BrJAC - N21

Forensic Analytical Chemistry

October – December 2018 Volume 5 Number 21

24 to 26 SEPTEMBER - 2019

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VisĂŁo Fokka - Communication Agency

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BrJAC publishes original, unpublished scientific articles and technical notes that are peer reviewed in the double-blind way.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.

BrJAC is Published by: VisĂŁo 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

Editorial Assistant Silvana Odete Pisani brjac@brjac.com.br Art Director: Adriana Garcia WebMaster: Daniel Letieri ISSN 2179-3433 digital

BrJAC’s website: www.brjac.com.br Like BrJAC on Facebook: https://www.facebook.com/brjachem

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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 / Postgraduate 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 / Department 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 / Department of Chemistry - Federal University of Maranhão, São Luís, MA, BR

Carlos Roberto dos Santos

Director of Engineering and Environmental Quality 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 / Department 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

Global Process Technology / Specialty Chemicals Manager - Braskem S.A. - Campinas, SP, BR

Marcos Nogueira Eberlin

Full Professor / School of Engineering - Mackenzie Presbyterian University, São Paulo, SP, BR

Maria das Graças Andrade Korn

Full Professor / Institute of Chemistry - Federal University of Bahia - Salvador, BA, BR

Ricardo Erthal Santelli

Full Professor / Analytical Chemistry - Federal University of Rio de Janeiro, RJ, BR

Contents

Br. J. Anal. Chem., 2018, 5 (21)

Editorial Editorial BrJAC: Eight Years Contributing to Analytical Chemistry Interview Professor Alice A. da Matta Chasin, who has extensive experience in Toxicology, with emphasis on Forensic Toxicological Analysis, recently gave an interview to BrJAC Point of View Forensic Analytical Chemistry

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Articles Bioactive Compounds, Antioxidant properties, and Metal Content Studies of Guava Fruit 8-18 by-products for Value Added Processing Development of Chromatographic Method for Determination of Impurities in Solid Dispersion 19-29 of Dasatinib Optical Analysis Authenticated Electrical Impedance Based Quantification of Aqueous 30-39 Naphthalene Features The 6th ENQFor&3rd SBCF Meeting discussed the Importance of Chemistry in Criminalistics 40-43 44-47 7th BrMASS and 4th BrProt brought together Scientists from Brazil and the World Sponsor Reports Forensics applications with Phenom desktop SEM Screening of 300 Drugs in Blood Utilizing Second Generation Exactive Plus High Resolution, Accurate Mass Spectrometer Microwave Sample Prep for heavy metals determination in cannabis plant, soil and water for medical applications

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Releases Phenom GSR – A Scanning Electron Microscope for Forensics Applications Exactive™ Plus Orbitrap Mass Spectrometer Ethos UP and Milestone Connect: High Performance Microwave Digestion Systems Pittcon Conference & Expo CHROMacademy Helps Increase your Knowledge, Efficiency and Productivity in the Lab SelectScience® Pioneers Online Communication and Promotes Scientific Success

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Notices of Books

82-83

Periodicals & Websites

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Events

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Acknowledgments

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Author’s Guidelines

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Editorial

Br. J. Anal. Chem., 2018, 5 (21), 1-1 DOI: 10.30744/brjac.2179-3425.2018.5.21.1-1

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BrJAC: Eight Years Contributing to Analytical Chemistry Lauro Tatsuo Kubota Full Professor Institute of Chemistry, University of Campinas - Unicamp Campinas, SP, Brazil kubota@unicamp.br When Brazilian Journal of Analytical Chemistry (BrJAC) was launched in 2010 to document some information that build the history of Analytical Chemistry from the novel discoveries to applications, which contributes to the great improvement in our life quality, it was a big challenge to be undertaken.

During these eight years, BrJAC has struggled to grow continuously and keeping the goals that were stablished at beginning, as a dream. Certainly, several improvements have been implemented in order to be better accepted by the people in the academia and industry. It was a privilege to be at the forefront of this project during these years as Editor-in-Chief, but now is time to go, leaving the responsibilities to other people to bring new energies and perspectives to the journal. I have to acknowledge all the people that were involved in this project with me during this time as well as all those who contributed to BrJAC. I am sure that Prof. Marco Aurelio Zezzi Arruda will do an excellent job for the BrJAC. It is important to stress that the success of BrJAC depends on the people who believe in this project and think it is important to the history of Analytical Chemistry. The reason for publishing interviews with renowned people is due to the importance to record the opinion and life history of the well succeeded and recognized people in Analytical Chemistry, showing their contributions to the area. In addition, the sections of point of view and letter are intended to record the opinion of the people about some subjects of Analytical Chemistry that provoke discussions. The articles about researches from the academia and companies are similar to those of any journal. Interesting themes are addressed in the journal, such as forensic science in this issue. I believe that BrJAC will continue to pursue the goals that were originally intended and planned, leaving a legacy for future generations. I hope you can enjoy reading this issue and many others, learning something from the history of Analytical Chemistry.

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Interview

Br. J. Anal. Chem., 2018, 5 (21), 2-5 DOI: 10.30744/brjac.2179-3425.2018.5.21.2-5

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Professor Alice A. da Matta Chasin, who has extensive experience in Toxicology, with emphasis on Forensic Toxicological Analysis, recently gave an interview to BrJAC Alice Aparecida da Matta Chasin Full Professor of Toxicology at the Faculty of Pharmacy and Biochemistry, Oswaldo Cruz, São Paulo, SP, Brazil alice.chasin@oswaldocruz.br Dr. Alice Aparecida Chasin held the position of Criminal Toxicologist of the Forensic Toxicology Nucleus of the Legal Medical Institute of São Paulo from 1976 to 2004. She was president of the Brazilian Society of Toxicology (SBTox) in the biennium 2004-2005. She has experience in toxicology with emphasis on toxicological analysis that applies mainly to the following subjects: drugs of abuse, crack, cocaine, cocaethylene, toxicology, ecotoxicology, toxicology forensics, and in the area of toxicology education. She also participated in research projects on drugs of abuse coordinated by the National Institute on Drug Abuse (NIDA). She was the representative of The International Association of Forensic Toxicologists (TIAFT) in Brazil during the period from 1995 to 2018. TIAFT is an association that aims to promote cooperation and coordination of efforts among members of this organization and to encourage research in forensic toxicology.

Her academic background includes a degree in Pharmacy and Biochemistry from the São Paulo State University - UNESP (1975), a master’s degree in Toxicology and Toxicology Analysis from the University of São Paulo - USP (1990) and a Ph.D. in Toxicology from the University of São Paulo – USP (1997). She has a Specialization in Drug Analysis conferred by WHO (World Health Organization - Narcotics Division) in 1994. During this period, she received scholarships to further her knowledge abroad in Salt Lake City, USA and in Buenos Aires, Argentina.

When was your first contact with the toxicology area? Did you have any influencer, for example, a teacher?

My first contact with toxicology was in college, during the undergraduate course of Pharmacy and Biochemistry. The professor of toxicology was great and motivated the students. But I also liked other disciplines a lot. Shortly before my graduation, a call for tenders was published to select forensic toxicologist experts for the Medico-Legal Institute of São Paulo (IML-SP). When I was recently graduated, I entered the contest, was approved, and started working as an expert in the so-called Forensic Toxicology Technical Service of the IML-SP. How was the beginning of your career? How do you see the progress of women in science?

Early in my career I fell in love with my job. Being able to speak for those who could no longer do so awakened me to the importance of forensic science and being able to participate in the development of forensic toxicology was extremely gratifying.

I see with great satisfaction the progress of women in science. We are many and we are strong (laughs). In Brazil, toxicology research is still carried out basically in public institutions, where admission is by contest and the number of women entering this career is very high.

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What has changed in the profile, ambitions, and performance of new forensic toxicology researchers since the beginning of your career?

In these 40 years in which I have been working with forensic toxicology, I realize that the experts, who were formerly technical professionals, started to behave as researchers and quite a significant number of experts have a master’s and doctoral degree in this area of action. That is, the interface between the practice of the craft and a strong academic background has consolidated more and more in recent years.

Could you briefly comment on recent developments in forensic and toxicological research?

Forensic research has grown significantly in Brazil and all of the world. Specifically, in relation to forensic toxicology, there has been a great advance in how the substances are identified using high resolution spectrometers, differentiating masses in the order of a thousandth of m/z. There has also been great progress in analytical measurements and the reliability of these measures. I explain, we went from an order of magnitude of ppm to ppb in analysis of in natura substances of toxicological interest, and from ppm to ppt in analyis of biological fluids that explain that there is an intoxication of medical-legal interest. This analytical possibility allowed matrices such as hair, nails, bones, etc. to be used as evidence of exposure.

Could you tell us how you got into the research area? Would you highlight any of your research works?

My entrance in the research area occurred when, after 10 years working with forensic toxicology at IML-SP, I decided to enter academic life, motivated by the demands that have arisen in professional work. In this sense, I would point out, for its historical value, the work of quantifying cocaine, its biotransformation products, and cocaethylene, which made possible the postmortem diagnosis of cocaine intoxication. I would also highlight the development of the analytical validations that allow the knowledge of analytical figures of merit, essential in the conduct of a legal procedure.

How do you keep yourself informed about the progress of scientific research in your area?

What is your opinion about the current progress of this research in Brazil? What are the recent advances and challenges in scientific research in Brazil?

I mainly follow the works of my former students, nowadays excellent researchers. As in all areas of science in Brazil, the development of research in the forensic and toxicological area is very difficult because of the government’s budget control over the state-level research promotion foundations, but also because of the bureaucracy involved to buy specific reagents and inputs, which can take years and seriously affect the start time or the completion of a research study. While a researcher in the US or Europe takes four weeks to plan and perform an in vitro test to find out what is the mechanism of action of a new drug, in Brazil that same study can take at least six months to a year because of the huge bureaucracy!

Today, the most recent advances in forensic toxicology research are analyses of new psychoactive substances (NPS), which are novel molecules with potent psychoactive properties that are synthesized and sold for recreational use. As there is a lack of information on the toxicity of these NPS, researchers in Brazil and in the world are conducting studies to find out how to identify these NPS in natura and in different biological materials and also to determine their detection time, their action in an organism, and the mechanism of this action, among other studies such as the influence of these NPS on driving a vehicle.

Could you mention some milestones of the breakthroughs in your area of expertise?

There are many. I would highlight the residue analyses performed thanks to the advancement of techniques that allow one to identify and quantify very low concentrations in biological material. I cite 3

Interview

the NPS as an example. If these NPS were synthesized in the 1980s - 90s, we would certainly have an extensive amount of work to just identify them. The advancement of analytical techniques has led to this identification.

Another milestone is the use of low volumes of biological samples for routine toxicological tests or even dry blood drop analysis, as for example in the Newborn Screening Test. To get an idea, an emergency toxicological analysis on a baby or a newborn needed a blood volume of the order of milliliters, and collecting that volume from a baby is actually very difficult. Today, volumes in the order of microliters are used; that is only one drop of blood and this facilitates the collection and reduces the suffering of the child and parents.

It is also important to highlight the decoding of exposure indicators, as well as the interpretation of their presence. Analysis of residues at trace levels of various chemical substances in the environment such as pesticides, drugs, metals, etc. indicate that we are polluting our planet more and more, year after year, and with this we are seeing an increase in the incidence of some diseases that were not prevalent in past decades.

And specifically, in the forensic area, I can highlight spectral interpretation techniques that allow the identification and quantification of NPS used as drugs of abuse without it being known that they are circulating in the illicit market. For example, there are cases, sometimes well-publicized in the media, in which a healthy young man who was at a party having fun had been found unconscious and had died in a hospital. The elucidation of the cause of death by an exogenous agent such as an NPS will serve to ensure that measures to control access to these NPS are adopted by regulatory agencies and that medical research is initiated in the search for a treatment and even an antidote for reversing intoxication.

There are in Brazil and in the world several meetings on the forensic and toxicology field. To you, how important are these meetings to the scientific community?

They are essential for any professional who works in these and other scientific areas. These are moments where important face-to-face contacts are established for discussions and for establishment of networking, partnership projects, thematic projects and, above all, the possibility of peer-driven professional growth. In these moments, the professionals are motivated to seek formal professional growth through the completion of post-graduate courses at the lato or stricto sensu level, eventually to carry out exchanges at other scientific centers, including abroad. Usually this perception of professional growth is accompanied by great satisfaction and joy, which is extremely gratifying.

You have already 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?

It is always wonderful to have your work recognized, especially when this recognition comes from your peers, those who work in the same area and know the difficulties of developing a quality work in this area of knowledge. Unfortunately, we live in a country where the sciences in general, and the forensic sciences in particular, are little recognized. These awards (already well-known in the societies representing toxicology and forensic science professionals) and the advent of TV serials related to the subject have propelled, through public opinion and the lay press, the forensic sciences that now integrate the thematic areas of main research funding agencies in Brazil.

For you, what is the importance of the funding support for the scientific development of Brazil?

It is fundamental. Without such support, any forensic research would be impossible. This is an area where any interference by the private sector is impossible but, in many cases, it can subsidize research in some way. In Brazil, research is, as a rule, maintained by funding agencies. We still do not have the culture of interaction between research institutes and universities with private companies, but specifically in forensic toxicology, this possibility does not apply.

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Interview

At the moment, the situation for scientific research in Brazil is one of decreasing investment.

How do you see this situation, and what would you say to young researchers?

I find this situation calamitous for ongoing research and for those that have not even started. One of the greatest challenges, which constitutes a barrier to Brazil becoming a scientific power, is the bureaucratization of the import policy of scientific material, such as analytical standards that, moreover, sometimes have prohibitive prices for a project. Added to this is the budget cut that has been affecting us since 2016 and which seems to have no prospect of being reversed. At times like this, with so many demonstrations of the importance of technical-scientific development in actions to prevent and solve problems, the finding that investment in research is not in the Brazilian government’s agenda further aggravates the situation in which we find ourselves.

However, my message to young researchers is this: Do not give up on your dreams! Seek academic education and publish articles, even if you consider that what is being produced has no relevance.

Expose your projects and studies to the scientific community and society. Participate in the meetings of professionals in your area. Discuss and participate as scientists and, especially, as citizens.

You have more than 30 years of dedication to the development of scientific research. Could you highlight what were the most important moments of these years?

There are several important moments that I could mention. One of the most important was my decision to pursue academic life when I was working for 10 years at the Legal Medical Institute. The job I was doing fascinated me. As I have already mentioned, being able to speak for those people who have died (what happens in the post mortem analyzes) has always thrilled me and very early I was aware that without science, it is impossible to carry out this task with such responsibility. I understood that in academic life I could reconcile these two aspects of my work, and it happened. To do the master’s and then the doctorate, both absolutely inserted in my professional area, was fundamental in my career.

Another important moment was the invitation for me to teach toxicology classes, initially at the Faculty of Pharmaceutical Sciences of Oswaldo Cruz Colleges, and then the invitation to be responsible for the forensic toxicology course in the postgraduate program at the University of SĂŁo Paulo and to integrate the adviser group of this program. I am very pleased to have been able to make this link between academia and practice, transforming academic knowledge to benefit society. However, without a doubt, I think that my greatest achievement was to have participated in the training of professionals who are now professors and experts who carry out their work with ethics and competence. This is my pride!

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Point of View

Br. J. Anal. Chem., 2018, 5 (21), 6-7 DOI: 10.30744/brjac.2179-3425.2018.5.21.6-7

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Forensic Analytical Chemistry Bruno Spinosa De Martinis Associate Professor (Forensic Chemistry) University of São Paulo Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto Department of Chemistry Laboratory of Forensic Toxicological Analysis martinis@usp.br The word “forensic” comes from the Latin language and it means public, to the forum or public discussion; argumentative, rhetorical, belonging to debate or discussion. A relevant, modern definition of forensic is relating to, used in, or suitable to a court of law. Any science used for the purposes of the law is a forensic science [1].

The forensic sciences are requested around the world to resolve civil disputes, to justly enforce criminal laws and government regulations, and to protect public health. Early on, forensic sciences became identified with law enforcement and the prosecution of criminal cases - an image reinforced by books, television, and movies. However, this is misleading because forensic sciences are objective, unbiased, and apply equally to criminal, civil, or other legal matters [1].

Forensic sciences encompass different disciplines, such as medicine, law, engineering, dentistry, biology, physics, and chemistry, among several others. In fact, Forensic Science is recognized itself as a multidisciplinary science. Forensic chemistry can be defined as the application of chemical expertise to assist in the resolution of legal cases. More specifically, forensic analytical chemistry is one of the most useful and instigating areas of investigation, and it has wide application in the analysis of licit and illicit drugs in different matrices; the analysis of adulteration of foodstuffs, such as olive oil and distilled beverages; fuels and vehicle chassis adulteration; and frauds in works of art and arson analysis.

Forensic analytical chemistry and its powerful methods of analysis, such as gas chromatography (GC), mass spectrometry (MS), high performance liquid chromatography (HPLC), thin layer chromatography (TLC), immunoassays, atomic absorption/atomic emission (AA/AE), inductively coupled plasma emission (ICP/AES) and mass spectrometry (ICP/MS), scanning electron microscopy (SEM), Fourier transform infrared spectrometry (FTIR), ultraviolet/visible spectrometry (UV/Vis), and electrophoresis, are now indispensable tools in the context of forensic expert activity as an auxiliary element of justice.

Among these applications, there is demand for the investigation of licit and/or illicit drugs in biological specimens, both in vivo and postmortem, especially considering the continuous emergence of new synthetic drugs and large drug abuse, which is responsible for serious public health problems throughout the world, greatly impacting youths.

Although the study of the science that studies toxic substances and their effects began in the early 1800s, the knowledge of poisons and poisonings had existed for thousands of years. Poisonings by opium, arsenic, and hydrocyanic acid had been reported in Europe during the Middle Ages. It was during that time that Philippus Aureolus Theophrastus Bombastus von Hohenhein, also known as Paracelsus, observed that any substance could be a poison, depending on the dose. In 1814, Mathieu Joseph Bonaventure Orfila, the head of the Department of Legal Medicine at the University of Sorbonne (France), made the first systematized and categorized study of some poisons and isolated arsenic from various postmortem biological samples. He was the pioneer in stating that a poison needs to be absorbed or enter the bloodstream to manifest its toxic effects [2].

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Point of View

In 1851, Jean Servais Stas developed the first effective analytical method for extracting alkaloids from biological samples. The procedure for the extraction was largely modified years later by Friedrich Julius Otto, who was able to isolate pure alkaloids. This analytical method became known as the StasOtto Method and it is still fundamental for drug extraction [2].

Licit and illicit drugs, including ethanol, cocaine, marijuana, amphetamines, benzodiazepines, and barbiturates, have long been the most studied analytes, especially in biological samples from living and dead individuals, such as blood, urine, viscera, and hair. More recently, new psychoactive substances, better known as NPSs, such as synthetic cannabinoids, substances derived from Fentanyl and Cathinones, have come into the spotlight of forensic analytical chemistry. In this scenario, it is crucial the research, development, and validation of new analytical techniques to attend the large demand from the modern society for the elucidation of forensic cases.

REFERENCES 1. https://www.aafs.org/ (accessed on February 25th, 2019).

2. Levine B. Postmortem Forensic Toxicology. In: Levine B. Principles of Forensic Toxicology, American Association for Clinical Chemistry, 1999, pp 1–12.

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Article

Br. J. Anal. Chem., 2018, 5 (21), pp 8-18 DOI: 10.30744/brjac.2179-3425.2018.5.21.8-18

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Bioactive Compounds, Antioxidant properties, and Metal Content Studies of Guava Fruit by-products for Value Added Processing Neela Emanuel*, Khushbu Sao, Aman Kaushik National Institute of Food Technology Entrepreneurship & Management (NIFTEM) Kundli, Sonepat, Haryana, 131028, India

Fruit processing industries generate enormous amount of by-product materials which can be a valuable source of bioactive compounds. Guava fruit’s by-products seed and pomace along with the peel were analyzed for antioxidant activity and bioactive compounds. In the present study the total phenolic content (TPC), evaluation of antioxidant activities using 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity, Ferric Reducing Antioxidant Power (FRAP), Total Flavonoid content (TFC) were determined. The quantification of some of the bioactive phenolics was carried out using HPLC. The pomace along with the peel extract contained highest phenolic content 432 mg gallic acid equivalent (GAE) per g and possessed strong antioxidant activity in antioxidant assay. The mineral and toxic metals characterization was carried out using ICP-MS method after extraction process. The compound extracted from these by-products can be utilized as nutraceuticals in food, food ingredients, formulation of new improved healthier food products and pharmaceutical industries.

Keywords: polyphenols, antioxidant properties, carotenoids, flavonoids, nutrients, minerals, toxic metals, healthier product INTRODUCTION “Psidium guajava” (Guava) is an important tropical fruit with high nutritious value [1]. It is an excellent source of Vitamins C, A, B2 (Riboflavin) and minerals like calcium, phosphorus, potassium, sodium and iron [2]. It contains about 180-300 mg of vitamins per 100 g of pulp and high level of about 50-300 mg per 100 g of ascorbic acid [3]. Ripe Guava fruits contain 14.0 percent total soluble solids (TSS); 0.3 percent acidity; 7.0 percent fiber; 14.3 percent carbohydrate; 2.5 percent protein and 1.3 percent minerals [4-5]. It is also rich source of carotenoids and phenolic compounds which have been associated with diminished risk of diseases such as cardiovascular, certain carcinogenic disease, inflammation, hypertension, arthritis and neurodegenerative diseases [6-8].

