BrJAC-2017-V4-N14

Food Safety: the continuing challenge for analytical chemists in the area of residues and contaminants analysis

Jan. - Mar. 2017 Volume 4 Number 14

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BrJAC publishes original, unpublished scientific articles and technical notes that are peer reviewed in the double-blind way.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

Published by Visão Fokka Communication Agency Publisher Lilian Freitas MTB: 0076693/ SP lilian.freitas@visaofokka.com.br Advertisement Luciene Campos luciene.campos@visaofokka.com.br ISSN 2179-3425 printed www.brjac.com.br

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

Br. J. Anal. Chem., 2017, 4 (14)

Contents Editorial Global Feeding – The Role of Food Safety Science

1-1

Interview Professor Marcos Eberlin, who is the chief executive of the Brazilian Society of Mass Spectrometry, recently spoke to BrJAC about his work

2-4

Point of View Food Safety: the continuing challenge for analytical chemists in the area of residues and contaminants analysis

5-6

Letter Functional foods: The future of human nutrition today

7-7

Articles Development and validation of method for the determination of the benzodiazepines clonazepam, clobazam and N-desmethylclobazam in serum by LC-MS/MS and its application in clinical routine Fluorescent N-doped carbon dots from mustard seeds: one step green synthesis and its application as an effective Hg (II) sensor Determination of insecticides in different commercial formulations by gradient HPLC A low-cost device for sample introduction and determination of mercury by Cold Vapour Atomic Absorption Spectrometry – application for irrigation water and paddy soil

8-16

17-24

25-33 34-43

Technical Note Determination of inorganic contaminants in meat by ICP OES: a simple method to comply with Brazilian and Chinese market demands

Sponsor Report Determination of multiclass veterinary drug residues in meat, plasma, and milk on a quadrupole-Orbitrap™ LC-MS system

44-47

48-53

Features 1st Ibero-American & 6th BrMass Conference consolidated as the largest Mass Spectrometry congress in Latin America

55-57

18th ENQA - Analytical Chemistry Integrated into Society18th ENQA - Analytical Chemistry Integrated into Society

58-61

Instituto GAIA de Espectrometria (IGE) offers courses to strengthen ties between academia and the private sector

62-63

Releases Thermo Scientific iCAP TQ ICP-MS Redefining triple quadrupole ICP-MS

65-65

Thermo Scientific Q Exactive Focus New hybrid quadrupole-Orbitrap Mass Spectrometer

66-66

Events

67-67

Notices of Books

68-69

Author's Guidelines

70-72

Br. J. Anal. Chem., 2017, 4 (14), pp 1-1

Editorial

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Global Feeding – The Role of Food Safety Science Fernando Vitorino da Silva Chemistry Laboratory Manager Nestle Quality Assurance Center – Brazil Fernando.Silva2@br.nestle.com The vast majority of people will experience a foodborne disease at some point in their lives. This highlights the importance of making sure the food we eat is not contaminated with potentially harmful bacteria, parasites, viruses, toxins and chemicals. According with World Health Organization statistics, 1 in 10 people fall ill every year from eating contaminated food, and unfortunately 420.000 people die each year as a result. The most common symptoms of foodborne disease are stomach pains, vomiting and diarrhea.

Long-term health problems, such as cancer or neurological disorders, also are associated to food contaminated with heavy metals, pesticides and naturally occurring toxins. The result is a fear of food that can effect on food choices among a wide section of the population leading a high stress and high levels of anxiety on Society. Sometimes even when the risk is not real, the population perception shows concerns on food consumption.

Food can become contaminated at any point during production, distribution and preparation. Everyone along the production chain, from producer to consumer, has an important role to play in order to ensure the food we eat does not cause diseases. To increase Food Safety, several professionals from different areas need to ensure use of best in class practice codes for production, stocking, retail and distribution of food. The union of players from different parts of civil society is the mechanism to engage everyone on the way to have a safe food available for human feeding.

From stand point of view of quality assurance, food safety can be summarized as a branch of science that has focus on quality food preservation by removing and/or avoiding contaminants with potential risk for human health across food chain. The contemporary Food Industry has this science as a pillar to produce reliable foodstuffs overwide. From a perspective of business, that's requires full control of crop on harvest at farm level, knowledge of production process, efficient raw material selection and modern methods form monitoring naturally occurred contaminants and formed process contaminants. Regarding this last topic, analytical science needs to delivery answers for quality testing by providing solutions for food safety demands. Several contaminants can be listed as target analytes, however each new day, the progress of instrumental analysis provides mechanism for better understanding of toxicity related to new pathogen species founded, degradation compounds and metabolites, process contaminants and advanced new science use on food production, such as nanotechnology. All this issues have been efficiently addressed by mass spectrometry, PCR analysis and separation techniques that become more and more popular on food industry quality control laboratories. Definitely, food safety science become more abroad and quality control schemes needs to follow this evolution. This direction of science brings more empowerment for consumer through creation of conscientious about risk and best practices to get a safe food.

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Br. J. Anal. Chem., 2017, 4 (14), pp 2-4

Interview

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Professor Marcos Eberlin, who is the chief executive of the Brazilian Society of Mass Spectrometry, recently spoke to BrJAC about his work Marcos Nogueira Eberlin Full Professor at the Organic Chemistry Department of the Institute of Chemistry, University of Campinas (UNICAMP), SP, BR eberlin@iqm.unicamp.br / http://thomson.iqm.unicamp.br At the head of the ThoMSon Laboratory, located at the Institute of Chemistry, University of Campinas, SP, BR, the Brazilian, chemist, scientist, Professor Marcos Eberlin, maintains an active presence in the national and international scientific community. At the height of its 25 years, the laboratory has trained about 200 students, who now work in their own MS laboratories in universities or as MS specialists in companies in Brazil and worldwide. In addition, some 800 papers have already been published, which makes ThoMson a reference in MS in the world.

With a vivid personality, Eberlin motivates everyone with his positive energy and joy of living.

