0.7

0.83

0.189

99.458

* The average of 5 determinations. ** The average of 15 determinations.

Table VIII. Batch method used to determine CFT, CFX, and CFM in pharmaceutical preparations. Cephalosporin

Ceftriaxone injection 1.0 g Pharma Roth

Cefotaxime injection 1.0 g Pharma Roth

Cefepime injection 1.0 g Turkey iLAC A.S.

Taken -1 mg L

Found -1 mg L

Relative Error %

Recovery %

RSD%*

10.33

3.3

103.3

0.13

25.04

0.15

100.1

0.03

37.94

-0.15

99.85

0.25

19.85

-0.767

99.44

0.421

29.53

-3.5

96.5

0.289

39.63

-0.912

99.08

0.213

19.86

-0.7

99.3

0.56

25.05

0.18

100.2

0.42

40.15

0.378

100.38

0.26

RSD%**

0.14

0.31

0.41

*The average of 5 determinations. ** The average of 15 determinations

Table IX. Application of the CPE method to the determination of CFT and CFX in pharmaceutical preparations. Cephalosporin

Ceftriaxone injection 1.0 g Pharma Roth

Cefotaxime injection 1.0 g Pharma Roth

Taken -1 mg L

Found -1 mg L

Relative Error %

Recovery %

RSD%*

3.0

2.99

-0.33

99.66

1.23

4.0

3.91

-2.16

97.84

0.98

5.0

4.88

-2.27

97.71

0.78

2.5

2.43

-2.4

97.6

0.3

3.5

3.56

0.597

100.596

0.267

4.5

4.48

1.86

101.86

0.225

*The average of ﬁve determinations. ** The average of 15 determinations.

RSD%**

0.99

0.264

Cloud Point Extraction Spectrophotometric Method for Determination of Three Types of Cephalosporin via Diazotization Reactions with Different Reagents

Article

Table X. Comparison of the proposed method with some spectrophotometric methods. Type of reagent

Linearity -1 mg L

Limit of detection -1 mg L

Recovery%

RSD%

Ref.

Q uercetin

80 – 400

41.86

100.3

----

p-dimethyl amino benzaldehyde

5 – 25

0.0217

99.6

< 2.0

Folin-Ciocalteu

2 – 36

----

99.3 – 101.4

± 0.983

naphthoquinone-4-sulfonic (NQS)

0.2 – 1.2

0.0465

97.09 – 99.3

----

Eriochrome black-T

10 – 30

0.154 0.07

100.21

0.260

triphenyltetrazolium chloride (TTC)

10 – 50

1.097

99.43 – 100.42

----

Hg2(NO3)2

3.6 – 40

1.2

100.19

1.451

CONCLUSION In this study, a fast, economical, and easy-to-operate method is presented, which is based on micro cloud point extraction for the preconcentration and determination of three types of cephalosporins, CFX, CFT, and CFM that react with β-naphthole, phenol, and resorcinol, respectively, to form an azo dye in alkaline medium, and are measured at λmax 545, 500, and 515 nm, respectively. This is the ﬁrst report for the determination of these drugs with the cloud point procedure. Triton X-114 was used as a non-ionic and green extractant solvent. In comparison to similar methods of extraction, CPE is less expensive, more environmental friendly and faster. In this paper, we coupled our CPE method with a spectrophotometer equipped with microcells. Therefore, we successfully determined these drugs in pure and pharmaceutical formulations by use of spectrophotometric instrumentation that is simple, fast, inexpensive, and mostly available in common laboratories th

Manuscript received May 22, 2017; 1 revised version received July 21, 2017; 2 revised version received Sept. 10, 2017; manuscript accepted Sept. 19, 2017.

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Analitica Latin America Conference and Expo discussed important issues for the Development of Science and Technology in Analytical Chemistry The Analitica Latin America Conference and Exposition 2017 took place between September 26 and 28 of 2017, at the Convention Center São Paulo Expo, São Paulo, SP, Brazil.

Opening of the 5th Analitica Congress Photo: Analitica Latin America

This was the 5th edition of the Analitica Latin America Conference and had, as the main objective, the interaction of the academic environment with the industrial sector. "The aim of this scientiﬁc meeting is to bring knowledge and expertise to the professionals of the area, since the chemical sector is one of the main axes of the economy that form the Brazilian GDP, whether as a cosmetics, pharmaceutical, petrochemical or agronomic industry. We hope to contribute to the growth of this sector”, said NürnbergMesse Brazil president João Paulo Picolo. In order to receive more than 300 congressmen, Prof. Dr. Pedro Vitoriano Oliveira, the meeting chairman, from the Institute of Chemistry - University of São Paulo (IQ-USP), João Paulo Picolo, President of NürnbergMesse Brazil, and Wolfgang Kranz, International Vice President of NürnbergMesse Germany were present. The programming grid was well received by the participants. Researchers, professors and professionals who were present participated in lectures and debates with unpublished contents and found future perspectives for their projects and applications. Some of these researchers spoke with BrJAC, such as Prof. Dr. Ronaldo Censi Faria, from the Federal University of São Carlos (UFSCar), who presented a lecture titled "Detection of Biomarkers for the Early Diagnosis of Diseases". The researcher presented his research project and development of ultra-sensitive immunoassays, based on the use of nanomaterials, for the detection of biomarkers. Congressmen in the auditorium where the lectures were presented - Photo: Analitica Latin America

The topics covered in the 5th edition of the Analytical LA Conference were: life sciences, contaminants and essential elements in food, electrochemistry and electroanalytical, energy, ethanol and biofuels in general, atomic and molecular spectrometry, mass spectrometry, analytical instrumentation, samples, chemometrics and separation techniques. These topics were discussed throughout the three days of the conference, through 21 lectures presented by internationally renowned researchers.

Feature The development of low-cost analytical instrumentation - including screen-printing disposable electrodes, paper-based sensing devices and microfluidic devices - for the detection of biomarkers at low blood concentrations can positively impact public health costs. The aim of this study, carried out in partnership with Barretos Cancer Hospital (Barretos, SP, Brazil), is to make an early diagnosis of diseases to anticipate their treatment. Using cancer as an example and as a driving illness of their research, Prof. Ronaldo Faria drew attention to the late diagnosis in patients: "Nowadays, the identification of cancer is made through the manifestation of symptoms in the patient, which can end up happening late. In Brazil, we have fewer identified cases of tumors compared to the United States and Europe. However, mortality in our country is much higher", he explains. "With this project, it is possible to identify the manifestation of the tumor in the early stages, facilitating treatment and fighting the cases most prone to death", he concluded. Prof. Dr. Maurício da Silva Baptista, from the Institute of Chemistry-University of São Paulo, presented a lecture titled "Manipulation of nanoparticles and their applications for sun protection". In the presentation, Prof. Mauricio Baptista used a chart classifying the wavelength ranges of sunlight as 'bad' and 'good' and explained that the purpose of his research is to make the 'bad' side include visible light, which is also harmful. "Until recently people were unaware of the risks of UVA rays - it was a discovery. Now they need to know that the visible rays are also harmful and that many cases of skin cancer start from this type of exposure", argued Prof. Baptista. The Analitica LA Conference program also had sessions of specific discussions by the Brazilian Society of Toxicology (SBTox), the Brazilian Society of Mass Spectrometry (BrMASS) and the Brazilian Society of Food Science and Technology (sbCTA). In these sessions, lectures were also presented with contents of the highest importance and quality, given by researchers who are at the most advanced frontiers of world scientific development. A total of eleven lectures were offered in these three specific sessions. Another great highlight of the program at the Analitica LA Conference was the presence of two foreign researchers, who were invited to join the so-called Pittcon Session. They were: Prof. Dr. Steven Ray of the State University of New York, Buffalo, USA, who presented the lecture entitled "Distance of Flight Mass Spectrometry and Solid State Ion Detectors"; and Dr. Jhanis Gonzalez of the Lawrence Berkeley National Laboratory, who presented the lecture entitled "Laser Ablation-Based Techniques: An Ideal Toolbox for Direct Chemical Analysis of Solids". It is worth remembering that it was Analitica Latin America's great initiative to enter into a partnership with the Pittcon Conference and Expo. This partnership includes the participation of foreign researchers in the Analitica LA Conference, as well as the presentation of a Brazilian Session in Pittcon, which allows showing the world the development of science in Brazil. At Pittcon 2018, a Symposium on Analytical Chemistry in Brazil, titled "Development of Omics Sciences in Brazil" will be presented. The presentation of lectures presented by internationally renowned researchers did not stop here. Congressmen and Analitica LA Expo visitors were also able to participate in the Circuit of Knowledge and Innovation, which approached diverse topics, ones current and directed at different areas of activity. Circuit of Knowledge and Innovation The Analitica LA Conference and Exposition, this important and already established meeting point of professionals in the area of analytical chemistry, presented the 3rd edition of the Circuit of Knowledge and Innovation, a place that promotes relevant and qualified content, debates and the exchange of experiences among visitors, speakers and exhibitors. The Circuit of Knowledge and Innovation covered the following parallel activities: LiveLab, Space NANOSolutions, Q-Lounge, I Symposium on Analytical Innovation, and poster sessions.

Feature LiveLab

LiveLab in activity - Photo: Analitica Latin America

The entire power grid, gas, exhausts and furniture infrastructure in the traditional LiveLab was assembled in just three days, and it worked perfectly. The laboratory pleased the participants, with live demonstrations of theory in practice. A gas chromatograph coupled to a mass spectrometer (GCMS), a comprehensive two-dimensional gas chromatograph (GCxGC), liquid chromatograph (UHPLC), ion chromatograph (HPIC), plasma optical emission spectrometer (ICP OES) and microwave sample digestor were installed. Six parallel experiments were carried out in parallel, for 340 participants, during the three days of the event.

NANOSolutions Space In the new NANOSolutions space, set up in partnership with the Nano Trade Show, in addition to the content, visitors found products for nanotechnology applications, which were presented in lectures. The NANOSolutions space received two speakers that addressed how nanotechnology can be applied in various fields of industry, such as cosmetics, pharmaceuticals, textiles or food. The Prof. Dr. Koiti Araki, from the Institute of Chemistry - University of São Paulo (IQ-USP), and Eduardo Caritá, from the Mikron Functional Company, made complementary presentations, each one in its area of expertise. Dr. Araki presented projects that are currently being tested at IQ-USP and explained how they can help improve the lives of the population, such as nanotechnology-based functional pigments that can be used in clothing to control odor or help to inhibit allergies. "I think it is very important to bring this subject to the public because nanotechnology is at the frontier of what is most advanced in knowledge and it is important that it be discussed. The cool thing here is to join the academy with the private production sector and demonstrate that together we can make many strides in this area. Only when we leave our comfort zone can we accomplish much greater things, "said Koiti Araki. Eduardo Caritá spoke about nanotechnology in the food industry, emphasizing the regulatory phase that, Congressmen attending lecture in the new according to him, is still a taboo. On the application NANOSolutions space - Photo: Analitica Latin America of nanotechnology, he exemplified its presence in aromas, product consistencies, reduction of waste and in aid to analytical techniques, as in the control of the use of pesticides in foods. "With nanotechnology, it is possible to increase the area of application and in turn, reduce contamination and environmental impact," Caritá said. During the three days of the fair, visitors could have access to the best in nanotechnology content, as well as having 12 booths exclusively for companies in this segment. A total of 16 lectures were presented by members of the academy and companies in the sector, on topics such as development tools, medicinal applicability, materials and techniques of manipulation and analysis, all aimed at the nano universe. Q-Lounge In the Q-Lounge space, the pharmacist and university professor Dr. Ana Lúcia Jacques Faria promoted a deep and dynamic reflection on the necessary changes in the corporate world. With the use of songs, 39

Feature excerpts from films and poems, with artists such as Elis Regina, Charles Chaplin and Cora Coralina, she invited the public to reflect on the need for changes in organizational forms in the face of the strong changes taking place around the world in the job market. The space had 13 lectures from six companies, with topics focused on biological protection, material hygiene, validation rules, organizational scenario, data integrity, and manipulation and analysis techniques. Promoting discussions and exchanges of experiences between visitors and exhibitors, the Q-Lounge pleased everyone by addressing current issues. I Symposium on Analytical Innovation This Symposium, held in partnership with the Brazilian Academy of Pharmaceutical Sciences (ACFB), aimed to present the development stage of clinical, pharmacological and toxicological researches, and of the analytical methodologies of drugs, cosmetics, food, and health products in general. Information was presented on legislation and incentives for research in the field of control analysis. Academics and experts delivered unique and innovative lectures on innovation for health professionals. Prof. Dr. Lauro D. Moretto, President Emeritus of the ACFB, curated this symposium. Posters with scientific works

Exhibition of posters with scientiﬁc works Photo: Analitica Latin America

As usual, in addition to lectures, this edition of the Analitica LA Conference had an area for a poster exhibition on studies developed in universities, research centers and industries, as a way to disseminate information and to increase academicindustrial interactions. The presentation of scientiﬁc works on posters allowed the congressmen the opportunity to discuss various research topics, and to expand and generate a new vision about the content presented at the congress. In total, 109 posters of scientiﬁc works were presented, of which, 16 were chosen for oral presentation in the program of the Analitica LA Conference.

The poster evaluated as the best work presented at this session of the Conference was that of Alan Lima Vieira, entitled "Laser-induced electric discharge as a strategy to improve sensitivity in the determination of phosphorus in fertilizers by LIBS". Alan L. Vieira is a PhD student in the postgraduate program in chemistry of the Institute of Chemistry - São Paulo State University (UNESP) and is guided by Prof. Dr. José Anchieta Gomes Neto, from the same university. The award grants the doctoral student registration, accommodation and airfare to the Pittcon Conference and Expo 2018, which will take place from February 26 to March 1 in Orlando, FL, USA. Pittcon is the world's leading annual conference and exposition on laboratory science. The conference was born in Pittsburgh in the year 1950 and attracts more than 16,000 attendees from industry, academia and government from over 90 countries worldwide. Pittcon's target audience is not just 'analytical chemists' but all laboratory scientists - anyone who identifies, quantifies, analyzes or tests the chemical or biological Aerial view of Pittcon Conference and Expo properties of compounds or molecules, or who Photo: Disclosure manages these laboratory scientists. With more than 2,000 technical sessions, Pittcon makes it easy to connect researchers and the latest research and developments in their field, and present a global perspective from leading scientists around the world. 40

Feature With satisﬁed visitors, congressmen and exhibitors, NürnbergMesse Brazil's portfolio director, Diego Carvalho, made a positive analysis of the event. "Analitica Latin America, for us, is a product of extreme importance, because we know how it helps in the integration of several sectors. The analytical chemistry sector suffered from the crisis that still affects Brazil, and for this reason, the Analitica LA Conference & Expo has become an even more important platform for business and communication. We are always looking for ways to reinvent ourselves and continue to thrive", he said.