Various processed products which were produced from Guava include beverages, syrup, juicewine, dehydrated and canned products. Guava processing industries generate large amount of the waste byproducts as peel, pomace and seed which contain valuable reusable bioactive compounds [8]. Recently, more emphasis is based on the extraction of the valuable products and their utilization as functional food, health supplement, neutraceutical product, food additives, food ingredients, medicinal and cosmetics [9-15]. The byproducts can be further processed to develop food products such as jams, jelly, velva frozen dessert and fruit leather [4,16].

Guava fruit contain enormous amount of essential nutrients and micronutrients such as phytochemical compounds, minerals, fibers and vitamins which are useful for the human health [7,11]. Recent study has shown that the fruit peel and seed contain considerable amount of phenolic compounds and ascorbic acid [17]. The role of the peel is to protect the inner portion of the fruit from pest and microorganisms [18]. Seed as the by-product of the fruit such as citrus, apricot kernel, and mango from food processing industry have been reported to contain phenolic compounds [19]. There is greater need to find different ways to utilize these byproducts containing bioactive compounds having antioxidant activity as food 8

* neela.emanuel@gmail.com https://orcid.org/0000-0002-5086-2851

Bioactive Compounds, Antioxidant properties, and Metal Content Studies of Guava Fruit by-products for Value Added Processing

Article

additives or supplements which eventually will increase the opportunity for commercial exploitations.

The extracted nutrients and polyphenols can be utilized for the further development of the functional food by addition as natural antioxidants during the processing of the food to improve health status. They can also be utilized as nutraceuticals in the medicinal form of pills, capsules or liquids. In both forms they provided demonstrated physiological benefits, improve health, and diminish disease risk through prevention [20]. Food enriched with natural antioxidants helps in the prevention of the development of diseases caused by oxidation stress. The exploitation of the byproducts of food processing industries as a source of functional compound and their application in food is promising field which require interdisciplinary research of food technologists, food chemists, nutritionists, and toxicologists.

Nutraceuticals and functional foods provide a means to reduce the increasing burden on the health care system by continuous preventive mechanisms. The interest in nutraceuticals and functional foods continues to grow and is powered by progressive research efforts to identify properties and potential applications of nutraceutical substances, coupled with public interest and consumer demand [21-22].

Nutrients present in guava fruits both pink and white varieties are plentiful and diverse. However they contain both essential minerals and toxic metals over a wide range of concentrations. The transition metals like Fe, Cu, Mn, and Zn occur naturally and they serve as plant nutrients depending upon their concentrations. On the other hand Pb, Cr, Ni, Hg, and many other heavy metals are indirectly distributed as a result of human activities which could be very toxic even at low concentrations [23].

Guava by-products are valuable raw materials as a source of polyphenols and nutrients. Thus, the present study was undertaken to assess total phenolic content, evaluate antioxidant activities, quantification of individual bioactive phenolic compounds, nutrients and toxic metal contaminants present in the peel, leftover pomace along with the seeds of guava waste. These products were taken for the study as they are generally generated from the fruits processing industries as waste byproducts and that can be utilized further for the formulation of various products and help in sustainable and stable growth of the food industries. The subsequent development of low cost technologically viable approach to convert guava by-products into nutraceuticals, food ingredients and the development of new healthier food products would help in reduction and disposal of byproducts and the production of value added food stuff.

MATERIAL AND METHODS Chemical Reagents All the reagents used were analytical grade and obtained from Sigma Aldrich Inc., Thermo Fisher, Merck, Fluka and Milli-Q water. The glassware was thoroughly cleaned before use.

Sample preparation Fresh guava both white and pink varieties of high quality with no visible scratch or spoilage were purchased in the spring season from Azadpur Mandi in Delhi. The by-products were obtained after extracting juice of guava containing guava peel, pulp and seed. The by-products of guava samples were lyophilized, homogenized, and stored at -20 ยบC in a sample bottle protected from light. Moisture content was determined for samples using standard method [24]. Total protein, ash content, crude fiber, and total fat content were determined according to the standard method of analysis [25].

Extraction process The extraction of the phenolic content was carried out by the following procedure: 2.56 g of powdered, freeze dried samples were weighed on analytical microbalance and transferred to centrifuge tubes.

Two step extractions were carried out and were combined for the subsequent analysis. In the first step the weighed sample was mixed with 20 mL ethanol:water (50:50, pH 2) solution and homogenized using orbital shaker incubator (New Brunswick Scientific) at 300 rpm for 60 minutes. The supernatant solution was separated and kept aside and the residue was used for the second step extraction. The 9

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Emanuel, N.; Sao, K.; Kaushik, A.

remaining residue sample was treated with 20 mL of acetone:water mixture (70:30, v/v) and extraction was carried out as mentioned in the first step. The supernatant was recovered and mixed with the first step solution and diluted to 100 mL. The extracts obtained were evaluated for the presence of various phytochemicals by chemical tests using standard procedures [26-28].

Physicochemical analysis Qualitative Phyto-chemical Analysis The extracted sample solutions were analyzed qualitatively for the presence of bioactive compounds by using standard method [29].

Determination of Total Phenolic Content (TPC) Total phenolic content in the sample solution was determined by Folin-Ciocalteau reagent [30]. Equal amount of extracted sample solution and diluted Folin-Ciocalteau reagent were mixed together and kept for few minutes followed by addition of 1 mL sodium carbonate solution (7.5% w/v) and incubated at room temperature for one and half hours in the dark. The absorbance of the resulting solution was measured at 760 nm using UV-Vis spectrophotometer. Gallic acid was used as a standard. The results were expressed as mean ± standard deviations of mg of gallic acid equivalent 100 g-1 extract.

Total Flavonoid Contents Total flavonoid contents in the sample solution were determined using the aluminum chloride colorimetric assay. The 1 mL sample extract, 2 mL distilled water and 0.3 mL of 5% NaNO2 solution were combined in 10 mL volumetric flask and kept at room temperature for 5 minutes. In this solution, 3 mL of 10% AlCl3 solution was added followed by 2 mL of 1 M NaOH solution and the total volume was made up to 10 mL with distilled water. The absorbance of the solution was measured at 510 nm using a spectrophotometer. The concentration of flavonoid compound was estimated by calibration curve using quercetin as flavonoid reference. The results were expressed as mean ± standard deviations of mg of quercetin equivalent 100 g-1 extract.

DPPH Free Radical Scavenging Capacity Antioxidant activities were measured in triplicate for the extracted sample solution using DPPH assay.

DPPH assay absorb strongly at wavelength 517 nm which is the λmax. When the phenolic compounds in the extract react with the stable DPPH radical, it remove the free radical which lead to the color change from blue complex to light yellow. The color change depends on the intrinsic concentration of available antioxidant and its rate of reactivity towards DPPH. The degree of reduction in absorbance measurement is indicative of the radical scavenging power of the extract. The measurement of the DPPH radical scavenging activity was performed according to described methodology [31]. The samples were treated with the stable DPPH radical in the extracted solution. To the 1 mL sample solution in test tube was added 5 mL of absolute ethanol followed by 1.0 mL of 0.5 mM DPPH radical solution.

Absorbance was measured at 517 nm after 60 min of reaction using a UV-Vis spectrophotometer (Shimadzu 2600). The control solution was prepared by mixing ethanol (5 mL) and DPPH radical solution (0.5 mL). The scavenging activity percentage (AA %) was determined using the equation: DPPH free radical scavenging activity (%) = (Absorbance Control−Absorbance Sample / Absorbance Control) ×100. The quality of the radical scavenging property of the sample extracts were determined by calculating IC50 value. IC50 (concentration providing 50% inhibition) values were calculated from percentage disappearance versus concentration plot.

Ferric Reducing Antioxidant Power Assay (FRAP) The FRAP assay was performed according to Benzie and Strain with some modifications [32]. The following stock solutions were prepared for the analysis: 300 mM acetate buffer pH 3.6, 10 mM TPTZ 10

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(2,4,6-tripyridyl-s-triazine) solution in 40 mM HCl, and 20 M FeCl3.6H2O solution. The fresh reagent was prepared from the stock solution by adding 25 mL acetate buffer, 2.5 mL TPTZ solution, and 2.5 mL FeCl3.6H2O solution and warmed to 37 ºC before use. Sample extracts were allowed to react with FRAP solution in the ratio 1:20 for 30 min in the dark. The molar absorbance was measured for the product formed [ferrous tri-pyridyltriazine complex] at 593 nm. Results were expressed as mg per 100 g extract weight (mg/100 g).

HPLC Analysis of Phenolic Compounds Phenolic Compounds were separated and quantified in the extracted sample solution using HPLC-2707 Water system equipment with thermostatically controlled column oven and a diode array detector-2996. Samples and mobile phase were filtered with Advantech filter and analyzed by using reverse phase HPLC. Optimal separation was achieved using isocratic elution on a C18 column (250 x 4.6 mm x 5 µm) at a flow rate of 1.1 mL/min at 35 ºC. The mobile phase used was methanol containing 0.2% phosphoric acid. All samples were injected three times using automatic sample injector. The spectral data of signals from PDA detector was measured at 360 nm. The external standard method was used for identification and quantification of phenolic compounds and expressed as mg/100 g of extract [33].

Proximate Analysis The proximate analysis was determined by standard method [34]. Moisture, ash, protein, crude fiber and lipid content were determined by AOAC method. The samples were measured in triplicate for all samples.

Determination of metals The metals were determined after drying the sample on a hot plate followed by ashing in the Muffle furnace (Metek Scientific Co. Ltd.). All glassware were thoroughly cleaned using freshly prepared 10% (v/v) HNO3 for about 48 h and finally rinsed with double deionized water (DDW).

Samples were initially dried at 65 ºC for 48 h and brought to room temperature in a desiccator. About 1 g of dried and well homogenized sample was weighed accurately in a porcelain crucible and 2 mL of 6 M HNO3 was added. Mineralization was carried out in three stages: (i) the sample was initially heated in a muffle furnace at 100 ºC to evaporate excess reagents; (ii) further, sample was heated at 250 ºC for 30 minutes; (iii) followed by heating at 450 ºC for 4 hours. The sample was brought to room temperature in desiccator. The minerals and contaminants were brought into the solution by adding few drops of HNO3 and DDW and quantitatively transferred to 50 mL measuring flask and diluted with DDW. All samples were analyzed by ICP-MS. Statistical Analysis Each test assay was done three times from the same extract in order to determine their reproducibility.

Results were expressed as mean values ± standard deviation. Comparisons were performed by analysis of variance (ANOVA). The correlations among the data were calculated using Pearson’s correlation coefficient (r) and p<0.05 was considered as significantly different. Duncan’s test was used to determine significant differences.

RESULTS AND DISCUSSION Phytochemical Screening The sample extracts of pink and white guava fruits were tested for the presence of alkaloids, steroids, tannins, saponins and glycosides. The qualitative approach was used and expressed as (+) for the presence and (−) for the absence of phytochemicals.

The phytochemical screening of sample extracts of pink and white guava samples indicate the 11

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presence of secondary metabolites such as flavonoids, saponins, terpenoids, steroids, and coumarins (Table I). Alkaloids were detected in the peel extract but not in the seed extract. Due to the medicinal properties of these phytochemical compounds the screening was carried out [35-42].

Table I. Secondary metabolite detection Phytochemicals

Peel

Seeds

Flavonoids

+

+

Tannin

+

+

Alkaloids

+

-

Phlobatannins

-

-

Saponins

+

+

Terpenoids

+

+

Steroids

+

+

Coumerins

+

+

Total Phenolic Content The total phenolic content (TPC) of white and pink guava peel and seed extracts is tabulated in Table II. Phenolic compounds present in large amount in peel waste in fruits are considered to be an important constituent due to their antioxidant activity [43]. TPC expressed as gallic acid equivalent (GAE), varied from 57 to 432 mg GAE/100 g in guava. In the present study TPC was found to be 339.9 mg GAE/100 g in peel byproduct and in seed byproduct was 57.2 mg GAE/100 g and similarly for pink flesh guava was 432.06 mg GAE/100 g and 74.62 mg GAE/100 g of peel and seed byproduct respectively. The highest (p≤0.05) TPC were obtained for peel byproducts and the lowest for the seed byproducts. Total phenolic content were correlated with the antioxidant activity mentioned in Table II. These results are comparable with those obtained by Dasgupta et al. [44] and Dorman et al. [45] where they reported direct relationship between total phenolic content and antioxidant activity in many plant species.

Table II. Total phenolic content, total flavonoid content and antioxidant activity of peel and seed of white and pink guava fruit Samples

TPC (mg/100 g)

TFC (mg/100 g)

IC50 (mg/100 g)

FRAP (mg/100 g)

Guava Peel (white)

339.9 ± 0.3a

32.4 ± 0.6b

21.6 ± 0.2b

24.2 ± 0.2b

Guava Seed (white)

57.2 ± 0.4c

15.6 ± 0.5a

27.9 ± 0.8a

30.6 ± 0.3a

Guava Peel (pink)

432.1 ± 0.7b

33.0 ± 0.5b

20.5 ± 0.2b

19.6 ± 0.3c

Guava Seed (pink)

74.6 ± 0.5d

16.5 ± 0.3c

26.5 ± 0.5a

28.5 ± 0.4a

The values of individual compounds were the mean ± standard deviation (n =3) and values followed by different letters in the same column are significantly (p<0.05) different from each other.

Total Flavonoids Content The flavonoid content of the extracts was investigated due to the power of these compounds to reduce oxidative damage to cells as demonstrated in several studies [44]. The flavonoids content of extracts was calculated as quercetin equivalent (QE). The total flavonoids content (TFC) in peel and seed extract was found to be 32.4 mg QE/100 g and 15.6 mg QE/100 g of white variety of guava and for pink guava was 33.0 mg QE/100 g and 16.5 mg QE/100 g. TFC ranged from 15.6 mg QE/100 g to 12

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33.0 mg QE/100 g in white guava and pink guava peel and seed extract. The higher (p ≤ 0.05) TFC yield were obtained for peel byproducts and the lowest for the seed byproducts. The TFC and antioxidant activity were also correlated as shown in Table II which indicates flavonoids might be responsible for its activity [47]. No significant (p ≤ 0.05) difference was found among guava white and guava pink peel extract in TFC. DPPH Activity The DPPH free radical scavenging by antioxidants is due to the hydroxyl group present in the sample. The large number of hydroxyl groups lead to greater radical scavenging activity. Phenolic compounds generally have significant scavenging effects for DPPH free radical [48-50]. The IC50 value for the sample extract defined as the concentration of extract causing 50% inhibition of absorbance was calculated. Lower the IC50 value, the higher the antioxidant power [51-52]. The results of IC50 of different samples of guava fruits are summarized in Table III. The IC50 value ranged from 20.5 – 27.9 mg/100 g in white and pink guava peel and seed extract. Lower value in case of guava peel extract indicates the higher antioxidant power as compared to guava seed extract. The scavenging activity of sample extracts were in the order: guava peel (pink) > guava peel (white) > guava seed (pink) > guava seed (white). This is also in accordance with the reported literature where the values were 32-33 µg/g for the white variety and 15-16 µg/g of red variety of guava fruit flesh extract. Guava peel extract exhibited the higher (p<0.05) antioxidant activity as compared to guava seed extract. The antioxidant activity of guava peel extract was similar with the whole guava fruit pomace and peel extract [53]. There is strong correlation with the TPC and TFC (r2 = 0.516).

Ferric Reducing Antioxidant Power (FRAP) Activity The reducing antioxidant power of sample depends on its electron transfer ability towards the FRAP reagent. There was increase in absorbance which indicates its reductive ability. The reduction of ferrictri-pyridyl triazine to the ferrous complex develops an intense blue color, which was measured at λ max of 593 nm. The external standard calibration curve was used for the quantification. The result of the ferric ion reducing activities of the extracts is the presented in Table II. The result shows that the value is 24.2 and 30.6 mg FeSO4 equivalent /100 g peel and seed of white guava and 19.6 and 28.5 mg/100 g for peel and seed of red guava. The value indicates the excellent reducing ability. It has been found that there is strong correlation between TPC and antioxidant activity as determined by FRAP and DPPH. The same correlations were also reported in fruit juices [54]. The difference in antioxidant activity as determined by DPPH and FRAP measurements is attributed to a relative difference in the ability of antioxidant compound to quench peroxyl radicals and to reduce the reagents (DPPH/iron).

HPLC Analysis The extracted peels, seed and pomace samples were analyzed using HPLC instrument with diode array detector. The peaks in HPLC chromatogram were well separated indicating that the used HPLC conditions were optimum resulting in better efficiency of separation. Peaks were identified based on the retention time, and the concentrations of four phenolic components were determined by using external standard method. The phenolic compounds and flavonoids identified and quantified by HPLC are shown in Table III. The result showed the phenolic compound varied in both white and pink variety of guava peel and seed. Among the phenolic compound chlorogenic acid present in fruit peels and seeds were in order: guava white > guava pink and it was higher in peel as compared to seed.

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Table III. Individual bioactive compounds in the white guava seed and peel, and pink guava seed and peel analyzed by HPLC (mg/100 g) Guava Sample

Peel–pink

Peel-white

Seed-pink

Seed-white

Myricetin

18.3 ± 0.2c

21.7 ± 0.2b

7.4 ± 0.4d

7.3 ± 0.1d

Apigenin

459.3 ± 3.2b

448.8 ± 1.9d

64.1 ± 0.3a

59.7 ± 0.8c

Quercetin

1.3 ± 0.3a

0.9 ± 0.3b

ND

ND

Chlorogenic acid

55.3 ± 0.6d

58.3 ± 2.1a

26.8 ± 0.5b

29.0 ± 0.7c

The values of individual compounds were the mean ± standard deviation (n =3). Data with different letters in the same row is significantly difference at level p<0.05. ND is not detected.

In the present study it was observed that the flavonoids present were higher in pink variety as compared to white variety and were higher in peel as compared to seed. The amount of quercetin was found to be in the range of 0.9 – 1.3 mg/100 g. An appreciable amount of quercetin was detected in the peel extracts of both white and pink guava and not detectable in the seed. Apegenin was found in appreciable amount in both peel and seed of guava white and pink varieties and it ranges from 448.8 – 459.3 mg/100 g in white and pink guava peel and ranges from 59.7 – 64.1 mg/100 g in white and pink guava seeds. Myricetin, with its three adjacent hydroxyl groups was one of the most active antioxidants. The poly-phenolic myricetin is a flavonol found in white and pink guava peel and seed.

The amount ranges from 18.3 – 21.7 mg/100 g in white and pink guava peel and in seed it ranges from 7.3 - 7.4 mg/100 g. Several studies had correlated that flavonoids are the main contributor for plant’s antioxidant activity [55,56]. The strong oxidant activity by the guava peel extract may be attributed to the abundance of apigenin, myricetin, and chlorogenic acid.

Proximate Analysis Proximate analysis of the seed and peel is done in order to know the nutrient content. According to Cabral et al. [57], the food value of guava fruit was 0.9% to 2% protein, 0.1% to 0.5% fat, 2.8% to 5.5% fibre along with minerals and vitamins in the whole guava fruit. Nutrient content of the peel and seed as shown in Table IV is significant and comparable.

Table IV. Proximate composition of guava seed and peel Parameters

Peel

Seed and pomace

Moisture% (Dry basis)

3.49 ± 0.12

a

2.77 ± 0.24b

Total Protein

0.91 ± 0.07a

0.70 ± 0.11c

Crude fiber

0.29 ± 0.03c

0.49 ± 0.09b

Crude Fat

0.06 ± 0.01a

0.08 ± 0.02a

0.024 ± 0.006a

0.011 ± 0.005c

Ash

The values of individual compounds were the mean ± standard deviation (n =3) and values followed by same letters within the rows are not significantly (p<0.05) different from each other.

The total protein content of peel and seed was 0.91% and 0.70%; fat content was 0.06% and 0.70%.

The fat content is more in seed as it contains essential fatty acid; similarly, crude fiber was 0.2930/g in peel as compared to 0.4857/g in seed; ash indicates the mineral content which is 0.024% and 0.011% in dry peel and seed of guava respectively.

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Trace metals and mineral analysis Certain metals such as Cu, Fe, Mn and Zn are the naturally occurring essential elements. Other metals such as Pb, Cr, Ni, As, and Hg are considered to be very toxic at very low concentrations. The amount of Cu, Fe, Mn, Zn, Pb, Cr, Ni, As, V, and Hg in the peel and seed of guava white and pink variety were determined using ICP-MS method. Table V shows the concentrations of all these metals. The toxic elements were present in less than 0.1 mg/L in all the samples which is below the toxic limit guide lines for fruits given by the regulations. The essential elements such as Fe, Mn, Cu, and Zn were present in all these samples. The range of Fe, Cu, Mn and Zn concentrations were 0.50 – 1.10, 0.10 – 0.30, 0.13 – 0.42, and 0.20 – 0.30 mg L-1 respectively.