The passion for chemistry came as a child, when a teacher showed him how fascinating chemistry can be. Since then he has traced his way into the world of chemistry, specializing in mass spectrometry. Also to his pride, his daughter Dr. Lívia Eberlin followed in his footsteps and has been conquering his space. Graduated from UNICAMP, she received several awards, including the Nobel Laureate Signature Award 2014 from the American Chemical Society – a coveted prize, which bears the signature of all Nobel laureates in chemistry. A Brazilian researcher has never before won this award.

Dr. Eberlin is also an award-winning researcher. He recently had the honor of being the first Latin American scientist to receive the J.J. Thomson medal. The medal is the main honor of the mass spectrometry area in the world, offered by the International Foundation of Mass Spectrometry (IMSF). He also received the award “Zeferino Vaz de Reconhecimento Acadêmico” (2002) and the Scopus-Capes Award (2008) for excellence in publications and staff training.

Another line of research advocated by him is the theory of Intelligent Design. Since 2014 he has been president of the Brazilian Society of Intelligent Design. This theory analyzes the latest scientific data on the events that gave birth to the Universe and to living beings. The researchers evaluate the feasibility against the data, of two possible causes for the Universe and Life: natural forces or the action of an intelligent mind. The theory then maintains, after this careful analysis, that the characteristics of the Universe and living beings are contrary to the action of natural processes, and better explained by an intelligent cause.

Marcos Eberlin is currently chief executive of the Brazilian Society of Mass Spectrometry (BrMASS), which today represents Brazilian mass spectrometry. He is also an associate editor of Jounal of Mass Spectrometry (JMS) of John Wiley & Sons Ltd. How your career as a chemist started? And what motivated you to be a chemist?

When I was about to finish first grade, I had a chemistry teacher who showed to me how fascinating chemistry is, talking about the use of it in many aspects of our daylife. I was also given a very special game as a birthday gift that was called “O Pequeno Químico” [The Little Chemist] and I was again fascinated with the simple but beautiful chemistry that this minilab showed to me. Then for high school I enter a technical course at the “Colégio Técnico Industrial Conselheiro Antonio Prado – COTICAP” in Campinas, SP, BR and my carrier started.

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Interview What could you say about your supervisor, Graham Cooks from Purdue University, USA? How important he was in your career?

Graham Cooks is - for me - by far the most talented mass spectrometrist ever. He is amazing, and has made numerous contributions to MS in many different fields by using many different approaches. He is a great inventor, his mind is always full of great ideas, and a master in detecting new opportunities and the best way to solve analytical problems. He is also a great leader, giving to his students all the freedom to think, to plan and to make things happen. Students at his hands are formed as researchers, not technicians. He is the best! I have learned from him most of what I am as a supervisor, and group leader, and I am always trying to follow his footsteps when thinking of new ways to develop MS. What is your opinion about analytical chemistry? What advice would you give to a freshman analytical chemist?

Analytical chemistry is the art – I believe – to reveal in details the chemical composition of a sample. In regard to MS, analytical chemistry is the art of going to the molecular and atomic level and inspecting a sample with the “hands” of a mass spectrometer.

Everything in the material universe is made of atoms and molecules for “everything is chemistry”! Analytical Chemistry is therefore a very exciting journey into revealing the molecular secrets of our Universe, from nature to all man-made products!

It is indeed a great adventure and a very rewarding field in science. For the freshman I would say: discover new ways to “see” the ballet of molecules and atoms that compose the chemistry of our life and universe! Innovate! Try the craziest Professor Marcos Eberlin in the ideas! Make things as simple as possible in a way that brings ThoMSon Lab. Photo: Luciene Campos analytical chemistry to all!

What studies are you current doing in you major research fields: “Fundamental Studies in MS” and “MS techniques and its applications”?

I think by far the most relevant and innovative studies are in the development of new analytical protocols in clinical and microbiology diagnosis. Recently we developed a new MS protocol for fast and secure characterization of different fungi that attacks the cacao plantations in Brazil causing serious diseases as that known as “vassoura de bruxa”. We also have developed an automate MS protocol for screening uterus cancer that could be used by pathologist to replace the tedious eye-inspection procedures they use today in the Papanicolau (Pap) testing.

As the founder and current president of the Brazilian MS society, and the organizer of its conferences that became of the largest in the world, what is your feeling about analytical chemistry conferences in Brazil?

Science is made by people, and is made in a much better way if such people know well each other and decide to get together summing their abilities and instruments in the pursuit of joint projects.

Collaborations are the best strategy to countries where research funds are little to make the best possible use of funds. And there is no better way to motivate collaborations via conferences, and large conferences.

Brazil should invest heavily in this area.

What is your opinion about Brazilian mass spectrometry?

If one looks back to 1990, MS in Brazil was nearly no-existing. Only a few brave pioneers doing their best. But in two decades Brazilian mass spectrometry has tremendously flourished and is today very active with numerous mass spectrometrits working in Brazil in various fields, from analytical chemistry to biochemistry, clinical and medical applications, material science, forensics, natural products, organic chemistry, proteomics, fuel chemistry and so on. We currently count close to 3.5% of all MS Science in the world!

In your opinion what is the future of MS and what is the major contribution is still to offer to 3

Interview analytical chemistry?

I have no doubt here: we need to make mass spectrometers as easy to use and as cheap as a mobile phone! And the MS data as simple to interpret as an ordinary image. I use to say: “An image is worth a thousand spectra”. And there are many mass spectrometrists in Brazil and around the world pursuing this dream!

Last year during the 21st International MS Conference in Toronto - Canada, you received the Thomson medal. What could you say about it?

It was a tremendous honor for me but particularly to all scientists in South America and Brazil; particularly to all analytical chemists, and most particularly to all mass spectrometrists. The Thonsom medal is the greatest award one could be given in MS. If you look at previous winners, the list shows many great scientist including Nobel Prize winners. I received the medal but the medal was the international recognition of a great number of people and institutions in Brazil that contribute for the creation of the ThoMSon laboratory at UNICAMP and provided a perfect environment in which we could explore the chemical world with the hands of a mass spectrometer.

How was the experience to be president of the international MS society from 2009 up to 2014?

In 2020, the international MS conference will be for the first time ever in South America, that is in Brazil (Rio de Janeiro). What is your feeling about this conference?