Analitica Latin America Exposition Even in a difficult year in Brazil for the industry, with historic cuts in the budget of public and private laboratories, the 14th edition of Analitica Latin America overcame the difficulties, showed its strength and presented a surprising result. Consolidated as the best business platform and content source in the industry, Analitica Latin America 2017 had a 10% increase in visitation compared to the last edition, held in 2015. More than 500 brands of exhibitors and the main representatives of the market attended the exhibition, which was held in an area of around 14,000 m². There were three busy days of lectures, debates, presentations of new solutions, crowded booths and diversified attractions. The final result caught the attention of exhibitors, even for the oldest ones, and was considered very positive.

Entry into the exhibition area of Analitica Latin America - Photo: Analitica Latin America

Among the companies present in the 14th edition of Analitica Latin America were: Agilent Technologies Agilent presented three major innovations in the Brazilian market, one of them being Intuvo 9000 Gas Chromatograph (GC). Built with advanced technologies, the Intuvo 9000 streamlines the GC workﬂow with a simpliﬁed operator experience to reduce operational and maintenance costs often found with conventional GC systems. Another was Ultivo Triple Quadrupole LC/MS, which presents a transformative approach to LC/TQ and is the newest member of the Agilent Triple Quadrupole family. Analitica Latin America exhibition area - Photo: São Paulo Expo

At the core of Ultivo lie the Agilent's Triad Technologies (Tt) - Vortex Collision Cell, Cyclone Ion Guide, and VacShield. These innovations afford technicians the sensitivity, robustness, reliability, and performance required for the day-in, day-out challenges of high-throughput sample analysis in the applied markets. And the third was the 1260 Infinity II LC system that features latest technology in HPLC, delivers highquality results for an affordable price, and is the flexible instrument choice for operational efficiency. The 1290 Infinity II LC embodies the next generation of liquid chromatography with ultrahigh performance for superior outcomes. For more information on Agilent technologies, visit www.agilent.com 41

Feature Allcrom has been operating for more than 28 years in the Brazilian market and currently provides more than 16 thousand items among equipment, consumables and accessories. Its objective is always to offer the best chromatography products and services to laboratories. At Analitica Latin America 2017, Allcrom presented the following novelties: Progeny portable Raman analyzer, Leman's gas generator running in real time with analysis being performed at the booth without the need for gas cylinders, supercritical extraction from Applied Separation, dissolutants Labindia, peristaltic and vacuum pumps, GC columns for rapid The exhibition was attended by more than 500 analysis of fatty acids (FAMEs) and immobilized chiral exhibiting brands - Photo: São Paulo Expo columns, both from Phenomemex. For more information on Allcrom technologies, visit www.allcrom.com.br

Allcrom

Veolia Water Technologies Veolia brought to Analitica Latin America the newest water purification solution: Purelab Chorus. The company launched the biggest project in ELGA LabWater's seventy five year history. Purelab Chorus is the first modular water purification system designed to fit individual laboratory applications, budgets and configurations. It delivers all grades of purified water and provides a scalable, flexible, customized solution, offering a selection of dispensing, storage and installation options. Modular elements can be positioned independently or combined to minimize footprint. The innovative modular chassis, with its requirement to load-bear high volumes of water and fit multiple components, improved the user experience. The visually iconic design elements, such as ambient communication via the Halo Dispensers, help to differentiate the ELGA units from those of competitors. Veolia also highlighted other ELGA technologies such as: Purelab Ultra, Purelab Classic, Purelab Flex, Purelab Option-Q, Purelab Micra, Purelab Pulse, Centra 200 and Centra 60. For more information on Veolia technologies, visit www.veoliawatertech.com Waters Technologies Waters presented Xevo TQ-S micro, a triple quadrupole mass spectrometer with high performance from a compact design with rapid, reliable and reproducible quantitative data. Revolutionary design has produced a tandem quadrupole instrument with a small footprint that delivers consistent low levels of quantitation with a wide dynamic range. Waters presented also the Xevo G2-XS QTof, which was designed for the scientist who needs to identify, quantify and confirm the broadest range of compounds in the most complex and challenging samples. X e v o G2 - X S p r e s e n ts m a x i m u m r o b u s tn e s s with no compromise in performance, class-leading real-world quantitative sensitivity, highest quality, most comprehensive qualitative information, flexibility to adapt to changing needs, and it is accessible to experts and non-experts alike. For more information on Waters technologies, visit www.waters.com 42

The ﬁnal result of Analitica Latin America attracted the attention of the exhibitors, even the oldest ones, being considered very positive Photo: Waters Technologies Brazil

Feature Thermo Fisher Scientific At Analitica Latin America, Thermo Fisher Scientific has unveiled new solutions from its portfolio of instruments, consumables, software and services that make complex analysis more accessible. Some of the Thermo Fisher's highlights were: The new Nicolet™ iN™ 5 FTIR microscope, which was designed with point-and-shoot simplicity to provide fast identification of unknown materials and contaminants that may affect product quality. Nicolet™ iN™ 5 FTIR has the same robustness and quality already established in the Nicolet™ iN™10 FTIR. Both feature optical setup allows you to simultaneously view your sample while collecting chemical information which guarantees the data is from the area you are viewing. ISQ™ EC Single Quadrupole Mass Spectrometer, which achieves limits of detection (LOD) in the single-digit-parts-per-billion (ppb) range. For analyses requiring peak confirmation, the ISQ EC MS provides mass-to-charge (m/z) confirmation for separated compounds to give confidence in the results. The Thermo Scientific TSQ Triple Quadrupole LC-MS Systems, which offer segmented quadrupole analyzers with high resolution single reaction monitoring (SRM), while their enhanced detectors achieve optimal performance across a wide mass range in every application domain. The TSQ Altis™ achieve ultimate sensitivity and robustness without compromise, and enables every analytical laboratory to successfully address its most demanding applications. The TSQ Quantis™ meets every targeted quantitation workflow requirement with an ease-of-use that ensures quality data from every user, every molecule, and every analytical lab. For more information on Thermo Fisher Scientific technologies, visit www.thermofisher.com Nova Analitica Nova Analitica also presented its new line of products from the acquisition of Anacom Scientific's business. The product line of Milestone Srl, which includes microwave digestion, extraction and synthesis systems, microwave ashing furnace, acid distillers for the production of ultra-pure acids for spectrometry, and direct mercury analyzer was presented. Also presented were the new electron microscopy line, which includes desktop Scanning Electron Microscopy products manufactured by PhenomWorld, and Atomic Force Microscopy manufactured by Park Systems. Nova Analitica also unveiled its complete line of chromatography and spectrometry consumables sold through a direct sales channel and an online store.

For more information about Nova Analitica products, visit www.analiticaweb.com.br The new Convention Center São Paulo Expo was an excellent option made by the organization of Analitica Latin America th

In order to access the other companies that were present at the 14 Edition of Analitica Latin America Exposition visit www.analiticanet.com.br/en/exhibitors The 14th edition of Analitica Latin America Expo also had two international pavilions, one from Germany with 12 exhibitors and the other from China with 32 exhibitors.

Feature In addition to the growth in the number of participants, the public was qualiﬁed, diversiﬁed and attentive to the main innovations of analytical chemistry, such as cosmetics, pharmaceutical, food, agribusiness, among others. Friedel Nimax, a business manager of NürnbergMesse Brazil, celebrated the achievement and was very optimistic for the next edition of this event. "In 2017 we delivered a new fair, with a strong investment in quality knowledge. We have been able to bring together the main players in the market. We are sure that in 2019 the success will be even greater", he said.

The 14th edition of Analitica Latin America overcame difﬁculties, showed its strength and closed the year with a surprising result - Photo: São Paulo Expo

The Analitica Latin America Conference & Exposition next edition is already scheduled, from September 24 to 26, 2019, at the Convention Center São Paulo Expo, São Paulo, SP, Brazil.

Br. J. Anal. Chem., 2017, 4 (17), pp 45-47

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International Institute of Chromatography offers Specialization Courses in several areas of Chromatography The International Institute of Chromatography (IIC), founded on August 24, 2004, has as its main goal of disclosing chromatography and related techniques, in all of its forms. For this, IIC periodically offers update courses on chromatography. It also holds scientiﬁc congresses, such as COLACRO (Latin-American Congress of Chromatography and Related Techniques) and SIMCRO (Brazilian Symposium on Chromatography and Related Techniques). The IIC is also responsible for publication of the scientiﬁc journal Scientia Chromatographica (ISSN 1984-4433). "The activities of the IIC involve the participation of several Brazilian and foreign professionals, who have extensive knowledge and experience, both in the development of the technique, equipment, columns and other accessories, as well as in their applications" explained the founder of the Institute, Prof. Dr. Fernando Mauro Lanças.

Prof. Dr. Fernando Lanças giving a class in International Institute of Chromatography – Photo: I.I.C/Facebook In addition to the courses and events, IIC participates in research and development projects funded by research promotion agencies, such as the São Paulo Research Foundation (FAPESP), the National Council for Scientific and Technological Development (CNPq) and others. It also has a partnership with The Mediterranean Separation Science Foundation and with several companies and research centers in Brazil and abroad. In 2011, the Institute established a new teaching standard in the area of chromatography and related techniques: the Mikhael Tswett School of Chromatography. The School of Chromatography consists of a s et of events, which aim to give the users of chromatography and its related techniques (mass spectrometry, sample preparation, electrophoresis, etc.) highly qualified training, consisting of courses, lectures, workshops, discussions, laboratory practical sessions, etc. These events are organized to encompass state-of-the-art knowledge in the separation technique and correlates. All courses of the School of Chromatography contain theoretical and practical classes coordinated and offered by professionals of great practical experience in the area. At the end of each course, IIC delivers a diploma of course completion for all students. The creation of the School of Chromatography is of extreme importance for the area, since, according to Prof. Lanças, the undergraduate curriculum of chemistry colleges does not cover this discipline, and sometimes only superficially presents it. 45

Feature Therefore, IIC fulﬁlls the role of assisting in the speciﬁc training of a large number of professionals to be absorbed by both academic and corporate sectors. "Often, IIC receives Doctoral and Master's degree students from prestigious universities, who come to the courses in order to complement both the theoretical and practical training received at the institution of origin" said Lanças.

IIC prepares the student to work in any type of project and with easy adaptation to any brand and equipment model. Photo: I.I.C/Facebook

According to the professor, the IIC, in practice, does not compete with the equipment manufacturers, whose courses are more oriented to training on the operation of a speciﬁc instrument and software rather than to the fundamentals of the technique. Nor does it compete with universities, which offer a short introduction to the subject in their undergraduate or graduate courses. To carry out all of its courses, which include several specialties within the area of chromatography and the coupling of this technique with mass spectrometry, in addition to courses on sample preparation and others, IIC courses have about four gas chromatographs, four liquid chromatographs (including U-HPLC), an LC-MS/MS system and a GC-MS/MS system, all with automatic samplers and modern data-processing systems. In addition, IIC also has a sample preparation lab that employs traditional techniques and modern techniques, such as Solid Phase Micro-Extraction (SPME), Stir Bar Sorptive Extraction (SBSE), Micro Extraction by Packed Sorbent (MEPS) and others.

IIC offers courses in initiation, updating, and specialization in several areas of chromatography - Photo: I.I.C/Facebook 46

Feature Check the 2018 courses offered by IIC:

Month

Date

Discipline

Hours

March

14 – 16

Liquid Chromatography (HPLC)

April

18 – 20

Modern Liquid Chromatography

May

8 – 10

Modern Gas Chromatography

June

3–6

Liquid Chromatography coupled to Mass Spectrometry

August

22 – 24

Modern Liquid Chromatography

September

19 – 21

Modern techniques for sample preparation

November

27 – 30

Liquid Chromatography coupled to Mass spectrometry

December

5–7

Advanced Liquid Chromatography

Br. J. Anal. Chem., 2017, 4 (17), pp 48-52

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US EPA 3546 Milestone Srl, Sorisole, BG, IT This paper discusses the use of Ethos X Microwave Extraction System utilizing fastEX24 rotor with disposable glass vials to extract organic pollutants from certified soils during a recovery study following US EPA Method 3546. Samples were analyzed using GC-MS. Keywords: Microwave solvent extraction, sample preparation, priority pollutants, GC-MS, US EPA SW846 Method 3546. INTRODUCTION The United States Environmental Protection Agency's (USEPA) Test Methods for Evaluating Solid Waste (SW846) provides a comprehensive source of information on sampling, sample preparation, analysis, and reporting. US EPA 3546 is a Microwave-Assisted Solvent Extraction (MASE) procedure for extracting water insoluble or slightly water soluble organic compounds such as organochlorine pesticides, semivolatile organics, PAHs, PCBs, phenoxyacid herbicides, phenols, dioxins, and furans from soils, clays, sediments, sludges, and solid waste. This method was formally included in SW846 in 2008 [1] and most of these compounds have been identified by the US EPA as priority pollutants. MASE results in a rapid sample preparation technique that enables extractions with reduced amounts of solvents while working at higher temperatures and pressures. The process consists in a partitioning of the compounds of interest from the sample matrix into the solvent within a closed vessel. This accelerates the extraction process, yielding results equivalent to the standard Soxhlet method, but in a fraction of the time and using significantly less solvent. Milestone's new Ethos X benchtop microwave extraction system offers the ability to extract up to 24 samples simultaneously. With the new fastEX 24 rotor, Ethos X is fully compliant with US EPA 3546 (100115 °C and 50-150 psi). In addition, disposable glass vials can accommodate sample up to 30 g of sample if needed, thereby improving the limit of quantitation (LOQ) for analysis. This exceeds by far both the throughput and sample size capabilities of all the other automated techniques, such as pressurized fluid extraction. MASE also uses far less solvent than conventional Soxhlet extractions. This combination of performance and reduced solvent usage provides for the lowest cost per test of any technique available. Today, it is possible to process up to 30 g of sample, thanks to the introduction by Milestone of the largest ever 145 mL Weflon vessel in combination with the largest ever 100 mL disposable glass vials that fits inside the new fastEX 24 vessel. This vial improves productivity by providing inexpensive, disposable glass tubes that eliminate the need to clean vessels between batches. The synergy between the Weflon construction and the contactless temperature control in all positions ensure perfect temperature uniformity and make fastEX24 a unique and innovative solution for the extraction of contaminants from soils, providing unmatched ease of use and low running costs. MASE has definitely become the preferred technique used by the most analytical laboratories for priority pollutants. MATERIALS AND METHODS Instrument · Milestone Ethos X microwave system equipped with fastEX-24 extraction rotor · 100 mL disposable glass vials (PN GB00122) · Gas chromatograph with Mass Spectrometer detector (GC-MS) · Analytical balance · Vials for collection of extracts · Glass funnels for filtration · Glass fiber filters 48

Sponsor Report Standard and reagents Pesticide grade or grade solvents and chemicals must be used in all tests. Samples should be extracted using a solvent system that gives optimum, reproducible recovery of the analytes of interest from the sample matrix, at the concentration of interest. The choice of extraction solvent will depend on the analytes of interest. Generally the most applied solvent mixtures are acetone-hexane and acetone methylene chloride as recommended in the EPA analytical methods. Table I. Recommended solvents and EPA analytical methods by analyte of interest Analyte

EPA analytical method

Solvent mixture

Semivolatile Organics

8270

1:1 acetone– hexane or methylene chloride

PCBs

8082

1:1 acetone– hexane or methylene chloride

PAHs

8270, 8100

1:1 acetone– hexane or methylene chloride

Phenols

8151

1:1 acetone–hexane and phosphate buffer

Chlorinated Pesticides

8081

1:1 acetone– hexane or methylene chloride

Organophosphorus pesticides

8141

1:1 acetone–hexane and phosphate buffer

Chlorinated herbicides

8141

1:1 acetone– hexane or methylene chloride

Dioxins and Furans

Sodium sulfate anhydrous, silica gel and glass wool or paper ﬁlter were used in the work up procedure. According to the analytical method surrogate and internal standard could be used. Sample information The sandy loam soil standard reference material LGC6115 was used for the determination of PAHs and PCBs [2]. The certiﬁed standard reference mineral oil contaminated sediment sample BAM-U015b was used for the determination of TPH [3]. Analytical Procedure Samples, wet or dried and ground, were weighed directly into the 100 mL extraction disposable glass vials. An aliquot of the surrogate solution was added to the samples just prior to solvent addition then, the glass vials were closed. According to the moisture content, the best suitable built-in method was chose. The extraction procedure so described follows the detailed method provided by U.S. EPA SW-846 Method 3546. Table II. Suggested solvent volumes according to the used sample amounts Sample amount (g)

Solvent mixture (mL)

Up to 10

10-20

20-30

Sponsor Report Table III. Microwave Program Step

Time (min)

Power (W)

Temperature (°C)

up to 1600*

110

up to 1600*

110

*The power applied depends on the moisture content. Dedicated methods are pre-loaded in the ETHOS X software according to the moisture content.