Table V. Metal concentrations in both white guava seed and peel, and pink guava seed and peel (Psidium guajava) extract (mg L-1) Sample

V

Cr

Mn

Fe

Ni

Cu

Zn

As

Hg

Pb

WP

<0.1

<0.1

0.41±0.04b

0.60±0.03b

<0.1

0.10±0.01a

0.20±0.06c

<0.1

<0.1

<0.1

WS

<0.1

<0.1

0.13±0.02a

0.50±0.01a

<0.1

0.30±0.04c

0.30±0.02c

<0.1

<0.1

<0.1

RP

<0.1

<0.1

0.42±0.06b

1.10±0.04c

<0.1

0.10±0.01a

0.30±0.03c

<0.1

<0.1

<0.1

RS

<0.1

<0.1

0.24±0.04c

0.60±0.02b

<0.1

0.30±0.02c

0.30±0.02c

<0.1

<0.1

<0.1

WP = white guava peel; WS = white guava seed; RP = pink guava peel; RS = pink guava seed. The values of individual compounds were the mean ± standard deviation (n =3) and values in the same column followed by different letters are significantly (p<0.05) different from each other.

CONCLUSION The waste byproduct of fruits and vegetables is already been a major issue in the food sector in the developing countries. To compensate the losses occurring in the fruit processing industry, it is required that the proper waste management is followed and directing the energy in the right direction. The observed results indicate that its bioactive compound content and antioxidant activity as determined by performing the various assays like free radical scavenging activity through DDPH assay, and FRAP assay are found to be excellent. The FRAP activity shows that it is 24.2 and 30.6 mg FeSO4 equivalent/100 g peel and seed of white guava and similarly 19.6 and 28.5 mg/100 g for peel and seed of red guava. The IC50 value ranged from 20.5 – 27.9 mg/100 g in white and pink guava peel and seed extract. The results of TPC show that there is 339.9 mg GAE/100 g of peel and that of seed is 57.2 mg/100 g and similarly for pink flesh guava is 432.1 mg GAE/100 g and 74.6 mg GAE/100 g of peel and seed respectively. Result of total flavonoid content (TFC) in peel and seed extract is 32.4 mg/100 g and 15.6 mg QE/100 g of white variety of guava and that of pink guava is 33.0 mg QE/100 g of guava.

The above results show that phenolic content of the seed, peel and pomace of guava fruit are present in significant amount indicative of its antioxidant activity.

The nutrient content observed in the peel and seed of the white and pink flesh guava indicates its excellent food value. The total protein content of peel and seed is 0.91% and 0.70%; fat content is 0.06% and 0.70% - the fat content is more in seed as from the literature review it contains essential fatty acid in it; similarly, crude fiber is 0.2930/g in peel as compared to 0.4857/g in seed; ash indicates the mineral content which is 0.024% and 0.011% in dry peel and seed of guava respectively. It indicates that along with the good nutrient content it has excellent bioactive compound with significant antioxidant activity.

The bioactive compounds present in by-product of the fruit processing can be utilized for various other purposes like value added processing, as functional food, food additives, therapeutic applications, for fortifications and many more.

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Emanuel, N.; Sao, K.; Kaushik, A.

Manuscript received: 05/21/18; revised manuscript received: 07/29/18; revised manuscript for the 2nd time received: 09/07/18; revised manuscript for the 3rd time received: 09/25/18; revised manuscript for the 4th time received: 10/25/18; manuscript accepted: 12/19/18; published online: 02/07/19.

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Br. J. Anal. Chem., 2018, 5 (21), pp 19-29 DOI: 10.30744/brjac.2179-3425.2018.5.21.19-29

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Development of Chromatographic Method for Determination of Impurities in Solid Dispersion of Dasatinib Chandrakant Sojitra1,2,3*, Ajay Tehare1, Chintan Dholakia1, Padmaja Sudhakar2, Sameer Agarwal3* and Kumar K. Singh1 Cadila Healthcare Limited, API Division, Sarkhej-Bavla N.H. Nº 8 A, Changodar, Ahmedabad – 382 210, India 2 Department of Chemistry, Faculty of Science, M. S. University of Baroda, Baroda – 390 002, India 3 Zydus Research Centre, Cadila Healthcare Ltd. Sarkhej-Bavla N.H. Nº 8 A, Moraiya, Ahmedabad – 382 210, India

1

An accurate, fast, precise and economic gradient reverse phase high performance liquid chromatographic (RP-HPLC) method was developed for quantitative determination of process and degradation related impurities in the solid dispersion of dasatinib drug substance. The optimum separation was achieved by Sunniest C18, 250 x 4.6 mm, 5 µm column at 35 °C. The mobile phase A was 20 mM ammonium acetate buffer (pH 5.0) and mobile phase B was composed of methanol:buffer:acetonitrile (90:5:5) (%, v/v/v); the chromatographic analysis was performed with gradient condition detecting the related substances at wavelength 310 nm at flow rate of 1.2 mL/min. The resolution for dasatinib and six related components was found to be greater than 2.0 for any pair of impurities. The stability indicating nature of the method was demonstrated by performing force degradation studies. Significant degradation was observed when the solid dispersion of dasatinib was subjected to oxidation, thermal and photo degradation, while the drug substance was stable in acid and alkali degradation. Relative standard deviation obtained for the system precision and method precision studies was less than 5%.

The accuracy of the method was demonstrated by performing recovery studies through spiking studies.

The developed method was validated for linearity, specificity, accuracy, precision, limit of detection, limit of quantitation and robustness studies; it can be used in quality control for commercialization of solid dispersion of dasatinib drug substances and performing stability studies.

Keywords: Dasatinib, chromatographic method, stress testing, method validation.

INTRODUCTION Dasatinib is an inhibitor of multiple tyrosine kinases, inhibiting the growth of chronic myeloid leukemia and acute lymphoblastic leukemia cell lines overexpressing BCR-ABL [1,2]. Dasatinib is an approved drug, sold under the brand name Sprycel, and is a chemotherapy medication used for the treatment of chronic myelogenous leukemia and acute lymphoblastic leukemia [3].

Stability testing is an integral part of the new drug development process because it provides quality of drug substances in different storage condition having variable temperature and humidity.

As per the International Conference on Harmonization (ICH) Guideline Q1A(R2) [4], the shelf life of any drug substances is determined by stability studies. The quantification of impurities and dasatinib API is required to be determined using stability indicating chromatographic method, as suggested by the previously mentioned ICH guideline and United State Pharmacopoeia (USP) [5]. However, a few methods have been used for quantification of major tyrosine kinase inhibitors i.e. imatinib, dasatinib and nilotinib in human plasma [6-17].

*chandrakant.sojitra@yahoo.com; sameeragarwal@zyduscadila.com https://orcid.org/0000-0002-7039-3028

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Development of Chromatographic Method for Determination of Impurities in Solid Dispersion of Dasatinib

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Table I. Chemical structure of dasatinib and its impurities Impurity particulars

Chemical structure N

H N

Dasatinib

Cl

NH

S

N

O

N

Cl

OH

N

Source

N-(2-chloro-6-methylphenyl)-2-((6(4-(2-hydroxyethyl) piperazin-1yl)-2-methylpyrimidin-4-yl) amino) thiazole-5-carboxamide

Target API

N-(2-chloro-6-methylphenyl)-2-((2methyl-6-(piperazin-1-yl) pyrimidin-4yl) amino) thiazole-5-carboxamide

Degradation impurity

4-(6-((5-((2-chloro-6-methylphenyl) carbamoyl) thiazol-2-yl) amino)2-methylpyrimidin-4-yl)-1-(2hydroxyethyl) piperazine 1-oxide

Degradation impurity

2-amino-N-(2-chloro-6-methylphenyl) thiazole-5-carboxamide

Key Starting Material

4-(2-(4-(6-((5-((2-chloro-6methylphenyl)carbamoyl)thiazol-2yl)amino)-2-methylpyrimidin-4-yl) piperazin-1-yl)ethoxy)-4-oxobutanoic acid

Degradation impurity due to HPMC

2-((6-chloro-2-methylpyrimidin-4-yl) amino)-N-(2-chloro-6-methylphenyl) thiazole-5-carboxamide

Intermediate

2-(4-(6-((5-((2-chloro-6methylphenyl) carbamoyl)thiazol2-yl)amino)-2-methylpyrimidin-4-yl) piperazin-1-yl)ethyl acetate

Degradation impurity due to HPMC

N

H N

Imp-1

N

IUPAC name

NH

S

N

O

N

N

NH

-

Cl O

Imp-2

O + N

H N

S

N

N

NH

N

Cl

O

N

N

O O

S

O Cl

N

H N Cl

HN

N N H

N

Imp-5

NH2

S

N

Imp-4

N

N

H N

Imp-3

OH

NH

S

N

O Cl

N

O

Imp-6

Cl O NH

N

H N

S N

N N

N

O

OH O

Several methods have been reported to determine only the process related impurities [18-20].

Moreover most of these methods are not validated and stability indicating. Thus, a novel robust method for the evaluation of all the process and degradation related impurities of solid dispersion of dasatinib 20

Sojitra, C.; Tehare, A.; Dholakia, C.; Sudhakar, P.; Agarwal, S.; Singh, K. K.

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drug substance is needed. In the present study we herein report the development and validation of a stability indicating chromatographic method for determination of process and degradation related impurities in solid dispersion of dasatinib drug substance by evaluating Response Factor (RF) values of each impurity as per validation of the ICH guideline. The developed method will be of high importance for the commercial production of dasatinib, an efficient oncological drug, with over $ 2000 million market size.

Dasatinib is not present in any of the pharmacopeia till date. Since impurities in drug substance could cause toxic effects in the patients, thus the guidelines on impurities in new drug substance (Q3AR2) have been issued by ICH. Therefore, a stability-indicating RP-HPLC method was developed for the quantitative determination of dasatinib and its six impurities including process and degradation impurities i.e. Imp-1, 2, 3, 4, 5 and 6 (Table I). This method was successfully validated according to the ICH guidelines (validation of analytical procedures: test and methodology Q2). Dasatinib received marketing approval by the European Medicines Agency in November 2006 and was approved by the U.S. Food and Drug Administration in June 2006.

MATERIAL AND METHODS Samples and Reagents Dasatinib standards and samples were synthesized in API Division, Cadila Healthcare Ltd. (Ahmedabad, India), by a route of synthesis previously described [21]. HPLC grade acetonitrile and methanol, analytical grade ammonium acetate, acetic acid solution, and hydrogen peroxide solution (30%) were purchased from Merck Specialities Pvt. Ltd. (Mumbai, India). High purity HPLC grade water was prepared by using Millipore Milli-Q Plus water purification system, Bradford, PA, USA. Preparation of Solid Dispersion of Dasatinib The solid dispersion of dasatinib was prepared by mixing dasatinib with hydroxypropyl methylcellulose (HPMC) in the ratio of 70:30 (%, w/w) [21].

HPLC Chromatographic Conditions The chromatographic experiments were performed on a Waters HPLC system with photodiode array detector. The detector wavelength was 310 nm and data processed using Empower 3 software, version builds 3471. The column used for chromatography was Sunniest C18 250 x 4.6 mm, 5 µm particle size.

The optimum separation was achieved using a gradient mode. Mobile phase A was 20 mM ammonium acetate (pH 5.0); mobile phase B was mixtures of methanol, buffer and acetonitrile in a ratio of 90:5:5 (%, v/v/v). The flow rate was 1.2 mL/min. The gradient program is presented in Table-II. The column temperature was maintained at 35 °C. The injection volume was 15 µL. A typical chromatogram of dasatinib and its six impurities is shown in Figure 1.

Table II. Gradient program of the HPLC method Time (min)

% Mobile phase-A

% Mobile phase-B

0.01

50

50

23

45

55

42

33

67

50

10

90

65

10

90

68

50

50

75

50

50

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Article

Development of Chromatographic Method for Determination of Impurities in Solid Dispersion of Dasatinib

LC-MS Chromatographic Conditions An electrospray LC-MS system (Shimadzu Prominence HPLC coupled with Triple Quadrupole Mass Spectrometer LCMS-8040 with lab solution software, version 5.72, Japan) was used for identification of degradant impurities formed during the stress testing studies. Chromatography was performed on Sunniest C18 250 x 4.6 mm, 5 µm particle size column from ChromaNik Technologies Inc. (Made in Japan) using mobile phase consisting of mobile phase A (20 mM ammonium acetate pH 5.0) and mobile phase B (mixture of methanol, buffer and acetonitrile in a ratio of 90:5:5%, v/v/v) at a flow rate of 1.2 mL/min. The LC gradient program has been applied as per Table II. The column temperature was maintained at 35 °C. Methanol:water:ACN in the ratio of 50:30:20 (%, v/v/v) was used as a diluent.

Injection volume was 15 µL. The analysis was carried out by using electrospray ionization mode (+ve and -ve). The capillary voltage at 3500 V and collision Energy -35 V. Desolvation temperature is 250 °C with nebulizing gas flow rate 180 L/h.

Figure 1.Typical chromatogram of dasatinib and its impurities.

Preparation of Solutions Standard stock solutions for validation Dasatinib stock solutions were prepared with a concentration of 1000 µg mL-1 for dasatinib in diluent, and a stock solution of impurities composite was prepared (a mixture of Imp-1, Imp-2, Imp-3, Imp-4 and Imp-6) at a concentration of 100 µg mL-1. Imp-5 first stock was prepared in dimethyl sulfoxide (DMSO) and second stock was prepared 100 µg mL-1 in a diluent.

Sample solution Dasatinib test solutions were prepared in a concentration of 1000 µg mL-1 in diluents, sonicated for 5 min to dissolve and were further analyzed by HPLC. Stress Degradation Studies Specificity can be performed by analysis of the sample spiked with process and degradation related impurities; no interference was observed at the retention time of interest analytes. Stress studies were performed at concentration of 1000 µg mL-1. Degradation was performed under stress condition of UV light (254 nm), heat (105 °C), acid (1.0 N HCl at 60 °C), base (1.0 N NaOH at 60 °C) and oxidation (3% H2O2 at 25 °C) to evaluate the capability of the proposed method to separate dasatinib and all impurities including process and degradation products. For thermal and photo stress studies, the study period was 24 h, whereas for acid hydrolysis approximately 1 h; alkali hydrolysis 1 h and oxidation 1.5 h. The purity of each peak was checked using PDA detector and the purity angle was found to be less than the purity threshold, directly demonstrated that peak is pure. Mass balance of each condition stressed samples 22

Sojitra, C.; Tehare, A.; Dholakia, C.; Sudhakar, P.; Agarwal, S.; Singh, K. K.

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was calculated by addition of %content of dasatinib + %known impurities + %unknown impurities in %, w/w.

Method Validation Protocol The proposed method was validated for the determination of related substances in the solid dispersion of dasatinib by HPLC as per ICH guidelines [22]. The limit of detection (LOD) and limit of quantification (LOQ) for dasatinib and all six impurities were determined by signal to noise ratio of 3:1 and 10:1 respectively.

RESULTS AND DISCUSSION Optimization of Chromatographic Method The main criteria for developing chromatographic method was that it must be stability indicating and easy to perform routine analysis in quality control laboratory. The first step for method development was the selection of wavelength. Analysis was performed by using diode array detector for selection of wavelength and to check homogeneity of peaks. The wavelength for analysis and quantification was selected based on UV spectrum of each impurity and analyte peak. Each peak is showing two UV maxima at about 220 nm and 320 nm. 310 nm was selected as cross section wavelength of Impurity-3 and all other peaks. Reference overlay UV spectra has been provided in Figure 2. Thus detection wavelength was selected as 310 nm for related substance analysis. For a method development, spiked solution of dasatinib was used. Initially, experiments were based on the available literature, gradient method using Cosmosil BDS C18 column (100 mm × 4.6 mm, 3.5 µm particle size) with the mobile phase composed of triethyl amine buffer solution pH 6.5±0.05 and solvent mixture (methanol, acetonitrile) in 50:50 (%, v/v) [11]. But in this method, Imp-2 and Imp-4 peaks were merged with dasatinib peak and Imp-6 peak was split. Hence, a gradient method trial was taken with 250 mm × 4.6 mm, 5 µm particle size column (Waters SunFire C18) and found that only Imp-4 peak was merged with dasatinib peak.

As dasatinib has a pKa of 10.95 calculated by ACD Chem (SciFinder), to check the effect of pH on the resolution of impurities, methods were tried using buffers at different pH ranging from 4.5 to 7.5 without changing the ionization pH range of dasatinib i.e. pKa ±1.5 [23]. For optimizing pH of the buffer, an HPLC column, Waters SunFire C18 (250 mm × 4.6 mm, 5 µm particle size) was selected. Spike solution was injected with the above mentioned buffers having different pH as mobile phase A and mixture of methanol and acetonitrile in a ratio of 80:20 (%, v/v) as mobile phase B with gradient elution and it was observed that at pH below 4.5 and above 5.5, Imp-4 and dasatinib merged each other, but separation was achieved for all the six impurities and dasatinib at pH 5.0±0.5. So finalized that method is sensitive to pH. It was thus decided to use ammonium acetate-acetic acid buffer of pH 5.0 as mobile phase A, and methanol:buffer:ACN in the ratio of 90:5:5 (%, v/v/v) as mobile phase B with a gradient as per Table II, at a flow rate of 1.2 mL/min. In above trial Imp-5 and Imp-6 were eluted in the gradient hump. To overcome this, the gradient was increased to 75 min, 5% buffer was added to mobile phase B and Sunniest C18 (250 mm×4.6 mm, 5 µm particle size) column used for baseline smoothening at the retention time of Imp-5 and Imp-6. Using these chromatographic conditions, significant separation (>2.0) for all the six impurities and dasatinib was achieved. The retention time of dasatinib was 20 min.

The typical spike chromatogram of dasatinib and its impurities is presented in Figure 1. It was confirmed that no blank interference observed at the retention time of any of the impurities and dasatinib. LC-MS analysis of impurities was performed as per section “HPLC Chromatographic Conditions” as instrument condition.

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Development of Chromatographic Method for Determination of Impurities in Solid Dispersion of Dasatinib

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Figure 2. Reference overlay UV spectra of dasatinib and its impurities.

In the developed HPLC conditions, system suitability parameters like tailing factor, USP theoretical plates and USP resolution were evaluated for dasatinib and its six impurities (Figure 1). USP theoretical plates for all impurities was more than 5000, USP tailing factor for all the impurities was less than 1.5 and USP resolution between any pair of impurities were more than 2.0 (Table III).

Table III. System suitability parameters Imp particulars

RT (min)

RRT

USP resolution

USP tailing factor

USP theoretical plates

%RSD

Imp-3

6.67

0.30

-

1.10

7964

1.22

Imp-1

12.75

0.58

15.37

1.23

10108

0.92

Imp-2

17.89

0.82

9.30

1.04

13629

1.35

Dasatinib

21.78

1.00

6.00

1.04

15084

1.25

Imp-4

28.97

1.33

9.88

1.01

23763

1.65

Imp-5

41.28

1.90

18.35

1.02

79760

1.42

Imp-6

48.48

2.23

14.80

0.98

239541

1.72

Method validation Precision The precision of the method was determined by intermediate precision and method precision studies.

In both the studies %Relative Standard Deviation of peak areas for all the impurities were less than 5.0%. These results demonstrate that the method is precise (Table IV).

Limit of Detection and Limit of Quantitation LOD and LOQ for dasatinib and all six impurities were determined by signal to noise ratio of 3:1 and 10:1 respectively. LOD and LOQ values were reported in Table IV. Method precision study was also conducted at the LOQ level and calculated the %RSD for the areas of each Impurity. Accuracy at LOQ level was confirmed by injecting three different preparations of dasatinib spiked with impurities at LOQ level and calculated %, w/w recoveries of each impurity.

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Table IV. Regression, precision, LOQ and LOD data Imp Particulars

Regression equation (y)

R2 value

Method Precision (%RSD)

Intermediate precision (%RSD)

(Âľg mL )

(Âľg mL )

Slope (b)

Intercept (a)

Correlation coefficient

Imp-3

0.06

0.21

39934

703

1.000

0.9997

1.49

1.20

Imp-1

0.07

0.23

47555

-250

1.000

0.9994

1.91

1.10

Imp-2

0.04

0.15

38955

1195

1.000

0.9990

1.64

0.80

Dasatinib

0.05

0.18

50006

2332

0.999

0.9980

N.A.

N.A.