To preside IMSF was for me also a great honor and pleasure. I decide to “change the face” of IMSF and was able to implement several new programs such as the international MS schools (the 1st was in Siena, nd Italy with a large delegation of Brazilian students and the 2 was organized in Natal, Brazil) and was responsible to implement International MS conferences “outside Europe”. I, as the IMSF president, nd presided the first ever IMSC conference in Kyoto, Japan and the election committee for the 2 in Toronto, Canada. I – as the BrMASS president – also presided the committee that get the IMSF approval to hold the 3rd international MS conference in Rio - 2020. This conference will be great and historical. BrMASS plans to organize the largest and best ever MS conference in Rio 2020 and we have started plans and actions to do it! th

st

The 6 BrMASS (Brazilian MS conference) was last year organized in conjunction with the 1 Ibero American MS conference. As a BrMASS founder and its current president, how important it was to organize these joint conferences? rd The BrMASS conferences have become the 3 largest MS conferences in the world but by attracting people mainly from Brazil and South America. Last year, despite the strong economic and political crisis in Brazil, we dared to make the conference larger and a little more international and proposed to a number of Ibero American societies to join us. It was again a great success! Close to 1500 people attended the conference and they all enjoyed it very much! Lecture rooms were full of people and the atmosphere was of great enthusiasm and optimism. The conference place in Barra da Tijuca, Rio de Janeiro is outstanding an in all, a superb environment for new and international collaborations was stetted up which is certainly to produce major gains for the further development of MS in Brazil.

You are the founder of the ThoMSon laboratory at UNICAMP. Last year the lab turned 25 years old. How were those 25 years there?

We started literally from scratch. There was no lab, no walls, no roofs. Only an empty field. There was also no instruments, no students. But we dreamed a giant dream and worked hard to make it true! In the th celebration of the 25 anniversary of the ThoMSon laboratory it was a great pleasure to look back in time and see how much we did. A great selection of state-of-the-art instrumentation in MS. Close to 200 students graduated from the lab and a large new generation of Brazilian mass spectrometrists were formed, and are now working in their own MS laboratories in universities or as MS experts in companies in Brazil and worldwide. We have exported mass spectrometrist to USA, South America and Europe. We developed many new MS techniques. We published close to 800 manuscripts. We established the ThoMSon lab as a reference center for MS in the world. It was unthinkable but we made it!

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Br. J. Anal. Chem., 2017, 4 (14), pp 5-6

Point of View

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Food Safety: the continuing challenge for analytical chemists in the area of residues and contaminants analysis Renato Zanella Full Professor Laboratory for Pesticide Residue Analysis (LARP) Chromatography and Mass Spectrometry Research Group (CPCEM) Chemistry Department, Federal University of Santa Maria, Santa Maria-RS, Brazil renato.zanella@ufsm.br Over the past several years, there has been an increase in consumer concern about food safety with a high number of publicized incidents related to a variety of residues and contaminants in food around the world. In addition to being a public health issue, the presence of dangerous substances in food for human consumption also leads to undesirable economic problems. Therefore, the safety and quality of food products is a growing problem for consumers, governments and producers. The occurrence of residues and contaminants is one of the most important problems in food safety and public and private organizations should strive to minimize this risk. On the other hand, as a result of the occurrence of high-risk substances in food, which can be hazardous to human health, there is a growing demand for new analytical methods that allow a quick and effective sample preparation combined with a reliable determination of these compounds.

In addition to inorganic and biological contaminations, there is a large variety of organic compounds found in food as a consequence of their allowed use or due to natural processes or from anthropogenic activities.

Organic compounds that appear in food because of their application during production or storage (i.e. pesticides and veterinary drugs) are classified as residues, whereas compounds that have been generated naturally during production, storage or food processing (i.e. mycotoxins and marine biotoxins) or by anthropogenic activities (i.e. dioxins and dioxin-like polychlorinated biphenyls) are called contaminants.

Although some useful tools are available nowadays to prevent, mitigate or control food contamination, the total elimination of residues and contaminants in food is not possible. In order to protect consumers from these health risks, national and international organizations have established regulations setting maximum residue levels (MRLs) for residues and contaminants in food. For prohibited compounds or their metabolites, minimum required performance limits (MRPLs) are established, indicating the levels that the analytical methods need to achieve to be accepted for control programs. In general, MRPL values are around 1 µg -1 kg . The development and validation of suitable analytical methods, including both recognized and newly identified contaminants, are very important to assure reliable food residue and contaminant testing.

The number of residues and contaminants, including newly emerging contaminants, is continuously growing, which implies an increasing demand for their analysis in raw materials of animal or plant origin and food commodities, leading to a continuing challenge to analytical chemists to establish new multiclass methods to analyze a wider range of compounds with high selectivity and sensitivity. Furthermore, typical difficulties such as complexity and variety of matrix composition of food samples, low concentration of compounds and low required MRLs also complicate the simultaneous analysis of several classes of compounds. In addition, the cost-effectiveness of analytical methods is an important issue for laboratories and the main way to reduce costs is to maximize the number of analytes that can be determined by a method. Therefore, the development of more generic extraction procedures, considering the different physicochemical characteristics of the compounds, combined with multiclass determination can reduce the number of analyses per sample reducing time and cost.

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Point of View The QuEChERS method (acronymic name for quick, easy, cheap, effective, robust and safe), with several modifications available currently, is the most widely applied sample preparation procedure for pesticide multiresidue analysis in food [1]. The procedure is based on an extraction with acetonitrile, which can be acidified, followed by partition of the organic phase by salt addition. After centrifugation, the organic phase is separated and a clean-up step based on dispersive solid phase extraction (d-SPE) using a small amount of one or a combination of sorbents, selected considering the amount of matrix coextractives, such as pigments and lipid content. The development of new sorbents has allowed the obtainment of cleaner extracts even for very complex matrices.

In the last decade, the majority of current residue and contaminant analyses have relied on the high sensitivity and selectivity of ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) with a triple quadrupole analyzer operating in the selected reaction monitoring (SRM) mode. This technique allows the determination of hundreds of compounds in one analysis. More recently, liquid chromatography coupled to high resolution mass spectrometry has been used for screening purposes, as well for identification of non-target and unknown compounds, requiring effective sample preparation procedures [2].