After the extraction, samples were filtered on glass fiber filters and sodium sulfate anhydrous, and the vials were rinsed with additional solvent aliquots. Extracts and rinsates were collected together. Quantification PCBs and PAHs analyses of the soil extract were performed according to the following method. Injection was through a splitless injector in a GC-MS equipped with VF-17-MS 30 m × 0.25 mm i.d. capillary columns -1 with 5 m guard column. The injector was maintained at 280 °C. The injection was 2 μL at 2 mL min flow rate. The oven was hold at 80 °C for 2 min, from 80-300 °C at 20 °C min-1 than hold for 29 min at 300 °C. The detector worked with electron impact chemical ionization mass spectrometer. TPHs analyses of the soil extract were performed according to the UNI EN 16703 method. Injection was through on-column injector in a GC-FID equipped with Select Mineral Oil 15 m × 320 μm i.d. (film 0.1 μm) -1 columns. The injector was maintained at 320 °C. The injection was 1 μL with 2 mL min flow rate. The oven -1 was hold at 70 °C for 2 min, from 70-320 °C at 30 °C min . The FID detector was programmed at flow -1 -1 -1 rates of 400 mL min air and 30 mL min H2, make up 30 mL min He. RESULTS AND DISCUSSION Results from extractions of sandy loam soil and sediment sample are shown in Table IV through IX. The tables show the recovery and the RDSs (%) for PCBs, PAHs and TPH content of these matrices. Recoveries for all compounds are in the range 70-120% of the certified standard reference material. The results demonstrate the efficiency of the Ethos X as sample preparation method for the determination of contaminants. Ethos X provides extracts with the lowest solvent usage and significant time compared to all the other extraction technique. Table IV. PCBs recovery from 1 g sandy loam soil standard reference material (LGC6115) (n=4) Certified value -1 (mg kg )

Ethos X -1 (mg kg )

Recovery (%)

RSD (%)

PCB 101

1.75

PCB 118

116

4.94

PCB 138

0.2

PCB 153

3.2

PCB 180

9.6

104

2.6

PCB Congener

Table V. Semivolatile organics, TPH recovery from 1g certiﬁed standard reference mineral oil contaminated sediment sample (BAM-U015b) (n=4)

Analyte

Certified value -1 (mg kg )

Ethos X -1 (mg kg )

Recovery (%)

RSD (%)

TPH

920 ± 100

841.8

91.5

2.4

Sponsor Report Table VI. PAHs recovery from 1 g sandy loam soil standard reference material (LGC6115) (n=4) Certified value -1 (mg kg )

Ethos X -1 (mg kg )

Recovery (%)

RSD (%)

Phenanthrene

178 ± 6

200.72

113

4.52

Fluoranthene

312 ± 7

297.29

5.41

Benz[a]anthracene

36 ± 1

33.40

2.09

Benzo[a]pyrene

0.13 ± 0.02

0.16

123

11.5

Benzo[ghi]perylene

0.33 ± 0.06

0.25

0.3

Analyte

Table VII. Recovery of TPH from solid waste sample (1g) – Ethos X compared to Soxhlet extraction (n=4) Analyte

Soxhlet -1 (mg kg )

Ethos X (Recovery % of Soxhlet)

RSD (%)

TPH

11354 ± 122

111

5.2

Table VIII. Recovery of PCBs from solid waste sample (1 g) Ethos X compared to Soxhlet (n=4) Analyte

Soxhlet -1 (mg kg )

Ethos X (Recovery % of Soxhlet)

RSD (%)

PCB 28

4.09

5.2

PCB 52

3.70

4.8

PCB 95

2.46

6.2

PCB 99

1.40

3.1

PCB 101

3.18

2.6

PCB 105

1.22

6.4

PCB 114

0.07

7.3

PCB 118

2.68

2.0

PCB 123

0.07

114

5.6

PCB 126

0.16

118

4.2

PCB 128

0.55

3.4

PCB 138

1.79

8.3

PCB 146

0.25

116

6.2

PCB 151

0.17

105

7.4

PCB 153

1.46

6.1

PCB 156

0.29

110

7.9

PCB 157

0.10

100

6.5

PCB 169

0.45

104

3.1

PCB 170

0.41

2.2

PCB 180

0.36

7.7

PCB 183

0.20

2.3

PCB 187

0.35

100

5.3

PCB 189

0.21

114

4.7

PCB 77+149

2.37

4.9

PCB 81+110

7.03

6.1

Sponsor Report Table IX. Recovery (n=4) of PCDD and PCDF from sandy soil standard reference material BCR-529 (2 g) Certified value -1 (mg kg )

Ethos X -1 (mg kg )

Recovery (%)

RSD (%)

2,3,7,8-TCDD

4500±0.6

4236

3.4

1,2,3,7,8-PeCDD

440±0.05

515

117

2.8

1,2,3,4,7,8-HxCDD

1220±0.21

1298

106

3.1

1,2,3,6,7,8-HxCDD

5400±0.9

4610

2.1

1,2,3,7,8,9-HxCDD

3000±0.4

2522

1.9

2,3,7,8-TCDF

78±0.013

2.7

1,2,3,7,8-PeCDF

145±0.028

116

3.5

2,3,4,7,8-PeCDF

360±0.07

329

2.6

1,2,3,4,7,8-HxCDF

3400±0.5

3402

100

1.9

1,2,3,6,7,8-HxCDF

1090±0.15

1082

3.8

1,2,3,7,8,9-HxCDF

22±0.010

3.6

2,3,4,6,7,8-HxCDF

370±0.05

445

120

2.2

Analyte

CONCLUSIONS The ETHOS X enables simultaneous solvent extraction of up to 24 samples (from weighing to ﬁltration steps) in only 40 minutes. This in turns means that it is able to extract over 200 samples in 8 hours workday. Contamination, memory effects, and cleaning are completely eliminated due to the use of disposable glass vials. The use of contactless temperature control ensures high reproducibility and full recovery of the target analytes for full compliance with EPA 3546. Moreover, thanks to the unique design, ETHOS X ensures reliable extraction also on difﬁcult samples such as solid waste. The ETHOS X with all its unique features fully addresses the need of environmental laboratories in terms of productivity, ease of use, running costs, and extraction quality.

REFERENCES 1. Federal Register, Vol. 73, No. 2, Thursday, 3 January 2008, Notices, pages 486-489. 2. https://www.lgcstandards.com/DE/en/Soil-PCBs-and-PAHs/p/LGC6115# 3. https://www.lgcstandards.com/DE/en/Mineral-oil-contaminated-sediment/p/BAM-U015B

This sponsor report is the responsibility of Milestone Srl.

Br. J. Anal. Chem., 2017, 4 (17), pp 53-65

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Routine Quantitative Method of Analysis for Pesticides using GC Orbitrap Mass Spectrometry in accordance with SANTE/11945/2015 Guidelines Dominic Roberts1, Samanta Uclés Duque2, Amadeo Fernández-Alba2, Paul Silcock1 1 Thermo Fisher Scientific, Runcorn, United Kingdom 2 European Union Reference Laboratory for Pesticide Residues in Fruits and Vegetables, University of Almeria, Spain Quantitative performance of an Orbitrap™ mass spectrometer was evaluated for the routine analysis of GC-amenable pesticides in fruits and vegetables following SANTE/11945/2015 guidelines using full scan acquisition. The GC-MS system used provides routine high-mass resolving power up to 60,000 (m/z 200) full width at half maximum (FWHM) with scan speeds suitable for GC peaks to facilitate the detection of trace compounds in the presence of high matrix components. Keywords: Pesticides, QuEChERS, Complex matrices, GC Orbitrap Mass Spectrometry, Quantitation, Accurate Mass, TraceFinder. INTRODUCTION The international trade in food commodities has enabled a wide variety of fruits and vegetables to be made available year round. However, this also creates a challenge for food safety regulators who seek to ensure a safe food supply chain, particularly with regard to the potentially hundreds of different pesticides in use across the globe. The European Union (EU) has some of the most stringent pesticide residue regulations. In 2008, it implemented regulation EC No. 396/2005 [1], which sets default maximum residue levels (MRLs) at 10 μg/Kg for all pesticide/commodity combinations for which no substantive MRL had been set. Further to this, in 2009, the pesticide safety review EU 91/414/EEC [2] led to the approval of approximately 250 pesticides and effectively set the permissible level for all other pesticides to the default limit (10 μg/Kg). Recently, at the beginning of 2016, the latest version of the SANTE/11945/2015 guidance document on analytical quality control and validation procedures for pesticide residues in food and feed took effect [3]. This document describes the method validation and analytical quality control (AQC) requirements to support the validity of data reported within the framework of official controls on pesticide residues and used for checking compliance with maximum residue levels (MRLs), enforcement actions, or assessment of consumer exposure. It is intended for use by Official control laboratories in Europe, but in practice it is used by pesticide laboratories worldwide. Implementation of the stringent requirements presents a major challenge to testing laboratories who seek to provide accurate and cost competitive services.

Pesticide residue testing requires detection using both liquid and gas chromatographic techniques typically coupled with triple quadrupole mass spectrometers. These analytical techniques can cover the range of compounds that need to be monitored with the required sensitivity and selectivity. However, they are limited to detecting pesticides that are measured at the time of acquisition and require careful method optimization and management to ensure selected ion monitoring windows remain viable. In recent years, high-resolution Orbitrap mass spectrometry has provided an alternative to MS/MS techniques with additional analytical advantages [4]. With high-resolution mass spectrometry (HRMS), the default acquisition mode is untargeted (full-scan) making it simple to manage and potentially allows for an unlimited number 53

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Routine Quantitative Method of Analysis for Pesticides using GC Orbitrap Mass Spectrometry in accordance with SANTE/11945/2015 Guidelines

of pesticides to be monitored in a single injection. In addition to this, full-scan data analysis provides access to supplementary identification points such as spectral matching and enables retrospective interrogation of samples to additionally search for emerging pesticides or other contaminants that were not considered at the time of acquisition. In this study, the quantitative performance of the Thermo Scientific™ Exactive GC Orbitrap™ mass spectrometer was evaluated for the routine analysis of GC-amenable pesticides in fruits and vegetables following SANTE/11945/2015 guidelines using full scan acquisition. The Exactive GC-MS system provides routine high-mass resolving power up to 60,000 (m/z 200) full width at half maximum (FWHM) with scan speeds suitable for GC peaks to facilitate the detection of trace compounds in the presence of high matrix components. MATERIALS AND METHODS Sample Preparation Tomato, leek and orange were purchased from a local supermarket and extracted following a citrate buffered QuEChERS procedure. Briefly, 10 mL of acetonitrile was added to 10 g of homogenized sample and shaken for 4 minutes. A mixture of salts was added and the centrifuge tube shaken for 4 minutes and centrifuged for 5 minutes at 3700 rpm. Supernatant (5 mL) was transferred to a 15 mL PTFE centrifuge tube containing magnesium sulphate and 125 mg of PSA. The extract was shaken in a vortex mixer and centrifuged as above. The final acetonitrile extracts (1 g/mL) were used as blank matrix. The calibration series was prepared by taking 100 μl of acetronitrile blank matrix and drying under a stream of nitrogen to complete dryness. The sample was reconstituted in 100 μl ethyl acetate containing the appropriate concentration of pesticides. Three calibration series of 51 pesticides were prepared in tomato, leek and orange at concentrations equivalent to 0.5, 1, 2, 5, 10, 20, 50, 100, 200 and 500 μg/Kg. The 51 pesticides included in the study cover a wide range of chemical classes and, with the three matrices, it generated a total of 153 pesticide/ matrix combinations. To assess compound linearity, the matrix matched calibration series were analyzed first, followed by ten replicate injections of the 10 μg/Kg sample for each matrix. To assess repeatability over an extended period of time, the 10 μg/Kg tomato standard was further injected 100 times from the same vial. Instrument and Method Setup In all experiments, an Exactive™ GC Orbitrap™ mass spectrometer was used. Automatic sample injection was performed using a Thermo Scientific™ TriPlus™ RSH™ autosampler, and chromatographic separation was obtained using a Thermo Scientific™ TRACE™ 1310 Gas Chromatograph and a Thermo Scientific™ TraceGOLD™ TG-5SilMS 30 m x 0.25 mm I.D. x 0.25 μm film capillary column with a 5 m integrated guard (P/N:26096-1425). Additional details of instrument parameters are given in Table I and Table II.

Roberts, D.; Duque, S. U.; Fernández-Alba, A.; Silcock, P.

