Laboratory Focus April/May 2014

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Exploring DNA Damage and Genotoxicity Page 7

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April/May 2014 Volume 18, Number 2

Keeping climate change in CHECK Page 11

R&D News.......................... 1 Pharma Notes..................... 5 Appointments..................... 6 New Products................... 15 Calendar........................... 17 App Reviews...................... 18

Joint efforts to study the atmospheric effects of biofuels combustion

NRC’s CT-133 research aircraft exits the hangar for a morning of final prep and fueling for flights later in the day.

Image Credit: NASA / Peter Merlin

The National Research Council of Canada (NRC) has signed a collaborative agreement with the National Aeronautics and Space Administration (NASA) to study the

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atmospheric effects of emissions from jet engines burning alternative fuels. With this cooperative work, NRC will take part in NASA’s ACCESS II project, the alternative

fuel effects on contrails and cruise emissions.

The ACCESS-II experiments have already commenced at NASA’s Armstrong Flight Research Center in Edwards, CA. Testing involved the deployment of NRC’s CT-133 aircraft to Palmdale, CA, to fly alongside aircrafts from NASA and the German Aerospace Center. The objective of the experiments is to obtain inflight airborne emission measurements and contrail characteristics from aircraft burning both conventional jet fuel and blended alternative fuels.

The collaboration on ACCESSII will result in the collection of complementary and unique flight test data that will be shared and reported to the International Forum for Aviation Research.

This important research will aid in the qualification and ready acceptance of the use of biofuels in aviation and open the door to future collaborations on alternative fuels tests.

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news Made in Canada vaccine approved for human trials A vaccine invented at the University of Guelph has been approved for human clinical trials by the U.S. Food and Drug Administration (FDA).

The Phase 1 clinical trial plan will utilize the cGMP sterile conjugated vaccine against Campylobacter jejuni– one of the leading bacterial causes

of food-borne illness in the world.

The vaccine, which was jointly developed by the Naval Medical Research Center (NMRC) and Prof. Mario Monteiro (University of Guelph), was produced in lyophilized vial form by Canadian CRO Dalton under contract with

The Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc. (HJF). The project was funded through a National Institute of Allergy and Infectious Diseases (NIAID) grant awarded to Dr. Patricia Guerry. Dr. Guerry is a Microbiologist specializing in Campylobacter research at

the Naval Medical Research Center (NMRC).

Under the contract with HJF, Dalton scaled up the conjugation and purification process of the protein conjugate vaccine, aseptically filled the vaccine into vials and lyophilized them.

Continued on page 3

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news New technology to facilitate shipping and storage of unstable biological agents without refrigeration

Professor Mario A. Monteiro and his research group

Continued from page 3

Approval for the Phase 1 challenge study with the vaccine was received from the U.S. FDA in February, and the human subject analysis phase started April 21, 2014. Phase 1 studies are expected to continue through January 2015.

According to the U.S. Centers for Disease Control and Prevention (CDC), Campylobacter, especially C. jejuni, is a leading cause of bacterial diarrheal worldwide. In the US, it is estimated to cause 1.3 million

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April/May 2014

human illnesses every year. These illnesses frequently occur as a result of travel, and are popularly referred to as “Travelers’ Diarrhea”. The diarrhea is frequently bloody, and is accompanied by abdominal pain and fever, and occasionally nausea and vomiting. No vaccine is currently available.

To see this story online visit http://www.laboratoryfocus. ca/?p=2388

A McMaster University team, funded

by Sentinel Bioactive Paper Network and NSERC, have found a way to ship and store chemically unstable agents and the preservatives to improve their shelf-life in pre-measured tablets. No refrigeration. No loss of chemical potency. No fancy tools.

The researchers working in the Biointerfaces Institute and Department of Chemical Engineering at McMaster University in Hamilton, ON, including Sana Jahanshahi-Anbuhi (lead author), Kevin Pennings, Vincent Leung, Meng Liu, Carmen Carrasquilla, Balamurali Kannan, Yingfu Li, Robert Pelton, John Brennan and Carlos Filipe used tablets made of pullulan, a natural polysaccharide that dissolves in water but resolidifies into a film when it dries. Pullulan is the only material that meets all the criteria for shipping agents: ships easily, protects against thermal and chemical damage, dissolves in water, releases the agent and does not interfere with the test.

The research was published by the German Chemical Society in its journal, Angewandte Chemie.

“The ability to keep highly unstable agents such as acetylcholinesterase (AChE) stable for months at room temperature has never been demonstrated before,” confirms Filipe.

“It now becomes possible to take reagents into the field, store them in remote locations under ambient conditions, and then use them when needed,” notes Brennan.

Instead of preparing a fresh batch of solution for each test, all the officials have to do now is drop a tablet of AchE into the sample, followed by a tablet of indoxyl acetate, and wait for the sample to turn faint blue or remain colourless if malathion is present.

Paper-based biosensors (bioactive paper) offer one of the best approaches for monitoring pesticides because of their low-cost, simplicity and rapid response time. The film-forming propContinued on page 4

Bioengineers at the University of Rome Tor Vergata and the University of Montréal have used DNA to develop a tool that detects and reacts to chemical changes caused by cancer cells and may one day be used to deliver drugs to tumour cells. The findings were published under the title: “Programmable pH triggered DNA nanoswitches,” in the Journal of American Chemical Society.

The researchers’ nanosensor measures pH variations at the nanoscale determining how acidic (a higher pH level) or alkaline (a lower pH level) it is. Many biomolecules such as enzymes and proteins are strongly regulated by small pH changes. These changes affect in turn biological activities such as enzyme catalysis, protein assembly, membrane function and cell death. There is also a strong relation between cancer and pH. Cancer cells often display a lower pH compared to normal cells: the pH level inside cancer cells is higher than it is outside. “In living organisms, these small pH changes

typically occur in tiny areas measuring only few hundred nanometers,” says senior author and professor, Chemistry Department of the University of Rome, Tor Vergata Francesco Ricci.

“Developing sensors or nanomachines that can measure pH changes at this scale should prove useful in the fields of in-vivo imaging, clinical diagnostics and drug-delivery.”

“DNA represents an ideal material to build sensors or nanomachines at the nanometer scale” adds co-senior author Alexis Vallée-Bélisle, a professor at the University of Montréal’s Department of Chemistry and Department of Biochemistry. “By taking advantage of a specific DNA sequences that form pH-sensitive triple helix, we have designed a versatile nanosensor that can be programmed to fluoresce only at specific pH values.”

Fluorescence is the emission of radiation, including visible light, caused by an exchange of energy.

“This programming ability represents a key feature for clinical applications –we can design a specific sensor to send a fluorescent signal only when the pH reaches a specific value which

Alexis Vallée-Bélisle

Researchers use DNA to build tool that may shine light on cancer

is, for example, characteristic of a specific disease,” adds first author Andrea Idili.

In the future, this recently patented nanotechnology may also find applications in the development of novel drug-delivery platforms that release chemiotherapeutic drugs only in the

vicinity of tumour cells.

The research was supported by the European Research Council and the Natural Sciences and Engineering Research Council of Canada.

To see this story online visit http://www.laboratoryfocus. ca/?p=2390

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April/May 2014 Laboratory Focus www.laboratoryfocus.ca

news Continued from page 3

A McMaster University team, funded by Sentinel Bioactive Paper Network & NSERC, have found a way to ship and store chemically unstable agents and the preservatives to improve their shelf-life in pre-measured tablets. No refrigeration. No loss of chemical potency. No fancy tools. (Photo credit McMaster University) erty of pullulan has been used in some unique applications in the pharmaceutical and food industries, such as breath fresheners and food additives.

Pullulan coatings applied to food packaging can act as oxygen barriers to prolong the shelf-life of various foods.

Pullulan has been shown to preserve the viability of bacteria under certain storage conditions.

To see this story online visit http://www.laboratoryfocus. ca/?p=2392

BC announces support for cellulose filaments research The government of British Columbia’s says it is contributing $2.25 million to cellulose filaments (CF) research as part of an existing R&D program focused on non-traditional applications of CF that are of interest to, and most beneficial for BC - specifically for the province’s northern bleached softwood kraft (NBSK) pulp producers.

A highly innovative wood-fibre based

biomaterial, CF is expected to have an immediate impact on Canada’s forest industry due to its capacity to be integrated into other materials and to its high strength, light weight and flexibility.

CF will be used in a wide range of applications as a lightweight strengthening additive to produce lower cost commercial pulps, papers, packaging,

(L-R) Jean Hamel, VP, FPInnovations, Pulp, Paper & Bioproducts; Hon. Andrew Wilkinson, Minister of Technology, Innovation & Citizens’ Services; Hon. Steve Thomson, Minister of Forests, Lands and Natural Resource Operations; Pierre Lapointe, President and CEO, FPInnovations. (CNW Group/FPInnovations).

tissues and towels. Looking to the future, CF may be combined with many materials to create high value products ranging from flexible packaging and films, to structural and non-structural panels in building construction.