In summary, one of the most important trends in food safety is the development of generic methods able to extract as many contaminants as possible and to detect all of them simultaneously. The combination of QuEChERS, UHPLC, MS/MS and HRMS, provides a powerful tool to achieve this goal. Moreover, some compounds, mainly ionic or unstable substances, are very difficult to analyze and are determined by specific methods. The development of easier methods for these compounds is very important.

Considering the current situation and the importance of the analyses of residues and contaminants to guarantee food safety, in my point of view, the challenge for analytical chemists is to continue working to improve analytical tools to meet the requirements established by legislations, minimizing cost, time and complexity in analyses.

Point of View

1. Prestes, O.D., Friggi, C. do A., Adaime, M.B., Zanella, R. QuEChERS - A modern sample preparation method for pesticide multiresidue determination in food by chromatographic methods coupled to mass spectrometry. Quim. Nova, 2009, 32, 1620-1634. DOI: http://dx.doi.org/10.1590/S010040422009000600046 2. Zanella, R.; Prestes, O.D.; Bernardi, G.; Adaime, M.B. Chapter 5 - Advanced Sample Preparation Techniques for Pesticide Residues Determination by HRMS Analysis. pp. 131-164. In: Applications in High Resolution Mass Spectrometry: Food Safety and Pesticide Residue Analysis. Edited by R. Romero-González and A.G. Frenich, ISBN: 978-0-12-809464-8, 2017 Elsevier, Amsterdam. DOI: http://dx.doi.org/10.1016/B978-0-12-809464-8.00005-1

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Br. J. Anal. Chem., 2017, 4 (14), pp 7-7

Letter

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Functional foods: The future of human nutrition today

Renata Aparecida Soriano Sancho Food Science PhD / Laboratory of Bioflavors and Bioactive Compounds, Department of Food Science, Faculty of Food Engineering, UNICAMP, Campinas, SP, BR. renatasancho@gmail.com

Glaucia Maria Pastore Full Professor / Laboratory of Bioflavors and Bioactive Compounds, Department of Food Science, Faculty of Food Engineering, UNICAMP, Campinas, SP, BR. Research area: Functional Foods, Bioactive compound of plants in Brazilian diversity. glaupast@fea.unicamp.br

Nowadays the relationship between food and health is well established. Among the several possibilities for improving health, functional foods stand out. They are able to provide benefits that go well beyond the basic nutrition, such as favoring and improving human health and reducing the risk of several chronic noncommunicable diseases, most of them associated with the aging process.

Almost 40 years after the emergence of the concept of functional food in Japan, the theme is more and more current. The growing interest in this class of foods is leveraged by the identification of food components responsible for the potential benefits, the mechanisms of action involved and their possible human applications. Composing this picture is the public interest and demand for better health and well-being.

Brazil, the first country in Latin America to establish legislation on functional foods, has its regulations established by the National Health Surveillance Agency (ANVISA), which defines functional and health claims for specific foods.

Functional foods are a heterogeneous class which includes whole foods, beverages, fermented milks, yogurts, fruits, margarines, among others. According to the bioactive components presented, they can be classified into prebiotics/probiotics/symbiotics, antioxidants, fatty acids, phytosterols and dietary fibers.

One of the most important functional aspects of food is related to the presence of prebiotics. Since the importance of diet in modulating the intestinal microbiota has been put in evidence, prebiotics, as a substrate for beneficial bacteria, have become prominent both for the academic research and food industry. Although non-digestible carbohydrates are considered prebiotic, recent studies present new alternatives in the modulation of the intestinal microbiota, such as polyphenols and vitamins.

Therefore new challenges and pathways are being opened up for research, concerning the identification of new candidates for prebiotics as well as new mechanisms of action for this class of foods.

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Br. J. Anal. Chem., 2017, 4 (14), pp 8-16

Article

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Development and Validation of Method for the Determination of the Benzodiazepines Clonazepam, Clobazam and N-Desmethylclobazam in Serum by LC-MS/MS and its Application in Clinical Routine Maria Elisa R. Diniz*, Nathan L. Dias, Breno P. Paulo, Fabrício V. Andrade, Elvis C. Mateo, Alessandro C. S. Ferreira Department of Research and Development, Institute Hermes Pardini, Av. Das Nações, 2448 – Distrito Industrial, CEP: 33200-000, Vespasiano – MG, Brazil

Clonazepam and clobazam are potent benzodiazepines derivatives that have been used primarily as anticonvulsants. Methods based on liquid chromatography tandem mass spectrometry (LC-MS/MS) are considered the gold standard in therapeutic monitoring. In this work a simple, rapid and sensitive LC-MS/MS method for the determination of clonazepam, clobazam and N-desmethylclobazam in serum was developed and validated. The method consisted in a simple liquid-liquid extraction, in which 200 µL of serum containing the internal standard (temazepam or stable isotopes of clonazepam and clobazam) were treated with ethyl acetate and subjected to LC-MS/MS analysis using positive electrospray ionization.

Chromatographic separation was performed on a C18 column and isocratic mobile phase containing -1 methanol:water:acetonitrile (50:30:20, v/v/v) with 0.05% of formic acid at 400 μL min . The linear analytical -1 -1 range of the procedure was between 10.0 and 160.0 ng mL for clonazepam, 25.0 and 525.0 ng mL for -1 clobazam and 100.0 and 5,000.0 ng mL for N-desmethylclobazam. The sample dilution of 1:2 and 1:10 for clobazam/N-desmethylclobazam and 1:2 for clonazepam was validated for the samples that exceed the method linearity. Relative standard deviations for intra and inter-day precision were lower than 4.3% for clonazepam, 7.5% for N-desmethylclobazam and 9.7% for clobazam. The recovery range was between 95 and 109% for all analytes. The LC-MS/MS method has been developed and validated successfully for the quantitative analysis and therapeutic monitoring of these benzodiazepines. The method has been applied successfully in the clinical laboratory routine.

Keywords: Benzodiazepines, LC-MS/MS, Validation, Therapeutic Drug Monitoring.