Imp-4

0.07

0.21

32346

1332

0.999

0.9980

3.27

1.52

Imp-5

0.08

0.25

35119

3659

1.000

0.9991

1.43

1.10

Imp-6

0.15

0.30

20198

571

0.998

0.9979

3.63

2.40

LOD

-1

LOQ

-1

Accuracy The accuracy of the method was determined by spiking of all impurities at four different level i.e. LOQ level, 50% level, 100% level and 150% level of specification limit (0.15%). Recovery for all six impurities was found within the range of 80 to 120%. Recovery study and method precision results are reported in Table V. Table V. Results of accuracy and method precision Accuracy Level

Imp-1

Imp-2

Imp-3

Imp-4

Imp-5

Imp-6

LOQ recovery LOQ R-1

92.2

114.1

106.2

105.2

109.6

95.8

LOQ R-2

101.3

106.8

94.5

112.0

109.8

102.1

LOQ R-3

82.7

99.5

95.6

85.5

106.2

95.2

50% level recovery 50% Level R-1

104.4

110.3

104.9

107.3

106.1

94.3

50% Level R-2

98.1

105.0

97.9

110.2

108.7

96.1

50% Level R-3

95.6

104.2

102.1

108.4

98.4

97.3

100.8

97.2

97.6

100% level recovery (method precision) Spike solution-1

102.3

105.5

100.0

Spike solution-2

104.6

105.1

104.1

99.6

101.0

96.4

Spike solution-3

105.0

105.0

101.1

100.9

100.8

93.9

Spike solution-4

101.2

101.6

103.0

103.0

100.2

104.0

Spike solution-5

104.2

105.5

103.3

100.3

99.8

96.8

Spike solution-6

105.6

105.6

102.1

108.6

100.6

95.2

109.6

108.5

106.8

109.8

103.6

107.8

150% level R-2

110.1

109.7

106.7

106.6

104.1

85.9

150% level R-3

108.4

107.8

105.0

103.6

102.7

108.5

150% level recovery 150% level R-1

Linearity and Range Linearity of method was calculated at seven levels ranging from LOQ to 150% (i.e. 0.30, 0.45, 0.75, 25

Development of Chromatographic Method for Determination of Impurities in Solid Dispersion of Dasatinib

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1.20, 1.50, 1.80 and 2.25 µg mL-1) to the specification level of 0.15%, while for drug substance calibration curve was obtained for dasatinib in the concentrations ranging from LOQ to 150% (i.e. 0.20, 0.30, 0.50, 0.80, 1.00, 1.20 and 1.50 µg/mL) to the specification level of 0.10%. The correlation coefficient was found more than 0.990 for all the impurities. The slope, correlation coefficient and y-intercept values have been reported in Table IV, which specified linear method.

Robustness We have performed robustness study as per ICH guideline of analytical method validation, which covers most of the critical variables of analytical method. For demonstrate the robustness of developed method spiked sample was analyzed by altered chromatographic conditions (flow rate, pH and column temperature). No significant difference in quantification and resolution which specify that the developed method is robust. Results of robustness study were reported in Table VI. Table VI. Results of robustness study Parameter/variation

USP resolution Imp-3

Imp-1

Imp-2

Dasatinib

Imp-4

Imp-5

Imp-6

5.6

15.37

9.30

6.00

9.88

18.35

14.80

a. 1.15

5.7

15.8

9.8

6.2

10.1

18.9

15.2

b. 1.25

5.4

15.2

9.1

5.8

9.4

18.0

14.5

a. 32

5.6

15.7

9.8

6.3

10.0

18.7

15.0

b. 38

5.3

15.1

9.0

5.6

9.2

17.9

14.8

a. 4.9

5.2

15.3

9.3

5.9

10.8

18.0

14.7

b. 5.1

5.5

15.3

9.8

6.0

7.0

18.1

15.2

As such conditions Flow rate (mL/min)

Temperature (°C)

Buffer pH

Specificity and stress testing studies For specificity study, each Imp-1 to Imp-6, HPMC and dasatinib were injected separately. Spiked sample was also injected and from diode array detector purity plots extracted for each impurities in the spiked sample. 1000 µg mL-1 dasatinib solution was injected for each stress testing studies. Degradation was not observed when the solid dispersion of dasatinib was subjected to alkali degradation (1 N NaOH heat at 60 °C for 1 h) and acid degradation (1 N HCl heat at 60 °C for 1 h) conditions. 15.0% degradation was observed when the drug was subjected to oxidation (3% H2O2 for 1.5 h) leading to the formation of Imp- 2, thermal degradation (105 °C approximately 24 h) leading to the formation of Imp4 and Imp-6, and UV degradation (254 nm for 24 h) leading to the formation of Imp-1 and Imp-6. The degradation products that were formed during the stress studies were confirmed by co-injecting with the standard solution with stressed samples. Results from stress testing studies were reported in Table VII. An assay was calculated against dasatinib standard solution. The mass balance was calculated for each condition stressed samples and was found to be in the range of 95-105% which confirms that the developed method was stability-indicating. Depending on the chemistry and structure of molecule it may undergo degradation in specific strength of acid, base or peroxide at specific temperature only, hence only some specific conditions influence the degradation of drug substance.

Peak purity was checked in the degraded sample and calculate purity angle and purity threshold for the known impurity are reported in Table VIII. This table shows that purity angle is less than purity threshold for known impurity peak formed due to degradation.

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Table VII. Summary of stress testing results Time

Temp.

Assay (%, w/w)

RS by HPLC % degradation

Mass balance (% assay + % deg. products)

Remarks/observation

A control sample (untreated)

-

-

100.6

0.27

100.8

NA

HCl, 1.0 N (acid degradation)

1h

60 °C

100.8

0.22

101.0

No significant degradation observed

NaOH, 1.0 N (base degradation)

1h

60 °C

100.9

0.25

101.1

No significant degradation observed

Oxidation by 3.0% H2O2

1.5 h

25 °C

84.7

12.26

96.9

Imp-2 was formed

Thermally treated

24 h

105 °C

98.1

0.68

98.79

Imp-4 and Imp-6 impurities were formed

UV treated (254 nm)

24 h

25 °C

99.7

0.64

100.4

Imp-1 and Imp-6 impurities were formed

Degradation condition

Table VIII. Peak purity results Degradation condition

Imp-1

Imp-2

Imp-4

Imp-6

Dasatinib

Purity angle

Purity threshold

Purity angle

Purity threshold

Purity angle

Purity threshold

Purity angle

Purity threshold

Purity angle

Purity threshold

Oxidation

-

-

0.079

0.219

-

-

-

-

0.291

0.522

Thermal

-

-

-

-

0.263

0.347

0.524

0.643

0.631

1.104

0.259

1.144

-

-

-

-

0.692

1.647

0.417

1.001

UV Treated

Relative Response Factor The slope of each impuritiy and dasatinib was calculated from seven levels linearity. Relative response factor was calculated by a slope of dasatinib divided by the slope of respective impurity.

Relative Response Factor for all six impurities has been reported in Table IX, which is not more than 3.0 for any impurity. Thus developed method was highly efficient, as quantification has been done by a diluted standard solution of dasatinib and no need to inject all impurity standards. Quantification of impurities was done by multiplying response factor of respective impurity.

Table IX. Relative Response Factor Impurity Particulars

Response Factor

Imp-3

1.25

Imp-1

1.05

Imp-2

1.28

Dasatinib

1.00

Imp-4

1.55

Imp-5

1.42

Imp-6

2.48

27

Article

Development of Chromatographic Method for Determination of Impurities in Solid Dispersion of Dasatinib

CONCLUSION An accurate, selective and sensitive gradient RP-HPLC method has been developed and validated as per regulatory guideline for the determination of process and degradation related impurities for the oncology drug, dasatinib. In addition, the developed method is cost effective as there is no need to inject expensive impurities standard solution during method validation. Taken together, developed RPHPLC method demonstrated precise, economical and commercially viable quantitative determination of dasatinib impurities which will also be useful for industrial scale manufacturing.

Acknowledgment The authors would like to acknowledge the management of Cadila Healthcare Ltd. for support and encouragement. ZRC Communication Nº 565.

Compliance with ethical standards The authors declare that they have no conflict of interest.

Manuscript received: 08/08/18; revised manuscript received: 10/31/18; revised manuscript for the 2nd time received: 12/31/18; manuscript accepted: 01/09/19; published online: 01/28/19.

REFERENCES 1. Ali, M. Mol. Diagn. Ther., 2016, 20, pp 315-333. doi: 10.1007/s40291-016-0208-1 2. Miura, M. Bio. Pharm. Bull., 2015, 38, pp 645-654. doi: 10.1248/bpb.b15-00103 3. Drug Bank: Dasatinib (DB01254).

4. International Conference on Harmonization. ICH Q1A(R2). Stability Testing of New Drug Substances and Products, 2003.

5. The United States Pharmacopoeia 39th ed., US Pharmacopoeia Convention, MD. 2017.

6. Zeng, J.; Cai, H. L.; Jiang, Z. P.; Wang, Q.; Zhu, Y.; Xu, P.; Zhao, X. L. J. Pharm. Anal., 2017, 7, pp 374-380. doi: 10.1016/j.jpha.2017.07.009 7. Pirro, E.; De Francia, S.; De Martino, F.; Fava, C.; Ulisciani, S.; Cambrin, G.; Racca, S.; Saglio, G.; Di Carlo, F. J. Chromatogr. Sci., 2011, 49 (10), pp 753-757. doi: 10.1093/chrsci/49.10.753 8. Gonzalez, A. G.; Taraba, L.; Hranicek, J.; Kozlik, P.; Coufal, P. J. Sep. Sci., 2017, 40, pp 400-406. doi: 10.1002/jssc.201600950 9. Prinesh, P.; Gananadhamu, S.; Veeraraghavan, S.; Rambabu, A.; Kanthi Kiran, V. S.; Swaroop Kumar, V. V. S. Anal. Methods., 2014, 6, pp 433-439. doi: 10.1039/C3AY41287C 10. D’Avolio, A.; Simiele, M.; De Francia, S.; Ariaudo, A.; Baietto, L.; Cusato, J.; Fava, C.; Saglio, G.; Di Carlo, F.; Di Perri, G. J. Pharm. Biomed. Anal., 2012, 59, pp 109-116. doi: 10.1016/j.jpba.2011.10.003 11. Lankheet, N.; Hillebrand, M.; Rosing, H.; Schellens, J.; Beijnen, J.; Huitema, A. Biomed.

Chromatogr., 2013, 27 (4), pp 466-476. doi: 10.1002/bmc.2814 12. Hesham, K.; Motiur, R. A. F. M.; Mohammed, K. Dasatinib. In: Brittain, H. G. (Ed.). Profiles of Drug Substances, Excipients and Related Methodology. Elsevier, San Diego, CA, 2014, 39, Chapter 4, pp 205-237.

13. De Francia, S.; D’Avolio, A.; De Martino, F.; Pirro, E.; Baietto, L.; Siccardi, M.; Simiele, M.; Racca, S.; Saglio, G.; Di Carlo, F.; Di Perri, G. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci., 2009, 877, pp 1721-1726. doi: 10.1016/j.jchromb.2009.04.028 14. Andriamanana, I.; Gana, I.; Duretz, B.; Hulin, A. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci., 2013, 926, pp 83-91. doi: 10.1016/j.jchromb.2013.01.037 15. Catarina, J.; Victor, D.; J. Electroanal. Chem., 2015, 752, pp 47-53. doi: 10.1016/j.jelechem.2015.06.006 28

Sojitra, C.; Tehare, A.; Dholakia, C.; Sudhakar, P.; Agarwal, S.; Singh, K. K.

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16. Couchman, L.; Birch, M.; Ireland, R.; Corrigan, A.; Wickramasinghe, S.; Josephs, D.; Spicer, J.; Flanagan, R. Anal. Bioanal. Chem., 2012, 403, pp 1685-1695. doi: 10.1007/s00216-012-5970-2 17. Gotze, L.; Hegele, A.; Metzelder, S.; Renz, H.; Nockher, W. Clin. Chim. Acta., 2012, 413, pp 143149. doi: 10.1016/j.cca.2011.09.012 18. Balaji, N.; Sultana, S. Int. J. Pharm. Sci., 2016, 8 (10), pp 209-216. doi: 10.22159/ijpps.2016v8i10.14020 19. Sunil, L.; Sajid, S.; Dhramveer Singh, S.; Nishikumar, N.; Radhiah Che, R.; Nurul Syazwani, N.; Mohd Zulfadli, M. Int. J. Chem. Sci., 2017, 15 (4) p 177.

20. Sunil, L.; Dhramveer Singh, S.; Nishikumar, N.; Radhiah Che, R.; Sajid Syed, S. Int. J. Sci. and Res., 2017, 6 (11), pp 51-58. doi: 10.21275/ART20177411 21. Dwivedi, S. P.; Singh, K. K.; Singh, N. A.; Patil, A. U.S. 9,249,134 B2, 2016, Cadila Healthcare ltd, Ahmedabad, India.

22. International Conference on Harmonization. ICH Q2 (R1) Validation of Analytical Procedures: Text and Methodology, 2005.

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Br. J. Anal. Chem., 2018, 5 (21), pp 30-39 DOI: 10.30744/brjac.2179-3425.2018.5.21.30-39

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Optical Analysis Authenticated Electrical Impedance Based Quantification of Aqueous Naphthalene Shramana Roy Barman1, Subhadip Chakraborty2, Aniruddha Mukhopadhyay1, Sanatan Chattopadhyay2* 1

Department of Environmental Science, University of Calcutta, 35 Ballygunge Circular Road, Kolkata 700019, India 2 Department of Electronic Science, University of Calcutta, 92A.P.C. Road, Kolkata 700009, India

Graphical Abstract

Schematic representation of the system under investigation and variation of its electrical and optical properties with varying concentration of naphthalene.

Polyaromatic hydrocarbons (PAH) are organic compounds with fused benzene rings that have toxic and mutagenic properties, and have been well documented for their detrimental effect on animal and plant health. Naphthalene, a two-ring PAH, is a common water pollutant which has been linked to the disruption of immune system as well as deformation of red blood corpuscles in human and thereby raising substantial concerns. The present study quantitatively estimates naphthalene in its aqueous solution by employing electrical impedance spectroscopy (EIS), which is simultaneously supported by UV-vis spectral analysis. Naphthalene solutions of varying concentrations (0.2-1 ppm) are prepared and subjected to EIS as well as UV-visible spectroscopy. For the EIS based studies, the data is recorded for the frequency range of 1 KHz - 1 MHz and electrical parameters such as capacitance, conductance and admittance are observed to increase from 3.5 pF - 7.2 pF, 2.3 µS - 6.1 µS and 2.4 µS - 27.3 µS, 30

* c_sanatan@yahoo.com / scelc@caluniv.ac.in https://orcid.org/0000-0002-9038-5332

Optical Analysis Authenticated Electrical Impedance Based Quantification of Aqueous Naphthalene

Article

respectively, with varying naphthalene concentration in its solution. On the other hand impedance values are observed to decrease with the same. Naphthalene is itself a non-polar molecule and the formation of instantaneous dipoles originating from the interaction of such molecules governs the overall dielectric nature of the solution. UV-vis spectroscopic measurements of the solutions reveal the characteristic absorbance maxima at 216 nm for all the concentrations under investigation. Absorbance values are observed to increase with the relative strength of naphthalene in the solution and such values vary in the range of 0.02 - 0.16 au for the peak obtained. Thus by corroborating the electrical and the optical parameters, this study establishes a quick and handy method for detection of naphthalene in aqueous solutions and ascertains a framework for further work that can be done to formulate a naphthalene sensing device.

Keywords: Naphthalene, electrical impedance spectroscopy, instantaneous dipoles, UV-vis spectral analysis, system energy.

INTRODUCTION Global industrialization has triggered a large scale abuse of natural resources and chemicals over the last few decades. This rapid development and commercialization have resulted in extensive pollution of water bodies in and around the industrial areas and cities since the dumping of toxic waste into water bodies is a common malpractice. This pollution of water bodies has become a prominent environmental issue due to the ever increasing demand for potable water, especially in heavily populated cities. Trace amount of compounds like pesticides, dyes, phenols surfactant, polyaromatic hydrocarbons, heavy metals etc. have been often found in drinking water [1-4].

Polyaromatic hydrocarbons (PAHs) constitute a group of organic compounds that contain two or more fused aromatic ring in their chemical structure [5]. They are environmentally persistent and their structure ranges from simple naphthalene to complex coronene with varying extent of toxicity [6].

PAHs are generally released into the environment due to the incomplete combustion of fossil fuels, improper disposal of waste, burning of tobacco and plastics, extensive use of pesticides etc. [7]. The infusion of these compounds into water bodies is attributed to oil spillage accidents, discharges from various industries, wet and dry atmospheric fallouts and surface runoff [5]. PAHs can easily form associates with various suspended particles within the aquatic system and thus become persistent, enabled of bio-magnification [8]. Most of the PAHs are sparingly soluble in water and thus form bio recalcitrant and persist in the environment for a long time. PAHs have been scheduled by various government agencies as pollutants of prime concern [9]. PAHs have been proven to be toxic, carcinogenic as well as mutagenic in nature and can act as potential immunosuppressant. Due to their lipophilic nature PAHs can easily penetrate the cell membrane altering their morphology and can interfere with enzyme activity. It has also been proven that PAHs can have detrimental effect on the humoral immunity [6,10]. Naphthalene is one of the most common PAHs discharged in the environment [11]. It is a simple two-ringed structure which has been linked to reduced immunity and destruction of red blood cells. Naphthalene reportedly can also disrupt functions of the cell membrane and interfere with enzyme functions [12]. Owing to its hazardous characteristics the United States Environmental Protection Agency has restricted the maximum PAH level in drinking water to be 0.2 ppb [13]. However, the discharge and level of various PAHs often go undetected and PAH level as high as 1.5 ppm has also been reported by previous studies [14].

Considering the hazardous impact posed by naphthalene on human health, the removal of the same from waste water using various methodologies is of primary concern. A vital step prior to the removal of naphthalene is its detection. Various biomarkers have been used to detect different PAHs in aquatic and marine organisms such as mussels, Atlantic cods, brown trout, bivalves etc. [15-17]. Traditional methods such as gas chromatography, mass spectrometry, capillary electrophoresis etc. for detecting aqueous PAH cannot be performed on site which is the need of the hour. Recently, a substantial amount 31

Article

Barman, S. R.; Subhadip Chakraborty, S.; Mukhopadhyay, A.; Sanatan Chattopadhyay, S.

of research is focused on onsite quick detection of PAHs in water. Electrochemical techniques such as formulation of self-assembled monolayer for detection of pyrene [18], fabrication of cadmium/aluminum layered electrodes for detection of anthracene [19] as well as preparation of polymer based molecularly imprinted sensors coupled with fluorescence and mass-sensitive transducers for the detection of PAHs [20] have been carried out in recent past. However amongst all other techniques, impedance spectroscopy has flourished as a potential contender in analytical measurements since last decade [21] due to its rapid turn out, simplicity and cost-effectiveness. In electrical impedance spectroscopy, several electrical parameters such as impedance, capacitance, reactance and conductance are monitored with a frequency sweep. The changes in these parameters are attributed to the variation of effective dielectric constant of the system caused by density gradient of the analyte to be estimated qualitatively or quantitatively.

The present study elucidates the quantitative estimation of naphthalene, a common urban water pollutant, using electrical impedance spectroscopy. Dielectric analysis, being the pivotal factor for such technique, is rigorously studied in this work considering the formation of instantaneous dipole moment of naphthalene which is inherently non-polar in nature. The electrical data thus generated have been supported optically by spectrophotometric analysis. This is the first known attempt towards designing a system for rapid electrical detection of naphthalene from its solution.

MATERIALS AND METHODS Chemicals used The naphthalene (C10H8) used for the present study has been procured from Merck, India. The other chemicals used were of analytical grade and obtained from the same.

Sample preparation Stock solution of 1.0 ppm naphthalene was prepared using distilled water and further dilutions of 0.2 ppm, 0.4 ppm, 0.6 ppm and 0.8 ppm were prepared just before conducting the experiments.

Spectrophotometric analysis Spectrophotometric analysis of the sample is performed by using a UV-vis spectrophotometer (LAMBDA 1050 UV/VIS/INR) [22]. Samples are scanned from 170 nm to 500 nm using a quartz cuvette of path length 10 mm. The optical densities (ODs) are recorded and used for data analysis. From the Beer Lambert’s law the molar absorption coefficient can be calculated using the Equation (1):

A = ɛcl (1)

where, A represents absorbance value, ɛ is the molar absorptivity coefficient (L mol-1 cm-1), c represents concentration (mol L-1) and l is the path length (cm).

Circuit analysis of the electrical measurement set-up The electrical measurement of the samples containing naphthalene in different strengths is performed using a computer-interfaced LCR meter (TEGAM, Model 3550), where a parallel-plate conductivity cell, with unity cell constant, is connected to the meter. The schematics of the experimental system and its electrical equivalent circuit are depicted in Figure 1.

32

Optical Analysis Authenticated Electrical Impedance Based Quantification of Aqueous Naphthalene

Article

Figure 1. (a) Schematics of the experimental system and (b) its electrical equivalent circuit.

The equivalent circuit consists of two capacitors (Cdl) and a resistor (Rsol) in series and the third capacitor (Csol) is in parallel with the series combination, as depicted in Figure 1(b). Cdl is associated with the parallel plates/electrodes and represents the double-layer capacitance on the surface of electrodes and the liquid. The dielectric nature of the solution contributes to the solution capacitance and resistance, represented by Csol and Rsol respectively.

Electrical measurements are performed at an applied AC bias of 1 V amplitude (peak to peak) and the LCR meter is well-equipped with noise reduction techniques.