Sponsor Report Table I. GC and Split/Splitless injector conditions

TRACE 1310 GC Parameters Injection Volume (mL): Liner:

Table II. Mass spectrometer conditions

Exactive GC Mass Spectrometer Parameters 1

Transfer line (°C):

280

LinerGOLD™ single taper*

Ionization type:

280

Ion source (°C):

250

Electron energy (eV):

Inlet (°C): Carrier Gas (mL/min):

He, 1.2

Oven Temperature Program

Acquisition Mode:

Full-scan

Temperature 1 (°C):

Mass range (Da):

50-550

Hold Time (min):

1.5

Resolving power (FWHM at m/z 200):

60,000

Temperature 2 (°C):

Lockmass, column bleed (m/z):

Rate (°C/min):

Hold Time (min):

1.5

Temperature 3 (°C):

280

Rate (°C/min):

Hold Time (min):

Temperature 3 (°C):

300

Rate (°C/min):

Hold Time (min):

207.03235

*P/N: 453A1345-UI

Data Processing Data were acquired using the Thermo Scientific™ TraceFinder™ software. This single platform software package integrates instrument control, method development functionality, and qualitative and quantitationfocused workflows. For target analysis a compound database for the 51 pesticides was prepared using the Thermo Scientific™ Orbitrap GC-MS Contaminants Library containing compound name, quantification ion and identification ions, accurate masses, retention times and elemental compositions of molecular ion and fragment masses. For the generation of extracted ion chromatograms a mass extraction window of 5 ppm was used. RESULTS AND DISCUSSION The objective of this study was to evaluate the analytical performance of the Exactive GC system for the routine analysis of pesticides in three different sample matrices following SANTE requirements. The sample types chosen (tomato, leek and orange) provided both easy and difficult matrices that are typically encountered in routine testing. To illustrate, the varying sample complexity total ion chromatograms with fixed Y-axis are shown in Figure 1.

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Routine Quantitative Method of Analysis for Pesticides using GC Orbitrap Mass Spectrometry in accordance with SANTE/11945/2015 Guidelines

Figure 1. Full scan Total Ion Chromatogram (TIC) of orange, leek and tomato extracts with y axis ﬁxed at 4.0 e9 showing the complexity of the sample matrices used in this study.

The leek matrix is clearly the most complex matrix and this is where high-mass resolution is required to extract target analytes from background chemical noise. The QuEChERS generic sample extraction technique employed in routine testing produces complex extracts containing high and variable concentrations of matrix components depending on the sample type. The lack of selectivity during sample preparation needs to be compensated for by a selective instrumental analysis. This was achieved using high-mass resolving power of the Exactive GC system (60k @m/z 200). This capability in combination with a full-scan acquisition increases the scope of the analysis without the need for optimization of acquisition parameters, as is the case with targeted analyses. For routine pesticide screening, the HRMS processing software needs to be fast, accurate and customizable. TraceFinder meets all of these requirements and was used to process each batch of calibration standards and ten replicates in less than five minutes. In TraceFinder, the results are presented to the user in a table format and data flags are used to quickly identify which pesticides are positive and which criteria are satisfied. Flexible reporting options means that data can be either exported to other software packages or reported directly from within TraceFinder. Identification to Guideline Requirements One aim of the analysis was to determine the limit of detection (LOD), limit of identification (LOI), linearity and peak area repeatability for all of the pesticides in all three matrices. Although the LOD is not discussed in the SANTE guidelines, it is useful to know the limit of detection of the quantifier ion as it is used in forming the calibration series that will ultimately be used in determining the concentration of a detected pesticide in a sample. This assessment was made by evaluating the matrix matched calibration series and the repeat injections at 10 μg/Kg for each matrix. The LOD was defined as the presence of a peak with S/N (peak to peak) >3 in the extracted ion chromatogram (XIC) of the main quantifier ion of a pesticide. For the determination of the LOI the SANTE/11945/2015 guidance document was followed. This requires that the following criteria are satisfied for a positive identification: (i)Two ions are detected for each pesticide with mass accuracy <5 ppm and peak S/N > 3 (ii)Retention time tolerance of ± 0.1 minutes compared with standards in the same sequence (iii)Ion ratio within ± 30% of the average of calibration standards from the same sequence (iv)Optional: For higher confidence in identification additional criteria can be used such as full-scan spectra, isotope pattern matching and additional fragment ions

Roberts, D.; Duque, S. U.; Fernández-Alba, A.; Silcock, P.

Sponsor Report All of the pesticides were identified following the regulatory criteria (LOI) in all of the matrices at or below 5 μg/Kg (Tables III-V) with the exception of chlorothalonil in leek, which is known to suffer losses due to interaction with sulphur compounds in the leek matrix [5]. The majority of the 153 pesticide/matrix combinations (79%) had an LOI ≤2 μg/Kg. The calculated LODs are summarized in Figure 2 which shows that the LOD for 93% of the pesticide/matrix combinations was ≤1 μg/Kg. Having multiple identification points and limits of detection well below the MRL increases the confidence in identifications and minimizes false negative and positive results. Using highly efficient electron ionisation (EI) in combination with fullscan acquisition provides the opportunity to use multiple diagnostic ions for the identification of pesticides.

Figure 2. The limit of detection (LOD) and limit of identiﬁcation (LOI) for pesticides/matrix combinations.

The Exactive GC system generates standard EI spectra that are highly reproducible and library searchable (nominal or high resolution MS libraries). This facilitates detection and identiﬁcation of pesticides based on spectral matching. Additional compounds can be quickly added to the compound database as chemical formulas can be easily assigned to accurate mass fragment ions due to the high mass accuracy of the Orbitrap analyzer. Reliable Quantitation Quantitative linearity was assessed using matrix matched standards across a concentration of 0.5-500 μg/Kg. In all cases, the coefﬁcient of determination (R2) was >0.99 for each pesticide from its LOD to 500 μg/Kg in the three matrices, an example of the TraceFinder browser showing propazine is given in Figure 3. One exception to this, possibly due to analyte adsorption, was fenpropidin which was linear up to 200 μg/Kg. Accurate quantitation is reliant upon a number of factors, one of which is an acquisition speed fast enough to provide at least 12 points across chromatographic peak. At a resolution of 60,000 the Exactive GC system has a scan speed of approximately 7 Hz. An example is shown in Figure 4 for the peak of chlorobenzilate which has 38 points across the 6 second peak.

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Routine Quantitative Method of Analysis for Pesticides using GC Orbitrap Mass Spectrometry in accordance with SANTE/11945/2015 Guidelines

Figure 3. TraceFinder browser showing positively identified pesticides, extracted ion chromatogram and calibration graph (propazine as an example). Sub-ppm mass accuracy for propazine across the calibration range and in replicates of 10 μg/Kg. Identification criteria information is available and flagged when out of tolerance.

Figure 4. Extracted ion chromatogram of chlorobenzilate (m/z 251.0025 ±5 ppm mass window) acquired at 60,000 resolution (FWHM at m/z 200) in leek spiked at 10 μg/Kg showing ~38 scans/peak (peak width 6 sec). Sub 1 ppm accurate mass is achieved for each individual scan (every third scan labeled). Average RMS mass difference of 0.6 ppm across the peak.

Roberts, D.; Duque, S. U.; Fernández-Alba, A.; Silcock, P.

Sponsor Report Table III. Summary of method performance results for pesticides in leek 2 LOD LOI R LOD-500 Mass Accuracy Pesticide (μg/Kg) (μg/Kg) (μg/Kg) at LOI (ppm) 2-phenylphenol 0.5 1 0.9986 -0.53 Acrinathrin 2 5 0.9975 -0.68 Azoxystrobin 1 5 0.9961 0.1 BHC, Alpha 0.5 1 0.9993 -0.6 BHC, beta 0.5 1 0.9992 0.8 BHC, gamma 0.5 2 0.9986 -0.8 Bifenthrin 0.5 0.5 0.9989 -0.5 Biphenyl 0.5 0.5 0.9986 -0.9 Bromopropylate 0.5 1 0.9973 0.3 Bupirimate 0.5 1 0.9979 -0.4 Chlorobenzilate 0.5 2 0.9979 1.04 Chlorothalonil ND* ND* Chlorpropham 0.5 2 0.9991 0.7 Chlorpyrifos 1 5 0.999 0.1 Chlorpyrifos-methyl 0.5 2 0.9988 0.5 Cyhalothrin 1 2 0.9954 -0.6 Cypermethrin I-IV 5 5 0.9962 0.5 DDD p,p’ 0.5 2 0.9982 0.7 DDE p,p’ 0.5 1 0.9988 0.41 DDT o,p 0.5 2 0.9982 0.7 DDT p,p’ 0.5 5 0.9962 0.1 Diazinon 1 2 0.9983 -0.34 Dichlorvos 0.5 1 0.9991 -0.5 Dieldrin 2 5 0.992 0.3 Endosulfan sulfate 1 5 0.999 -0.2 Endosulphan alpha 2 5 0.994 -0.2 Endosulphan beta 2 5 0.9982 -0.4 Etofenprox 2 5 0.9978 -0.1 Fenitrothion 2 2 0.9968 0.1 Fenpropidin 0.5 5 0.9986 -0.3 Fenpropimorph 0.5 5 0.9977 -1.1 Fenvalerate SS,RR 0.5 2 0.9954 0.6 Fipronil 0.5 1 0.9979 0.2 Hexachlorobenzene 0.5 1 0.9985 1.1 Iprodione 0.5 5 0.9975 0.4 Kresoxim-methyl 0.5 2 0.9989 0.36 Metalaxyl 2 5 0.9989 -0.91 Myclobutanil 0.5 5 0.9987 -0.96 Oxadixyl 1 2 0.9983 0.34 Parathion-methyl 1 5 0.9985 0.61 Pendimethalin 2 5 0.9989 0.98 Pirimicarb 0.5 2 0.9991 -0.28 Procymidone 1 1 0.9988 0.26 Propazine 0.5 2 0.9988 -0.62 Pyrimethanil 0.5 1 0.9984 -0.31 Terbuthylazine 0.5 1 0.9985 -0.19 Tetramethrin 1 5 0.9991 -0.23 Tolclofos-methyl 0.5 1 0.9991 0.55 Trifluralin 1 1 0.9963 -0.52 Triphenylphosphate 1 2 0.9979 0 Vinclozolin 0.5 2 0.9987 -0.6

Leek 10 μg/Kg (%RSD) n=10 2.5 6 6.3 4.4 4.4 4.5 4 3.3 6.4 5.1 3.8 2.9 4.6 4.1 6.9 7.9 4.7 3.5 4.4 4.2 3.5 4.1 3.6 5.9 9.1 7.5 6.2 6.6 4.1 2.8 6.5 6.3 3 7.5 4.3 4.9 5 6 4.8 6.5 3.1 5.9 2.9 3.6 4 5.4 2.5 3.9 6 4.6

*Chlorothalonil known to degrade in leek. 59

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Routine Quantitative Method of Analysis for Pesticides using GC Orbitrap Mass Spectrometry in accordance with SANTE/11945/2015 Guidelines

The results of the 10 replicate injections at 10 μg/Kg in all three matrices are presented in Figure 5. All detected pesticides had RSD% of less than 10%, well below the 20% threshold requirement in the SANTE guidance document. This shows that the Exactive GC system operated in full-scan at 60k resolution has the selectivity and sensitivity required to analyse pesticides in a robust manner well below the respective MRLs.

Figure 5. Peak area repeatability (%RSD) for 10 μg/Kg (n=10) for each pesticide in the three matrices studied. SANTE guideline of 20% threshold shown is also indicated.

Robust Mass Accuracy Acquiring reliable accurate mass measurements is critical when detecting low level pesticides in complex sample matrices. Low mass errors allow selectivity to be maintained through the use of narrow mass extraction windows during data processing and help ensure positive detections are robust. The low mass errors observed with the Exactive GC system are enabled through the high-mass resolving power that is able to discriminate between matrix interferences and target analyte ions. When the resolution is insufficient, the mass profile of two ions overlap, which results in the incorrect assignment of the mass of the target compound. This is demonstrated in Figure 6 where the leek 10 μg/Kg matrix standard was analysed at resolving powers of 15K, 30K and 60K. The zoomed mass spectra show the quantifier ion for pyrimethanil and a matrix ion of a similar mass causing interference. At 15K and 30K, the pyrimethanil ion is not resolved resulting in poor mass accuracy of 10.1 and 6.3 ppm respectively. However, the ions are sufficiently resolved at 60K resulting in the expected sub 1 ppm mass accuracy. Without this level of mass resolution this pesticide would have failed the SANTE identification criteria of <5 ppm and would have been a false negative (reported as not detected). This supports previous a report that a resolving power of 60k (at 200 m/z) is required in some cases to ensure the highest selectivity [6].

Roberts, D.; Duque, S. U.; Fernández-Alba, A.; Silcock, P.

Sponsor Report Table IV. Summary of method performance results for pesticides in orange Pesticide 2-phenylphenol Acrinathrin Azoxystrobin BHC, Alpha BHC, beta BHC, gamma Bifenthrin Biphenyl Bromopropylate Bupirimate Chlorobenzilate Chlorothalonil Chlorpropham Chlorpyrifos Chlorpyrifos-methyl Cyhalothrin Cypermethrin I-IV DDD p,p' DDE p,p' DDT o,p DDT p,p' Diazinon Dichlorvos Dieldrin Endosulfan sulfate Endosulphan alpha Endosulphan beta Etofenprox Fenitrothion Fenpropidin Fenpropimorph Fenvalerate SS,RR Fipronil Hexachlorobenzene Iprodione Kresoxim-methyl Metalaxyl Myclobutanil Oxadixyl Parathion-methyl Pendimethalin Pirimicarb Procymidone Propazine Pyrimethanil Terbuthylazine Tetramethrin Tolclofos-methyl Trifluralin Tri-phenylphosphate Vinclozolin

LOD (μg/Kg) 0.5 1 1 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 1 5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 1 1 1 0.5 0.5 1 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5

LOI (μg/Kg) 0.5 5 5 0.5 1 0.5 0.5 0.5 1 0.5 0.5 0.5 2 1 1 5 5 2 0.5 2 5 0.5 0.5 2 2 5 2 2 2 5 2 2 0.5 1 1 1 1 2 2 2 2 1 2 1 1 1 5 1 1 1 1

R LOD-500 (μg/Kg) 0.997 0.9956 0.9977 0.9984 0.9985 0.9989 0.9972 0.998 0.9985 0.9987 0.9982 0.9987 0.9981 0.9982 0.9989 0.9963 0.9986 0.9986 0.9989 0.9988 0.9967 0.999 0.9983 0.9989 0.9986 0.9987 0.9988 0.9937 0.998 0.993* 0.9924 0.9919 0.9983 0.999 0.9983 0.9984 0.9991 0.9977 0.9983 0.9988 0.9978 0.9976 0.9977 0.9981 0.9935 0.999 0.9979 0.9986 0.9974 0.9977 0.999

Mass Accuracy at LOI (ppm) -0.1 -0.42 -0.1 -0.6 -0.6 -0.21 -0.7 -0.37 -0.16 0.36 0.37 0.42 -0.13 0.1 0.38 -0.6 -0.5 -0.1 0 0.14 -0.11 0.51 0.29 0.5 1.2 -1.2 0.4 0.4 0.1 1 -0.44 0.37 -0.8 -0.17 -0.5 0.43 -0.8 -0.2 0.46 -0.3 1 -0.65 0.1 0.3 -0.3 -0.2 -0.41 0.78 0.56 0.28 0.5

Leek 10 μg/Kg (%RSD) n=10 2.7 5.7 7.9 3.7 3.3 2.7 4.2 3.9 5.4 4.8 3.9 6.5 2.7 2.5 3.1 6.9 6.7 4.1 2.2 3.1 4.1 3 4 3 6.1 9.8 3.8 4.5 5 7.5 5.4 7.3 5.1 3.5 7.1 3.8 4.4 5.1 5.1 4.9 3.8 3.4 3.6 0.3 3.3 3.7 5.7 2.5 4.7 4.3 3.1

*LOD 200 μg/Kg 61

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Routine Quantitative Method of Analysis for Pesticides using GC Orbitrap Mass Spectrometry in accordance with SANTE/11945/2015 Guidelines

Figure 6. Effect of resolving power on mass accuracy of the diagnostic ion of Pyrimethanil at 10 μg/Kg in leek acquired at different resolutions of 15K, 30K and 60K. At 15K and 30K the Pyrimethanil ion is not resolved from the interfering matrix ion resulting in poor mass accuracy assignment.