“This announcement is a shining example of how collaboration and targeted investment in research and development can positively impact traditional markets while leading to the development of innovative new products,” said Pierre Lapointe, president and CEO of FPInnovations. “Cellulose filaments are set to become a key element in the transformation of the Canadian pulp and paper industry enabling the industry to gain a foothold in non-traditional markets while building on its existing manufacturing capacity in forest-dependent communities across BC.” The potential initial market for CF as a strength reinforcing agent for traditional pulp and paper products is conservatively estimated at 125,000 tons per year in North America alone. In addition, a similar-sized, non-traditional market is forecast for thermoplastics, reinforced plastics, thermosets, adhesives, non-woven fabric and coatings, representing a total revenue potential of $500 million per year for companies that make use of CF. To see this story online visit http://www.laboratoryfocus. ca/?p=2394

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Two Simon Fraser University psychologists have made a brain-related discovery that could revolutionize doctors’ perception and treatment of attention-deficit disorders.

The Journal of Neuroscience has just published a paper about the discovery

by John McDonald, an associate professor of psychology and his doctoral student John Gaspar, who made the discovery during his master’s thesis research. The discovery opens up the possibility that environmental and/ or genetic factors may hinder or suppress a specific brain activity that the researchers have identified as helping us prevent distraction.

“This is the first study to reveal how our brains rely on an active suppression mechanism to avoid being distracted by salient irrelevant information when we want to focus on a particular item or task,” said McDonald.

McDonald, a Canada Research chair in Cognitive Neuroscience, and other scientists first discovered the existence of the specific neural index of suppression in his lab in 2009. But, until now, little was known about how it helps us ignore visual distractions.

“This is an important discovery for neuroscientists and psychologists

10/11/2013 11:27:05 AM

Photo: SFU

Scientists discover brain’s anti-distraction system

because most contemporary ideas of attention highlight brain processes that are involved in picking out relevant objects from the visual field. It’s like finding Waldo in a Where’s Waldo illustration,” says Gaspar. “Our results show clearly that this is only one part of the equation and that active suppression of the irrelevant objects is another important part.”

The psychologists say their discovery could help scientists and health care professionals better treat individuals with distraction-related attentional deficits. The researchers are now turning their attention to understanding how people deal with distraction. They’re looking at when and why people can’t suppress potentially distracting objects.

To see this story online visit http://www.laboratoryfocus. ca/?p=2396

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Laboratory Focus April/May 2014

Photo: SFU

Pharma notes SQI Diagnostics Inc. (Toronto, ON) says it is being paid by an unidentified pharmaceutical company to develop and validate two custom multiplex anti-drug-antibody (ADA) assays. According to SQI, the customer will evaluate these two products using an established drug from its portfolio in order to assess the capabilities and performance of SQI’s technologies.

(82%), and peginterferon/ ribavirin ineligible/intolerant (82%) patients, including cirrhotic and non-cirrhotic patients (84% and 85%).

In the study the DCV plus ASV regimen was generally well tolerated. The Phase 3 multinational clinical trial included 116 sites in 18 countries, including countries that have a high prevalence of GT1b such as Korea and Taiwan.

Transition Therapeutics Inc. (Toronto, ON) has dosed the first patient in its Phase 2 clinical study of TT-401 (LY2944876), a drug candidate for the treatment of type 2 diabetes. The study is expected to enroll up to 375 subjects and will be performed by Transition’s development partner, Eli Lilly and Company (Lilly).

The objectives of the study will be to evaluate the safety and effectiveness of TT-401 compared to once-weekly exenatide extended release and placebo. The study is randomized, double-blind and placebo-controlled. It will include six study arms, four doses of TT-401, a placebo arm and a once-weekly exenatide arm. It will also include a 12-week blinded treatment period, where neither the participant nor the investigator will know which treatment each individual is assigned.

Helix BioPharma Corp.

(Aurora, ON) has submitted an Investigational New Drug (IND) application to the U.S. Food and Drug Administration (FDA) to initiate a Phase 1 clinical trial with L-DOS47.

L-DOS47 is Helix’s first immunoconjugate-based drug candidate in development based upon the company’s DOS47 technology, which is designed to use an innovative approach to modify the microenvironmental conditions of cancer cells in a manner that leads to their destruction. L-DOS47 is currently being clinically evaluated as a treatment for certain patients with nonsmall cell lung cancer. The study is entitled: “A Phase 1, Open Label, Dose Escalation Study of Immunoconjugate L-DOS47 in Combination with Standard Doublet Therapy of Pemetrexed/ Carboplatin in Patients with Stage IV (TNM M1a and M1b) Recurrent or Metastatic Non-Squamous Non-Small Cell Lung Cancer (NSCLC).”

Phase 3 results from Bristol-Myers Squibb’s (Mississauga, ON) global HALLMARK-Dual study were presented at the 49th annual meeting of the European Association for the Study of the Liver (EASL) in London, UK. The study focused on investigating the all-oral, interferon- and ribavirin-free regimen of daclatasvir (DCV), a NS5A inhibitor, and asunaprevir (ASV), a NS3 inhibitor, among genotype 1b hepatitis C virus (HCV) infected patients. The results showed that the 24-week regimen achieved an overall sustained virologic response (a functional cure) 12 weeks after the end of treatment (SVR12) among treatmentnaïve (90%), peginterferon/ ribavirin non-responder

Specialty pharmaceutical company Medicure Inc. (Winnipeg, MB) has entered into an arrangement with Knight Therapeutics Inc. that will see Knight Therapeutics provide advisory services to help advance Medicure’s U.S. specialty pharmaceutical business and corporate development initiatives. Knight Therapeutics Inc., headquartered in Montréal, QC, is a specialty pharmaceutical company focused on acquiring or in-licensing innovative pharmaceutical products for the Canadian and world markets.

BC-biotech company Xenon Pharmaceuticals Inc. is teaming up with Genentech to discover and validate new therapeutic targets and mechanisms for treating pain. The collaboration leverages Xenon’s Extreme Genetics discovery platform to focus on rare phenotypes where individuals have an inability to perceive pain or where individuals have non-precipitated spontaneous severe pain. It’s not the first time that Xenon has teamed up with Genentech, a member company of the Roche Group. The two sides previously formed a strategic alliance in 2012 for the discovery and development of compounds and companion diagnostics for the potential treatment of pain.

Pursuant to the terms of the

new agreement, both Xenon and Genentech will own the intellectual property arising out of the collaboration.

Xenon has also granted Genentech a time-limited exclusive right of negotiation on a target-by-target basis to form joint drug discovery collaborations. Financial terms of the agreement were not disclosed..

Amorfix Life Sciences (Mississauga, ON) has entered into a collaboration with Trellis Bioscience (San Fransisco, CA) to develop antibodies against misfolded CD38 protein as a treatment for haematological malignancies including leukemia and lymphoma.

CD38 is a protein that is highly expressed on the surface of a variety of white blood cells and has been implicated in a number of hematologic malignancies including multiple myeloma, chronic lymphocytic leukemia, B-cell lymphoma, acute lymphoblastic leukemia, acute myeloid leukemia, follicular lymphoma and mantle cell lymphoma.

Antibodies that bind to misfolded CD38 have the potential to only kill tumour cells and not normal cells through a variety of ways including the recruitment of the body’s immune system for complement-dependent cytotoxicity (CDC), antibodydependent cell-mediated cytotoxicity (ADCC), induced cell death (apoptosis) as

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well as by blocking or modulating CD38 enzymatic activity. The two sides hope that together, their technologies will enable the companies to isolate and develop fully human therapeutic antibodies that will target only cancer cells and not healthy ones. Under the terms of the collaboration, Amorfix will have an exclusive option to develop any resulting antibodies.

An affiliate of Cardiome Pharma Corp. (Vancouver, BC) has entered into a distribution agreement with UDG Healthcare plc, headquartered in Dublin, to fulfill orders and distribute BRINAVESS™ (vernakalant intravenous) in Ireland. The initial term of the agreement is for three years effective as of April 1, 2014.

Financial details have not been disclosed.

Warnex Inc. (Dorval, QC) has entered into a binding letter of agreement with DIAGNOS Inc. to merge with a wholly-owned subsidiary of Diagnos. As a condition to entering into a definitive agreement, Persistence Capital Partners, L.P., a healthcare private equity firm that holds approximately 52% of the issued and outstanding shares of Warnex, will enter into a lock-up agreement pursuant to which it will agree to vote its shares in favour of the transaction.