The capacitance of the system can be calculated using Equation (2): đ??ś=

đ?&#x153;&#x20AC;â&#x2C6;&#x2014; đ??´ đ?&#x2018;&#x2018;

(2)

where, É&#x203A;* is the complex permittivity of the system, given by É&#x203A;* = É&#x203A; - jĎ&#x192; / 2Ď&#x20AC;f , Îľ and Ď&#x192; being the real permittivity and the conductivity of the system and f being the applied frequency on the system. A and d denote the electrode area and the distance between two electrodes, respectively. Here j is the square root of negative one.

The equivalent impedance of such a circuit is given by Equations (3), (4) and (5):

(3)

where

đ?&#x2018;§1 =

đ?&#x2018;&#x2026;đ?&#x2018; đ?&#x2018;&#x153;đ?&#x2018;&#x2122; â&#x2C6;&#x2019;

đ?&#x2018;&#x2014; đ?&#x153;&#x2039;đ?&#x2018;&#x201C;đ??śđ?&#x2018;&#x2018;đ?&#x2018;&#x2122;

(4)

đ?&#x2018;&#x2014; 2đ?&#x153;&#x2039;đ?&#x2018;&#x201C;đ??śđ?&#x2018; đ?&#x2018;&#x153;đ?&#x2018;&#x2122;

and (5) đ?&#x2018;§2 = â&#x2C6;&#x2019;

where j is the square root of negative one In such type of systems, double layer capacitors become significant at very low frequencies, resistive part and the dielectric part of the solution dominates at mid and relatively higher frequency ranges respectively [23-26].

33

Article

Barman, S. R.; Subhadip Chakraborty, S.; Mukhopadhyay, A.; Sanatan Chattopadhyay, S.

RESULTS AND DISCUSSION Variation of electrical and optical parameters with relative strength of naphthalene in its aqueous solution Figures 2(a), (b), (c) and (d) represent the variation of impedance, capacitance, conductance and admittance, respectively with varying strength of naphthalene. It is apparent from the Figure 2 that the system impedance decreases with naphthalene concentration whereas capacitance, conductance and admittance are observed to increase with the same. Impedance values are observed to vary from 40 kΩ to 436 kΩ for the frequency range 1 kHz to 1 MHz and the relevant capacitance and conductance values change from 3.5 pF - 7.2 pF and 2.3 µS – 6.1 µS, respectively. For a fixed frequency, the increase of capacitance with naphthalene concentration is attributed to the formation of instantaneous dipole moment inside the system. Coefficient of regression (R2) values and line equations for the various measurements are summarized in Table I.

Figure 2. Variation of electrical parameters with relative strength of naphthalene in its aqueous solution; (a) represents variation in impedance; (b) depicts the variation of capacitance; (c) shows the variation conductance; and (d) represents the variation admittance with varying strength of naphthalene.

Naphthalene is a non-polar molecule and exhibits London forces as a result of the correlated movements of the electrons as they interact with each other [27]. Electrons of the adjacent molecules always repel each other which results in the redistribution of electron density in a molecule [28]. Such redistribution of electron density consequently causes fluctuations in their polarity structure, and thereby forming instantaneous dipoles as illustrated in Figure 3. Spontaneous formation of a dipole induces a polarity in the neighboring molecules. Increment of the non-polar molecule concentration in the system enhances the formation of instantaneous dipoles and hence the net effective dipole moment of the system increases [18]. Such increase of net dipole moment augments the system polarization, and hence the effective permittivity of the solution under investigation, which consequently raises the capacitance of the system.

34

Optical Analysis Authenticated Electrical Impedance Based Quantification of Aqueous Naphthalene

Article

Table I. Coefficient of regression (R2) values and line equations for the various measurements Impedance 1 KHz

10 KHz

100 KHz

1 MHz

Coefficient of regression

R² = 0.9107

R² = 0.8728

R² = 0.9276

R² = 0.7633

Equation of line

y = -3E+5x + 46878

y = -2.9E+5x + 45583

y = -1.6E+5x + 31620

y = -0.7E+5x + 4848.6

Capacitance Coefficient of regression

R² = 0.9295

R² = 0.8846

R² = 0.8733

R² = 0.785

Equation of line

y = 3E-13x + 4E-12

y = 8E-14x + 4E-12

y = 1E-13x + 3E-12

y = 9E-14x + 3E-12

Conductance Coefficient of regression

R² = 0.8998

R² = 0.9011

R² = 0.9101

R² = 0.9385

Equation of line

y = 3.54E-07x + 1.7E-06

y = 3.57E-07x + 1.8E-06

y = 3.56E-07x + 1.9E-06

y = 3.52E-07x + 2.7E-06

Admittance Coefficient of regression

R² = 0.929

R² = 0.930

R² = 0.9644

R² = 0.9719

Equation of line

y = 4E-07x + 2E-06

y = 3.7E-07x + 1.7E-06

y = 3.4E-07x + 2.8E-06

y = 7E-07x + 2E-05

Figure 3. Redistribution of electron cloud in naphthalene molecule and instantaneous dipole and induced dipole.

Dielectric constant or relative permittivity is a fundamental property of a system depending upon its constituents. Capacitance is directly proportional to dielectric constant for a fixed geometrical dimension of the measuring system. Impedance is a complex parameter which depends upon the internal reactance (or, the capacitive impedance) due to the capacitance. Increment in capacitance consequently lowers the capacitive reactance and hence the impedance of the system. Admittance and

35

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Barman, S. R.; Subhadip Chakraborty, S.; Mukhopadhyay, A.; Sanatan Chattopadhyay, S.

conductance are inversely proportional to the system impedance, and hence exhibit incremental trends with increasing naphthalene concentration.

UV-vis spectroscopic measurements are performed on the samples with varying naphthalene concentration of 0.2 - 1.0 ppm and plotted in Figure 4. A peak is obtained at 216 nm for all the concentrations of naphthalene observed from the plots. Absorbance values are observed to increase with the relative strength of naphthalene in the solution and such values vary in the range of 0.02 - 0.16 au for the peak obtained. This increment can be attributed to the increased amount of capacity of the system to absorb light in the UV-vis spectral region with addition of naphthalene content in the solution.

It is apparent from the figures that the nature of variation of the experimentally obtained absorbance values follows the Beer-Lambert law.

Figure 4. (a) UV absorbance spectrum of naphthalene solutions of different strengths; (b) Variation of absorbance with varying naphthalene concentration.

As discussed earlier, formation of instantaneous dipoles inside the system increases with the addition of naphthalene, which in turn enhances the effective polarization and hence the randomness of the system. Consequently, system entropy and thereby the internal energy of the system get increased.

UV-vis spectral analysis confirms increment of absorbance with compositional variation of naphthalene, and thus indicates an increase in the total absorbed energy of the system which corroborates the physical observation from the dielectric study of the system with varying strength of naphthalene.

Determination of the coefficient of sensitivity The slope of the curve as shown in Figure 2(a) describes the change in impedance for a variation of naphthalene concentration of a unit ppm. Hence, a factor in terms of impedance may be defined to signify the sensitivity of the system:

(6)

where, m and Z indicate the naphthalene concentration and impedance of the system respectively.

The bar operator on m and Z gives their respective average values for all relevant experimental data available. Figure 5 depicts coefficient of sensitivity of the present measurement system, calculated by using Equation (6), for the frequencies under consideration. It is apparent from the figure that coefficient of sensitivity decreases with the frequency of external electric field applied to the system.

36

Optical Analysis Authenticated Electrical Impedance Based Quantification of Aqueous Naphthalene

Article

Figure 5. Plots showing the coefficients of sensitivity in terms of impedance for the frequencies under consideration.

Statistical analysis All the experiments are performed thrice a day for three days and the data is represented as mean Âą standard deviation (SD). Microsoft Excel 2007 is used for calculations and analysis of data. The measured data displayed uncertainty of â&#x2030;¤ 2%.

Electrode stability To evaluate the stability of the measuring system, impedance values relevant to a fixed naphthalene concentration (0.2 ppm) and frequency (1 KHz) are measured for three days at an interval of one day to ensure the reproducibility of the electrode performance. It has been observed that the impedance values exhibit a variation in activity only by 1.5% and there by justify the robustness of the experimental system. Figure 6 shows the relevant impedance values for three days.

Figure 6. Plots of the impedance values corresponding to a fixed naphthalene concentration (0.2 ppm) and frequency (1 KHz) measured for three days.

37

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Barman, S. R.; Subhadip Chakraborty, S.; Mukhopadhyay, A.; Sanatan Chattopadhyay, S.

CONCLUSIONS Electrical parameters including the impedance, admittance, capacitance and conductance of naphthalene solution of varying strength are measured by employing impedance spectroscopy in this study. Formation of instantaneous dipoles in the system is observed to govern the effective dielectric behavior of the naphthalene solution. Spontaneous formation of a dipole induces a polarity in the neighboring non-polar constituent molecules. Increment of the non-polar molecule concentration in the system augments the formation of instantaneous dipoles and hence the effective dipole moment of the system increases. Coefficient of sensitivity in terms of impedance is also calculated for the measuring system, from which a decent change in impedance is observed for a variation of naphthalene concentration of a unit ppm. Spectrophotometric analyses of the solutions are also conducted and a prominent physical corroboration of the optical and electrical data has been established. This study provides a deterministic approach towards quantifying naphthalene in its solution in terms of its electrical properties. Such study promotes the development of electrical/electronic sensors based on impedance spectroscopy, which would be an excellent tool for sensing hazardous PAHs in terms of robustness, rapidity and cost effectiveness.

Conflicts of interest The authors hereby declare that they have no conflict of interest.

Manuscript received: 9/14/18; revised manuscript received: 11/28/18; manuscript accepted: 12/21/18; published online: 1/28/19.

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Optical Analysis Authenticated Electrical Impedance Based Quantification of Aqueous Naphthalene

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15. Beyer, J.; Aarab, N.; Tandberg, A. H.; Ingvarsdottir, A.; Bamber, S.; Børseth, J. F.; Velvin, R. Mar. Pollut. Bull., 2013, 69 (1-2), pp 28-37. doi:10.1016/j.marpolbul.2013.01.001 16. Vincze, K.; Scheil, V.; Kuch, B.; Köhler, H. R.; Triebskorn, R. Environ. Sci. Pollut. Res., 2015, 22 (15), pp 11822-11839. doi:10.1007/s11356-015-4398-6 17. Bolognesi, C.; Cirillo, S. Curr Zool., 2014, 60 (2), pp 273-284. doi:10.1093/czoolo/60.2.273 18. Muñoz, J.; Crivillers, N.; Mas-Torrent, M. Chem. Eur. J., 2017, 23 (61), pp 15289-15293. doi:10.1002/chem.201703264 19. Qiao, X.; Wei, M.; Tian, D.; Xia, F.; Chen, P.; Zhou, C. J. Electroanal. Chem., 2018, 808, pp 35-40. doi:10.1016/j.jelechem.2017.11.063 20. Dickert, F. L.; Tortschanoff, M.; Bulst, W. E.; Fischerauer, G. Anal. Chem., 1999, 71 (20), pp 45594563. doi:10.1021/ac990513s 21. Chattopadhyay, S.; Chakraborty, S.; Das, C.; Saha, R. Recent Progresses on Micro-and NanoScale Electronic Biosensors: A Review. In Chakraborty, S. Mukherjee, P. (Ed.) Nanospectrum: A Current Scenario, Allied Publisher, India, 2016, pp 19-40.

22. Barman, S. R.; Banerjee, P.; Das, P.; Mukhopadhayay, A. Int. J. Eng. Water Res., 2018, pp 1-13. doi:10.1007/s42108-018-0001-4 23. Chakraborty, S.; Das, C.; Saha, R.; Das, A.; Bera, N. K.; Chattopadhyay, D.; Karmakar, A.; Chattopadhyay, D.; Chattopadhyay, S. J. Electr. Bioimp., 2015, 6, pp 10-17. doi:10.5617/jeb.2363 24. Chakraborty, S.; Das, C.; Bera, N. K.; Chattopadhyay, D.; Karmakar, A.; Chattopadhyay, S. J. Electroanal. Chem., 2017, 784, pp 133 -139. doi:10.1016/j.jelechem.2016.11.055 25. Das, C.; Chakraborty, S.; Acharya, K.; Bera, N. K.; Chattopadhyay, D.; Karmakar, A.; Chattopadhyay, S. Talanta, 2017, 171, pp 327-334. doi:10.1016/j.talanta.2017.05.016 26. Chakraborty, S.; Das, C.; Karmakar, A.; Chattopadhyay, S. Adv. Mater. Proc., 2016, 1, pp 25–31. doi: 10.5185/amp.2016/106 27. Eisenschitz, R.; London, F. Z Phys., 1930, 60, pp 491-527. doi:10.1007/BF01341258 28. Wagner, J. P.; Schreiner, P. R. Angew. Chem., 2015, 127 (42), pp 12446-12471. doi:10.1002/ ange.201503476

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Br. J. Anal. Chem., 2018, 5 (21), pp 40-43

The 6th ENQFor & 3rd SBCF Meeting discussed the Importance of Chemistry in Criminalistics The congress that brought together the 6th National Meeting of Forensic Chemistry (ENQFor) and the 3rd Meeting of the Brazilian Society of Forensic Sciences (SBCF) took place at the Convention Center of Ribeirão Preto (SP, Brazil) from November 4 to 8, 2018, with support from the National Institute of Forensic Science and Technology (INCT Forense).

Opening Ceremony of the 6th ENQFor & 3rd SBCF Meeting (Photo: Henry de Martinis)

A brief history of the National Meeting of Forensic Chemistry: although it is a subject that arouses a lot of interest in society, like the large audience of several television series such as the famous C.S.I. Crime Scene Investigation, in which the routine work of forensic scientists is presented, the application of chemistry in the field of criminology is still a new line of research in Brazil. The first bachelor’s degree in forensic chemistry was created in 2006 by the Chemistry Department of the Faculty of Philosophy Sciences and Letters of Ribeirão Preto, University of São Paulo (FFCLRP-USP). The need to create a forensic event was evidenced by a significant increase in the number of forensic chemistry papers presented at national congresses. As spaces for forensic chemistry were not yet offered, such works were allocated in other areas. Thus, researchers at the Chemistry Department of FFCLRP-USP decided to offer the Brazilian scientific community an event dedicated to the presentation of research works in forensic chemistry as well as a discussion of important topics in criminalistics. In 2008 the 1st National Meeting of Forensic Chemistry (1st ENQFor) was created. Since then this event has taken place every two years and has always been organized by FFCLRP-USP and by the Brazilian Society of Forensic Sciences (SBCF). Nowadays, ENQFor is an important landmark for the dissemination of forensic chemistry in Brazil.

The main objective of the 6th ENQFor & 3rd SBCF Meeting was to promote a discussion forum on the advances in forensic sciences through conferences, lectures, coordinated sessions, technical lectures, workshops, short courses, poster presentations, and the engagement of all participants in talks, discussions, and social gatherings during the event.

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Poster Discussion Session (Photo: Henry de Martinis)

Perspectives on forensic chemistry research in Brazil as well as suggestions for further narrowing the gap between the university and criminal investigation expertise were discussed in this event. It was five days of great learning and dissemination of forensic science in Brazil, with presentations by national and international speakers.

Some of the speakers present are listed below: Prof. Dr. Carmen Cinira Santos Martin, who is a retired professor in the Department of Pathology and Legal Medicine at the Faculty of Medicine of Ribeirão Preto (FMRP), USP and one of the founders of the project that culminated in the creation of the Legal Medicine Center (CEMEL) in Ribeirão Preto, SP, Brazil.

Prof. Dr. Ashraf Mozayani, the executive director of forensic sciences and a professor at Texas Southern University. Prior to this position, she was the crime lab director and chief toxicologist for the Harris County Institute of Forensic Science, Houston, TX, USA. Dr. Carlos Eduardo Palhares Machado, current head of the External Expertise Area of the National Institute of Criminalistics (INC) in the Brazilian Federal District.

MSc. Rodrigo Mayrink, who is responsible for carrying out criminal investigations related to the crimes of trafficking in wild animals, animal maltreatment, and fraud in animal products, as well as environmental damage to the biotic environment in the field of mining, agricultural and livestock enterprise industries. He was responsible for coordinating the work of expert witness to verify damages to the fauna in the case of the rupture of the “Fundão” dam, which occurred on November 5, 2015, in Mariana, MG, Brazil.

Prof. Dr. Alice Aparecida da Matta Chasin is Full Professor of Toxicology and Coordinator of the Health Area of the Postgraduate Center of Oswaldo Cruz Colleges. She is a specialist in abuse drugs with a title conferred on her by the UN (United Nations).

In addition to all the academic content offered, event participants also had the opportunity to put their knowledge of Crime Scene Investigation (CSI) into practice. From experts to students, they were able to analyze a crime scene and draw their conclusions.

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Remake of a Crime Scene (Photo: Henry de Martinis)

The event also had an exhibition area in which companies presented their products and innovations and advanced technologies in the area. Among these companies were the following: Agilent, Bruker, Waters, Analitica, Shimadzu, and LAS of Brazil.

Booths of some Companies in the Exhibition Area (Photo: Henry de Martinis)

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Members of the Brazilian Journal of Analytical Chemistry team were also present at the event and held a drawing for some journalâ&#x20AC;&#x2122;s printed issues and mascot.

Winners of the Raffles promoted by BrJAC, and Members of the Journal Team (Photo: Henry de Martinis)

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Br. J. Anal. Chem., 2018, 5 (21), pp 44-47

7th BrMASS and 4th BrProt brought together Scientists from Brazil and the World The 7th Conference of the Brazilian Mass Spectrometry Society (7th BrMASS) was held simultaneously with the 4th Brazilian Congress of Proteomics (4th BrProt) in Rio de Janeiro between December 8 and 12, 2018. During these scientific meetings, participants had the opportunity to listen and exchange ideas with national and international researchers representing the best of proteomics and mass spectrometry worldwide.

Prof. Dr. Marcos N. Eberlin and guests (Photo BrMASS)

The 7th BrMASS focused on the advances in instrumental mass spectrometry (MS) and the state-ofthe-art of its applications. It was promoted and coordinated by Prof. Dr. Marcos Nogueira Eberlin, who is currently a researcher at the University of Campinas (Unicamp), SP, Brazil and also a professor and coordinator of the Mackenzie Research Group in Science, Faith, and Society at Discovery-Mackenzie, an interdisciplinary research center of the Mackenzie Presbyterian University, São Paulo, SP, Brazil.

The 4th BrProt promoted discussions on current knowledge and future trends in proteomics, as well as on teaching and research and development of new technologies and techniques applied to the analysis of biomolecules. In one of the courses offered in this scientific congress, the bases of knowledge in topdown proteomics in both native and denaturing modes were addressed.

In the five days of the meeting, participants at all levels (undergraduate, postgraduate, postdoctoral, and professionals – both young and experienced) could deepen their knowledge about MS. The event included courses, plenary lectures, MS user meetings, oral presentations of selected papers, poster sessions, a round-table for debate among experienced professionals, and more. The participants were also able to attend social events, such as a fantastic Hawaiian-style party.

Among the important researchers present at the event was Dr. Livia Schiavinato Eberlin, Assistant Professor in the Chemistry Department at the University of Texas at Austin, USA, who gave the opening lecture entitled “The MasSpec Pen Technology to Guide Surgical Procedures: Hype or Hope?”.

During the opening lecture, Livia Eberlin spoke about the research she has developed in the field of mass spectrometry. The researcher explained that she and her team have developed a pen that can identify if a patient has cancer in 10 seconds, a much shorter time than the currently available technique which takes two hours. “Normal tissues and cancerous tissues have a completely different composition. With the technology we developed, we are able to analyze these profiles in a matter of seconds,” explained Dr. Eberlin.

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Prof. Dr. Livia Ebelin presenting the opening lecture of the congress (Photo “Divulgação”)

This innovative research has recently provided Livia Eberlin with a MacArthur Foundation Fellowship, sometimes called a “genius” award. The prestigious, no-strings-attached, five-year fellowship awards $625,000 to each recipient.

MasSpec Pen, a portable probe that can non-destructively analyze human tissue samples to identify cancer (Photo “Divulgação”)

Each year, the MacArthur Foundation selects talented individuals who have shown exceptional originality in and dedication to their creative pursuits. The award aims to help talented people from all over the world who have stood out “creatively” in areas such as computing, science, and the arts. The idea is to allow these talented people to develop their projects with “comfort and freedom”.

“The cool thing about MacArthur is that it’s really a complete surprise to winners. They are superdiscrete and do not reveal who made the nominations of scientists. The only thing they revealed to me is that they take into consideration at least 30 letters of recommendation”, said Livia Eberlin.

The event had the participation of several national and international speakers of undoubted relevance for research in the area.

One of the researchers present was Prof. Dr. Giancarlo La Marca from the University of Florence, Italy, who lectured on “The use of mass spectrometry in pediatric clinical chemistry: the case of newborn screening”.

Prof. Dr. Mario Palma from São Paulo State University (UNESP) was also present and spoke on the 45

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theme “System biology strategies to reveal the toxic secret of spider webs”.

Other important researchers in the MS area such as Prof. Dr. Norberto Peporini (USP), Prof. Dr. Alessandra Sussulini (UNICAMP), Prof. Dr. Marco Aurélio Zezzi Arruda (UNICAMP), Prof. Dr. Renato Zanella (UFSM), Prof. Dr. Francisco de Assis Paiva Campos (UFCE), and Prof. Gilberto Barbosa Domont (UFRJ) also honored the event.