The mass accuracy was assessed for all 51 pesticides at their LOI and the results are shown graphically in Figure 7. The mass error values did not exceed 1.2 ppm for any of the analytes, well below the guideline limit of 5 ppm delivering the highest conﬁdence in accurate and selective detection. In pesticide analysis, it is also essential that the instrument is able to maintain mass accuracy across the complete range of possible analyte concentrations encountered. It would not be acceptable if a high concentration pesticide violation was missed due to detector saturation. On the Exactive GC system, the Orbitrap analyzer is protected from saturation through the use of automatic gain control (AGC) which regulates the number of ions entering. This ensures that, no matter what concentration is encountered, the mass accuracy performance is preserved. This is demonstrated in Figure 8 that shows the mass accuracy for four pesticides at concentrations ranging from 0.5 to 500 μg/Kg in leek matrix is always < 1 ppm.

Figure 7. Mass difference measurements at the LOI level for each pesticide across the three matrices.

Figure 8. Mass accuracy measurements across the concentration range (0.5-500 μg/mL) for four pesticides in leek. Mass accuracy is maintained at sub 1 ppm level.

Roberts, D.; Duque, S. U.; Fernández-Alba, A.; Silcock, P.

Sponsor Report Table V. Summary of method performance results for pesticides in tomato 2 LOD LOI R LOD-500 Mass Accuracy Leek 10 μg/Kg Pesticide (μg/Kg) (μg/Kg) (μg/Kg) at LOI (ppm) (%RSD) n=10 2-phenylphenol 0.5 0.5 0.9999 -0.71 2.1 Acrinathrin 1 5 0.9915 -0.34 5.5 Azoxystrobin 1 2 0.9938 -0.1 7.8 BHC, Alpha 0.5 0.5 0.9984 -0.46 3.6 BHC, beta 0.5 0.5 0.9984 -0.21 2.6 BHC, gamma 0.5 0.5 0.9984 -0.63 2.6 Bifenthrin 0.5 0.5 0.9981 -0.75 3.5 Biphenyl 0.5 0.5 0.9977 -0.37 3.2 Bromopropylate 0.5 1 0.9939 0.37 4.6 Bupirimate 0.5 0.5 0.9969 -0.51 3.6 Chlorobenzilate 0.5 0.5 0.9982 0.43 2.5 Chlorothalonil 0.5 0.5 0.9985 1 1.9 Chlorpropham 0.5 0.5 0.999 0.7 1.6 Chlorpyrifos 0.5 0.5 0.999 0.14 2.3 Chlorpyrifos-methyl 0.5 0.5 0.999 0.81 2.2 Cyhalothrin 0.5 1 0.999 -0.76 4.7 Cypermethrin I-IV 5 5 0.997 -0.5 6 DDD p,p' 0.5 1 0.9974 0.1 3.2 DDE p,p' 0.5 0.5 0.9995 0.35 2.5 DDT o,p 0.5 1 0.997 0.34 2.7 DDT p,p' 0.5 5 0.9923 -0.17 2.4 Diazinon 0.5 0.5 0.9991 -0.68 2.2 Dichlorvos 0.5 0.5 0.9987 -0.11 2.1 Dieldrin 0.5 2 0.9988 0.21 2.7 Endosulfan sulfate 1 2 0.9975 0.15 1.9 Endosulphan alpha 1 2 0.9993 0.19 1.9 Endosulphan beta 1 2 0.9981 -0.64 4.8 Etofenprox 1 5 0.9982 -0.37 6.5 Fenitrothion 0.5 2 0.9943 0.49 2.4 Fenpropidin 0.5 2 0.999 0.36 8.5 Fenpropimorph 0.5 5 0.999 0.51 4.5 Fenvalerate SS,RR 0.5 2 0.991 0.31 6.7 Fipronil 0.5 0.5 0.9949 0.36 3.1 Hexachlorobenzene 0.5 1 0.9993 0.54 2.8 Iprodione 0.5 1 0.9964 0.39 5.6 Kresoxim-methyl 0.5 0.5 0.9984 0.36 3.1 Metalaxyl 0.5 1 0.9993 -0.53 3.5 Myclobutanil 0.5 2 0.9984 0.4 2.1 Oxadixyl 0.5 1 0.9985 0.46 4.6 Parathion-methyl 0.5 1 0.9974 0.73 2.2 Pendimethalin 0.5 2 0.9936 0.62 2.2 Pirimicarb 0.5 0.5 0.9992 -0.37 2 Procymidone 0.5 1 0.9984 0.58 2.3 Propazine 0.5 0.5 0.9989 -0.12 2.2 Pyrimethanil 0.5 1 0.998 0.13 1.4 Terbuthylazine 0.5 0.5 0.9989 -0.12 2.2 Tetramethrin 0.5 5 0.9948 -0.41 4 Tolclofos-methyl 0.5 1 0.9992 0.78 1.6 Trifluralin 0.5 0.5 0.9947 0.76 1.9 Tri-phenylphosphate 0.5 0.5 0.9968 -0.1 4.8 Vinclozolin 0.5 0.5 0.9991 0.9 2.1

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Routine Quantitative Method of Analysis for Pesticides using GC Orbitrap Mass Spectrometry in accordance with SANTE/11945/2015 Guidelines

Real World Performance In a high-throughput routine pesticide analysis laboratory, mass spectrometry instruments are in near constant operation and it is essential that they provide the same level of performance over an extended period of time. To evaluate the performance of the Exactive GC system over a longer period, a tomato extract at 10 μg/Kg was repeatedly injected (n=100) from a single vial. Prior to commencing analysis, a new injector liner was installed, the source tuned and the MS calibrated. No further interventions were made during the 66 hours of continual operation. The results showed that the system, from injector to MS, provided outstanding performance. Figure 9 shows the peak area response of hexachlorobenzene, vinclozolin and triﬂuralin at 10 μg/Kg in tomato over the 100 injections, with RSD% of 5.3, 4.6 and 3.8%, respectively. Furthermore, the mass accuracy stability remained <1.2 ppm (99% ≤1ppm) for the duration of the analysis without further mass calibration (Figure 10).

Figure 9. Repeat injections (n=100) of a tomato extract spiked at 10 μg/Kg showing that the sensitivity is maintained over the 66 hours of continual operation.

Figure 10. Mass accuracy (ppm) over 100 injections for hexachlorobenzene, vinclozolin and triﬂuralin in tomato extract at 10 μg/Kg. Data was acquired with same liner and without further calibration of the mass spectrometer or tuning of the source.

CONCLUSIONS The results of this study demonstrate that the Thermo Scientiﬁc Exactive GC Orbitrap high-resolution mass spectrometer, in combination with TraceFinder software, is a high performance analytical system that delivers robust and sensitive performance for routine pesticide analysis in fruits and vegetables in complete accordance with SANTE guidance document. - 99.3% of the pesticide/matrix combinations were detected below the MRL with excellent linearity and meeting the required performance criteria. Importantly, the scope of the analysis is increased by acquisition in full-scan with targeted data processing with a compound database. 64

Roberts, D.; Duque, S. U.; Fernández-Alba, A.; Silcock, P.

Sponsor Report - Acquisition at 60,000 FWHM resolution dramatically reduces matrix interferences and increases conﬁdence in results when screening for pesticides in complex sample matrices. Consistent sub ppm mass accuracy was achieved for all compounds over a wide concentration range ensuring that compounds are detected with conﬁdence at low and high concentration levels. - Repeated injections of a tomato matrix at 10 μg/Kg showed that the system is able to maintain a consistent level of performance over an extended period of time as is demanded by a routine testing laboratory.

REFERENCES 1. http://ec.europa.eu/food/plant/docs/plant_pesticides_mrl_guidelines_wrkdoc_11945_en.pdf 2. http://eur-lex.europa.eu/legal-content/EN/TXT/?uri=URISERV:l13002a 3. SANTE/11945/2015. Guidance document on analytical quality control and method validation procedures for pesticides residues analysis in food and feed. Supersedes SANCO/12571/2013. Implemented by 01/01/2016. 4. Kellmann, M.; Muenster, H.; Zomer, P.; Mol, H. J Am Soc Mass Spectrom, 2009, 20, pp 1464-1476. 5. Belmonte, V. N.; Retamal, M.; Martinez-Uroz, M. A.; Mezcua, M.; Fernandez-Alba A. R.; de Kok, A. Analyst, 2012, 137 (10), pp 2513-2520. 6. Mol, H. G. J.; Tienstra, M.; Zomer, P. Anal Chim Acta, 2016, 935, pp 161-172. doi:10.1016/j.aca.2016.06.017

This sponsor report is the responsibility of Thermo Fisher Scientiﬁc.

Br. J. Anal. Chem., 2017, 4 (17), pp 66-78

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Fast Routine Analysis of Polar Pesticides in Foods by Suppressed Ion Chromatography and Mass Spectrometry 1

Katerina Boušová , Cees Bruggink , Michal Godula 1 Thermo Fisher Scientific, Special Solutions Center, Dreieich, Germany 2 Thermo Fisher Scientific, Special Solutions Center, Breda, Netherlands The goal of this work was to develop and test an IC-MS/MS multi-residue method that can be applied for high-throughput screening and quantitation of polar pesticide residues and their metabolites in food matrices below the current legislative requirements. Keywords: Glyphosate, AMPA, polar pesticides, pesticide residues, IC-MS, TSQ Endura, Integrion. INTRODUCTION The presence of very polar ionic pesticides in surface and drinking water, as well as food and beverages, has become a controversial issue in recent years. The development of genetically modified crops tolerant to glyphosate and glufosinate, for example, promoted the use of these broad spectrum herbicides. In addition, glyphosate is used as a crop desiccant to suppress weeds in parks and at roadsides. Consequently, these pesticides often occur in foods as residues and in the environment as contaminants of surface waters. There are concerns about their potential adverse effects on human health, such as their potential carcinogenicity [1], although the latest toxicological assessments do not predict risks for humans under normal conditions or environmental exposures [2]. Current regulations offset maximum residue levels (MRLs) of glyphosate and its metabolite aminomethylphosphonic acid (AMPA) at 100 ng/L in drinking water. In food and beverage samples, higher MRLs typically apply, ranging generally from 10 μg/kg for food intended for consumption by children up to hundreds of mg/kg in other matrices [3]. The analysis of glyphosate and other polar compounds presents a difficult analytical challenge. Their polarity does not allow the direct analysis by reversed-phase HPLC, so alternative methods need to be applied. Derivatization of glyphosate prior to analysis [4] or application of specific chromatographic columns, such as the Thermo Scientific™ Hypercarb™ column, are the common approaches [5]. With both of these approaches, poor method robustness and questionable results are often reported in laboratories, especially when the method is applied in routine high-throughput analysis of samples with rather complex matrix composition. Recent developments in ion chromatography and mass spectrometry offer many advantages for the analysis of very polar substances. Ion chromatography is the preferred separation technique for polar ionic analytes, such as anions, cations or small polar analytes (metabolites), and sugars. Mass spectrometry, namely in triple quadrupole MS/MS systems, offers very low detection limits and high detection selectivity when operated in selected reaction monitoring (SRM) mode. The system robustness allows the analysis of food and environmental samples. The aim of this work is to develop and validate an IC-MS/MS method for direct analysis of polar ionic pesticides in food samples and to assess its applicability under routine conditions. MATERIALS AND METHODS Sample Preparation For sample preparation, an optimized method was used that was developed by the EU Reference Laboratory for Residues of Pesticides in Stuttgart, Germany [5]. Since a different chromatographic technique was used, a small change improving the efficiency was implemented. A different volume of nonacidified cold methanol was used for sample extraction. 66

Fast Routine Analysis of Polar Pesticides in Foods by Suppressed Ion Chromatography and Mass Spectrometry

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Homogenized samples of lettuce, oranges, and wheat flour were extracted first with water and then with cold methanol. The sample extracts were then centrifuged and, after filtration through syringe filters, were injected into the IC-MS/MS system (Figure 1). Plasticware was used instead of glassware to minimize losses of polar pesticides by adsorption onto glass.

Figure 1. Schematic of the method.

Chemicals · Deionized water (Thermo Scientific™ Barnstead™ EASYpure™ II water system) · Methanol (99.9% purity, LC/MS grade, Fisher Chemical™, Optima™) · Methanol (99.9 % purity, HPLC grade, Fisher Chemical) · Thermo Scientific™ Pierce™ Triple Quadrupole Calibration Solution, ext. mass range Apparatus · ULTRA-TURRAX® High speed blender · ULTRA-TURRAX Rotor/Stator - Dispensing tool · ULTRA-TURRAX Plug-in coupling (dispersing element) · Waring® laboratory blender with timer · Fisherbrand™ Compact balance · Sartorius™ analytical balance Instruments All instruments used are from Thermo Scientific™ · TSQ Endura™ Triple Quadrupole MS · Dionex™ Integrion™ HPIC™ System · Dionex™ EGC KOH Eluent Generator · Dionex™ AERS™ 500 Anion Electrolytically Regenerated Suppressor 500 – 2 mm · Dionex™ AS-AP Autosampler · Dionex™ CR-ATC 600 · Dionex™ AXP-MS Auxiliary pump (make-up flow) · Dionex™ AXP-MS Auxiliary pump (AERS regeneration) · Heraeus™ Multifuge™ X3 Centrifuge Automatic Pipettes All automatic pipettes used are Finnpipette™ from Thermo Scientific™ · Novus Electronic 1–10 μL · Novus Electronic 10–100 μL 67

Boušová, K.; Bruggink, C.; Godula, M.