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aPPointments

Pivotal Therapeutics Inc announces the resignations of two of its directors, Dr. John P. Nicholson, Jr. and James Connolly. Additionally, Dr. George Jackowski has stepped down as chairman effective May 13, 2014 but remains a director and the chief scientific officer of the company. John S. Gebhardt, an independent director of the company, has been appointed as nonexecutive chairman to fill the vacancy. A member of Pivotal’s board of directors since 2011, Gebhardt has over thirty years of experience in the financial and securities industry. A former managing director at Knight Capital Group, he also worked at PaineWebber for over 20 years where he was a managing director, prior to it becoming part of UBS Financial Services.

Molecular diagnostics company Xagenic Inc. has hired Dr. Linda McAllister as its new chief medical officer. Dr. McAllister brings a track record of accomplishments in clinical medicine, academic research and medical device/diagnostics companies. Prior to joining Xagenic, she was the chief medical officer at Cellscape, a prenatal genetic testing company. Previously, she was chief medical officer and VP of R&D at PharmaJet where she led R&D efforts to support the submission of several successful 510(k) filings for a needle-free injection device system. She also led the regulatory and development efforts at Arbor Vita, a therapeutic and diagnostic company, where she obtained a 510(k) clearance on a diagnostic test for Avian flu and launched a global clinical trial for a diagnostic test to detect HPV. Earlier in her career, Dr. McAllister contributed to the development and launch of new molecular technologies at Roche Diagnostics, Celera Diagnostics and Affymetrix. She also served as adjunct clinical professor in the department of Medicine at UCSF from 1992 to 2005.

Vicki Bebeau has joined Neovasc Inc. in the role of vice president, clinical affairs, a newly-created position. She brings to Neovasc more than 18 years of experience in clinical trial planning and management having worked at St. Jude Medical, Boston Scientific and Medtronic.

Novartis Pharmaceuticals Canada Inc. has named Timothy Maloney as its new country head and president. Since joining Novartis in

at the Business Council of British Columbia where he directs the Council’s work on economic, fiscal, tax, environmental, regulatory and human capital issues of interest to the province’s business community.

Finlayson previously served as vice president of research at Canadian Council of Chief Executives.

Timothy Maloney 2003, he has held various positions in the U.S. and global commercial organizations. After leading the U.S. cardiovascular business, he moved to Switzerland to lead the $7-billion global cardiovascular and metabolism franchise. In 2011, he successfully launched the company’s Primary Care Franchise, contributing to the success of treatments for diabetes and hypertension, as well as building a world-class portfolio of products for respiratory diseases.

Maloney will also immediately start contributing to the overall Canadian research-based pharmaceutical industry by way of his appointment to the board of directors of Rx&D, Canada’s Research-Based Pharmaceutical Companies. Prior to joining Novartis, Maloney held sales and marketing leadership positions with Pfizer and its predecessor companies, Upjohn and Pharmacia.

Joseph Garcia and Jock Finlayson have been appointed to the Genome BC board of directors.

Garcia is a partner at Blake, Cassels and Graydon LLP where he practises in the securities and commercial areas. His practice includes all types of merger and acquisition and corporate finance transactions including public and private equity financings, take-over and issuer bids, share and asset acquisitions and divestitures, independent counsel to investment dealers and boards of directors, and all regulatory and stock exchange compliance work.

His clients are primarily in the life sciences and technology sectors.

Prior to his career in law, Garcia worked in corporate finance with a national investment bank and in clinical research with a multi-national pharmaceutical company. He currently is also a member of the board of directors of LifeSciences BC. Jock Finlayson is executive vice president and chief policy officer

The Ontario Hazelnut Association (OHA) has named Elliott Currie as its new executive director. Currie is currently a professor in the department of management at the University of Guelph. In his role, Currie will lead many of the programs that have been started by the OHA. These include the business development mandate to encourage growers to start hazelnut orchards in southern Ontario, develop business assistance programs for the sector and to oversee the administration of the association.

AbCelex Technologies has added Ron Meeusen, Matthew Bell, and Paul Dick to its board of directors. Dr. Meeusen is a managing director of Cultivian Sandbox and co-founder and managing partner of Cultivian Ventures. He has over 30 years of experience in bringing new technologies and products to market, leading major corporations in agricultural chemicals, field crop and vegetable genetics and breeding, animal health, novel foods and industrial materials. Prior to forming Cultivian, Meeusen led the expansion of the biotechnology research and development program of Dow AgroSciences, and founded a successful biopharmaceutical company, Immuneworks, Inc. He represents Cultivian on the board of directors of Allylix, Proterro and Rivertop Renewables, and represents Cultivian Sandbox on the board of EnEvolv. Bell is a principal of Cultivian Sandbox and has over nine years of technology commercialization and start-up experience through his time with the University of Michigan’s Office of Technology Transfer where he was directly involved with the formation activities of over 20 startup companies. Prior to working at the University, he spent over 10 years in the agriculture industry working for several production Ag supply companies including Growmark Incorporated where he was involved with their precision farming services group.

Dr. Dick has 25 years of experience in the animal health industry including product development,

regulatory affairs, business development, commercialization and general management. Other roles in this sector have involved both large and medium multinationals and small start-up companies, in various capacities including COO of Naturagen Health Solutions within Elanco and CEO of Chemaphor involved in development of pharmaceuticals and veterinary natural health products. He has also served a number of organizations including as director of the Canadian Animal Health Institute, president of the Ontario Veterinary Medical Association, and peer reviewer of NSERC strategic grants.

Nick Borrelly is joining Sernova Corp. in a consultant corporate development role. Borrelly has over 25 years’ experience in corporate/ business development as well as marketing and sales in the pharmaceutical (Ciba-Geigy, Novartis and Sanofi-Aventis) and biopharma industries. As president of Camargue Consulting, he specializes in development, evaluation and inlicensing of technologies, initiation of strategic alliances with multi-national corporate partners, and leading sales and marketing teams for commercial stage products. His past roles include manager, business development – licensing & acquisitions with Sanofi Aventis Pharma Canada; vice president, business development for MNLpharma (U.K.) Ltd.; and vice president, business development for CV Technologies/ Afexa Life Sciences Inc. Baycrest Health Sciences announces that Garry Foster has been appointed as president and CEO of the Baycrest Foundation.

Foster joined the Foundation in September 2013 on an interim basis and is the former chair of the Baycrest board of directors. His other past roles include vice-chair of Deloitte, member of the board of trustees of Calloway REIT, chair of the board of Cogniciti Inc., a Fellow Chartered Accountant and a Member of the Institute of Corporate Directors and has served some of Canada’s largest and most prestigious companies. His experience includes advising companies on governance, mergers, acquisition and a wide range of financial activities.

Medical diagnostic company Miraculins Inc. announces that David Eichler has stepped down from the company’s board of directors.

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Laboratory Focus April/May 2014

feature

by Joseph Hwang, Ph.D.

Exploring DNA Damage and Genotoxicity

DNA damage in cells is inevitable. It has been estimated that up to one million DNA changes occur per cell per day in response to environmental changes and byproducts of normal metabolism.1 If not repaired, lesions in critical genes such as tumor suppressors can impede a cell’s normal functions and increase the likelihood of tumour formation, as in the case of skin cancer.

Table 1

Figure 1

Genotoxic (G) and nongenotoxic (NG) compounds.

Simplified schematic showing the DNA damage/genotoxicity pathway. Analytes detected using the MILLIPLEX® MAP 7-plex DNA Damage/Genotoxicity Kit are highlighted in the schematic.

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April/May 2014 Laboratory Focus www.laboratoryfocus.ca

Feature

Genotoxicity describes the capacity of chemical agents to cause DNA damage within a cell, leading to mutations and potentially cancer. Genotoxicity tests are routinely used in the pharmaceutical industry to determine whether a pharmaceutical compound induces genetic damage, which can cause a wide range of problems including cancer and inherited birth defects.

A cell’s response to DNA damage involves many complex pathways and mechanisms, collectively called the DNA damage response. Once initiated, these pathways ultimately lead to repair of the DNA damage or initiation of apoptosis. The DNA damage response plays a crucial role in maintaining the function, genomic stability and viability of the cell and organism at large. Dysfunctions in the DNA damage response are implicated in many disease states, including cancer, premature aging, tissue toxicity and neurodegenerative disease.