Homage Session One of the highlights of the congress was the delivery of the José Manoel Riveros Medal for Mass Spectrometry (formerly called BrMASS Medal). This medal is intended to give recognition to the scientists who have provided relevant service in the scientific advancement and diffusion of mass spectrometry in Brazil. It pays homage to Prof. Dr. José Manoel Riveros Nigra (USP) who is one of the great pioneers in mass spectrometry in Brazil, as well as a researcher of great world renown. He is and active collaborator in the Brazilian Society for Mass Spectrometry (BrMASS) and has worked for several administrations as its president and currently is president emeritus.

The following were honorees of the 7th BrMASS: Prof. Dr. Enio Frota da Silveira, who is a full professor of the Pontifical Catholic University of Rio de Janeiro, RJ, Brazil. His research is in the area of surface physics (condensed matter physics), with emphasis on ionic desorption produced by ion-surface collision and by photon-matter interaction (UV laser or synchrotron light).

He works with time of flight mass spectrometry (TOF-MS) with ion accelerators (MeV), 252Cf-PDMS and MALDI.

Prof. Dr. Gilberto De Nucci, who is a full professor at the following institutions: Department of Pharmacology of the Faculty of Medical Sciences of the University of Campinas (UNICAMP), Department of Pharmacology of the Institute of Biomedical Sciences of the University of São Paulo (USP), University Brazil, and Metropolitan University of Santos (UNIMES). His involvement with mass spectrometry was the introduction and dissemination of its use in bioequivalence and pharmacokinetic studies in humans. In this particular area, he has published seventy-five papers in international journals.

Prof. Dr. Gilberto Domont, who has a very extensive and prominent academic career, is a member of the Editorial Board of the Journal of Proteomics and Associate Editor of the Journal of Proteome Research. He is on the Board of Directors of the Human Proteome Organization (HUPO) and is the principal investigator of the consortium that manages the Chromosome-centric Human Proteome Project (C-HPP) - Biology/Diseases. He leads the Human Proteome Project for chromosome 15 created by HUPO. Currently, he is a Professor Emeritus of the Federal University of Rio de Janeiro, RJ, Brazil and has been working in Mass Spectrometry and Proteomics Applied to Biological Systems.

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Prof. Dr. Rodinei Augusti is a full professor at the Chemistry Department of the Federal University of Minas Gerais, Belo Horizonte, MG, Brazil. He develops work on the application of mass spectrometry in several areas, such as monitoring reactions of environmental interest and quality control of alcoholic beverages.

He is adviser to the following journals: Rapid Communications in Mass Spectrometry, Journal of Mass Spectrometry, Analytical Chemistry, Inorganic Chemistry, Journal of the Brazilian Chemical Society, Analyst (London), Journal of the American Society for Mass Spectrometry, and “Química Nova”.

Parallel to the scientific congress was the ExpoCenter with the presence of sponsoring companies such as: Thermo, Analitica, Waters, Sciex, Shimadzu, Bruker, LECO, Peak, Agilent, and Allcrom. The companies took full advantage of the exhibition area with booths that enriched the meeting even more.

In addition, companies hosted hospitality suites with live music and cocktails.

Booths of the companies sponsoring the 7th BrMass (Photo Lilian Freitas)

Members of the Brazilian Journal of Analytical Chemistry were also present at BrMASS & BrProt and during the event they held a drawing for some printed editions of the journal and for its mascot.

Winners of the drawing by BrJAC (Photo Lilian Freitas) 47

Sponsor Report This section is dedicated for sponsor responsibility articles.

Br. J. Anal. Chem., 2018, 5 (21), pp 48-56

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Forensics applications with Phenom desktop SEM Every forensic laboratory contains a number of optical and digital microscopes. This is one of the primary requirements when setting up a forensic laboratory to perform routine imaging tasks. In general, these optical devices are robust and easy to operate. However, the demand for magnifications beyond the optical range (>1000x) is increasing as the features of interest become smaller. Scanning Electron Microscopy (SEM) can go beyond the optical scale with very high magnifications and depth of focus.

Another advantage of SEM is the possibility to perform elemental analysis of microstructures. Combined with electron imaging, X-ray analysis is a very powerful tool for understanding the composition and structure of materials.

To step up from an optical device to a traditional SEM can however put a large dent in the budget of a forensic laboratory. Besides the considerable system investment, it requires special lab facilities and a dedicated trained operator.

The use of SEM in the forensic market can be of great value. The diversity of samples and applications is broad. The Phenom can investigate the majority of these samples. The Phenom desktop SEM combines the best of the optical and electron optical world. The Phenom provides useful images up to 45,000x magnification with high depth of focus. It is as easy to use as a typical laboratory-grade optical microscope and is therefore accessible for any forensic examiner in the lab. Some of the key applications are listed below.

Gun Shot Residue (GSR) GSR is the residue deposited on and around the body of the shooter after a bullet has been fired.

Detection of a significant amount of residue, therefore, is a powerful piece of forensic evidence that the particular person was very near to or even holding the gun when it discharged. Particles containing lead, antimony and barium are considered characteristic of traditional GSR. The most definitive method for determining whether a particle is characteristic of or consistent with GSR is by its elemental profile.

Particle analysis made by the Phenom proX desktop SEM with an energy-dispersive X-ray spectroscopy detector (EDS) can be the most powerful tool for forensic scientists in establishing proximity to a discharged firearm and/ or the contact with a surface exposed to GSR. Figure 1 shows an image of a potential Gun Shot Residue particle (6400x magnification). GSR particles can quite easily be recognized based on compositional information from the Phenom back scatter detector. The particle can be further analyzed by using the Phenomâ&#x20AC;&#x2122;s Energy Dispersive X-Ray Detector (EDS).

Figure 1. Image of a potential Gun Shot Residue particle (6400x magnification).

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Figure 2 is a screenshot that shows the Phenom EDS spot analysis results from the particle in the form of spectrum peaks and an output table containing the elements and weight percentage. The particle is identified as (traditional) gunshot residue due to the presence of Lead (Pb), Antimony (Sb) and Barium (Ba).

Figure 2. Phenom EDS spot analysis results from a traditional gunshot residue.

Figure 3 is a screenshot that shows an EDS analysis of gun powder (gun: dagsx 9x19 np). Both the police in the Netherlands and in Germany use Gadolinium in their gun powder for easy recognition. The electron image is shown top right. The Gd peak can clearly be seen in the spectrum at 6.056 keV. The weight percentage is also displayed in the output table (3.6%).

Figure 3. EDS analysis of gun powder (gun: dagsx 9x19 np).

Traffic accidents Scanning electron microscopy is often used to investigate forensic evidence where a traffic incident has resulted in serious injury. With the Phenom desktop SEM, forensics investigators can easily image a light bulb filament and perform elemental analysis. If a vehicle headlight, rear light or indicator was on at the time of an accident, the lamp filament would be hot and glass particles would melt onto it.

Different glass particles can be shown and analyzed with the desktop SEM and used as evidence when investigating causes of accidents.

The safety belt of a car will reveal whether or not it was in use during the moment of impact. Animal hair found on the vehicle could point to the cause of a collision. Car paint flakes can be very useful in providing evidence of hit and run accidents.

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The screenshot in Figure 4 shows the Phenom image and elemental analysis of a light bulb. When a car head- or rear light or indicator is on, the lamp filament becomes hot and at the time of a crash glass particles will melt onto the filament. EDS spot analysis from the highlighted position (spot 1) inside the image clearly shows the presence of SiO2 (glass). The other 2 particles that can be seen attached to the filament have the same gray level as the identified glass particle and can be considered as glass particles as well. Material contrast based on gray level is very powerful when imaging with the back-scatter detector.

Figure 4. Phenom image and elemental analysis of a light bulb. EDS spot analysis from the highlighted position (spot 1) shows the presence of SiO2 (glass).

The other 2 particles can also be considered as glass particle.

Small car paint flakes can often be found at the crime scene following a hit and run accident. Car paint consists of different layers. Comparisons are made between flakes from a suspect vehicle and the specimen in order to find a match. Figure 5 shows a paint flake revealing various paint layers. The arrows represent the positions where a spot mode analysis has been made.

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Figure 6 is a screenshot showing an overview of a car paint flake that has been mapped.

Elemental mapping reveals the distribution of the different elements within the sample. The different elements can be highlighted by coloring.

Figure 6. Screenshot showing an overview of a car paint flake that has been mapped.

Animal hair found in a car could be the cause of a so called â&#x20AC;&#x153;one vehicleâ&#x20AC;? accident. Hairs are composed primarily of the protein keratin and can be defined as slender outgrowths of the skin of mammals. Each species of animal possesses hair of a characteristic length, color, shape and root appearance, and internal microscopic features that distinguish one animal from another. Figure 7 shows images taken using the charge reduction sample holder which makes it possible to image non-conductive hair in its original state (no sputter coating is required).

(a) (b) Figure 7. (a) Cat hair; (b) Dog hair

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Crime scene investigation If a crime scene contains microscopic samples such as hair, diatoms or pollen, these can be easily identified using electron microscopes.

Diatoms and pollen analysis can be useful in forensic science by helping identify the provenance of individuals, clothing or materials recovered from investigation sites. Forensic hair analysis can be of crucial value in a criminal investigation when identifying criminal suspects and victims or when associating them with a specific location. Hairs can be transferred during physical contact; their presence can associate a suspect to a victim or a suspect/victim with a crime scene. Comparison of the microscopic characteristics of questioned hairs to known hair samples helps determine whether a transfer may have occurred.

Hair Forensic hair analysis can be used for: • Identifying criminal suspects • Identifying crime victims • Associating a victim or suspect with a location • Determining the type of crime committed Hairs can be transferred during physical contact; their presence can associate a suspect to a victim or a suspect/victim to a crime scene. The types of hair recovered and the condition and number of hairs found all impact on their value as evidence in a criminal investigation. Comparison of the microscopic characteristics of questioned hairs to known hair samples helps determine whether a transfer may have occurred.

In humans, hairs found on the head, pubic region, arms, legs and other body areas have characteristics that can determine their origin. Most information can be derived from head hair. Differences in cross section, the outer layer (cuticle), central canal etc. can indicate the person’s origin (African, European, Asian).

(a)

(b)

Figure 8. Hair of European (a) and Asian (b) origin including a point-to-point measurement made with Phenom.

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Biological evidence Diatoms are unicellular organisms that live in open water. Diatom analysis can be of further use in forensic science by identifying the provenance of individuals, clothing or materials from sites of investigation.

In cases of drowning, the inhalation of water causes penetration of diatoms into the system and blood stream, and thus their deposition into the brain, kidneys and other organs. If the victim was dead before the body was submerged, the transport of diatom cells to various organs is prevented because of a lack of circulation.

The type of diatoms that have been found can be used to pinpoint the location at which the drowning occurred. The presence of specific diatoms in the cloth of a body can reveal whether a body has been moved.

Figure 9. Images of diatoms.

Diatoms generally range in size from approx. 2-200 Îźm which can make it difficult to recognize them using light microscopy. A desktop SEM is the perfect solution due to its higher magnification and depth of focus. Sample preparation is similar to that used with optical devices.

Pollen is a fine to coarse powder containing the microgametophytes of seed plants. Pollen grains come in all shapes and sizes and never decompose. Pollen can tell a lot about where a person or object has been because specific locations can have a distinctive collection of pollen species. Pollen evidence can also reveal the season in which a particular object picked up the pollen. For instance, a dead body may be found in a wood, and the clothes may contain pollen that was released after death, but in a place other than where the body was found. That indicates that the body was moved.

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Birch pollen

Grass pollen

Taxus pollen

Pollen Figure 10. Images of pollen.

Mechanical evidence Most crimes are committed using tools such as knives, crow bars, screwdrivers and wire cutters.

When criminals use these tools to commit a crime they can leave behind marks or damage to the material they come into contact with. The marks made are generally lines (called striations) and are caused because of imperfections on the surface of the tool. Cutting fibers with a knife will be different from cutting with a pair of scissors. Gunshots will also damage the fibers in a specific way.

As shown on Figure 11, the back-scatter imaging detector inside the Phenom can be used in two modes: 1) Compositional mode (full mode). This gives maximum signal resulting in material information by means of contrast differences. Different materials can be recognized based on the gray scale level.

2) Topographical mode. This gives a different kind of shading resulting in a topographical image (3D effect). This mode makes the surface structure more clearly visible.

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Figure 11. Images obtained in two different modes: Compositional mode (left image) and Topographical mode (right image).

Figure 12. Fiber ends from textile which have been cut by a knife.

Asbestos In the 20th century, asbestos was often used in construction materials. Forensic investigation of suspected asbestos-related deaths includes a life-time occupational history, a complete autopsy, and identification of the asbestos fiber tissue burden.

(a)

(b)

Figure 13. (a) The screenshot shows the Phenom EDS analysis results from crocidolite, also known as â&#x20AC;&#x153;blue asbestosâ&#x20AC;?; (b) Blue asbestos.

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Other SEM applications in forensics: • Classifying minerals in soil • Detection of small pieces of bone in debris • Analyzing explosive residues • Examining micro-traces of foreign material in forensic pathology and anthropology Phenom-World Phenom-World offers a wide range of desktop-based electron microscopes and accessories. The Phenom-World products are intuitive to use, fast to create results and built to high quality standards.

From the entry level pure series through to the high-end pro series, all Phenom electron microscopes share ease of use, unmatched time to results, and worry-free system ownership.

The Phenom desktop SEM is a true walk-up tool for many forensic experts who investigate their own samples in first line. Forensic applications of SEM are found mostly in areas where there is a need for good imaging at high magnifications in combination with elemental analysis. This is the case in areas where small particles of relatively heterogenic character and with a complex composition play a major part, for example gunshot residue, and pyrotechnical post-explosion residues.

These analyses can be accomplished with the Phenom proX desktop SEM – a powerful all-inone electron microscope with fully integrated EDS technology. The Elemental Identification software available for the Phenom proX allows researchers to program multiple point analysis and identify any hidden elements within any sample.

This sponsor report is the responsibility of Thermo Fischer Scientific.

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Br. J. Anal. Chem., 2018, 5 (21), pp 57-64

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This section is dedicated for sponsor responsibility articles.

Screening of 300 Drugs in Blood using 2nd Generation Exactive Plus High-Resolution, Accurate Mass Spectrometer and ExactFinder Software Kristine Van Natta, Marta Kozak, Xiang He Thermo Fisher Scientific, San Jose, CA

The purpose of this study was to evaluate the Thermo Scientific Exactive Plus high performance bench-top mass spectrometer for drug screening of whole blood for forensic toxicology purposes. Whole blood samples were processed by precipitation with ZnSO4/ methanol. Samples were injected onto an HPLC under gradient conditions and detected on an Exactive™ Plus mass spectrometer. Results were analyzed with Thermo Scientific ExactFinder software. Over 400 drugs were detected at LODs ranging from 5-100 ng/mL in whole blood.

INTRODUCTION Forensic scientists and toxicologists need to search for many different compounds in samples of human blood. Endogenous matrix components and the wide variety of possible compounds make the task daunting. The second generation Exactive Plus high-resolution accurate mass spectrometer with fast polarity switching enables identification of compounds in a wide chemical space with minimum interference from endogenous compounds. Additionally, full scan data allows for retrospective analysis of data for previously unknown compounds.

MATERIALS AND METHODS Sample Preparation Mix 50 µL of blood with 5 µL of internal standard solution containing reserpine and tolbutamide-d9.

Add 150 µL of 40 mM ZnSO4 in 66% methanol and vortex immediately. Place samples in a -20 °C freezer for 20-30 minutes. Centrifuge at 13,000 rpm for 10 minutes. Transfer supernatant to an autosampler vial, cap, and inject 20 µL onto HPLC system.

Liquid Chromatography The HPLC used is a Thermo Scientific Accela 600 pump with Accela™ open autosampler. Mobile phases are 10 mM ammonium acetate in water (A), 0.1% formic acid in acetonitrile (B), and acetonitrile:1propanol:acetone (45:45:10) (C). The HPLC column used is a Thermo Hypersil GOLD, 5 µm, 100 x 3 mm run under the gradient shown in Table I. Table I. HPLC Gradient Method Start (min)

Sec

Flow (mL/min)

%A

%B

0.00

60

0.50

95

5

1.0

660

0.50

100

12.0

60

0.75

100

13.0

30

1.00

13.5

60

2.00

95

5

14.5

30

0.50

95

5

%C

100

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Mass Spectrometry Compounds are detected on a Exactive Plus high performance bench-top mass spectrometer equipped with an Orbitrapâ&#x201E;˘ mass analyzer. A schematic diagram of the Exactive Plus instrument is illustrated in Figure 1.

Figure 1. Schematic diagram of the Exactive Plus high-resolution accurate mass benchtop mass spectrometer.

An atmospheric pressure chemical ionization (APCI) probe was used as an ion source. The instrument was operating in alternating positive and negative full-scan and all-ion fagmentation (AIF) mode with fast polarity switching at a resolution of 70,000 to 35,000 (FWHM) at m/z 200.

The Exactive Plus has an S-lens for improved ion transmission. The performance and robustness of the Automatic Gain Control (AGC) is improved using a C-Trap charge detector (CTCD). The higherenergy collisional dissociation (HCD) cell collects fragment-rich spectra similar to those generated in the collision cell of a triple stage quadrupole instrument. Relevant scan and source parameters are shown in Tables II and III. Table II. Scan Parameters for Exactive Plus Mass Spectrometer Parameter

Value

Full MS Micro scans

1

Resolution

70,000

AGC Target

3e6

Maximum IT

200 msec

Scan Range

m/z 120-1200

AIF Micro scans

1

Resolution

35,000

AGC Target

3e6

Maximum IT

100 msec

NCE Scan Range

35.0 m/z 80-1200

Parameters are the same for positive and negative mode.

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Table III. Source Parameters for APCI Probe Parameter

Value

Sheath Gas

35

Aux gas

15

Sweep gas

1

Discharge current

4

Capillary temp

320

S-Lens RF Level

60

Vaporizer Temp

350

Study Design To determine LOD for each compound, aliquots of whole blood were spiked with sets of compound at 5, 10, 20, 50, and 100 ng/mL. Matrix effects were determined by processing a sample spiked in water in place of blood.

Data Analysis Chromatograms are reconstructed with a mass tolerance of 5 ppm. Compounds are identified based on exact mass and retention time; confirmation is with fragments and isotopic pattern using ExactFinderâ&#x201E;˘ software. Figure 2 shows the ExactFinder method used in this study along with the compound database.

Figure 2. ExactFinder processing method and database.

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RESULTS AND DISCUSSION Over 400 different compounds were tested in this study: 487 were detected at 5 ng/mL, 39 at 10 ng/ mL, 10 at 20 ng/mL, 18 at 50 ng/mL, and 7 at 100 ng/mL. Figure 3 shows a representative chromatogram.

Fast polarity switching enabled identification of both positively and negatively charged species in one analytical run. Average mass accuracy was <3 ppm. Table IV shows the limits of detection of compounds in this study.

Figure 3. Chromatograms reconstructed with a 5 ppm mass window at 5 ng/mL.