Sponsor Report · Novus Electronic 30–300 μL · Novus Electronic 100–1000 μL · Novus Electronic 0.5–5 mL · Novus Electronic 100–1000 μL · F1 1000–10,000 μL Consumables All consumables used are from Thermo Scientific™ · Dionex™ IonPac™ AS24 Analytical column (2 x 250 mm) · Dionex™ IonPac™ AG24 Guard column (2 x 50 mm) Pesticide standards · Ethephon, Sigma-Aldrich®; P/N: 45473 · HEPA (2-hydroxyethylphosphonic acid), LGC Standards®; P/N: CA13230200 · Glufosinate, Sigma-Aldrich®; P/N: 45520 · N-Acetyl-glufosinate, LGC Standards®; P/N: CA14031500 · MPPA (3-Methylphosphinicopropionic acid), LGC Standards®; P/N: XA15141200AL · Glyphosate, Sigma-Aldrich®; P/N: 45521 · AMPA (aminomethylphosphonic acid), Sigma-Aldrich®; P/N: 324817 · Phosphonic acid (phosphorous acid), Sigma-Aldrich®; P/N: 389025000 · N-Acetyl-AMPA (N-Acetyl-aminomethylphosphonic acid), LGC Standards®; P/N: DRE-C10205150 · Fosetyl-Al (Fosetyl-aluminium), LGC Standards®; P/N: CA13940000 Proficiency test material · T19186 red grape purée, FAPAS®; P/N: T19186QC Preparation of standards Stock standard solutions Stock standard solutions were prepared individually by dissolving the analytes in methanol, acidified methanol, water, acetonitrile, or mixtures. Table I shows the used solvents and concentrations in the stock solutions. Plastic flasks and stoppers were used for preparation of stock solutions of glyphosate and AMPA, compounds that tend to interact with glass surfaces. Solutions were stored at -20 °C. The frozen solution was allowed to thaw at room temperature before further dilutions were made. Table I. Stock standard solutions Standard

Solvent

c (μg/mL)

Ethephon

CH3OH + 1% formic acid

1000

CH3OH

1000

H2O/CH3OH (2:1)

1000

CH3OH

1000

MPPA*

ACN

Glyphosate

H 2O

1000

AMPA

H 2O

100

Phosphonic acid

H 2O

1000

N-Acetyl-AMPA

H 2O

100

H2O/CH3OH (3:1)

100

HEPA Glufosinate N-Acetyl-Glufosinate

Fosetyl-Al

*MPPA was purchased as the ready standard solution in acetonitrile with concentration of 10 μg/mL. 68

Fast Routine Analysis of Polar Pesticides in Foods by Suppressed Ion Chromatography and Mass Spectrometry

Sponsor Report

Working standard solutions The working standard solution of 10 compounds (c = 1 mg/L) was prepared by diluting individual stock standard solutions into water. The solution should be prepared freshly every time before use. The solution stored in a plastic tube was used for the spiking of samples in recovery experiments and for the preparation of calibration standards. Sample preparation Homogenization A representative amount (100–150 g) of the sample (lettuce, orange, or flour) was homogenized in the blender. For fruits and vegetables, cryogenic milling (e.g. using dry ice) is preferred to minimize degradation, reduce particle size, and to improve homogeneity as well as residue accessibility. However, the classic blender was used in our work because no adverse effects were observed. For dry commodities (e.g. flour), fine grinding is recommended (e.g. particle size < 500 μm). Extraction and filtration Homogenized sample (10 g) was accurately (+/- 0.01 g) weighed into a 50 mL plastic centrifuge tube. Then, 10 mL of water was added and the tube was shaken vigorously for 5 minutes on the horizontal shaker. Afterwards, 30 mL of cold methanol (~5 °C) was added, and the sample was again agitated for 1 minute on the shaker. The samples were then centrifuged for 5 minutes at 4000 rpm and 5 °C, filtered (PES, 45 μm), and injected into the IC-MS/MS system. The 2 mL plastic vials were used to avoid possible analyte adsorption to the surface of glass walls, hence improving analyte recovery. To improve recovery in wheat flour samples, respective extraction times may need to be extended (up to 15–20 min with water and 5–10 min with methanol). Instrument and method setup The instrument system comprised a metal-free Dionex Integrion ion chromatograph and a Dionex AS– AP autosampler coupled to a TSQ Endura mass spectrometer (Figure 2). The chromatographic separation was carried out using a polymer-based Dionex IonPac AS24 column with guard in the 2 mm format. Instrument parameters and settings are shown in Table II. The hydroxide eluent was prepared in-situ using an eluent generator, the Dionex EluGen KOH cartridge and a Dionex CR-ATC II, preventing the use of external chemicals. After separation, the eluent passed the electrochemically regenerated AERS suppressor, where the cations from both the eluent and the sample were replaced with hydronium ions, effectively neutralizing the high pH eluent and rendering it compatible with a mass spectrometer. No external chemical regenerants were needed, as an external pump delivered water feeding the electrolytic process to continuously regenerate the suppressor membranes. In order to improve desolvation, a second pump added methanol as a make-up solvent at a low flow rate before entering the mass spectrometer (Figure 2). Table II. IC conditions

Parameter

Setting

Mobile Phase:

KOH (Gradient conditions, Table III-a)

Eluent Source:

Eluent Generator

Analytical Column:

Dionex IonPac AS24 (2 x 250 mm) with guard column

Suppressor:

Dionex AERS 500–2 mm (External water mode, Table III-b)

Flow Pump 1 (AERS Regeneration): Make-Up Solvent: Flow Pump 2: Injection Volume: Column Temperature: Flow Rate:

1.2 mL/min CH3OH 0.1 mL/min 10 μL 21 °C 0.3 mL/min

Boušová, K.; Bruggink, C.; Godula, M.

Sponsor Report Table III-a. Gradient conditions

Table III-b. Suppressor conditions

Time (min) 0

Concentration of KOH in Eluent (mM) 25

Time (min) 0

0.2

Suppressor Current (mA) 32

15.4

11.1

100

15.5

12.5

100

12.6

Figure 2. IC-MS/MS system.

Mass spectrometer conditions Data acquisition was performed in selected reaction monitoring mode (SRM). All SRM traces (parent, quantifier, and qualifier ions) were individually tuned for each target analyte injecting the corresponding standard solution (10 mg/L). The mass spectrometer conditions are shown in Table IV and SRM parameters for analyzing targeted analytes are shown in Table V. Data was acquired and processed using Thermo Scientific™ TraceFinder™ 4.0 software allowing easy building of the acquisition and processing methods for high-throughput quantitative analysis with improved data reviewing and reporting. Table IV. Mass spectrometer conditions Parameter Ionization Mode: Scan Type: Polarity:

Heated Electrospray (H-ESI) SRM Negative ion mode

Spray Voltage:

2500 V

Sheath Gas Pressure:

20 Arb

Aux Gas Pressure:

5 Arb

Ion Sweep Gas Pressure:

0 Arb

Capillary Temperature:

329 °C

Vaporizer Temperature:

400 °C

Dwell Time:

10 ms

Q1/Q3 Resolution: Collision Gas Pressure (CID) Gas:

Setting

0.7 1.5 mTorr

Source Fragmentation:

Use Calibrated RF Lens:

Yes

Fast Routine Analysis of Polar Pesticides in Foods by Suppressed Ion Chromatography and Mass Spectrometry

Sponsor Report

Table V. IC-MS/MS parameters for selected reaction monitoring transitions Compound

AMPA

Glyphosate

Ethephon

Fosetyl-Al

Glufosinate

MPPA

Phosphonic Acid

HEPA

N-acetyl-AMPA

N-acetylglufosinate

R.T. (min)

5.70

9.95

7.35

3.95

5.51

6.40

3.84

5.72

5.76

5.67

Polarity

Neg

Transition Type

Precursor (m/z)

Product (m/z)

Collision Energy

Quantifier

110.1

79.2

Qualifier 1

110.1

63.3

Qualifier 2

110.1

81.3

Quantifier

168.1

63.3

Qualifier 1

168.1

79.2

Qualifier 2

168.1

81.2

Qualifier 3

168.1

94.2

Quantifier

143.0

107.2

Qualifier 1

143.0

79.2

Qualifier 2

145.0

107.1

Quantifier

109.1

81.2

Qualifier 1

109.1

79.2

Qualifier 2

109.1

63.2

Quantifier

180.1

95.2

Qualifier 1

180.1

78.2

Qualifier 2

180.1

85.3

Qualifier 3

180.1

102.1

Quantifier

151.1

133.0

Qualifier 1

151.1

107.2

Qualifier 2

151.1

63.3

Qualifier 3

151.1

79.2

Quantifier

81.2

79.2

Qualifier 1

81.2

63.3

Qualifier 2

81.2

81.0

Quantifier

125.1

79.2

Qualifier 1

125.1

95.2

Qualifier 2

125.1

63.3

Quantifier

152.0

110.0

Qualifier 1

152.0

63.0

Qualifier 2

152.0

79.0

Quantifier

222.3

136.1

Qualifier 1

222.3

63.3

Qualifier 2

222.3

59.2

Neg

Note: RF lens values are not optimized for the individual transitions (calibrated RF lens value is used).

Boušová, K.; Bruggink, C.; Godula, M.

Sponsor Report Mass spectrometer calibration - extended mass range (EMRS) versus classic (with polytyrosine) Since the target analytes are small molecules with product ions after fragmentation < 100 Daltons, it is recommended to calibrate the mass spectrometer with the Thermo Scientific™ Pierce™ triple quadrupole, EMRS, calibration solution. It consists of 14 components (mass range from 69 m/z to 2800 m/z) for the calibration in both positive and negative ionization modes. This solution improves mass accuracy and transmission compared to conventional polytyrosine tune solutions, especially in the low m/z range where many of the polar pesticides are found. Identification and quantification Identification of the pesticides was indicated by the presence of three or four transition ions measured in SRM mode corresponding to the retention times (± 2.5%) of the corresponding standards. The measured peak area ratios for qualifier and quantifier ions must be in close agreement with ratios of the standards [6] as shown in Table VII. The quantifier and qualifier ions were selected among the product ions produced by the fragmentation of the selected precursor ion on the basis of the intensity and selectivity. Matrixmatched calibration was utilized for the quantification of the target pesticides in the samples. A calibration curve was plotted as the peak area is a linear function of the concentration of the analyte. RESULTS AND DISCUSSION The objective of this study was to evaluate the possibility of IC-MS/MS application for fast routine analysis of polar pesticides and their metabolites in food extracts. Various analytical parameters were assessed and the results of these experiments are described. Samples and quality control materials For recovery and repeatability experiments, blank matrices of lettuce, orange and flour were used. The absence of the target analytes was checked by repeated measurements of the food products purchased in local food stores. After homogenization the blank food samples were weighed and fortified at desired levels with working standard solution. Sample preparation was performed as described above. In addition, the method's accuracy was assessed using a FAPAS T19186 test sample of red grape containing a known amount of ethephon. Matrix effect Strong sample matrix effects were identified comparing calibration curves obtained using matrix-matched calibration standards with those derived from the use of matrix free standards. The significant difference for both slopes and intercepts (> 20%) obtained for all analytes-calibrations and all investigated matrices strongly suggested the use of matrix matched calibrations. The influence of matrix can be observed by comparing chromatograms of spiked orange sample (Figure 3 and Figure 4) and the related chromatograms of standard mixture (Figure 5).

Fast Routine Analysis of Polar Pesticides in Foods by Suppressed Ion Chromatography and Mass Spectrometry

Sponsor Report

Figure 3. SRM chromatograms of multiple transitions in orange sample spiked with 10 pesticides at level 100 Îźg/kg.

Figure 4. SRM chromatograms of orange sample spiked with 10 pesticides at level 10 Îźg/kg.

Boušová, K.; Bruggink, C.; Godula, M.

Sponsor Report

Figure 5. SRM chromatograms of standard mixture in solvent (water) with 10 pesticides at concentration 100 μg/L.

Matrix matched calibration & linearity Matrix-matched calibration standards were prepared from blank extracts (Table VI). It is recommended to add an aliquot volume of a working standard to fresh blank extract and use this solution for the preparation of matrix-matched calibration. The linearity of the calibration curves for all target compounds was demonstrated for the concentration range from 0 to 600 μg/kg; correlation coefﬁcients obtained ranged from 0.985 to 0.990. Table VI. Preparation of matrix-matched calibration standards. Final volume = 1 mL; working standard solution concentration = 1000 μg/L Cal. STD 1

Cal. STD 2

Cal. STD 3

Cal. STD 4

Cal. STD 5

Cal. STD 6

Cal. STD 7

c (μg/kg) in Matrix

100

200

500

600

c (μg/L) in Vial

2.5

12.5

125

150

V (μL) of Working Standard Solution

2.5

12.5

125

150

1000

997.5

987.5

975

950

875

850

V (μL) of Blank Extract

Selectivity Due to the SRM mode used for the measurements, the selectivity was confirmed based on the presence of the transition ions (quantifier and two qualifiers) at the retention times corresponding to those of the respective pesticides. The measured peak area ratios of qualifier/quantifier are within ± 30% (relative) of average of calibration standards from the same sequence, defined in Reference 6 when compared to the standards (Table VII).

Fast Routine Analysis of Polar Pesticides in Foods by Suppressed Ion Chromatography and Mass Spectrometry

Sponsor Report

Table VII. Ion ratios (Quan/Qual1 and Quan/Qual2) in matrix and standard mixture at level 10 μg/kg (μg/L) Ion Ratio Standard Mix – Quan / Qual1 0.73

Standard Mix – Quan / Qual2 0.43

Lettuce – Quan / Qual1 0.76

Lettuce – Quan / Qual2 0.47

Oranges – Quan / Qual1 0.70

Oranges – Quan / Qual2 0.48

Flour – Quan / Qual1 0.72

Flour – Quan / Qual2 0.48

Ethephon

0.50

0.33

0.53

0.38

0.50

0.33

Fosetyl-Al

0.38

0.13

0.30

0.27

0.29

0.28

0.19

Glufosinate

0.46

0.65

0.53

0.56

0.47

0.62

0.47

0.58

Glyphosate

0.70

0.49

0.74

0.46

0.74

0.45

0.77

0.48

HEPA

0.13

0.31

0.12

0.40

0.11

0.34

0.14

0.36

MPPA

0.40

0.06

0.34

0.08

0.36

0.06

0.43

0.09

N-acetyl-AMPA

0.48

0.14

0.49

0.16

0.50

0.15

0.47

0.18

N-acetylglufosinate

0.26

0.23

0.21

0.27

0.21

0.26

0.25

0.29

Phosphonic acid

0.31

0.49

0.14

0.43

0.33

Compound Compound AMPA

The agreement between ion ratios should be within the permitted tolerance, which is deﬁned in SANTE 11945/2015 [6]. Note: *Ethephon was not detectable in the wheat ﬂour samples, **Ion Qual2 is coeluting with interference of the same m/z.