INTRODUCTION Benzodiazepines (BZD) are psychoactive drugs commonly used for treatment of anxiety, insomnia, and psychological disorders, and are the most frequently prescribed medications worldwide. Although overdoses can happen, BZD are considered relatively safe. However, its safety decreases when co-administered with alcohol, sedatives, antidepressants and neuroleptics [1,2].

Clobazam is a 1,5-benzodiazepine used successfully worldwide since 1970 as an anxiolytic and antiepileptic drug [3]. The activity of clobazam is attributed to both the parent drug and N-desmethylclobazam, one of its metabolite [4]. Clonazepam is a 1,4-benzodiazepine widely used as anticonvulsant agent and for treatment of epilepsy in adults and children [5]. Clonazepam is considered safe by addiction medicine specialists, but it has been frequently abused as a street drug [6].

The development of methods for quantifying the drugs concentrations in biological fluids made possible to study the relationship between drug dosage, drug concentration in body fluids and pharmacologic effects [7]. Therapeutic drug monitoring (TDM) is important for avoiding the adverse effects of drug interactions with other drugs as well as optimizing the dosage in individual patients [8].

Liquid chromatography tandem mass spectrometry (LC–MS/MS) is an increasingly important tool in TDM due to its great sensitivity and specificity compared to other techniques and the possibility that some *mariaelisaromanelli@yahoo.com.br

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Diniz, M.E.R.; Dias, N.L.; Paulo, B.P.; Andrade, F.V.; Mateo, E.C.; Ferreira, A.C.S.

Article of the methods can quantify multiple drugs simultaneously, which can be beneficial for patients that use more than one anticonvulsant [9]. Benzodiazepines have similar structure and they can coelute with another compound or metabolite mainly due to the administration of different drugs during a treatment.

Thus, the specificity of LC-MS/MS is a differential factor in therapeutic monitoring of benzodiazepines.

The aim of this work was the development of a method for therapeutic monitoring of clonazepam, clobazam and N-desmethylclobazam in serum using LC-MS/MS and its application in clinical laboratory routine.

MATERIALS AND METHODS Chemicals Clonazepam, clobazam, temazepam, and the stable isotopes clonazepam-d4 and clobazam-8-chloro 13 isomer- C6, used as internal standard, were obtained from Cerilliant (Round Rock, TX, USA).

N-desmethylclobazam was obtained from LGC Standards (Luckenwalde, Germany). Ethyl acetate was purchased from Sigma-Aldrich (St. Louis, MO, USA). Formic acid, ammonium hydroxide 25%, HPLC-grade acetonitrile and HPLC-grade methanol were supplied by Merck (Darmstadt, Germany).

Internal standards, calibrators and controls Stock solutions of clonazepam and clobazam at a concentration of 0.1 mg mL-1 were prepared in methanol and the working solutions were prepared in methanol:water (80:20, v/v). Stock solution of N-desmethylclobazam at a concentration of 0.1 mg mL-1 was prepared in acetonitrile and the working solutions were prepared in acetonitrile:water (80:20, v/v). Stock solutions of clonazepam-d4 and 13 -1 clobazam-8-chloro isomer- C6 at a concentration of 0.01 mg mL and temazepam at a concentration of 0.1 mg mL-1 were prepared in methanol and the working solutions were prepared in acetonitrile.

Calibration curve and quality controls were prepared in the same biological matrix as samples by spiking the matrix with the working solutions of clonazepam, clobazam and N-desmethylclobazam to obtain the same concentrations approved in the linearity test and covering the linear range, respectively.

Instrumentation Clonazepam, clobazam and N-desmethylclobazam were determined using a Quattro Micro tandem mass spectrometer triple quadrupole analyzer with positive electrospray ionization (Waters, Milford, MA) and an HPLC Alliance HT System (Waters, Milford, MA).

The chromatographic parameters were optimized performing one injection after another until achieving the best result. The mobile phase composition was varied as to obtain better response and chromatographic profile, and a reduced run time. Therefore, methanol, acetonitrile, water, ammonium hydroxide, formic acid and acetic acid were tested in different proportions. The column temperature and injection volume were also varied.

-1 The mass spectrometer conditions were optimized by direct infusion of a solution containing 1.0 µg mL of each analyte separately in an aqueous solution of methanol 50%, in order to provide greater analyte response.

Sample preparation Two hundred microliters of serum were spiked with 50 μL of internal standard (temazepam or stable isotopes of clonazepam and clobazam). The pH was adjusted to 9.0 with ammonium hydroxide 5% and then submitted to stirring for 5 s. 1,000 μL of ethyl acetate were added and the mixture was submitted to a vigorous stirring for 40 s using a vortex. It was centrifuged (5 min at 14,000 rpm) and the supernatant was transferred to a 5 mL test tube, and evaporated with a vacuum concentrator. The extract was reconstituted with 200 μL of acetonitrile:water (80:20, v/v) and 10 µL were injected in the chromatographic system.

9

Article

Development and validation of method for the determination of the benzodiazepines clonazepam, clobazam and N-desmethylclobazam in serum by LC-MS/MS and its application in clinical routine

Method linearity and limits The linearity was evaluated using a serum pool spiked in concentrations of 10.0; 40.0; 70.0; 100.0; -1 -1 130.0 and 160.0 ng mL for clonazepam, 25.0, 125.0, 225.0, 325.0, 425.0 and 525.0 ng mL for clobazam -1 and 100.0; 1,000.0; 2,000.0; 3,000.0; 4,000.0 and 5,000.0 ng mL for N-desmethylclobazam. The calibration curve levels including blank and zero level (blank spiked with internal standard) were extracted in triplicate and injected in simplicate.

The concentration of the lower and upper limit of calibration was proposed based in a reference value of these benzodiazepines in serum, and the linearity was assessed using ANOVA, Jackknife test for removing the outliers, and Brown-Forsythe test for evaluating residuals homoscedasticity [10].

The lower limit of quantification (LOQ) in this work was defined as the lowest point of calibration curve, and it should have relative standard deviation lower than 20% and accuracy within 80 and 120% of the nominal concentration [11]. Therefore, the LOQ was prepared in sextuplicate in three different days by three analysts. The limit of detection (LOD) was determined according to equation 1, where SD is the standard deviation of the intercepts of 5 calibration curves prepared in different days, and slope is these 5 curves slopes average.