FDA regulations require testing drug candidates for safety, efficacy, pharmacokinetics, toxicology, carcinogenicity and genotoxicity. The International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidelines on genotoxicity testing recommend two in vitro assays (such as the Ames test and the comet assay) and one in vivo assay (such as a micronucleus test (MNT)). These are the gold standards for genotoxicity testing and are offered as assay services from various companies. In addition, cellbased assays, such as the ATAD5 assay2 and high content assay/screening (HCA/HCS) services are also readily available. Although these assays are broadly used to determine if a drug is genotoxic, they provide limited mechanistic understanding of the cellular response to genotoxic compounds. To meet this need for mechanistic understanding of druginduced DNA damage response, a high-throughput assay to elucidate pathway changes within cells can be useful.

Signaling molecules in the DNA damage/genotoxicity pathway are regulated by phosphorylation, and understanding the role of this pathway requires the ability to simultaneously measure the phosphorylation status of multiple protein targets. Several assays to examine phosphorylation status are currently available, including Western blotting, ELISA, reverse phase arrays, quantitative cell imaging and mass spectrometry. Although some

of these platforms yield absolute, quasi-quantitative data, the assays are either limited to measuring only one analyte at a time, or are excessively difficult or expensive. In recent years, bead-based multiplex assays, such as those using Luminex xMAP® technology, have enabled the high-throughput measurement of phosphorylation levels of multiple proteins simultaneously, which give the advantages of reduced sample volume, time and cost compared to traditional methods.

The MILLIPLEX® MAP DNA Damage/Genotoxicity Kit (EMD Millipore, Cat. No. 621MAG) is a magnetic bead-based immunoassay that simultaneously detects seven proteins in the DNA damage/genotoxicity pathway in a single sample, enabling the measurement of phosphorylation changes (Figure 1).

This article demonstrates the utility of this assay in the analysis of DNA damage/genotoxicity in two cancer cell lines, HepG2 and HEK293. All analytes were detected with strong specificity, sensitivity and precision.

In addition, genotoxic compound screening shows the utility of this kit in drug discovery and development research.

Methods Tissue Culture HepG2 and HEK293 cells were cultured according to ATCC® guidelines in recommended media. Cells were plated at 50,000 cells per well in a 96-well plate. Twenty-four hours after plating, cells were treated with fresh complete media. After another 24 hours, cells were treated with designated genotoxic and nongenotoxic compounds (listed in Table 1) for a predetermined time period.

Figure 2

Phosphorylated proteins were simultaneously detected in HeLa, Jurkat and A549 cells.

(2A) Immunoprecipitation (IP) of proteins was performed with capture beads and detected by Western blotting with the biotinylated detection antibodies. Lanes correspond to: (1) untreated HeLa cell lysate, (2) camptothecin-treated A549 cell lysate and (3) anisomycin-treated Jurkat cell lysate. (2B) Lysate titrations were performed on Jurkat cells treated with 25 μM anisomycin (4 hours) and A549 cells treated with 5 μM camptothecin (overnight). The signal is represented as Median Fluorescent Intensity (MFI).

(2C) Intra- and inter-assay coefficients of variation (CVs) were calculated and reported as percentages (n=16).

Figure 3

Sample Preparation Immediately prior to harvest, media were collected and centrifuged. Cells were lysed, and the samples were collected and incubated with gentle rocking and centrifuged. Lysate supernatants were transferred into new tubes. Protein concentration in untreated samples was determined by a bicinchoninic acid (BCA) assay.

Using unstimulated sample protein concentration as an estimator, samples were diluted in assay buffer to provide a concentration of approximately 20 μg/well of a 96-well plate.

Signals from the compound screening studies were all normalized to β-Tubulin signal using the β-Tubulin MAPmate™ assay (EMD Millipore, Cat. No. 46-713MAG).

Dose response in HepG2 and HEK293 cells. DNA Damage/Genotoxicity Panel analytes were detected in HepG2 (3A) and HEK293 (3B) cells treated with genotoxic and nongenotoxic compounds ranging in concentration from 1 μM to 1 mM (on diagram, decreasing from left to right; 0.01 μM to 10 μM for TAX) for 48 hours. Median Fluorescent Intensities (MFI) were normalized to β-Tubulin and reported as fold change over the untreated control.

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Laboratory Focus April/May 2014

feature Microspheres We developed the MILLIPLEX® MAP 7-plex DNA Damage/Genotoxicity Kit by conjugating specific capture antibodies to magnetic microsphere beads purchased from Luminex Corporation.

Each set of beads is distinguished by different ratios of two internal dyes, yielding a unique fluorescent signature to each bead set. Capture antibodies were covalently coupled to the carboxylate-modified magnetic microsphere beads.

lines and tissues.

Changes in levels of phosphorylated Chk1, Chk2, H2A.X and p53, and total ATR, MDM2 and p21 were tested in HepG2 and HEK293 cells treated with genotoxic and nongeno-

toxic carcinogens (Table 1).

Changes in the DNA damage response were detected in a dose- (Figure 3) and timedependent (Figure 4) manner.

Because the panel enabled the simultaneous measure-

ment of multiple proteins, we could distinguish the varying effects of compounds that exerted their genotoxicity through varying mechanisms, as has been reported using gene expression profiling.3 For example, treatment

with compounds that caused DNA double-strand breaks, such as ETO, HQU and CIS, resulted in greatly increased phosphorylation of the cell cycle regulating kinases Chk1 and Chk2, indicating activation of checkpoint-me-

Immunoassay Protocol The multiplex assay was performed in a 96-well plate according to product instructions. The plate was first rinsed with 100 μL assay buffer. 25 μL of controls and samples and 25 of μL beads were added to each well. Plates were incubated overnight at 4°C. Beads were washed twice with assay buffer, and then incubated for one hour at room temperature with the biotinylated detection antibody cocktail. The detection antibody cocktail was replaced with 25 μL of streptavidinphycoerythrin (SAPE) and incubated for 15 minutes.

25 μL of amplification buffer was added and incubated for another 15 minutes. Then the SAPE/amplification buffer was removed and beads were resuspended in 150 of μL assay buffer. The assay plate was read and analyzed in a Luminex 200™ system, a compact unit consisting of an analyzer, a computer and software.

Results and Discussion The multiplex assay enabled the detection of phosphorylated Chk1, Chk2, H2A.X and p53, and total ATR, MDM2 and p21 with strong specificity, sensitivity and precision (Figure 2). The assay provided high specificity, indicated by the detection of proteins at the expected molecular weights as shown by immunoprecipitation/Western blot (Figure 2A). In addition, demonstrations of high signal-to-noise ratios (data not shown), sample linearity (Figure 2B) and precision (Figure 2C) lent support to the assay’s robustness. In addition, all analytes could be detected in human cell

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feature

figure 4

figure 5

time course of toxicity biomarker expression in heK293 cells. analytes were detected in heK293 cells treated with 1% Dmso, nongenotoxic compound (Dia) or genotoxic compounds (cis and eto) for 0, 0.5, 6, 16 and 24 hours. analyte protein concentrations are reported as fold change over the untreated control.

time course in hepg2 and heK293 cells. DNa Damage/genotoxicity panel analytes were detected in hepg2 (4a) and heK293 (4b) cells treated with genotoxic and nongenotoxic compounds for 0, 0.5, 6, 16 and 24 hours. median fluorescent intensities (mfi) were normalized to β-tubulin and reported as fold change over the untreated control.

diated pathways (Figure 3).4,5 Compounds that exerted genotoxic effects through other means, such as the microtubule-binding agent TAX or the DNA alkylators ENU and MMS, showed different patterns of pathway activation. Dose response data for ENU, MMS and TAX, for example, showed less dramatic phosphorylation of the Chk kinases accompanied by phosphorylation of p53 or histone H2A.X (Figure 3).

The p53 and histone H2A.X proteins are important players in the DNA repair pathway and can be phosphorylated in response to multiple types of DNA damage.

Measurement of time-dependent DNA damage response (Figure 4) also revealed differences in mechanism between different genotoxic compounds. While the doublestrand break-inducers, ETO and CIS, caused increasing phosphorylation of Chk1, Chk2, p53 and histone H2A.X with respect to time,

the DNA alkylator, MMS, caused an initial spike in Chk kinase and histone H2A.X phosphorylation that then diminished over time, but was accompanied by increased activation of p53. Again, this pattern may indicate initial checkpoint activation, which cells might have overcome, but was followed by checkpoint-independent DNA damage response.

As expected, LIM and DIA (nongenotoxic carcinogens) did not result in any significant changes in the analytes. Also as expected, little effect was seen with any of the compounds on total ATR, except at high doses of compounds that may have caused a general decline in cell health.

Finally, cell toxicity was not observed in HEK293 cells (Figure 5) using the MILLIPLEX® MAP Human Kidney Toxicity Panel 2 (EMD Millipore, Cat. No. RKTX2MAG-37K), as shown by the absence of any increase in the measured analytes

with respect to time.