Table IV. LODs for around 490 compounds Compound

LOD

Compound

LOD

Compound

LOD

Compound

LOD

1‐(3‐Chlorophenyl)piperazine

5

Deacetyl Diltiazem

5

Lormetazepam

5

Perimetazine

5

10‐hydroxycarbazepine

5

Demexiptiline

5

Loxapine

5

Phenacetin

5

Des(2‐hydroxyethyl)opipramol

5

LSD

5

Phenazone

5

5‐(p‐Methylphenyl)‐5‐phenylhydantoin

10

Desalkylflurazepam

5

Maprotiline

5

Pheniramine

5

6‐Methylthiopurine

5

Desipramine

5

Maraviroc

5

Phenobarbital

5

6‐Monoacetylmorphine

5

Desmethylcitalopram

5

MBDB

5

Phenylbutazone

7(2,3dihydroxypropyl)Theophylline

5

Desmethylclozapine

50

MDEA

5

Phenytoin

10

7‐Aminoclonazepam

5

Desmethyldoxepin

5

MDMA

10

Pholcodine

5

7‐Aminoflunitrazepam

5

Desmethylmirtazapine

5

Meclofenamic Acid

10

Pimozide

5

9‐hydroxy‐risperidone

5

Desmethylnefopam

5

Medazepam

5

Pindolol

5

Acebutolol

5

Desmethyltramadol

5

Medifoxamine

5

Pipamperone

5

Acenocoumarol

5

Dexamethasone

5

Mefloquine

5

Pipotiazine

5

Acepromazine

5

Dextromethorphan

5

Melatonin

5

Piroxicam

5

Aceprometazine

5

Dextropropoxyphene

Pizotifen

Acetazolamide

5

Dextrorphan

Ajmaline

5

Albuterol

20

Alfentanyl

5

6‐Acetylcodeine

60

100

5

5

Meperidine

5

10

Mephenesin

10

Diazepam

5

Mepivacaine

5

Pramipexole

5

Diazoxide

5

Meprobamate

5

Prazepam

5

Dibenzepine

5

Mescaline

Prazepam‐d5

5

10

Posaconazole

5 50

Sponsor Report

Compound Alfuzosine Alimemazine Allobarbital

LOD

Compound

LOD

5

Dihydrocodeine

5

Mesoridazine

5

Diltiazem

5

Metapramine fumarate

Dimetotiazine

5

Metenolone

10

Compound

LOD

5

Compound

LOD

Prednisolone

5

50

Prednisone

5

20

Primaquine

5

Alpha‐hydroxy‐midazolam

5

Diphenhydramine

5

Methadone

5

Primidone

10

Alprazolam

5

Dipyridamole

5

Methamphetamine

5

Procainamide

50

Amantadine

5

Disopyramide

Amidopyrine

5

Dobutamine

Amiloride

5

Amineptine

5

Amiodarone Amisulpride

5

Methandienone

100

100

Methaqualone

5

Domperidone

5

Methcathinone

10

Proguanil

5

Dosulepin

5

Methohexital

10

Promazine

10

5

Doxepine

5

Methoxsalen

5

Doxorubicin

5

Methoxyamphetamine

Amitriptyline

5

Droperidol

5

Methoxymethamphetamine

Amlodipine

5

Dropopizine

5

Methylene Blue

Amobarbital

5

Duloxetine

5

Methylparaben

Amoxapine

10

EDDP

5

Methylphenidate

Ampicillin

20

Efavirenz

5

Methylprednisolone

5

Prochlorperazine

5

Procyclidine

5

Promethazine

5

Prometryn

5

Propafenone

5

10

Propericiazine

5

10

Propizepine

5

5

Propranolol

5

10

Protriptyline

5

50 5

Amprenavir

5

EMDP

5

Metobromuron

5

Pseudoephedrine

Antipyrine

5

Enalaprilat

5

Metoclopramide

5

Pyrilamine

10 5

Apocodeine

5

Ephedrine

20

Metoprolol

5

Quinidine

5

Aprindine

5

Escitalopram

5

Metronidazole

5

Quinine

5

Aprobarbital

5

Esmolol

5

Mexiletine

5

Quinupramine

5

Astemizole

5

Estazolam

5

Mianserine

5

Raltegravir

5

Atazanavir

5

Ethyl Loflazepate

5

Miconazole

20

Ramipril

5

Atenolol

5

Ethylamphetamine

10

Midazolam

5

Ranitidine

5

Atropine

5

Ethylmorphine

5

Milnacipram

5

Repaglinide

5

50

Etifoxine

5

Mirtazapine

5

Riboflavin

5

Baclofen

5

Etodolac

5

Molsidomine

5

Rifabutin

5

Bendroflumethiazide

5

Etomidate

5

Morphine

5

Risperidone

5

Benperidol

5

Etoposide

5

Mycophenolic acid

5

Ritonavir

5

Benzoylecgonine

5

Felbamate

5

Nadolol

5

Ropivacaine

5

Bepridil

5

Felodipine

5

Nalbuphine

5

Roxithromycin

5

Betamethasone

5

Fenbufen

20

Nalorphine

5

Rufinamide

5

Betaxolol

5

Fenfluramine

5

Naloxone

5

Salicylamide

50

Bezafibrate

5

Fenofibrate

5

Naltrexone

5

Saquinavir

5

Biperiden

5

Fenoterol

5

N‐DemethylTrimipramine

5

Scopolamine

5

Bisoprolol

5

Fenspiride

5

N‐Desmethyl Clomipramine

5

Secobarbital

5

Brallobarbital

5

Finasteride

5

N‐Desmethyl Mephenytoin

5

Selegiline

5

Bromazepam

10

Flecainide

5

N‐Desmethylclobazam

5

Sertraline

5

Fluconazole

5

N‐Desmethylflunitrazepam

5

Sotalol

Flumethasone

5

N‐Desmethyltrimipramine

5

Stanozolol

10

20

AZT

Brompheniramine Buflomedil

5 10

5

Bupivacaine

5

Flunarizine

N‐Desmethylvenlafaxine

5

Strychnine

5

Bupropion

5

Flunitrazepam

5

Nebivolol

5

Sufentanil

5

Buspirone

5

Flunixin

5

Nefazodone

5

Sulfamethoxazole

5

Butabarbital

5

Fluoxetine

5

Nelfinavir

5

Sulindac

5

Butabital

5

Fluoxymesterone

5

Nelfinavir M8

5

Sultopride

5

Butalbital

5

Flupentixol

5

Netilmicin

5

Tacrine

5

61

Sponsor Report

Compound

LOD

Compound

LOD

Compound

LOD

Compound

LOD

Butorphanol

5

Fluphenazine

5

Nevirapine

5

Telmisartan

5

Cafeine

5

Flurazepam

5

Nialamide

50

Temazepam

5

Carbamazepine

5

Fluvoxamine

5

Niaprazine

5

Terbinafine

5

Carbamazepine epoxyde

5

Furosemide

50

Nicardipine

5

Terfenadine

5

Carbinoxamine

5

Galantamine

5

Nifedipine

5

Tetracaine

5

Carbutamide

5

Glafenine

5

Niflumic Acid

5

Tetrazepam

5

Carpipramine

5

Glibenclamide

5

Nimodipine

5

Theophylline

10

Carvedilol

5

Glibornuride

5

Nitrazepam

5

Theophylline‐7‐acetic acid

5

Celiprolol

5

Gliclazide

5

Nitrendipine

5

Thiopental

5

Nizatidine

5

Thioproperazine

Nomifensine

5

Thioridazine

5

Thioridazine sulfone

5

Tianeptine

5

5

Glipizide

5

Chloramphenicol

10

Glyburide

50

Chlordiazepoxide

5

Cetirizine

Haloperidol

5

Norbenzoylecgonine

5 20

Chlormezanone

20

Haloperidol‐d4

5

Norcocaine

10

Chloroquine

10

Heptabarbital

5

Norcodeine

5

Tiapride

5

Chlorpheniramine

10

Heroin

5

Chlorproethazine

5

Chlorpromazine

10

Chlorpropamide

5

Chlorprothixene Chlortalidone Cilazapril

Ticlopidine

5

Hexobarbital

10 5

Norpropoxyphene

50

Timolol

5

Hydrochlorothiazide

5

Norethandrolone

50

Tinidazole

5

Hydrocodone

5

Norfloxacin

5

Tofisopam

5

5

Hydromorphone

5

Norfluoxetine

5

Tolbutamide

5

5

Hydroquinine

5

Norfluvoxamine

5

Tolmetin

5

5

Hydroxychloroquine

5

Norketamine

5

Tramadol

5

Nordazepam

Cimetidine

5

HydroxyItraconazole

5

Nor‐LSD

5

Trans‐2‐Phenylcylopropylamine

Cinchonine

5

Hydroxyzine

5

Normaprotiline

5

Trazodone

5

Cinnarizine

5

Ibogaine

5

Normeperidine

20

Triamterene

5

Ciprofloxacin

5

Imatinib

5

Normesuximide

5

Triazolam

5

Cisapride

5

Imipramine

5

Normorphine

Clemastine

5

Indalpine

5

Noroxycodone

5

Trifluperidol

5

Clenbuterol

5

Indinavir

5

Northiaden

5

Triflupromazine

5

Clindamycin

50

Indometacin

5

Nortriptyline

5

Trihexyphenidyl

5

10

Trifluoperazine

10

50

Clobazam

5

Irebsartan

5

Norverapamil

5

Trimeprazine

5

Clomethiazole

5

Isocarboxazide

5

Noscapine

5

Trimetazidine

5

Clomipramine

5

Itraconazole

5

Ofloxacin

5

Trimethoprim

5

Clonidine

5

Ketamine

5

Olanzapine

5

Trimipramine

5

Clopidogrel

5

Ketoconazole

Ondansetron

5

Tropatepine

5

Clotiazepam

5

Ketoprofen

5

Opipramol

5

Urapidil

5

Orphenadrine

5

Valsartan

10

5

Cloxacillin

100

Ketorolac

50

Clozapine

5

Ketotifen

5

Oxatomide

5

Vecuronium

100

Clozapine N‐oxide

5

Lacidipine

50

Oxazepam

5

Venlafaxine

5

5

Lacosamide

5

Oxcarbazepine

5

Verapamil

5

Cocaine

10

Lamotrigine

5

Oxprenolol

5

Viloxazine

5

Codeine

5

LAMPA

5

Oxycodone

5

Vinbarbital

10

Colchicine

5

Levofloxacin

5

Oxymorphone

10

Vincristine

5

Cotinine

5

Levomepromazine

10

Pancuronium

50

Vinylbital

5

Cyamemazine

5

Levorphanol

10

Papaverine

5

Voriconazole

5

Cyclobenzaprine

5

Lidocaine

5

Paroxetine

5

Yohimbine

5

Cocaethylene

62

Sponsor Report

Compound

LOD

Compound

LOD

Compound

LOD

Compound

LOD

Cyclophosphamide

5

Linezolid

5

Penbutolol

5

Zimelidine

5

Cyproheptadine

5

Lisinopril

100

Penfluridol

5

Ziprasidone

5

Danazol

5

Lopinavir

5

Pentamidine

5

Zolpidem

5

100

Loprazolam

5

Pentazocine

5

Zonisamide

Dapsone

5

Loratadine

5

Pentobarbital

5

Zopiclone

5

Darunavir

5

Lorazepam

5

Perazine

5

Zuclopenthixol

5

Dantrolene

10

For targeted screening, ExactFinder uses parameters set in processing method to identify and confirm the presence of compound based on database values. Figure 4 shows data review results for one donor sample. In this method, compounds were identified by accurate mass within 5 ppm and retention time. Identity was further confirmed by isotopic pattern and presence of known fragments.

ExactFinder utilizes novel algorithms for background noise subtraction, peak detection and integration that require no user input.

Figure 4. ExactFinder results page showing XIC chromatogram reconstructed with 5 ppm mass window (a), isotopic pattern (b) and fragment ion confirmation (c). Fragment mass deviation is 1.5 ppm.

Matrix effects were observed to be compound dependent and were generally within Âą50%. Figure 5 shows peaks for hydrocodone in water and blood to compare effect of matrix on signal intensity.

Figure 5. XIC of hydrocodone in water (left) and blood (right) showing matrix effects.

63

Sponsor Report

CONCLUSION We have developed a screening method for over 400 compounds in whole blood with LODs of 5-100 ng/mL. Matrix effects are compound dependent and ±50%. Combination of high-resolution, accurate mass and fast polarity switching allows for rapid and confident identification in whole blood matrix for forensic toxicology purposes.

Acknowledgements We would like to thank Bénédicte Duretz of Thermo Scientific in France for supplying compound pools and Sarah Shugartsof UCSF Hospital for supplying donor samples.

This sponsor report is the responsibility of Thermo Fischer Scientific.

64

Sponsor Report This section is dedicated for sponsor responsibility articles.

Br. J. Anal. Chem., 2018, 5 (21), pp 65-68

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Microwave Sample Prep for heavy metals determination in cannabis plant, soil and water for medical applications Metals testing of agricultural samples and cannabis plant material with Milestoneâ&#x20AC;&#x2122;s Ethos UP high throughput microwave digestion system INTRODUCTION The medical cannabis industry is currently one of the fastest growing industries in the United States and is becoming more prevalent worldwide. Although systems for growing, production, and sale of medical cannabis and cannabis-related products are well established, regulation and enforcement of quality and safety testing have lagged behind. As the industry matures, many challenges have been encountered. One of those challenges is how to ensure safe products that are free from potential contaminants such as heavy metals.

Like all plants, cannabis absorbs metals from its environment, a result of normal plant metabolism.

Some of these metals are naturally occurring and leach into groundwater. Others precipitate in rainwater or may be introduced into the plantâ&#x20AC;&#x2122;s environment as constituents of fertilizers, pesticides, herbicides, and fungicides used to increase crop yield. Regardless of their prevalence, when metabolized, metals are absorbed and transported through the plant roots and into plant tissue. Cannabis is so effective at absorbing metals from its environment that it has been referred to as a hyperaccumulator of trace metals, including lead, cadmium, copper, chromium, arsenic, mercury and cobalt. This leads to concern that these elements may occur in high concentrations in cannabis plants.

State governments and private laboratories are focusing on product safety testing with special emphasis on As, Cd, Hg and Pb, as they are extremely hazardous to human health, even at low levels.

The combination of ICP-MS and Milestoneâ&#x20AC;&#x2122;s Ethos UP equipped with MAXI-44 high-throughput rotor enables digestion and testing of heavy metals in a wide variety of samples; from plant material and soil to water. The Ethos UP microwave digestion system incorporates all the benefits of closed vessel microwave digestion, making sample preparation fast, easy, and efficient. The Ethos UP with MAXI-44 helps speed up the sample preparation process, improves recovery of all elements (including volatiles) and reduces possible sources of contamination.

MATERIALS AND METHODS Instrument ETHOS UP The ETHOS UP meets many requirements of the new medical cannabis regulations. It offers several unique benefits including: high throughput to increase productivity; flexibility to digest a variety of matrices; intuitive software; industry leading safety. This microwave digestion system is a flexible and high performing platform used for trace element and routine determinations in many fields of analysis.

It is constructed of 18/8 stainless steel, features a built-in camera and can accommodate both highpressure and high-throughput rotors. Ethos UP includes 300 built-in digestion methods, which virtually eliminate method development. Additionally, the Ethos UP features Milestone Connect, which enables remote system control, 24/7 technical support and access to a comprehensive library of content developed especially for the analytical lab.

65

Sponsor Report

MAXI-44 High Throughput Rotor The MAXI-44 is a high throughput rotor capable of digesting a large number of soil, water and cannabis samples (up to 44 simultaneous), greatly improving lab throughput. The MAXI-44 is fully controlled by contact-less temperature and pressure sensors that directly control the temperature and the pressure of each vessel, thus assuring maximum safety and digestion quality.

Analytical Procedure Sample amount and acid mixture used for the microwave digestion Sample Cannabis plant material Soil (SRM 2711a) Water

Sample amount

Acid mixture

0.3 g

7 mL of HNO3 65%, 1 mL HCl 37%,1 mL H2O2

1g

9 mL of HNO3 65%, 3 mL HCl 37%

50 mL

5 mL of HNO3 65%

Microwave program used to digest samples Step

Time

Temp

Power

1

00:20:00

200 °C

1800 W

2

00:15:00

200 °C

1800 W

Final dilution: 50 mL with deionized water

Quantification

ICP-MS Instrumental Parameters

RF Power

1600 W

Dwell time per AMU

Sampling depth

10 min

Mode

Carrier gas

50 ms KED

0.8 L/min

Scan Mode

Peak hopping

Sweeps/Reading

20

Cell Gas A

0.6

Readings/Replicate

1

RP a

0

Number of replicates

3

RP q

0.25

Integration time

1000 ms

RESULTS AND DISCUSSION The performance of the Milestone Ethos UP equipped with MAXI-44 rotor was evaluated through a recovery study on samples of interest for the cannabis industry. Cannabis plant material, soil and 66

Sponsor Report

water were digested with Milestone’s Ethos UP equipped with MAXI-44 high throughput rotor and subsequently analyzed via ICP-MS. Cannabis plant material was fortified with a spike solution containing 20 ppb of As, Cd, Pb, Ag, Ba, Co, Cr, Cu, Mn, Ni, Se, V, Zn and 10 ppb of Hg. Water samples were fortified with a spike solution containing 5 ppb of As, Cd, Pb, Ag, Ba, Co, Cr, Cu, Mn, Ni, Se, V, Zn and Hg. A soil reference material (SRM 2711a) was also included in this study as a quality control sample. The analytical results are shown in Table I with recoveries of all elements between 80-120% and RSDs below 3%. This demonstrates the robustness and reproducibility of microwave digestion using the Ethos UP equipped with MAXI-44 technology.

Table I. Data of the recovery study

Cannabis Recovery plant material

(n=3) (%) RSD (%) Recovery

Water**

(n=3) (%) RSD (%)

As

Cd

Hg

Pb

Ag

Ba

Co

Cr

Cu

Mn

Ni

Se

V

Zn

95.4

92.7

101.1

89.3

87.8

95.9

92.7

91.9

90.2

-*

91.7

94.6

94.5

-*

1.3

2.2

1.5

2.6

2.1

1.2

2.2

2.0

1.7

-*

0.9

2.4

2.6

-*

102.4

98.8

98.6

94.6

92.4

101.3

102.5

97.9

93.9

96.7

103.4

107.2

97.1

98.4

1.1

1.6

0.8

2.1

2.2

0.9

0.7

1.4

0.6

0.4

1.4

1.9

0.9

1.8

*The ratio between spiked/unspiked concentration was too low. **In order to meet the requirements of EPA 3015A method, the digestion temperature for water samples was 175 °C.

Soil (SRM 2711a)**

As

Cd

Hg

Pb

Ag

Ba

Co

Cr

Cu

Mn

Ni

Se

V

Zn

Recovery (n=3) (%)

86.1

92.0

99.4

92.7

89.7

34.8**

86.7

31.8**

97.2

85.1

84.9

90.3

44.7**

90.4

RSD (%)

2.4

1.8

1.6

1.3

1.1

0.8

0.9

2.3

1.1

1.4

2.4

2.1

0.7

0.9

*In order to meet the requirements of EPA 3015A method, the digestion temperature for water samples was 175 °C. **The recoveries were calculated according to the total element content and they represent the leachable fraction (please, refer to NIST Certificate of Analysis for SRM 2711a for further details).

CONCLUSION The data shown in this technical note demonstrates full recovery of the most common elements occurring in cannabis plants.

All cannabis growing related products can be easily digested with the ETHOS UP equipped with MAXI-44, ensuring high quality digestion and high throughput. In addition to full analyte recovery, microwave digestion using Milestone’s Ethos UP provides the highest level of reproducibility, even for volatile elements such as As and Hg. Milestone’s ETHOS UP with MAXI-44 rotor offers multiple benefits for sample preparation for trace metals analysis and is the most suitable microwave digestion system for cannabis testing labs that require digestion of wide variety of samples such as plant material, soil and water.

This new era of acceptance and legalization has opened new opportunities for labs. Standardization of these methods for the industry will give regulators the resources they need to include sensible requirements for regulation and legislation that are being crafted to monitor and control the use of medical cannabis within the United States.

67

Sponsor Report

About Milestone Established in 1998, Milestone is headquartered in Italy and has offices in Germany, Switzerland, the United States, China, Japan and Korea. We operate worldwide through a network of over 100 exclusive distributors, all providing our customers premium application and service support. Milestoneâ&#x20AC;&#x2122;s mission is to help chemists by offering them the most advanced instrumentation for sample preparation and direct mercury analysis in the world. Our industry-leading technology, in combination with fast, responsive service and applications support, allows Milestone to support our goal of providing you the highest return on investment possible.

This sponsor report is the responsibility of Milestone.

68

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Phenom GSR – A Scanning Electron Microscope for Forensic Applications The Phenom SEM microscopes are intuitive to use, fast to create results and built to high quality standards.

Thermo ScientificTM Phenom GSR is a desktop Scanning Electron Microscope (SEM) that can be used for many forensics applications, such as automated gunshot residue (GSR) analysis, ballistics, paint analysis and fiber characterization. Moreover, the Phenom GSR is easy to set up and transport and can be relocated without difficulty. The system does not require any special facilities, such as compressed air, chillers, liquid nitrogen, EM shielding, cooling water, and has a low CO2 footprint (energy usage of maximum 300 Watt). Both software and hardware are fully integrated to enhance user-friendliness, reliability and analysis speed.

The Phenom GSR Desktop SEM comes with the following items: • Automated Gunshot Residue analysis and classification software package • Integrated BSED and EDS detector • Calibration sample The Phenom GSR desktop SEM is equipped with a CeB6 source that enables stable operation and has a typical operational life time of >1,500 hours, which is ideal from a usability, serviceability and uptime perspective. With a loading time of less than 1 minute and its fast stages, the Phenom GSR is the ideal tool for highly automated applications, such as automated gunshot residue analysis.

High throughput, reliable results Thanks to the fully motorized stage the Phenom GSR can handle a scan area of 100 mm x 100 mm.

The software uses the internal scan control of the SEM. This enables more accurate beam positioning which especially helps when revisiting the particle in the GSR verification phase.

GSR software is based on a four-step wizard to consistently set up the software in order to receive fast and reliable results from each run. The wizard is highly intuitive and allows the user to analyze multiple samples automatically. The GSR software complies with the current ASTM E1588 standard guide for GSR and is equipped with the standard layouts as provided by ENFSI. Fully integrated EDS Energy Dispersive Spectroscopy (EDS) allows users to analyze the chemical composition of their samples. Detailed chemical composition can be obtained from a micro volume via a spot analysis.

Step-by-step data collection The dedicated software package Element Identification (EID) is used to control the fully integrated EDS detector. Analysis has become as easy as imaging, since there is no need to switch between external software packages or computers. The CeB6 electron source in the Phenom is used to generate the highest X-ray count rate in its market segment, allowing fast results. The EID software package allows the user to identify nearly all materials in the periodic table, starting from Boron (5) and ranging up to Americium (95).