Precision and accuracy The precision and accuracy of the method was determined by analyzing fortified blank samples of lettuce, oranges, and wheat flour. The samples were fortified by addition of a calculated amount of working solution to the homogenized food matrix. Six replicates at three different concentration levels were prepared, after the sample was left 30 minutes to allow soaking of the standard into the matrix. The intermediate precision was determined by the analysis of two other sample sets, with six replicates prepared only at one concentration level (middle level) and measured over a period of two days. The results are shown in Table VIII to Table X. Additional accuracy was established for ethephon by analyzing FAPAS T19186 proficiency test material. The matrix was red grape purée and the obtained results were in the satisfactory range (Table XI). The most demanding sample matrix during this work was wheat flour, as low recovery was achieved in flour matrix for glyphosate and AMPA, and ethephon couldn't be analyzed at all. One possible way to improve the recovery for this troublesome matrix is to use labeled internal standards. Table VIII. Results of method precision (expressed as relative standard deviation – RSD (%)) at three different spike levels (n=6) Spiking Levels (μg/kg) Compound Compound

RSD (%)

All Three Matrices

Lettuce

Oranges

Flour

III

AMPA

200

500

Ethephon

200

500

Fosetyl-Al

200

500

Glufosinate

200

500

Glyphosate

200

500

HEPA

200

500

MPPA

200

500

N-acetyl-AMPA

200

500

N-acetyl-glufosinate

200

500

Phosphonic acid

200

500

17 75

Boušová, K.; Bruggink, C.; Godula, M.

Sponsor Report Table IX. Results of method accuracy (expressed as recovery) at three different levels (n=6) Recovery (%)

Compound

Lettuce

Oranges

Flour

III

AMPA

Ethephon

120

Fosetyl-Al

111

Glufosinate

101

Glyphosate

HEPA

118

MPPA

116

N-acetyl-AMPA

N-acetyl-glufosinate

Phosphonic acid

115

122

Table X. Method intermediate precision expressed as RSD (%) Intermediate Precision at Level II (%)

Compound

Lettuce Day 1

Oranges

Day 2

Day 3

Day 1

Day 2

Flour Day 3

Day 1

Day 2

AMPA

Ethephon

Fosetyl-Al

Glufosinate

Glyphosate

HEPA

MPPA

N-acetyl-AMPA

N-acetyl-glufosinate

Phosphonic acid

Note: Six sample replicates were prepared for each set at one level and measured during three days. Flour samples were measured in only two days due to time constraints. Table XI. Results of FAPAS proﬁciency test material – red grape purée T19186. Compound Ethephon

Assigned Value (μg/kg)

Average (μg/kg) RSD (%)

RSD (%)

REC (%)

629 ± 216

553

Limits of detection (LOD) and quantification (LOQ) Limits of detection and quantification were estimated following the IUPAC approach, which consists of analyzing the blank sample to establish noise levels and then estimating LODs and LOQs for signal/noise at 3 and 10, respectively. In addition, the sample injections' repeatability at the LOQ level has to be below 20% (expressed as RSD; n = 3). The LODs and LOQs are shown in Table XII. Finally, as shown in Table XIII, reported LOQs comply with currently valid pesticide maximum residue limits (MRL) defined by the EU and allow the use of the method in routine food control for the tested pesticides and matrices. In Figure 4 is shown the chromatogram of spiked orange sample at level 10 μg/kg, since this value is for most of the target pesticides LOD or LOQ.

Fast Routine Analysis of Polar Pesticides in Foods by Suppressed Ion Chromatography and Mass Spectrometry

Sponsor Report

Table XII. Limits of detection and quantiﬁcation of the method (LOD and LOQ) for lettuce, oranges, and wheat ﬂour shown Lettuce

Compound AMPA

Oranges

Flour

LOD (μg/kg)

LOQ (μg/kg)

LOD (μg/kg)

LOQ (μg/kg)

LOD (μg/kg)

LOQ (μg/kg)

20 Not detected

Ethephon

Not detected

Fosetyl-Al

Glufosinate

Glyphosate

HEPA

MPPA

N-acetyl-AMPA

N-acetyl-glufosinate

Phosphonic acid

Table XIII. Comparison of method detection limits and maximum residue limits deﬁned by EC 396/2005 [2] Lettuce

Compound AMPA

Oranges

Flour

MRL (μg/kg)

LOQ (μg/kg)

MRL (μg/kg)

LOQ (μg/kg)

MRL (μg/kg)

LOQ (μg/kg)

n.r.

n.d

1000

Not detected

75,000

2000

500

100

Glyphosate

100

500

10,000

HEPA

n.r.

MPPA

n.r.

N-acetyl-AMPA

n.r.

N-acetyl-glufosinate

n.r.

75,000

Ethephon Fosetyl-Al

Glufosinate

Phosphonic acid

Note: n.r.= not required 1 Fosetyl-Al = sum of fosetyl, phosphonic acid and their salts, expressed as fosetyl 2 Glufosinate ammonium = sum of glufosinate, its salts, MPPA, and NAG, expressed as glufosinate equivalents

CONCLUSIONS The reported in-house validated method enables the quantiﬁcation of ten polar ionic compounds or four ionic pesticides and their metabolites in different food matrices by coupling ion chromatography to a triple quadrupole mass spectrometer. In contrast to methods described in the literature, sample preparation was simpliﬁed and the use of ion chromatography speeds up the separation. This method can be recommended as a reliable and cost-effective solution for any routine lab dealing with the determination of polar pesticides and their metabolites in food samples.

Boušová, K.; Bruggink, C.; Godula, M.

Sponsor Report REFERENCES 1. International Agency for Research on Cancer, World Health Organization, Q&A on Glyphosate. https://www.iarc.fr/en/media-centre/iarcnews/pdf/Q&A_Glyphosate.pdf (last accessed October 11, 2016). 2. European Food Safety Authority, Pesticides Unit, EFSA explains the carcinogenicity assessment of glyphosate. https://www.efsa.europa.eu/sites/default/files/4302_glyphosate_complementary.pdf (last accessed October 11, 2016). 3. Regulation (EC) No 396/2005 of the European Parliament and of the Council of 23 February 2005 on maximum residue levels of pesticides in or on food and feed of plant and animal origin and amending Council Directive 91/414/EEC Text with EEA relevance. http://ec.europa.eu/food/plant/pesticides/eupesticides-database/public/?event=homepage&language=EN (last accessed October 11, 2016). 4. Hanke, I.; Singer, H.; Hollender, J. Anal Bioanal Chem. 2008, 391 (6), pp 2265-2276. doi: 10.1007/s00216-008-2134-5 5. European Commission, QuPPe Method. http://quppe.eu/ (last accessed October 11, 2016). 6. European Commission, Guidance document on analytical quality control and method validation procedures for pesticides residues analysis in food and feed. http://ec.europa.eu/food/plant/docs/pesticides_mrl_guidelines_wrkdoc_11945.pdf (last accessed October 11, 2016).

This sponsor report is the responsibility of Thermo Fisher Scientiﬁc.

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Notices of Books Foundations of Analytical Chemistry: A Teaching–Learning Approach Miguel Valcárcel Cases, Ángela I. López-Lorente, Ma Ángeles López-Jiménez - Authors August 2017, Springer This book offers a completely new approach to learning and teaching the fundamentals of analytical chemistry. A distinct feature of this novel book is its focus on the fundamental concepts and essential principles of analytical chemistry, which sets it apart from other books presenting descriptive overviews of methods and techniques. Read more…

Nanomaterials in Chromatography: Current Trends in Chromatographic Research Technology and Techniques Chaudhery Mustansar Hussain - Editor July 2018, Elsevier This book provides recent advancements in the wide variety of chromatographic techniques applied to nanotechnology. It includes chapters on such crucial topics as the use of nanomaterials in sample preparation and the legalization of nanomaterials, along with a section on reducing the cost of the analysis process, both in terms of chemicals and time consumption. Read more...

Mass Spectrometry - A Textbook 3rd Edition Gross, Jürgen H. - Author 2017, Springer This third edition of the highly successful textbook, acclaimed for its comprehensiveness, accuracy, and excellent illustrations and photographs now comes with updated coverage plus numerous didactical improvements. Read more…

Direct Analysis in Real Time Mass Spectrometry: Principles and Practices of DART-MS Yiyang Dong - Editor Nov 2017, Wiley DART-MS is a relatively new, but very fast evolving technology. Due to its versatility, it addresses ﬁelds of crucial importance to people and community, e.g. food or agricultural, forensic, industrial, environmental, medicinal and clinical analysis. Read more…

Books Handbook of Advanced Chromatography / Mass Spectrometry Techniques Michal Holcapek, Wm. Craig Byrdwell - Editors Sept 2017, Elsevier This book is a compendium of new and advanced analytical techniques that have been developed in recent years for analysis of all types of molecules in a variety of complex matrices, from foods to fuel to pharmaceuticals and more. Focusing on areas that are becoming widely used or growing rapidly, this is a comprehensive volume that describes both theoretical and practical aspects of advanced methods for analysis. Read more…

Targeted Biomarker Quantitation by LC-MS Naidong Weng, Wenying Jian - Editors July 2017, Wiley The ﬁrst book to offer a blueprint for overcoming the challenges to successfully quantifying biomarkers in living organisms. It provides a detailed guide for quantifying biomarkers in biological systems. It uses numerous real-world cases to exemplify key concepts, all of which were carefully selected and presented so as to allow the concepts they embody to be easily expanded to future applications, including new biomarker development. Read more…

Biosensors: An Introductory Textbook Jagriti Narang, C.S. Pundir - Editors April 2017, Pan Stanford This handbook has evolved from the authors' teaching and research experience in the ﬁeld of nanoparticle biosensing. It encompasses: study of the various characterizing techniques that help deduce the shape, size, and morphology of nanoparticles; and applications of nanoparticles in the ﬁeld of biosensors. It is a practical and user-friendly textbook that introduces the various basic principles and practical information that will help undergraduate and advanced-level students and researchers understand the science behind nanoscale sensing. Read more…

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Periodicals & Websites

Jan/Feb 2018 Volume 50, Nº 1

American Laboratory The American Laboratory® publication is a platform that provides comprehensive technology coverage for laboratory professionals at all stages of their careers. Unlike ® single-channel publications, American Laboratory is a multidisciplinary resource that engages scientists through print, digital, mobile, multimedia, and social channels to provide practical information and solutions for cutting-edge results. Addressing basic research, clinical diagnostics, drug discovery, environmental, food and beverage, forensics, and other markets, American Laboratory combines in-depth articles, news, and video to deliver the latest advances in their ﬁelds. Read more… LCGC

January 2018 Volume 36, Issue 1

Chromatographyonline.com is the premier global resource for unbiased, peer-reviewed technical information on the field of chromatography and the separation sciences. Combining all of the resources from the regional editions (LCGC North America, LCGC Europe, and LCGC Asia-Pacific) of award winning magazines, Chromatographyonline delivers practical, nuts-and-bolts information to help scientists and lab managers become more proficient in the use of chromatographic techniques and instrumentation, thereby making laboratories more productive and businesses around the world more successful. Read more…

Scientia Chromatographica

2017, Volume 9, Nº 3

Scientia Chromatographica is the first and to date the only Latin American scientific journal dedicated exclusively to Chromatographic and Related Techniques (Mass Spectrometry, Sample Preparation, Electrophoresis, etc.). With a highly qualified and internationally recognized Editorial Board, it covers all chromatography topics (HPLC, GC, SFC) in all their formats, in addition to discussing many related topics such as "The Pillars of Chromatography", Quality Management, Troubleshooting, Hyphenation (GC-MS, LC-MS, SPE-LC-MS/MS) and others. It also provides columns containing general information for the area, such as: calendar, meeting report, bookstore, etc. Read more… Select Science ® SelectScience promotes scientists and their work, accelerating the communication of successful science. SelectScience® informs scientists about the best products and applications through online peer-to-peer information and product reviews. Scientists can make better decisions using independent, expert information and gain easy access to manufacturers. SelectScience® informs the global community through Editorial Features, Event Coverage, Video and Webinar programs. Read more… Spectroscopy Spectroscopy's mission is to enhance productivity, efficiency, and the overall value of spectroscopic instruments and methods as a practical analytical technology across a variety of fields. Scientists, technicians, and laboratory managers gain proficiency and competitive advantage for the real-world issues they face through unbiased, peerreviewed technical articles, trusted troubleshooting advice, and best-practice application solutions. Read more…

January 2018 Volume 33, Issue 1 87

Br. J. Anal. Chem., 2017, 4 (17), pp 88-89

Events February 18 - 21 34th International Symposium on Microscale Separations and Bioanalysis (MSB 2018) Windsor Barra Hotel (Barra da Tijuca), Rio de Janeiro, RJ, Brazil www.msb2018.org

February 26 - March 1 PITTCON Conference and Expo 2018 Orange County Convention Center, Orlando, FL, USA pittcon.org/pittcon-2018/

February 26 - March 2 11th Winter Symposium on Chemometrics (WSC 11) "Avrora-Klub" Center, St. Petersburg, Russia wsc.chemometrics.ru/wsc11/

May 21 - 24 41th Annual Meeting of the Brazilian Chemical Society (41th RASBQ) Rafain Palace Hotel, Foz do Iguaçu, PR, Brazil www.sbq.org.br/41ra/

June 3 - 6 2nd Latin American Congress of Clinical and Laboratory Toxicology (II TOXILATIN) Federal University of Rio Grande do Sul, Porto Alegrre, RS, Brazil

June 19 - 22 th 40 International Conference on Environmental & Food Monitoring Santiago de Compostela, Spain www.iseac40.es

June 25 - 29 17th Conference on Chemometrics in Analytical Chemistry (CAC 2018) Halifax, Canada www.cac2018halifax.com

July 24 - 27 XII Workshop on Sample Preparation (XII WPA) Institute of Chemistry, University of São Paulo, São Paulo, SP, Brazil www3.iq.usp.br/

Books September 16 - 19 19th Brazilian Meeting on Analytical Chemistry (19th ENQA) 7th Ibero-American Congress of Analytical Chemistry (7th CIAQA) Complexo Acqua DiRoma, Caldas Novas, GO, Brazil enqa2018.com.br/ September 19-20 19th World Congress on Analytical & Bioanalytical Techniques Singapore analytika.pharmaceuticalconferences.com November 4 - 8 6th Brazilian Meeting on Forensic Chemistry (6th ENQFor) & 3rd Meeting of the Brazilian Society of Forensic Sciences (SBCF) Convention Center of RibeirĂŁo Preto, SP, Brazil www.sbcf.org.br November 12 - 15 XIII Latin American Symposium on Environmental Analytical Chemistry (XIII LASEAC) La Serena, Chile December 8 - 12 7th Brazilian Conference on Mass Spectrometry (7th BrMASS) Windsor Barra Hotel, Rio de Janeiro, RJ, Brazil http://www.brmass.com