These studies demonstrate that the DNA damage response is complex and involves more than the dysregulation of a single pathway.6 This conclusion further underscores the importance of simultaneous measurement of multiple phosphoprotein targets and demonstrates the utility of a new magnetic bead-based immunoassay in elucidating the mechanism of action for DNA damaging compounds.

References 1.

Lodish H et al. Molecular Biology of the Cell, 5th Edition. New York (NY): Freeman. (US); 2004.

2. Fox JT et al. High-throughput genotoxicity assay identifies antioxidants as inducers of DNA damage response and cell death.

Proc Natl Acad Sci U S A. 2012 Apr 3;109(14):5423-8.

3. Boehme K et al. Genomic profiling uncovers a molecular pattern for toxicological characterization of mutagens and promutagens in vitro. Toxicol Sci. 2011 Jul;122(1):185-97.

4. Niida H, Nakanishi M. DNA damage checkpoints in mammals. Mutagenesis. 2006 Jan;21(1):3-9.

5. Zhou BB, Bartek J. Targeting the checkpoint kinases: chemosensitization versus chemoprotection. Nat Rev Cancer. 2004 Mar;4(3):216-25.

6. Kastan MB. DNA damage responses: mechanisms and roles in human disease: 2007 G.H.A. Clowes

Joseph Hwang, Ph.D., Senior Research Scientist, Cell Signaling Group, EMD Millipore Corporation, joseph.hwang@emdmillipore.com Dr. Hwang develops new MILLIPLEX® MAP multiplex kits and single plex MAPmate™ assays for the Luminex xMAP® magnetic bead-based platform. He has more than 20 years of experience in cell signaling research, primarily in the area of insulin signaling.

To see this story online visit http://www. laboratoryfocus. ca/?p=2379

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Laboratory Focus April/May 2014

feature

b y : ly n n G a u K e r

Keeping climate change

in CHECK

Climate change is one of the most significant challenges our society faces. The Standards Council of Canada (SCC), through its Accreditation Program for GHG validators and verifiers, is putting the lid on greenhouse gas (GHG) emissions – and is making a difference for Canadians, Canadian and international businesses, and the environment. So too is SCC collaborator, ClimateCHECK.

A

ccording to Chantal Guay, SCC’s vice-president, Accreditation Services, “SCC’s accreditation program was launched to support the Government of Canada’s Sustainability Agenda for the reduction of GHG emissions, as well as to support provincial and territorial initiatives on climate change and clean air objectives.”

Today, she says, SCC’s accreditation program, recognized by and referenced in provincial regulations, continues to support government priorities with delivery of accredited verification of GHG emissions reports. “British Columbia, Alberta, Ontario and Québec have developed and put regulations into force for

mandatory reporting of GHG emissions by industry over the past several years.”

Innovative GHG management solutions Innovative GHG management solutions organization, ClimateCHECK, is collaborating with the SCC in the fight against climate change.

Launched in 2007, ClimateCHECK’s GHG experts have been major contributors to the development and application of GHG standards, protocols and programs. All of these items are used globally to establish best practices in the carbon markets and to spark growth in Canada’s environmental sector and for the economy.

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April/May 2014 Laboratory Focus www.laboratoryfocus.ca

feature

The good news, Guay points out, is that more opportunities will arise for innovative organizations offering environmental solutions.

“These opportunities will be generated through greater alignment with the United States at the standards and conformity assessment level, and through availability of a framework for mutual recognition among nations of accreditation.”

According to the organization’s co-founder and chief operating officer, Patrick Hardy, the organization helps corporations and industry associations across all economic sectors develop strategies, implement measurement, reporting and verification systems, and support clean technology demonstrations and other project solutions to manage GHG emissions. “We prepare our customers to meet government GHG regulations and to enter the carbon markets.”

Accrediting GHG validators and verifiers The organization’s key collaborations with the SCC dates back to 2007, when ClimateCHECK helped the SCC develop its new Accreditation Program for GHG validators and verifiers. ClimateCHECK’s role in assisting the SCC has been to evaluate the SCC’s customer processes, to

ensure they meet ISO/International Electrotechnical Commission standard 14065 Greenhouse gases – Requirements for greenhouse gas validation and verification bodies for use in accreditation or other forms of recognition, says Hardy.

Accreditation demonstrates that an organization has the competencies and systems to perform audits with the same consistent rigour and quality. As Hardy points out, “Accreditation can benefit organizations of all sizes.”

Evaluations and audits bring results

Value of accreditation

“Typically, we will perform the assessment and review of initial accreditation documentation, looking at compliance with ISO 14065,” he explains. “As well, we conduct initial on-site evaluations of the procedures and competencies at the head office of each SCC customer.” Afterwards, Hardy’s organization will perform witness audits with SCC’s customers at the facility that is being verified or validated. On a yearly basis, the organization undertakes surveillance assessments and audits for the SCC to consider for accreditation.

“Accredited organizations’ verification and validation procedures and tools are reviewed and assessed, ensuring that the work they perform is of high quality and meets the most up-to-date, good practices guidance,” he says. “There is continuous improvement of the accredited organization’s procedures throughout the accreditation cycle.”

Hardy says that SCC’s accreditation program allows GHG validation and verification organizations the opportunity to access the current provincially regulated GHG markets (British Columbia, Alberta, Ontario and Québec). The SCC also enables these organizations to become verifiers for voluntary GHG programs, such as The Climate Registry.

Solutions for industry and government ClimateCHECK’s expertise and innovations extend to the international marketplace, having co-founded the global Greenhouse Gas Management Institute (GHGMI), as well as the Collaborase web platform. GHGMI helps build capacity, and Collaborase supports collaboration and knowledge management of GHG-related standards. ClimateCHECK’s experience with ISO GHG standards has engaged customers around the world.

Future trends Guay says that GHG-related standards will continue to mature and evolve, and accreditation and conformity assessment bodies will need to adapt and keep pace with the changes. “More provincial/territorial and federal regulations are forthcoming, as are stricter emissions levels and greater focus on specific areas of industry.”

The good news, Guay points out, is that more opportunities will arise for innovative organizations offering environmental solutions. “These opportunities will be generated through greater alignment with the United States at the standards and conformity assessment level, and through availability of a framework for mutual recognition among nations of accreditation.”

Learn more about ClimateCHECK’s activities and SCC’s energy-related programs and initiatives, and come celebrate, at SCC’s World Accreditation Day 2014 networking breakfast event. Patrick Hardy will be the keynote speaker at SCC’s event on Monday, June 9, 2014, in Ottawa, Ontario, under the global theme, Delivering confidence in the provision of energy.

To see this story online visit http://www. laboratoryfocus. ca/?p=2377

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feature

by: JosePh m.weitZel, aGilent technoloGies inc.

Addressing the Emergence of Trace Organic Compounds in Wastewater, Surface and Drinking Water A large number of organic contaminants are appearing in worldwide water supplies at trace levels, prompting regulatory efforts to assess the threat that they pose to the environment and human health. These efforts require the ability to accurately measure parts per trillion concentrations in a costeffective and timely manner. Several advances in chromatography, mass spectrometry technologies and methods are helping to meet this challenge.

Rising concern A multitude of reports in recent years have highlighted the alarming emergence of trace organic compounds in various water sources, including industrial pollutants, agricultural runoff, personal care products and pharmaceuticals. Collectively referred to as trace organic contaminants (TOrCs) or contaminants of emerging concern (CECs), their presence in these water sources has been consistently reported for more than a decade.1 For example, a study by the U.S. Geological Survey (USGS) of 139 streams in the U.S. detected 82 chemicals in 80 per cent of the tested waterways in 1999 and 2000, and the most common chemicals were steroids, antibiotics, non-prescription drugs, caffeine and insect repellent. This is a serious threat to the environment, as miniscule amounts of estrogen present in municipal wastewater discharges have been shown to have the ability to decimate wild fish populations living downstream, in a study conducted in the Experimental Lakes Area (ELA) in northwestern Ontario, Canada.2 An Associated Press investigation in 2008 found that “a vast array of pharmaceuticals including antibiotics, anti-convulsants, mood stabilizers

and sex hormones have been found in the drinking water supplies of at least 41 million Americans”.3 Drugs were detected in the drinking water supplies of 24 major metropolitan areas.

These included anti-epileptic and anti-anxiety medications in a portion of the treated drinking water in Southern California, and 56 pharmaceuticals or byproducts detected in treated drinking water in Philadelphia, including medicines for pain, infection, high cholesterol, asthma, epilepsy, mental illness and heart problems.