Precision, accuracy and analyte recovery The precision, accuracy and analyte recovery were evaluated with a serum pool spiked in three different concentrations covering the linear range for each analyte and analyzed in sextuplicate. The concentrations -1 -1 evaluated were 30.0; 80.0 and 120.0 ng mL for clonazepam, 75.0, 250.0, and 400.0 ng mL for clobazam and 300.0, 2,500.0 and 3,800.0 ng mL-1 for N-desmethylclobazam.

The relative recovery was determined according to equation 2, where CF is the concentration determined in fortified sample, CU is the concentration determined in unfortified sample and CA is the concentration of analyte added.

Intra-day precision was determined by analysis of each level extracted in sextuplicate and processed within the same day of the preparation. Inter-day precision was assessed by determining the same levels of the controls above and prepared in three different days by three analysts. In this test, each analyst was responsible for preparing, injecting and analyzing data.

The accuracy was evaluated by the agreement between CA and CF, and was expressed by percentage.

The results of accuracy and recovery are acceptable if between 85% and 115% and the precision is acceptable if the relative standard deviation was lower than ±15% [11].

The sample dilution of 1:2 and 1:10 for clobazam/N-desmethylclobazam and 1:2 for clonazepam were also assayed. The criteria for approving the dilution were the same of the precision, accuracy and recovery.

Cross-talk and carry-over The chromatographic system was tested in order to verify occurrence of cross-talk and carry-over.

Cross-talk occurs when there is detection cross between analyte and internal standard, identical production or unintentionally monitored fragment ions [12], whereas carry-over occurs when the analytes present in a first sample are still detected in the injection of the subsequent sample [13].

10

Diniz, M.E.R.; Dias, N.L.; Paulo, B.P.; Andrade, F.V.; Mateo, E.C.; Ferreira, A.C.S.

Article In order to perform the test, two LLQ (lower limit of quantitation), four blank, two LLQ, two ULQ (upper limit of quantification), two blank, two ULQ, four blank, two ULQ without internal standard, and two zero were extracted and injected one at a time and sequentially.

Selectivity Sample blank was analyzed and its chromatogram was compared with the lower limit of quantification of each analyte in order to check the endogenous interferences at the retention time of clonazepam, clobazam, N-desmethylclobazam, and internal standard.

Method comparison studies The comparability of the methodologies LC-MS/MS and HPLC-UV was evaluated using 18 human serum samples contaminated with different concentrations of clonazepam, clobazam and N-desmethylclobazam. The HPLC-UV analysis for comparison study was performed by another clinical laboratory. The method performance using temazepam or clonazepam-d4 and clobazam-chloro-isomer-13C6 as internal standards was evaluated by analysis of 19 human serum samples for clonazepam, and 31 human serum samples for clobazam and N-desmethylclobazam. The results were compared using a paired t-test with 95% confidence interval. The tests were performed in a supplemental data analysis tool in Microsoft Excel 2013 program.

RESULTS AND DISCUSSION A LC-MS/MS method was developed and validated for determination of clonazepam, clobazam and N-desmethylclobazam in serum.

The precursor ions were monitored as [M + H]+ for all analytes and quantitative data were obtained by selected reaction monitoring (SRM). The monitored ions were 316→270 (m/z) for clonazepam, 320→274 (m/z) for clonazepam-d4, 301→259 (m/z) for clobazam, 307→265 (m/z) for clobazam-13C6, 287→245 (m/z) for N-desmethylclobazam and 301→255 (m/z) for temazepam. The mass spectrometric parameters selected were: source temperature of 120 ºC, desolvation temperature of 400 ºC, desolvation gas flow of 600 L h-1 and capillary voltage of 3,0 kV. The monitored ions and corresponding fragments voltages are listed in Table I. Table I. Mass spectrometric parameters for each benzodiazepine Precursor Product ion Dwell time Cone voltage Collision energy Benzodiazepine ion (m/z) (m/z) (s) (V) (eV) Clonazepam 316 270 0.20 40 25 Clonazepam-d4 320 274 0.20 40 25 Clobazam 301 259 0.20 35 20 13 Clobazam- C6 307 265 0.20 35 20 N-desmethylclobazam 287 245 0.20 35 20 Temazepam 301 255 0.20 25 20 Chromatographic performance was obtained with a Symmetry C18 column (75 mm x 4.6 mm x 3.5 μm particle size) (Waters, Milford, MA) using an isocratic mobile phase consisting of methanol:water:acetonitrile -1 (50:30:20, v/v/v) with 0.05% of formic acid pumped at a flow rate of 400 µL min . The column was maintained at 30 °C and the method had a chromatographic running time of approximately 6.0 min. The benzodiazepines were separated with a good resolution and no significant effects of carry-over and cross-talk were observed.

The comparability of using different internal standards, as described in the Material and Methods section, presented a good correlation between temazepam and the stable isotopes of clonazepam and clobazam.

11

Development and validation of method for the determination of the benzodiazepines clonazepam, clobazam and N-desmethylclobazam in serum by LC-MS/MS and its application in clinical routine

Article

The Pearson correlation, which measures the correlation between two variables, was greater than 0.98 for all analytes. The calculated Student's t-factor (tcalc) was 0.38 for clonazepam, 0.42 for clobazam and 0.40 for N-desmethylclobazam. The critical Student`s t-factor (tcrit) was 2.09 for clonazepam and 2.03 for clobazam and N-desmethylclobazam with a conďŹ dence interval of 95%. The value of tcalc is less than tcrit, therefore we accept the null hypothesis indicating that there is no signiďŹ cant difference between the use of stable isotopes of clonazepam and clobazam, and temazepam as internal standard. Thus, we can conclude that these internal standards have the same performance. However, temazepam cannot be applied as internal standard for patients that use temazepam or diazepam as medicine because temazepam is one of the metabolites of diazepam [14] and this fact restricts the application of the method.

Due to this, the labelled isotopes were selected as internal standard for this clinical application.