Br. J. Anal. Chem., 2017, 4 (17), pp 90-92

Acknowledgement The BrJAC's editors are grateful to all those who have refereed papers in the last two years (2016-17) using signiﬁcant time and effort to provide constructive inputs. Alessandra Sussulini – UNICAMP Aline Klassen – UNIFESP Ana Cristi Basile Dias – UnB Ana Valéria Cantú – UNICAMP André Fernando de Oliveira – UFV Anne Hélène Fostier - UNICAMP Auro Atsushi Tanaka – UFMA Boaventura Freire dos Reis – USP Carlos Roberto de Menezes Peixoto – FURG Carolina Maria Machado de Carvalho Andrade – SENAI Cassiana Carolina Montagner Raimundo – UNICAMP Cassiana Seimi Nomura – USP Cesar Ricardo Teixeira Tarley – UEL Christiana Andrade Pessoa – UEPG Cláudia Carvalhinho Windmoller – UFMG Cristina Donizeti Bernardes – FUNCESI Daniella Lopez Vale – UFRJ Diego Pereira dos Santos – UNICAMP Dirce Pozebon – UFRGS Djenaine de Souza – UFU Dosil Pereira de Jesus – UNICAMP Edenir Rodrigues Pereira-Filho – UFSCar Éder José dos Santos – TECPAR Edilson Benvenutti – UFRGS Eduardo de Almeida – USP Eduardo Mathias Richter – UFU Elen Romão Sartori Braz – UEL Eliana F. G. C. Dores – UFMT Elias Ayres Guidetti Zagatto – USP Elizabeth de Souza Nascimento – USP Emerson Schwingel Ribeiro – UFRJ Erik Galvão Paranhos da Silva – UESC Evandro Bona – UTFPR Fabio Augusto – UNICAMP Fábio Rodrigues Piovezani Rocha – USP Fernanda Veronesi Marinho Pontes – UFRJ Francisco José Krug – USP Gilberto Batista de Souza – Embrapa Pecuária Sudeste Joaquim de Araújo Nóbrega – UFSCar Jorge Eduardo de Souza Sarkis – IPEN José Luiz da Costa – UNICAMP Leandro Wang Hantao – UNICAMP 90

Acknowledgement Lucia Regina R. Martins – UTFPR Luiz Alexandre Sacorague – PETROBRAS Manoel Leonardo Martins – FURG Manoel Lima de Menezes – UNESP Marcelo Filonzi dos Santos – USP Marcia Foster Mesko – UFPel Márcia Miguel Castro Ferreira – UNICAMP Marcio Eduardo Vidotti Miyata – UFPR Marco Tadeu Grassi – UFPR Marcone Augusto Leal de Oliveira – UFJF Marcos Yassuo Kamogawa – USP Maria do Carmo Hespanhol – UFV Maria Josefa Santos Yabe – UEL Maria Valnice Boldrin – UNESP Marial Del Pilar Sotomayor – UNESP Mariela Monica Pistón Pedreira – Universidad de la República - Uruguai Marise Tenorio Wanderley Hübner – FIOCRUZ Mathieu Tubino – UNICAMP Mônica Teixeira da Silva – PETROBRAS Nadia Regina Rodrigues – UNICAMP Paulo Clairmont Feitosa de Lima Gomes – UNESP Paulo Roberto Filgueiras – UFES Pedro de Lima Neto – UFC Pedro Orival Luccas – UNIFAL Rachel Ann Hauser-Davis – CESTEH / ENSP / FIOCRUZ Rafael Arromba de Sousa – UFJF Reinaldo Teofilo – UFV Renato Zanella – UFSM Rennan Araújo – UFBA Ricardo Santelli – UFRJ Rita de Cassia Silva Luz – UFMA Rodinei Augusti – UFMG Rodolfo G. Wuilloud – Universidad Nacional de Cuyo - Argentina Rodrigo Alejandro Abarza Muñoz – UFU Rodrigo Moretto Galazzi – UNICAMP Ronaldo Censi Faria – UFSCar Ronei Jesus Poppi – UNICAMP Rosangela Gorni – Nestle Brasil Ltda. Rose Mary G. Naal – USP Sandro Navickiene – UFS Sergio Toshio Fujiwara – UEPG Solange Cadore – UNICAMP 91

Acknowledgement Susane Rath – UNICAMP Thiago R. L. Paixão – USP Tiago Santana Balbuena – UNESP Tiele Medianeira Rizzetti – UFSM Valderi Luiz Dressler – UFSM Verônica Maria de Araújo Calado – UFRJ Wanessa Melcher Mattos – USP Wendell Coutro – UFG

Br. J. Anal. Chem., 2017, 4 (17), pp 93-96

Author's Guidelines The Brazilian Journal of Analytical Chemistry (BrJAC) is a peer-reviewed scientific journal intended for professionals and institutions acting mainly in all branches of Analytical Chemistry. BrJAC is an open access journal which does not charge authors an article processing fee. Scope BrJAC is dedicated to professionals involved in science, technology and innovation projects in the area of analytical chemistry at universities, research centers and in industry. About this journal BrJAC publishes original, unpublished scientific articles and technical notes that are peer reviewed in the double-blind way. In addition, it publishes reviews, interviews, points of view, letters, sponsor reports, and features related to analytical chemistry. BrJAC's review process begins with an initial screening of the manuscripts by the editor-in-chief, who evaluates the adequacy of the study to the journal scope. Manuscripts accepted in this screening are then forwarded to at least two referees indicated by the editors. As evaluation criteria, the referees will employ originality, scientific quality and contribution to knowledge in the field of Analytical Chemistry, the theoretical foundation and bibliography, the presentation of relevant and consistent results, compliance to the journal's guidelines, and the clarity of writing and presentation. Brief description of the BrJAC sections · Articles: Full descriptions of an original research finding in Analytical Chemistry. Manuscripts submitted for publication as articles, either from universities, research centers, industry or any other public or private institution, cannot have been previously published or be currently submitted for publication in another journal. Articles undergo double-blind full peer review. · Reviews: Articles on well-established subjects, including a critical analysis of the bibliographic references and conclusions. Manuscripts submitted for publication as reviews must be original and unpublished, and undergo double-blind full peer review. · Technical Notes: Concise descriptions of a development in analytical method, new technique, procedure or equipment falling within the scope of BrJAC. Technical notes also undergo double-blind full peer review. The title of the manuscript submitted for technical note must be preceded by the words "Technical note". · Sponsor Reports: Concise descriptions of technical studies not submitted for review by referees. Sponsor responsibility documents. · Letters: Discussions, comments, suggestions on issues related to Analytical Chemistry, and consultations to authors. Letters are welcome and will be published at the discretion of the editor-in-chief. · Points of view: The expression of a personal opinion on some relevant subject in Analytical Chemistry. · Interviews: Renowned chemist researchers are invited to talk with BrJAC about their expertise and experience in Analytical Chemistry. · Releases: Articles providing new and relevant information for the community involved in analytical chemistry, and companies' announcements on the launch of new products of interest in analytical chemistry. · Features: A feature article gives to the reader a more in-depth view of a topic, a person or opinion of acknowledged interest for Analytical Chemistry. Manuscript preparation (download a template on the BrJAC website) The manuscript submitted to BrJAC must be written in English and should be as clear and succinct as possible. It must include a title, an abstract, a graphical abstract, keywords, and the following sections: Introduction, Methods, Results and Discussion, Conclusion, and References. Because the manuscripts are subjected to double-blind review, they must NOT contain the authors' names, affiliations, or acknowledgments. The manuscript must be typed in Arial font size 11 pt., and the lines numbered consecutively and double-spaced throughout the text, except in the figure captions, titles of tables and references. 93

Author's Guidelines The manuscript title should be short, clear and succinct, and a subtitle may be used, if needed. The abstract should include the objective of the study, essential information about the methods, the main results and conclusions. Then, three to five keywords must be indicated. The section titles should be typed in bold and subsections in italics. Graphics and tables must be numbered according to their citation in the text, and should appear close to the discussion about them. For figures use Arabic numbers, and for tables use Roman numbers. The captions for the figures must appear below the graphic; for the tables, above. The same result should not be presented by more than one illustration. For figures, graphs, diagrams, tables, etc. identical to others previously published in the literature, the author must ask for permission for publication from the company or scientific society holding the copyrights, and send this permission to the BrJAC editor-in-chief with the final version of the manuscript. The chemical nomenclature should conform to the rules of the International Union of Pure and Applied Chemistry (IUPAC) and Chemical Abstracts Service. It is recommended that, whenever possible, authors follow the International System of Units, the International Vocabulary of Metrology (VIM) and the NIST General Table of Units of Measurement. Abbreviations are not recommended except those recognized by the International Bureau of Weights and Measures or those recorded and established in scientific publications. If the abbreviations are numerous and relevant, place their definitions in a separate section (Glossary). The manuscript must include only the consulted references, numbered according to their citation in the text, with numbers in square brackets. It is not recommended to mention several references with identical statements - select the author who demonstrated them. It is recommended that references older than 5 (five) years be avoided, except in relevant cases. Include references that are accessible to readers. References should be thoroughly checked for errors before submission. Manuscripts must be submitted in conjunction with an analysis report of plagiarism obtained through anti-plagiarism software. BrJAC indicates CopySpider© 2013 freeware to support plagiarism checking analyzes. Download the CopySpider freeware: www.copyspider.com.br

Examples of reference formatting Journals 1. Arthur, K. L.; Turner, M. A.; Brailsford, A. D.; Kicman, A. T.; David A. Cowan, D. A.; Reynolds, J. C.; Creaser, C. S. Anal. Chem. 2017, 89, pp 7431- 7437 (DOI: 10.1021/acs.analchem.7b000940). The titles of journals must be abbreviated as defined by the Chemical Abstracts Service Source Index (http://cassi.cas.org/search.jsp). If a paper does not have a full reference, please provide its DOI, if available, or its Chemical Abstracts reference information. Electronic journals 2. Natarajan, S.; Kempegowda, B. K. LCGC North America, 2015, 33 (9), pp 718-726. Available from: http://www.chromatographyonline.com/analyzing-trace-levels-carbontetrachloride-drugsubstanceheadspace-gc-flame-ionization-detection [Accessed 10 November 2015]. Books 3. Burgot, J.-L. Ionic Equilibria in Analytical Chemistry. Springer Science & Business Media, New York, 2012, Chapter 11, p 181. 4. Griffiths, W. J.; Ogundare, M.; Meljon, A.; Wang, Y. Mass Spectrometry for Steroid Analysis. In: Mike, S.L. (Ed.). Mass Spectrometry Handbook, v. 7 of Wiley Series on Pharmaceutical Science and Biotechnology: Practices, Applications and Methods. John Wiley & Sons, Hoboken, N.J., 2012, pp 297-338. Standard methods 5. International Organization for Standardization. ISO 26603. Plastics — Aromatic isocyanates for use in the production of polyurethanes — Determination of total chlorine. Geneva, CH: ISO, 2017.

Author's Guidelines Master’s and doctoral theses or other academic literature 1. 6. Ek, P. New methods for sensitive analysis with nanoelectrospray ionization mass spectrometry. Doctoral thesis, 2010, School of Chemical Science and Engineering, Royal Institute of Technology, Stockholm, Sweden. Patents 2. 7. Trygve, R.; Perelman, G. US 9053915 B2, June 9 2015, Agilent Technologies Inc., Santa Clara, CA, US. Web pages 3. 8. http://www.chromedia.org/chromedia [Accessed 21 June 2015]. Unpublished source 9. Mendes, B.; Silva, P.; Pereira, J.; Silva, L. C.; Câmara, J. S. Poster presented at: 36th International 4. Symposium on Capillary Chromatography, 2012, Riva del Garda, Trento, IT. 10. Author, A. A. J. Braz. Chem. Soc., in press. 5. 11. Author, B. B., 2015, submitted for publication. 6. 7. 12. Author, C. C., 2011, unpublished manuscript. Note: Unpublished results may be mentioned only with express authorization of the author(s). Personal communications can be accepted exceptionally. Manuscript submission Three different PDF files, as described below, must be sent online through the website www.brjac.com.br I. A cover letter addressed to the editor-in-chief with the full manuscript title, the full names of the authors and their affiliations, the complete contact information of the corresponding author, including the ORCID iD, and the manuscript abstract. This letter must present why the manuscript is appropriate for publication in BrJAC, and contain a statement that the article has not been previously published and is not under consideration for publication elsewhere. The corresponding author must declare on behalf of all the authors of the manuscript any financial conflicts of interest or lack thereof. This statement should include all potential sources of bias such as affiliations, funding sources and financial or management relationships which may constitute a conflict of interest. When the manuscript belongs to more than one author, the corresponding author must also declare that all authors agree with publication in BrJAC. II.The manuscript file that must NOT mention the names of the authors or the place where the work was performed, but must include the title, abstract, keywords, and all sections of the work, including tables and figures, but excluding acknowledgments that will be included in the final paper upon completion of the review process. III. An analysis report of plagiarism on the manuscript. A Sponsor Report should be sent as a Word file attached to a message to the email brjac@brjac.com.br Revised manuscript submission Based on the comments and suggestions of the reviewers and editors a revision of the manuscript may be requested to the authors. The revised manuscript submitted by the authors must contain the changes made in the manuscript clearly highlighted. A letter without any author's information must also be sent with each reviewer's comment items and a response to each item. Copyright When submitting their manuscript for publication, the authors agree that the copyright will become the property of the Brazilian Journal of Analytical Chemistry, if and when accepted for publication. The copyright comprises exclusive rights of reproduction and distribution of the articles, including reprints, photographic reproductions, microfilms or any other reproductions similar in nature, including translations. Final Considerations

Whatever the nature of the submitted manuscript, it must be original in terms of methodology, information, interpretation or criticism. As to the contents of published articles, the sole responsibility belongs to the authors, and Br. J. Anal. Chem. and its editors, editorial board, employees and collaborators are fully 95

Author's Guidelines

exempt from any responsibility for the data, opinions or unfounded statements BrJAC reserves the right to make, whenever necessary, small alterations to the manuscripts in order to adapt them to the journal rules or make them clearer in style, while respecting the original contents. The article will be sent to the authors for approval prior to publication.

rd meeting of the

6 ENQ th

Brazilian Society of Forensic Sciences

For

National Meeting of Forensic Chemistry

Integrated Congress

04 to 08 november 2018 Convention Center - Ribeirão Preto - SP - Brazil

"The challenges of Forensic Sciences in the integration between know ledge, intelligence and expert technique" Organized by

Held by

Brazilian Society of Forensic Sciences UN

University of São Paulo IV

U PA S ID ADE DE SÃO

Brazilian Society of Forensic Sciences, Faculty of Philosophy, Sciences and Letters of Ribeirão Preto (FFCLRP), and University of São Paulo (USP)

http://www.sbcf.org.br

October â&#x20AC;&#x201C; December 2017 Volume 4 Number 17