A report in November of 2013 by the International Joint Commission, a US/Canada consortium that studies the Great Lakes, found that many of the prescription drugs and other newly emerging contaminants in sewage are not removed by treatment plants.4 Ten years of data from wastewater treatment plants around the world were reviewed to determine how well they removed more than 40 compounds that continue to appear in the Great Lakes. At least half of the compounds were likely to be removed by municipal waste water treatment plants, but many were not. A separate study found 32 pharmaceuticals and personal care prod-

figure 1

a novel online spe/lc/ms configuration for rapid automated analysis of water samples. automated online spe greatly reduces the sample volume and amount of sample preparation required.

ucts (PPCPs) in Lake Michigan. Thirty were detected in the sediment, with numerous PPCPs being detected up to 3.2 km offshore from the Milwaukee sewage outfalls.5

Protecting water quality The United States Environmental Protection Agency (EPA) and the United States Geological Survey (USGS) are working to improve the understanding of a number of CECs, particularly PPCPs and perfluorinated compounds. Since CECs are being found with increasing frequency in the world’s streams, lakes, and ground water, this improved understanding is necessary to measure the potential threat to the environment and human health. Some CECs are known endocrine disruptors, while others affect glucocorticoid activity. The synergistic effects of long term exposure to low doses of CECs is yet unknown.

As a result, the EPA has instituted Unregulated Contaminant Monitoring Rules (UCMRs) to assess the potential threat of CECs in the water supply.

The latest, UCMR3, requires monitoring for 30 contaminants (28 chemicals and two viruses) from 2013 to 2015. States, laboratories, and public water systems will participate in assessment monitoring, a screening survey, and prescreen testing.6 The UCMR program will provide EPA and other interested parties with scientifically valid data on the occurrence of these contaminants in drinking water, permitting assessment of the population being exposed and the levels of exposure. This data set is one of the primary sources of occurrence and exposure information the EPA uses to develop regulatory decisions for emerging contaminants.

The European Union Water Framework Directive (2000/60/EC) promotes sustainable water use, including the long-term reduction of wastewater contaminant discharges to the aquatic environment.7 It is supplemented by the Priority Substances

figure 2 the agilent 5975t ltm gc/msD system deployed in the field for onsite water testing.

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April/May 2014 Laboratory Focus www.laboratoryfocus.ca

feature Daughter directive (PSDD), which establishes Environmental Quality Standards (EQSs) for priority substances.

They define concentrations of chemical contaminants that are consistent with good chemical status in EU water bodies. A clearer understanding of the occurrence, concentration, and behavior in wastewater treatment of these substances is required in order to address the PSDD obligations.

To meet the challenge presented by the PSDD, UK Water Industry Research (UKWIR), in collaboration with the UK Environment Agency, has created the Chemical Investigations Programme (CIP) for the management and control of concentrations of priority substances. This program includes the quantification of risk and assessment of treatment options through the analysis of crude sewage, process streams, final effluents, and sludge.

by high performance liquid chromatography (HPLC) and detection and quantitation using a triple quadrupole mass spectrometer for tandem mass spectrometry analysis (MS/MS) using either positive and/or negative electrospray ionization (ESI). One such method, US EPA method 1694, is capable of detecting and quantitating 74 pharmaceuticals and personal care products (PPCPs) in water at minimum limits of quantitation ranging from one to 200 ng/L (http:// water.epa.gov/scitech/methods/cwa/ bioindicators/upload/2008_01_03_ methods_method_1694.pdf). However, many laboratories around the world are applying new technologies to provide methods that are fast, accurate, reproducible and validated.

These new methods require minimum sample volume, are automated, and can use new technologies to identify unknowns while detecting CECs at low parts per trillion concentrations.

New analytical technologies and methods are needed to meet the challenge

Automated online sample enrichment

The U.S. EPA has developed a variety of methods utilizing solid phase extraction of water samples, separation

One of the challenges to monitoring water sources for trace contaminants is the size of the sample and

Table 1 Measurement of Organic Contaminants at parts per trillion concentrations (ppt) in wastewater across several stages of treatment using online SPE on an Agilent 1200 LC/6460 Triple Quadrupole LC/MS Mass Spectrometer.

Compound Influent Secondary After Sand Chlorinated Effluent Filtration Effluent Atenolol

721

43

22

30

Caffeine

>8000

41

37

26

Trimethoprim

2030

61

47

8

Primidone

1676

514

443

670

Sulfamethoxazole

6552

3941

4223

62

Meprobamate

693

618

636

612

Simazine

<MRL <MRL <MRL <MRL

Ditiazem

158

78

85

71

Carbamezapine

375

307

310

275

PFBS

36

67

97

84

Fluoxetine

71

22

23

27

Atrazine

<MRL <MRL <MRL <MRL

DEET

1383

150

93

156

Bisphenol A

310

20

30

<MRL

Testosterone

45

<MRL <MRL <MRL

Naproxen

>8000

180

PFOA

<MRL <MRL <MRL <MRL

Estrone

<MRL <MRL <MRL <MRL

TCPP

1791

1338

1213

1299

Ibuprofen

>8000

88

55

55

PFOS

148

<MRL <MRL <MRL

Gemfibrozil

>8000

119

85

66

Triclocarban

765

25

32

11

Triclosan

2227

73

124

97

117

<MRL

Table 2 Identification of 36 pharmaceuticals found in surface waters across the US using an Agilent Q-TOF MS and accurate mass software tools. The average concentrations (in ppt) and the percentage of water samples in which they were found are included. Those highlighted in yellow were found most frequently.

Compound

% Detection in Average water samples concentration (ng/L)

1,7-Dimethylxanthine 10,11-Dihydroxy-carbamazepine

10 45

110 80

10-Hydroxy-carbamazepine

85

255

Atenolol

74

166

Bupropion

68

140

Caffeine

70

220

Carbamazepine

95

350

Cetirizine

82

70

Citalopram

79

85

Clarithromycin

75

46

Cotinine

22

40

Demethyl-dextrorphan

65

10

Des-venlafaxine

78

84

Dextrorphan

75

50

Diltiazem

69

47

Diphenhydramine

80

57

Erythrohydrobupropion

78

180

Erythromycin

55

137

Erythromycin Anhydrate

35

62

Fluoxetine

25

65

Gabapentin

44

54

Gemfibrozil

74

95

Hydroxy-bupropion

75

150

Ibuprofen

20

21

Lamotrigine

97

455

Metoprolol

91

237

Metoprolol acid

85

74

2N-glucuronide lamotrigine

68

95

Naproxen

64

22

Nor-citalopram

66

74

Propranolol

88

53

Sulfamethoxazole

95

320

Thiabendazole

75

188

Triclocarban

64

96

Trimethoprim

76

264

Venlafaxine

78

310

the sample preparation required for adequate detection. Conventional analytical approaches often require 1L of water, solid-phase extraction and extract concentration before instrumental analysis can be applied.

These methods require transportation of large sample volumes, are very labour intensive, and consume high volumes of organic solvents.

Online solid phase extraction (SPE) placed in front of the high performance liquid chromatography/mass spectrometer (HPLC/MS) flow path can dramatically reduce the volume of sample required for analyses, as well as minimize the amount of sample preparation required (Figure 1).

The result is robust and sensitive detection of ng/L levels of trace organic contaminants, using less than 2.0 mL of sample, rather than one litre. In addition, new faster scanning mass spectrometers allow simultaneous positive and negative electrospray ionization (ESI), which can result in significant

time savings. In a study reported by Shane Snyder, University of Arizona, 24 trace contaminants of emerging concern (CECs) were analyzed utilizing this online SPE-HPLC/MS/MS technique.8 Method reporting limits (MRLs) for these 24 organic contaminants were reported as low as 0.10 ppt (ng/L). Most recoveries ranged from 70 to 130 per cent, were comparable to conventional extraction, and had relative standard deviations lower than 10 per cent for most compounds. The method is robust, as it has been used to monitor the levels of these contaminants across the wastewater treatment process, from influent to chlorinated effluent (Table 1).

Recoveries were also comparable to traditional offline SPE methods. With a 19 minute cycle time, automated online SPE-HPLC/MS/MS is an ideal choice for fast, sensitive, reproducible, and robust analysis of trace organic contaminants in a variety of water sources.

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Laboratory Focus April/May 2014

Online SPE HPLC/MS/MS has also been used to analyze a variety of trace organics in water. These include herbicides, pesticides, pharmaceuticals, estrogens, and glyphosate in potable water. Sarah Gledhill of SouthEast Water (UK) reports on a validated online SPE LC/MS/MS method for detection of 17 chlorinated phenoxy acid herbicides and pentachlorophenol (PCP) in surface and treated water, using only 1.5 mL of sample.9 This method delivers <10 ng/L (ppt) limits of detection (LODs), as well as recoveries >95 per cent for most of the compounds. In addition to a reduction in sample volume, this method provides faster results at lower cost. The solid phase extraction cartridges are reusable, and less solvent is used for extraction of the sample. Finally, the results are more reproducible than their older method based on manual SPE, because the system is fully automated and less prone to operator error. This method meets the performance requirements set by the UK Drinking Water Inspectorate for standard deviation, bias, recovery, and total error and is accredited by the United Kingdom Accreditation Service (UKAS).