The chromatogram of a serum sample containing clonazepam, clobazam and N-desmethylclobazam performed with internal standard temazepam is showed in Figure 1(a) and the chromatogram of the analytes above, performed with stable isotopes as internal standard, is showed in Figure 1(b).

(a)

(b) Figure 1. Chromatogram of clonazepam, clobazam and N-desmethylclobazam in human serum using (a) temazepam as internal standard and (b) stable isotope as internal standards.

The sample extraction established in this work was very simple, robust and economic. The method was linear for all analytes in the ranges studied and it allows quantifying high concentration of N-desmethylclobazam. The dilutions 1:2 and 1:10 for clobazam/N-desmethylclobazam and 1:2 for clonazepam were assessed and successfully validated. This allowed us to quantify samples until 320.0 -1 -1 -1 ng mL for clonazepam, 5,250.0 ng mL for clobazam and 50,000.0 ng mL for N-desmethylclobazam with precision and accuracy acceptable. The correlation coefďŹ cient, parameters of calibration curves, and the detection limit determined according to equation 1 are described in Table II.

12

Diniz, M.E.R.; Dias, N.L.; Paulo, B.P.; Andrade, F.V.; Mateo, E.C.; Ferreira, A.C.S.

Article Table II. Linearity parameters of clonazepam, clobazam and N-desmethylclobazam

Slope

Intercept

Correlation Coefficient

Linear Range (ng mL-1)

LOQ (ng mL-1)

LOD (ng mL-1)

Clonazepam

0.023358

0.046095

0.997786

10.0 – 160.0

10.0

0.8

Clobazam

0.016541

-0.038457

0.998564

25.0 – 525.0

25.0

3.9

N-desmethylclobazam

0.009999

-0.095916

0.993020

100.0 – 5,000.0

100.0

34.8

Analyte

Several papers in literature have reported assays for BZD by LC-MS/MS in conventional matrices.

Although many of them provide more analytes like other BZD, antiepileptic and anticonvulsant drugs, they almost always focus in applying to forensic cases and not in TDM. Therefore, few works provide simultaneous analysis of active metabolites like N-desmethylclobazam [8, 15-17]. In our bibliographic search, we found three papers performing clonazepam, clobazam and N-desmethylclobazam by LC-MS/MS simultaneously in plasma [8], in serum [16] and whole blood [17], being only one performed in serum [16]. Other works performed with forensic objective do it only with clonazepam [18] or clobazam/clonazepam and do not perform N-desmethylclobazam [19-21]. A comparison between similar reported papers is described in Table III.

N-Desmethyl clobazam

Clobazam

Clonazepam

Table III. Comparative table with similar reported papers

Reference

THIS WORK

[16]

[8]

[17]

Matrix

Serum

Serum

Plasma

Whole blood

Extraction

Liquid-Liquid

SPE

Precipitation

SPE

Linearity (ng mL -1)

10.0 – 160.0

10.0 – 500.0

10.0 – 50.0

1.0 – 1000.0

Accuracy (%)

95.1 - 105.0

90.1 - 97.7

-

72.0

RSD (%)

3.5

4.0

-

6.0

Linearity (ng mL -1)

25.0 – 525.0

5.0 – 500.0

50.0 - 250.0

2.0 – 1000.0

Accuracy (%)

96.3 - 109.0

91.3 - 100.1

-

82.0

RSD (%)

5.3

4.6

-

4.0

Linearity (ng mL -1)

100.0 – 5000.0

5.0 – 500.0

250.0 – 1250.0

10.0 – 500.0

100.9 - 107.2

92.1 - 98.7

-

82.0

4.5

3.5

-

6.0

Accuracy (%) RSD (%)

A previously reported method analyzed 21 BZD and metabolites in serum by LC-MS/MS and showed low LOQ and good recovery [16]. However, they used solid phase extraction (SPE) for sample preparation, which is more expensive than LLE, and alleged that their method can be applied for TDM although the -1 linearity (5.0 – 500.0 ng mL ) is below the therapeutic window for N-desmethylclobazam (300.0 – 3,000.0 -1 ng mL ) [7].

13

Development and validation of method for the determination of the benzodiazepines clonazepam, clobazam and N-desmethylclobazam in serum by LC-MS/MS and its application in clinical routine

Article

The same is observed in another study, which analyses 33 BZD, metabolites and benzodiazepine-like substances in whole blood by SPE and LC-MS/MS [17]. The authors report a low linearity range for TDM of N-desmethylclobazam (10.0 – 500.0 ng mL-1) and suggest repeating the analysis after sample dilution for those out of linearity, which makes analysis more laborious and expensive. Another reported method analyses clonazepam, clobazam and N-desmethylclobazam and other antiepileptic drugs in plasma by LC-MS/MS [8]. They use protein precipitation with methanol and although their method is simple and fast to execute, they did not reach linearity acceptable for TDM. In our development, we tested sample extraction by precipitation with acetonitrile and, initially, it showed good results. However, the durability of the chromatographic column was lower, only 200 injections, and makes the analysis economically infeasible due to the high cost of the column. The liquid-liquid extraction with diethyl ether described for clonazepam analyses [22] and liquid-liquid extraction with ethyl acetate were tested. Both showed good recovery, however the extraction with ethyl acetate was selected considering the precision and easiness to work. The results for intra and inter-day precision presented in Table IV were below 10% for all analytes.

Table IV. Intra and inter-day precision and recovery of the LC–MS/MS method for clonazepam, clobazam and N-desmethylclobazam in serum.

N-desmethylclobazam

Clobazam

Clonazepam

Intra-assay (n=6)

14

Inter-assay (n=18)

Mean (ng mL -1)

Recovery (%)

RSD (%)

29.0

96.7

2.0

30.2

99.2

3.0

28.6

105.0

1.7

79.4

100.7

1.5

79.1

98.9

2.6

75.7

101.1

1.8

126.0

95.4

0.9

121.4

94.6

1.6

114.1

95.1

0.9

73.5

98.0

3.2

72.2

96.3

7.7

73.0

97.3

3.0

251.6

100.7

5.2

248.1

99.3

5.0

264.2

105.7

1.2

388.0

97.0

4.6

394.1

98.5

3.3

436.0

109.0

1.5

315.6

105.2

3.9

315.4

103.1

6.7

320.5

106.8

3.3

2523.4

100.9

3.5

2538.4

101.5

5.9

2612.4

104.5

2.8

3927.1

103.3

2.8

3950.6

104.0

2.8

4160.2

107.2

5.7

Mean (ng mL -1)

Recovery (%)

RSD (%)

29.3

100.3

3.2

78.1

100.2

2.9

120.5

95.0

4.3

72.9

97.2

4.8

254.7

101.9

4.8

406.0

101.5

6.2

317.2

105.0

4.6

2558.1

102.3

4.3

4012.7

104.8

4.7

Diniz, M.E.R.; Dias, N.L.; Paulo, B.P.; Andrade, F.V.; Mateo, E.C.; Ferreira, A.C.S.