70

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Exactive™ Plus Orbitrap Mass Spectrometer Thermo Scientific™ Exactive™ Plus Orbitrap Mass Spectrometer screens, identify and quantify compounds in complex samples rapidly and with confidence. This benchtop LC-MS system delivers high-resolution, accurate-mass (HR/AM) data and fast full-scan capabilities to increase your sample throughput and confidence in results. Easy-to-use and cost effective to operate, it is the ideal instrument for analytical laboratories performing forensic toxicology, food safety or environmental screening, clinical research, DMPK, metabolomics and biopharma (intact protein analysis) applications.

Quick, High-Confidence Results • Combination of high-resolution, accurate mass and fast polarity switching allows for rapid and confident identification for forensic toxicology purposes.

• HRAM and full-scan capabilities capture all sample data, all the time, enabling retrospective data analysis without the need to repeat experiments.

• Resolving power up to 140,000 FWHM eliminates isobaric interferences, increasing confidence in results when analyzing samples in complex matrices.

• Better than 1 ppm mass accuracy in full and AIF scan modes ensures confident compound identification.

• Fast scanning at 12 Hz, suitable for UHPLC applications.

• Rapid polarity switching maximizes information obtained.

• AIF and multiple dissociation techniques, including in-source CID and HCD, aid in compound identification.

• Extended mass range to 6,000 m/z enhances detection of singly charged small molecules and biomolecules.

• More than four orders of magnitude intrascan dynamic range and femtogram-level sensitivity enable detection of trace-level and high-abundance compounds in the same scan.

Easy-to-Use • HRAM and full-scan capabilities simplify MS method development.

• Intuitive operating software reduces learning curve.

• Compatibility with Thermo Scientific™ LCQUAN™, Mass Frontier™, and TraceFinder™ software speeds data analysis.

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Q Exactive Plus - Thermo Scientific

Identification, Quatification & Confirmation 'Quanfirmation' in a single analysis · Advanced quadrupole improves the quantification of low abundance ions. · Orbitrap - high resolution and exact mass. · The second generation Exactive Plus high-resolution accurate mass spectrometer with fast polarity switching enables identification of compounds in a wide chemical space with minimum interference from endogenous compounds.

· Full scan data allows for retrospective analysis of data for previously unknown compounds.

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ETHOS UP and MILESTONE CONNECT: High Performance Microwave Digestion Systems Open the door to a new Milestone! The new ETHOS UP is the most advanced microwave digestion systems Milestone has ever manufactured.

The new Milestone ETHOS UP microwave cavity has a volume in excess of 70 litres, by far the largest currently available; up to 44 samples can be accommodated, improving productivity and sample preparation throughput.

ETHOS UP is equipped with the most advanced yet easy to use reaction sensors for complete quality control of the digestion conditions.

In combination with our ‘vent-and-reseal’ vessel technology, the sensors ensure complete and safe digestions without any loss of volatile compounds Included with the brand-new ETHOS UP microwave digestion system is a unique web based application – Milestone Connect. The app provides up to date information and extended instrument control from outside the laboratory.

By adding the IP address of your network, operators will be able to control the ETHOS UP from outside the laboratory with remote monitoring of every sample in the digestion run and other information related to the system on any wifi-enabled mobile device. That ultimately helps to provide high quality sample preparation. The app works on various external devices such as PC, tablets or smartphones connected to the ETHOS UP. Users will be part of Milestone scientific community and will gain an exclusive access to Milestone contents: application notes, digestion tips and techniques, Milestone library, scientific articles, video tutorials, special offers, news and a help-on-line section.

Milestone know-how and 26-year experience in sample preparation are now available for chemists to provide instant support available 24 hours a day, 7 days a week.

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Pittcon Conference & Expo Pittcon is the worldâ&#x20AC;&#x2122;s leading annual conference and exposition for laboratory science.

Pittcon not only covers analytical chemistry and spectroscopy, but also showcases developments made in the field of food safety, environmental sciences, bioterrorism, pharmaceuticals, and more.

Pittcon attracts attendees from industry, academia and government from more than 90 countries worldwide. It is a great opportunity to network with colleagues.

Cutting Edge Research The high-caliber technical program features scientists from around the world.

With more than 2,000 technical sessions, Pittcon makes it easy for you to get connected to the latest research and developments from leading scientists from around the world.

The robust technical program offers the latest research covering a diverse selection of methodologies and applications.

Skill-Building Short Courses Offered at beginner and intermediate levels.

With more than 100 from which to choose, there are a wide variety of classes covering relevant analytical topics in food science, water/wastewater, environmental, life science, pharmaceutical.

Courses for broad-based application and general lab functions include lab management, quality control, technical writing, statistics, data management, and lab safety.

3 Day Expo This global exposition gives you the opportunity to get a hands-on look at the latest laboratory instrumentation, participate in live demos and product seminars, talk with technical experts, and find solutions to all your laboratory challenges.

For more information please visit https://pittcon.org

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The robust technical program oďŹ&#x20AC;ers the latest research in more than 2,000 technical presentations covering a diverse selection of methodologies and applications.

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Pittcon is the worldâ&#x20AC;&#x2122;s leading annual conference and exposition on laboratory science. Pittcon attracts attendees from industry, academia and government from over 90 countries worldwide.

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CHROMacademy Helps Increase your Knowledge, Efficiency and Productivity in the Lab

CHROMacademy is the world’s largest eLearning website for analytical scientists. With a vast library of high-quality animated and interactive eLearning topics, webcasts, tutorials, practical information and troubleshooting tools CHROMacademy helps you refresh your chromatography skills or learn something completely new.

A subscription to CHROMacademy provides you with complete access to all content including: • Thousands of eLearning topics covering HPLC / GC / Sample Prep / Mass Spec / Infrared / Basic Lab Skills / Biochromatography Each channel contains e-Learning modules, webcasts, tutorials, tech tips, quick guides and interactive tools and certified assessments. With over 3,000 pages of content, CHROMacademy has something for everyone.

• Video Training Courses Each course contains 4 x 1.5-hour video training sessions, released over 4 weeks, with full tutor support and certification.

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Lite members have access to less than 5% of CHROMacademy content.

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SelectScience® Pioneers Online Communication and Promotes Scientific Success since 1998

Working with Scientists to Make the Future Healthier 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.

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Some recent contributions from SelecScience® to the scientific community • Editorial Feature Forensics & Toxicology - In this special feature, SelectScience® brings together useful resources from across the fields of forensics and toxicology to help your lab workflow, minimize errors, and to give an insight into how the fields have developed and where they might be heading. Read this full text here • Application Notes ID Kit - A Rapid, Reliable Test for Heroin and Other Illicit Drugs As threats from heroin become ever more critical, you depend on fast, convenient, and reliable presumptive testing. Current test methods – notably colorimetric tests – are notoriously inadequate.

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Notices of Books Fundamentos de Química Forense – Uma análise prática da química que soluciona crimes (2nd Edition, 2019) Aline Thaís Bruni, Jesus Antonio Velho, Marcelo Firmino de Oliveira, Authors Publisher: Millennium, Campinas, SP, Brazil.

This book addresses forensic chemistry in the control of chemicals, drug trafficking, doping in sport, driving under the influence of alcohol, counterfeit beverages, environmental crime analysis, and others. The reader will also find the fundamentals of the application of forensic chemistry in the physico-chemical analysis of document fraud, crime scene traces analysis, unveil of suppressed characters of guns and vehicles, gun shot residue analysis, forensic analysis of DNA, among others. Read more Toxicologia Forense – Teoria e Prática (5th Edition, 2018) Marcos Passagli, Author Publisher: Millennium, Campinas, SP, Brazil This book provides a careful selection of technical fundamentals of Forensic Toxicology. It is not limited to technical-analytical aspects, but also discusses the action of licit and / or illicit psychoactive substances in the human body. This book is intended for specialization programs in Forensic Toxicology, Forensic Chemistry, Chemical Engineering, Pharmacy, Biochemistry, Biomedicine, Forensic Medicine and Law. Read more Chromatographic Techniques in the Forensic Analysis of Designer Drugs Teresa Kowalska, Mieczyslaw Sajewicz, Joseph Sherma, Editors Publisher: CRC Press, published February, 2018 There is a dramatic rise of novel drug use due to the increased popularity of socalled designer drugs. This thoughtfully constructed edited reference presents the main chromatographic methodologies and strategies used to discover and analyze novel designer drugs contained in diverse biological materials. The methods are based on molecular characteristics of the drugs belonging to each individual class of compounds, so it will be clear how the current methods are adaptable to future new drugs that appear in the market. Read more Field Confirmation Testing for Suspicious Substances Rick Houghton, Author Publisher: CRC Press, published April 2018 Divided into three sections, this book begins by exploring physical confirmation tests as measurement of temperature, vapor density, radioactivity, and other factors. The author then examines chemical confirmation tests suitable for field use, providing over 400 different analyses, most of which provide a colorimetric result. The book also includes a section on instrumentation. It offers an overview of the technologies used to analyze materials and presents the strengths and weaknesses of the technology.

The appendix provides two detailed sections on drug and explosives tests. Read more

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Introduction to Forensic Chemistry Kelly M. Elkins, Author Publisher: CRC Press, published October 2018 This book introduces chemical tests, spectroscopy, advanced spectroscopy, and chromatography to students. The second half of the book addresses applications and methods to analyze and interpret controlled substances, trace evidence, questioned documents, firearms, explosives, environmental contaminants, toxins, and other topics. The book looks at innovations in the field over time including the latest development of new discernible chemical reactions, instrumental tools, methods, and more. Read more Handbook of Smart Materials in Analytical Chemistry Miguel de la Guardia, Francesc A. Esteve-Turrillas, Editors Publisher: John Wiley & Sons, published January 2019 This comprehensive, two-volume handbook provides detailed information on the present state of new materials tailored for selective sample preparation and the legal frame and environmental side effects of the use of smart materials for sample preparation in analytical chemistry, as well as their use in the analytical processes and applications. It covers both methodological and applied analytical aspects, relating to the development and application of new materials for solid-phase extraction (SPE) and solid-phase microextraction (SPME), their use in the different steps and techniques of the analytical process, and their application in specific fields such as forensics, clinical sciences, pharmaceuticals, water, food and air. Read more

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Periodicals & Websites The Column Cover story in this issue: The Role of Chromatography in Entomotoxicology The detection of drugs or other toxic substances can be crucial to forensic investigations. However, if the investigation involves a heavily decomposed, skeletonized, or missing corpse, that information can be difficult to obtain, and may require the skills of a unique branch of forensic toxicology, the entomotoxicology.

To explain the role of chromatography in this field, The Column spoke to Paola A. Magni from Murdoch University, in Perth, Australia. Access this issue here American Laboratory The American Laboratory® publication is a platform that provides comprehensive technology coverage for lab professionals at all stages of their careers. Unlike singlechannel 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 Chromatographyonline.com is the premier global resource for unbiased, peerreviewed 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 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 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, 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, Video and Webinar programs. Read more

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Spectroscopy Spectroscopyâ&#x20AC;&#x2122;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, peer-reviewed technical articles, trusted troubleshooting advice, and best-practice application solutions. Read more

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Events 2019 February 11 - 13 Brazilian Congress of Materials Microscopy - XV Micromat Brazilian Nanotechnology National Laboratory (LNNano), Campinas, SP, Brazil https://www.sbmm.org.br/ February 28 – March 1th 10th Edition of International Conference on Analytical Chemistry London, UK https://analyticalchemistry.euroscicon.com/ March 17 - 21 PITTCON Conference and Expo 2019 Pennsylvania Convention Center, Philadelphia, PA, USA https://pittcon.org/pittcon-2019/ March 25-28 35th International Symposium on Microscale Separations and Bioanalysis CH2M HILL Alumni Center, Oregon State University, Corvallis, Oregon, USA https://msb2019.org/ May 12 – 18 43rd International Symposium on Capillary Chromatography & 16th GCxGC Syposium Hilton Fort Worth, Fort Worth, TX, USA https://www.isccgcxgc.com/ May 27 - 30 42nd Annual Meeting of the Brazilian Chemical Society (42nd RASBQ) Centro de Convenções Expoville, Joinville, SC, Brazil http://www.sbq.org.br/reunioes-anuais June 16 - 20 48th International Symposium on High-Performance Liquid Phase Separations and Related Techniques (HPLC 2019) University of Milano-Bicocca, Milan, Italy https://www.hplc2019-milan.org/ July 7-12 IUPAC 47th World Chemistry Congress Palais des Congrès, Paris, FR www.iupac2019.org July 14 - 19 Latin American Congress on Chromatography and Related Techniques (COLACRO XVII) Unit – Universidade Tiradentes, Aracaju, SE, Brazil https://www.colacro2019.com

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September 1 - 5 Brazilian Symposium of Electrochemistry and Electroanalysis - XXII SIBEE Convention Center Ribeirão Preto, Ribeirão Preto, SP, Brazil www.xxiisibee.com.br September 1 - 5 Euroanalysis XX - Europe’s Analytical Chemistry Meeting Istanbul, Turkey http://euroanalysis2019.com/ September 8 - 11 133rd AOAC Annual Meeting & Exposition Sheraton Denver Downtown Hotel, Denver, CO, USA http://www.aoac.org September 24 - 26 15th Analitica Latin America Expo & 6th Analitica Congress Centro de Exposições São Paulo Expo, São Paulo, SP, Brazil https://www.analiticanet.com.br/en October 28 - 31 XXI Brazilian Congress of Toxicology & XV The International Association of Forensic Toxicologists (TIAFT) Latin-American Regional Meeting Águas de Lindóia, SP, Brazil www.cbtox-tiaft2019.org/‎ November 5 - 8 9th International Symposium on Recent Advances in Food Analysis - RAFA 2019 Prague, Czech Republic http://www.rafa2019.eu November 6 - 8 XIV Latin American Symposium on Environmental Analytical Chemistry (LASEAC) & IX National Meeting on Environmental Chemistry (ENQAmb) Bento Gonçalves, RS, Brazil http://www.laseac2019.furg.br

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Acknowledgments BrJAC editors are grateful to all those who have reviewed papers in 2018 using significant time and effort to provide constructive inputs.

Affonso Celso Gonçalves Jr. - Universidade Estadual do Oeste do Paraná Alam Gustavo Trovó - Universidade Federal de Uberlândia Alessandra Sussulini - Universidade Estadual de Campinas Alex Domingues Batista - Universidade Federal de Uberlândia Ana Valéria Colnaghi Simionato Cantú - Universidade Estadual de Campinas Andréa Fernandes Arruda - Universidade Federal de Goiás Andrea Rodrigues Chaves - Universidade Federal de Goiás Anselmo Elcana de Oliveira - Universidade Federal de Goiás Arnaldo Alves Cardoso - Universidade Estadual Paulista Bruno Campos Janegitz - Universidade Federal de São Carlos Bruno José Gonçalves da Silva - Universidade Federal do Paraná Carla Sirtori - Universidade Federal do Rio Grande do Sul Carlos Alberto Perez - Laboratório Nacional de Luz Síncrotron Cassiana Carolina Montagner Raimundo - Universidade Estadual de Campinas Cezar Augusto Bizzi - Universidade Federal de Santa Maria Claudete Fernandes Pereira - Universidade Federal de Pernambuco Cyro Lucas Silva Chagas - Universidade Federal de Goiás Diego Pereira dos Santos - Universidade Estadual de Campinas Diogo Librandi da Rocha - Universidade Federal do ABC Edilson Valmir Benvenutti - Universidade Federal do Rio Grande do Sul Edson Nossol - Universidade Federal de Uberlândia Eduardo Costa de Figueiredo - Universidade Federal de Alfenas Elias Ayres Guidetti Zagatto - Universidade de São Paulo Emerson Schwingel Ribeiro - Universidade Federal do Rio de Janeiro Erik Galvão Paranhos da Silva - Universidade Estadual de Santa Cruz Fabiano Molinos de Andrade - Universidade Federal de Goiás Fábio Ferreira Gonçalves - Universidade Federal do Rio Grande Fábio Rodrigo Piovezani Rocha - Universidade de São Paulo Fernando Fabriz Sodré - Universidade de Brasília Gabriel Gustinelli Arantes de Carvalho - Universidade de São Paulo Gabriela Rodrigues Mendes Duarte - Universidade Federal de Goiás Geisamanda Pedrini Brandão Athayde - Universidade Federal do Espírito Santo Guilherme Miola Titato - Universidade de São Paulo Hadi Beitollahi - Int. Center for Science, High Technology & Environmental Sciences, Kerman, Iran Hudson Wallace Pereira de Carvalho - Universidade de São Paulo Isabel Cristina Sales Fontes Jardim - Universidade Estadual de Campinas Ivo Milton Raimundo Júnior - Universidade Estadual de Campinas Jez Willian Batista Braga - Universidade de Brasília Joaquim de Araújo Nobrega - Universidade Federal de São Carlos José Alberto Fracassi da Silva - Universidade Estadual de Campinas Juliana Naozuka - Universidade Federal de São Paulo Karoliny Almeida Oliveira - Universidade Federal de Goiás Kelly das Graças Fernandes Dantas - Universidade Federal do Pará Khrissy Aracélly Reis Medeiros - Pontifícia Universidade Católica do Rio de Janeiro Letícia Malta Costa - Universidade Federal de Minas Gerais Lívia Flório Sgobbi - Universidade Federal de Goiás 88

Liziara da Costa Cabrera - Universidade Federal da Fronteira Sul Lucas Mattos Duarte - Universidade Federal de Juiz de Fora Lucilene Dornelles Mello - Universidade Federal do Pampa Maciel Santos Luz - Instituto de Pesquisas Tecnológicas do Estado de São Paulo Márcia Guekezian - Universidade Presbiteriana Mackenzie Marco Tadeu Grassi - Universidade Federal do Paraná Marcone Augusto Leal de Oliveira - Universidade Federal de Juiz de Fora Maria Del Pilar Sotomayor - Universidade Estadual Paulista Maria Eliana Ribeiro de Queiroz - Universidade Federal de Viçosa Maria Isabel Ribeiro Alves - Universidade Federal de Goiás Mariela Pistón - Universidad de la República de Uruguay, Montevideo, Uruguay Marília Oliveira Fonseca de Goulart - Universidade Federal de Alagoas Mathieu Tubino - Universidade Estadual de Campinas Mauro Bertotti - Universidade de São Paulo Núbia Natália de Brito - Universidade Federal de Goiás Oleg Tkachenko - V. N. Karazin Kharkiv National University, Kharkiv, Ukraine Paula Fernandes de Aguiar - Universidade Federal do Rio de Janeiro Paulo Clairmont Feitosa de Lima Gomes - Universidade Estadual Paulista Pedro Vitoriano Oliveira - Universidade de São Paulo Regina Vincenzi Oliveira - Universidade Federal de São Carlos Renan Geovanny Oliveira Araújo - Universidade Federal da Bahia Renato Zanella - Universidade Federal de Santa Maria Ricardo Bettencourt da Silva – Universidade de Lisboa, Lisboa, Portugal Ricardo Queiroz Aucélio - Pontifícia Universidade Católica do Rio de Janeiro Roberta Cerasi Urban - Universidade Federal de São Carlos Rodinei Augusti - Universidade Federal de Minas Gerais Rodrigo Moretto Galazzi - Universidade Estadual de Campinas Ronei Jesus Popi - Universidade Estadual de Campinas Rose Mary Georgetto Naal - Universidade de São Paulo Sandro Navickiene - Universidade Federal de Sergipe Sergiane Caldas Barbosa - Universidade Federal do Rio Grande Sergio Toshio Fujiwara - Universidade Estadual de Ponta Grossa Sherlan Guimarães Lemos - Universidade Federal da Paraíba Silvana Ruella de Oliveira - Universidade de São Paulo Solange Cadore - Universidade Estadual de Campinas Susanne Rath - Universidade Estadual de Campinas Tânia Mara Pizzolato - Universidade Federal do Rio Grande do Sul Tatiana Dillenburg Saint´Pierre - Pontifícia Universidade Católica do Rio de Janeiro Thiago Regis Longo Cesar da Paixão - Universidade de São Paulo Tiele Medianeira Rizzetti - Universidade de Santa Cruz do Sul Tony Rogério Lima Dadamos - Universidade Estadual Paulista Valderi Luiz Dressler - Universidade Federal de Santa Maria Vanessa Nunes Alves - Universidade Federal de Goiás Waldomiro Borges Neto - Universidade Federal de Uberlândia William Reis de Araujo - Universidade Estadual de Campinas

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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 doubleblind 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-inchief.

• 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, an event, 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 90

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.

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).

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: 91

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.

Master’s and doctoral theses or other academic literature 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 7. Trygve, R.; Perelman, G. US 9053915 B2, June 9 2015, Agilent Technologies Inc., Santa Clara, CA, US. Web pages 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 Symposium on Capillary Chromatography, 2012, Riva del Garda, Trento, IT. 10. Author, A. A. J. Braz. Chem. Soc., in press.

11. Author, B. B., 2015, submitted for publication.

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 on 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.

Sponsor Reports should be sent as a Word file attached to a message to the email brjac@brjac.com.br

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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 authorâ&#x20AC;&#x2122;s information must also be sent with each reviewerâ&#x20AC;&#x2122;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 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.

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