Q-TOF Accurate Mass analysis enables highly sensitive and untargeted screening of pharmaceuticals in water Mike Thurman and Imma Ferrer at the University of Colorado demonstrated that a quadrupole time-offlight mass spectrometer (Q-TOF MS) can provide high confidence identification and quantitation of pharmaceuticals, enabling an untargeted LC/ MS screening method that has been used to characterize more than 100 pharmaceuticals and their metabolites in water sources at concentrations as low as parts per trillion10 (Table 2). Sophisticated software tools for molecular feature extraction, formula generation, molecular structure correlation, and accurate mass database matching are key to the success of the method. This approach is particularly useful for separating and identifying isobaric and isomeric compounds that can be difficult to analyze using other methods.

Mobile analysis can be used effectively to rapidly assess drinking water quality Circumstances can often dictate the need for onsite analysis to immediately confirm the safety of surface and drinking water. Such a need arose after a recent earthquake in China. Agilent Technologies was requested to provide onsite water quality monitoring, as rapid and reliable lab-quality results in the field were necessary in order to make decisions to avoid a national emergency from contaminated drinking water.

The Agilent 5975T LTM GC/MSD, a mobile combination gas chromato-

graph/mass selective detector (GC/ MSD) configured with a purge and trap concentrator and low thermal mass technology, was used as a testing station in this crisis.11 Although portable, it provided the accuracy and sensitivity required to assure compliance with regulatory standards for drinking water safety (Figure 2).

A method was developed on this portable system to rapidly analyze for 22 trace volatile organic pollutants that are regulated in drinking water.

While routine cycle time was reduced to five to 10 minutes, compared to more than 30 minutes with traditional laboratory methods, the minimum detection limits varied between 0.090.14 μg/l (ppb) for all 22 compounds These levels are below the acceptable detection limits for these compounds in drinking water in China. This method provides results with high sensitivity, accuracy, sensitivity, and reproducibility similar to results that can be obtained in a stationary lab, providing the versatility for analyses in remote disaster locations.

Conclusion Modern society generates a plethora of waste products containing a wide range of organic chemicals that are clearly making their way into surface and drinking water. These compounds are often present in trace amounts, even parts per trillion, and their impact on the environment and human health are unknown.

Regulatory agencies worldwide are making efforts to characterize the nature and concentrations of these compounds so that their threat to the environment and human health can be accurately assessed. These efforts require new technologies and methods to assure success in a meaningful timeframe. Recent advances in automated online sample enrichment, more sensitive and faster scanning mass spectrometers, Q-TOF and accurate mass software tools, and mobile analysis systems are helping to meet this challenge. Together they can reduce the time and sample volume needed to assess water sources, while providing accurate and reproducible detection of >200 trace organic contaminants simultaneously, at parts per trillion (ng/L) concentrations.

References 1. Anumol T., Merel S., Clarke B. O., Snyder S. A. Ultra high performance liquid chromatography tandem mass spectrometry for rapid analysis of trace organic contaminants in water. Chem Cent J. Jun 18;7(1):104 (2013).

2. Kidd K. A., Blanchfield P. J., Mills K. H., Palace V. P., Evans R. E., Lazorchak J. M., Flick R. W. Collapse of a fish population after exposure to a synthetic estrogen.

Proc Natl Acad Sci U S A. 104, 8897-8901 (2007).

3. Drugs in the drinking water, an

Associated Press Investigation: http://hosted.ap.org/specials/interactives/pharmawater_site/.

4. Arvai A., Flecka G., Jasim, S. Melcer H., Laitta M. T. Protecting our great lakes: assessing the effectiveness of wastewater treatments for the removal of chemicals of emerging concern.

Water Qual Res J Can In Press, doi:10.216/wgrjc 2013.104.

5. Blair B. D., Crago J. P., Hedman C. J., Klaper R. D., Pharmaceuticals and personal care products found in the Great Lakes above concentrations of environmental concern. Chemosphere 93, 2116-2123 (2013).

6. Unregulated Contaminant Monitoring Rule 3 (UCMR3),http:// water.epa.gov/lawsregs/relesreg/ sdwa/ucmr/ucmr3/index.cfm 7. Pollutants in Urban Waste Water and Sewage Sludge, http://ec.europa.eu/environment/ waste/sludge/pdf/sludge_pollutants_xsum.pdf 8. Anumol T., Snyder S., Mohsin S. B. Sensitive LC/MS Quantitation of Trace Organic Contaminants in Water with Online SPE Enrichment. Agilent Technologies Application Note 5991-1849EN (2013).

9. Gledhill, S. High Sensitivity Detection of Pesticides in Water Using Online SPE Enrichment.

feature Agilent Technologies Application Note 5991-0871EN (2012).

10. Ferrer I., Thurman E. M. High Resolution Mass Spectrometry (LC/Q-TOF/MS) for the Detection of Pharmaceuticals in Water.

Agilent Technologies Application Note 5991-3261 (2013).

11. Cooperation with the China Academy of Urban Planning and Design for Field VOC Measurement of Drinking Water Sources with the Agilent 5875T LTM GC/ MSD. Agilent Technologies Application Note 5991-3140EN (2013).

Mr. Weitzel is the Agilent Global Environmental Market Development Manager. Before being employed by Agilent, Joe had been employed as a municipal water utility chemist and the lab manager for an environmental contract testing laboratory. Joe has been with Agilent since 1984 in various sales and marketing positions.

To see this story online visit http://www. laboratoryfocus. ca/?p=2382

comPany & advertiser indeX COMPANY

PAGE

WEBSITE

amorfix life sciences ................................5.....................................www.amorfix.com bristol-myers squibb ................................5.................................. www.bmscanada.ca caledon labs ..........................................5............................www.caledonlabs.com canadian society for chemical ................................................................................. technology ..............................................4...........................www.chem-tech.ca/cct children’s miracle network...................17........www.childrensmiraclenetwork.ca cardiome pharma corp. ............................5.................................. www.cardiome.com Dalton pharma .........................................2...................................... www.dalton.com

eppendorf ....................................... 20 ......................... www.eppendorf.ca genome bc ..............................................6....................................www.genomebc.ca helix biopharma corp. ..............................5.......................... www.helixbiopharma.com Knight therapeutics inc. ............................5................................ www.gud-knight.com mandel ...................................................9......................................www.mandel.ca medicure inc. ...........................................5.................................. www.medicure.com mcmaster university ................................3....................................www.mcmaster.ca National Research council of canada ..........1................................. www.nrc-cnrc.gc.ca Neovasc inc. ............................................6....................................www.neovasc.com Novartis pharmaceuticals canada inc. ........6...................................... www.novartis.ca pivotal therapeutics inc. ...........................6...................... www.pivotaltherapeutics.us simon fraser university ............................4..............................................www.sfu.ca sQi Diagnostics inc. ..................................5........................... www.sqidiagnostics.com transition therapeutics inc. .......................5............... www.transitiontherapeutics.com university of guelph ..................................2..................................... www.uoguelph.ca vwr .......................................................2........................................ www.vwr.com Warnex inc...............................................5........................................www.warnex.ca

wyvern scientific ............................ 9 ....................... www.wyvernsci.com Xagenic inc...............................................6.....................................www.xagenic.com Xenon pharmaceuticals inc. .......................5...........................www.xenon-pharma.com

16

April/May 2014 Laboratory Focus www.laboratoryfocus.ca

New Products

Pumps

The new Masterflex® L/S® Compact Pumps with Miniflex™ Pump Head from Cole-Parmer is a complete system which includes a pump head and drive. Created for transferring, sampling or filling applications in laboratories and research settings, the new peristaltic pump system delivers repeatable low flow rates with flow ranges of 1.8 to 220 mL/min (depending on the model and tubing size selected). It operates with continuous tubing, eliminating leakage and the need for fittings or connections, thus providing a cleaner fluid path. Users can select from several formulations of tubing.

Automatic tubing retention makes the tube loading quick and painless—it takes less than 15 seconds.

The stackable Masterflex pump operates with single-turn speed control for forward or reversible

w:Layout 1 12/19/2012 10:01 AM Page 1

pumping and maintains speed setting when the pump is turned off. Select from single-channel and dual-channel models.

Web: www.ColeParmer.com/20923

Workstation Hamilton Robotics launches the Decapping Microlab® STAR workstation, a fully automated tube tracking, decapping and recapping workstation. It includes eight decapping modules for safe and secure sample preparation. By incorporating lab automation, researchers can speed up time to results and spend more time analyzing data instead of managing and monitoring complex workflows. In addition to decapping and recapping, the STAR Workstation transports

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tubes, mixes liquids in tubes, pipettes and tracks samples. Although timing depends on pipetting volumes, the entire process of tube and cap handling can be accomplished within a few minutes.

Up to two Decapping Modules can be placed on a STAR deck and are served by four twister channels on a slim arm, with up to eight 1 mL or four 5 mL channels on an additional slim arm. The decapping modules are completely controlled and monitored through VENUS software so that all steps can be tracked and handled automatically, thus increasing process reliability. The system can accommodate tubes with a diameter of 15 to 38 mm, with caps up to six mm larger in diameter than the tube, to ensure robust and versatile handling.

Web: www.hamiltonrobotics.com

Molecular Arrays Oxford Gene Technology (OGT) has expanded its range of research-validated CytoSure Molecular Arrays to investigate DNA copy number variation (CNV) underlying a variety of genetic disorders.

Designed and optimised in collaboration with experts at Emory Genetics Laboratory (Atlanta, GA), the arrays are the ideal complement to DNA sequencing, providing a particularly powerful tool for investigating the variety of aberrations underlying genetic disorders. The expanded CytoSure Molecular Array portfolio now enables detection of CNV in genes associated with over 20 genetic disorders, including cardiovascular, inherited eye, intellectual disability and neuromuscular disorders, as well as a range of inherited cancers.

In addition, genes covering each disorder can be combined to create bespoke custom arrays, or further customised by the addition of novel content to suit each individual research project. For the easy extraction of results from aCGH data, all CytoSure Molecular Arrays are supplied with OGT’s CytoSure Interpret Software.

Web: www.ogt.com

www.laboratoryfocus.ca Laboratory Focus

JUNE 2014 June 11-12 2014 Canadian Biosafety Symposium Venue: Winnipeg, MB Email: biorisk@icid.com Web: http://biosafety.icid.com

April/May 2014

July 30-August 1 Clinical Lab Expo Venue: Houston, TX Tel: 301-657-2768 Fax: 301-657-2909 Email: ascls@ascls.org Web: www.ascls.org

June 16-19

Calendar AUGUST 2014

17

OCTOBER 2014

August 13

October 5-8

LSO Golf Classic Venue: Toronto, ON Tel.: 416 426-7293 Email: admin@lifesciencesontario.ca Web: www.lifesciencesontario.ca

ABIC 2014 Venue: Saskatoon, SK Tel: 306-668-2650 Email: abic2014@agwest.sk.ca Web: www.abic.ca/abic2014/

Drug Discovery & Therapy World Congress 2014 Venue: Boston, MA Tel: 857-239-8855 Email: info@ddtwc.com Web: www.ddtwc.com/

June 16-19 Global Biotechnology Congress 2014 Venue: Boston, MA Tel: 857-239-8855 Email: info@globalbiotechcongress. com Web: www.globalbiotechcongress. com/

June 17-18 2014 Bloom Burton & Co. Healthcare Investor Conference Venue: Toronto, ON Tel: 416-640-7580 Email: bbloom@bloomburton.com Web: www.bloomburton.com/ conference/

June 19

June 21-23 Labcon 2014 Venue: Saskatoon, SK Tel: 1-800-263-8277 Web: http://labcon.csmls.org/en/

June 23-26 Bio International Convention Venue: San Diego, CA Tel: 202-962-6655 Web: convention.bio.org

JULY 2014 July 13-15 Biotechnology & Human Health Symposium 2014 Venue: Charlottetown, PEI Tel: 902-367-4400 Email: jennifer@peibioalliance.com Web: www.biotechnologyand humanhealth.com

Children’s MiraCle network funds

CritiCal priority needs for

14

Children’s

hospitals

aCross Canada

eaCh day, there are

4,900

Children

MedEdge 2014 Venue: Richmond Hill, ON Tel: 905-771-5483 Email: ecdev@richmondhill.ca Web: www.businessrichmondhill.ca/ mededge/

who rely on the support of Children’s MiraCle network

MeMber hospitals in Canada

What is Children’s Miracle Network?

Children’s Miracle Network® raises funds for 170 children’s hospitals in North America, 14 of which are in Canada. These hospitals, in turn, use the money where it’s needed the most.

When a donation is given, it stays in the community, ensuring that every dollar is helping local kids.

These donations have gone to support critical research and training, purchase life-saving equipment, and ensure excellence in care - all in support of our mission to save and improve the lives of children.

July 27-August 1 IUMS 2014 Venue: Montreal, QC Tel: 613-993-0414 Email: iums2014@nrc-cnrc.gc.ca Web: www.montrealiums2014.org/ contact_us_e.shtml

Learn more at: ChildrensMiracleNetwork.ca

18

Laboratory Focus April/May 2014 www.laboratoryfocus.ca

LABCON

pays a visit to the sunshine city Canada’s medical laboratory community is set to gather for three days next month as the sunshine city of Saskatoon, SK hosts LABCON 2014 (June 21 to 23 at TCU Place).

As the Canadian Society for Medical Laboratory Science’s (CSMLS) national conference, LABCON is the place for lab professionals, managers, directors, educators and students in Canada to share information, experience new technology and connect with colleagues from across the country. Essentially, it gets lab professionals out of the lab to refresh and refocus so they can return to the lab armed with new ideas, knowledge and energy.

Last year’s event was hugely successful with more than 500 attendees and positive feedback to new presentation formats, including roundtable discussions and panel presentations.

CSMLS has also announced two captivating plenary speakers already.

On June 21, Dr. Fergall Magee will lead a session titled: “Changing Practice in Laboratory Medicine”; while Dr. Michael Noble will lead a session on June 22 titled “Risk and Error and Costs in the Medical Laboratory.”

If you’ve never been to Saskatoon before, we encourage you to visit the Canadian Light Source Synchrotron, Intervac, as well as the other facilities that make up Saskatchewan’s bustling Innovation Place. And of course LABCON 2014 isn’t the only event we want you to circle on your calendars.

Back by popular demand, Genome Canada has announced it will once again be hosting its “Genomics: The Power and the Promise” event. Taking place November 24 to 26 at the Ottawa Convention Centre, the event hopes to build upon the last highly successful conference held two years ago, by bringing together world renowned genomics experts and visionaries from academia and industry. The theme for this year’s conference will be “Genomics and the Environment” and will explore ways genomics can help leverage Canada’s traditional resource sectors. It will also showcase how far we’ve come in our understanding of genomics, expanding its applications into areas beyond human health. Registration for the event is already open, and you can view program details and register at http://powerandpromise.event.com/2014.

app review Mahjong Chem

From Stetson University Academics For iPhone and iPad https://itunes.apple.com/se/app/mahjong-chem/ id416482474?l=en&mt=8 Mahjong Chem is a neat little app that lets you practice your chemistry skills, using the classic game Mahjong.

The free app falls under the ‘Education’ category, meaning it’s aimed more towards students than scientists. It’s a fun play for when you feel like testing your knowledge of the periodic table.

This app is meant for students or researchers developing their knowledge of chemistry. It’s not a lab aid. However, it’s a great download for students to learn the rules of basic chemistry. Bonus: the app features the classic Mahjong game for taking a break between study sessions.

S

C t i

Science360 for iPad

From National Science Foundation For iPad https://itunes.apple.com/us/app/science360-for-ipad/ id439928181?mt=8 Science360 is an iPad-only app that brings the wealth of informative and educational content from the U.S. government’s National Science Foundation (NSF) Science360 website to Apple’s tablet.

This free app won’t be much use in the lab but it’s highly recommended for just about anyone with even a glancing interest in science as the editorial content from Science360 come from every science discipline with lots of high res photos and videos that are easily sharable to your social networking site of choice built within the app.

So even though it won’t help at work, the wealth of content on it will keep you entertained at home.

D

Fo

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Ready for Reading Eppendorf PlateReader AF2200 The new Eppendorf PlateReader AF2200 is specially designed for UV/Vis and fluorescence readings in 6 to 384-well format.

The system is developed to simplify set-up procedures and data analysis.

This makes your daily lab work easier.

> Plate reader for UV/Vis absorption and fluorescence intensity (top and bottom reading) > Pre-programmed applications for quick start including basic data analysis > Eppendorf accessories from microplates to the micro-volume measuring plate

www.eppendorf.ca/detection • 800-263-8715 025.A1.0111.A © 2014 Eppendorf AG.

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