Article The average ranges of recovery of a spiked serum were 95.1 – 105.0% for clonazepam, 96.3 – 109.0% for clobazam and 100.9 – 107.2% for N-desmethylclobazam.

The relative standard deviation for the LOQ was between 1.8 and 9.3% and the accuracy was between 87.3 and 116.9% for all analytes.

The LC-MS/MS method developed in this work was compared with a reference HPLC-UV assay (n = 18) by Passing & Bablok method [23]. The graphical dispersion, the corresponding regression equation and the correlation coefficient for clonazepam, clobazam and N-desmethylclobazam were showed in Figure 2. The Pearson correlation was greater than 0.99 for all analytes suggesting that the methods are equivalent.

Figure 2. Graphical dispersion for HPLC-UV and LC-MS/MS methods for clobazam (a), N-desmethylclobazam (b) and clonazepam (c).

The method has been applied successfully in the clinical laboratory routine. So far, more than 1000 samples for clonazepam, clobazam and N-desmethylclobazam were analyzed.

CONCLUSIONS The parameters linearity, selectivity, repeatability, intermediate precision, accuracy, quantification limit and detection limit evaluated in the validation step were successfully determined. The developed method has high selectivity, demands a simple extraction procedure and short consumption of solvent. The results showed that this analytical methodology can be applied in clinical routine as a reference technique to drug therapeutic monitoring of clonazepam, clobazam and N-desmethyclobazam ensuring quality in the exam report. Despite the good correlation of temazepam with the stable isotopes, its use should be carefully checked for every single blood sample before the analysis avoiding incorrect results.

ACKNOWLEDGMENTS We would like to thank Instituto Hermes Pardini (Brazil) for the financial support and providing the required structure to perform this work.

Manuscript received Jul. 21, 2016; revised manuscript received Oct. 21, 2016; accepted Oct. 25, 2016.

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Article

Development and validation of method for the determination of the benzodiazepines clonazepam, clobazam and N-desmethylclobazam in serum by LC-MS/MS and its application in clinical routine

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Br. J. Anal. Chem., 2017, 4 (14), pp 17-24

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Fluorescent N-doped Carbon Dots from Mustard Seeds: One step Green Synthesis and its Application as an effective Hg (II) Sensor Roshni V. and Ottoor Divya Praveen* Department of Chemistry, Savitribai Phule Pune University, Ganeshkhind Road, Pune, 411007, India We have developed a complete green synthetic procedure based on ultra-sonication for the synthesis of fluorescent N-doped carbon dots (C.dots) from mustard seeds. Commonly, mustard oil is generated by the pyrolysis of mustard seeds leaving behind a carbon rich black residue. Using simple and environment friendly ultra-sonication method the C.dots were generated from the mustard seeds which exhibited a high degree of water solubility. C.dots showed bright blue fluorescence and excitation dependent emission properties. The average size distribution of C.dots was in the range of 3-6 nm. The obtained C.dots were effectively used as a sensor for Hg (II). Series of metal ions which has a hazardous impact on the ecological system have been taken for the analysis and it was observed that the fluorescence of C.dots got remarkably quenched by Hg (II). Fluorescence quenching was studied using standard Stern-Volmer quenching model. Limit of detection was found to be 2.03 µM of Hg (II).

Keywords: Carbon dots, C.dots, fluorescence, mustard seeds, metal ions, quenching, ultra-sonication.

INTRODUCTION In the recent past, considerable attention has been given to nano science especially carbon based nano materials due to their diverse applications [1]. The use of the environmental friendly carbon nano particles in the place of chemical substances for optical sensing is widely appreciated. Among them, Carbon dots (C.dots) have got a remarkable position. C.dots are generally small and oxygenous carbon nanoparticles with near spherical geometry with size below 10 nm. They can be a promising candidate in place of traditional semiconductor nanoparticles (Q-dots) that can cause toxicity and potential environmental concerns due to the use of heavy metals in them [2]. The fascinating attributes of C.dots such as valuable photoluminescence, good bio compatibility, tuneable emission wavelength, excellent photo stability, and possibility of surface modification etc. enables them to be used as promising applicant for sensing applications. Along with the above properties, the properties such as low toxicity and biocompatibility hold up the use of C.dots for biological applications [3].

Some of the recent literature work deals with the synthesis of C.dots from natural sources: Chandra and co-workers had used naturally occurring carbohydrates like sucrose [4]; Yang and co-workers used chitosan [5]; Hsu & Chang used citric acid [6]; Zhou and co- workers had experimented with milk [7]; etc.

Apart from optical sensing, C.dots have found widespread applications in the areas of photo catalysis [8-9], bio imaging [10] and optoelectronics [11] etc. In the past few years various synthetic methods were also experimented in the fabrication of C.dots. Out of this, hydrothermal, microwave and ultra-sonication etc. has obtained prominence due to its low-cost, efficient and facile nature [12-13].

Monitoring the metal pollution in the environment is a current necessity and optical methods are found to be more suitable due to their sensitivity and low detection levels. Out of the various metal ions present, mercury is considered to be a potential pollutant due to its toxic effect in the ecosystem [14]. *divya@chem.unipune.ac.in 17

Fluorescent N-doped carbon dots from mustard seeds: One step green synthesis and its application as an effective Hg (II) sensor

Article

Several studies were reported earlier which illustrated the detection of mercury qualitatively and quantitatively [15-16]. High selectivity, sensitivity, speed of determination etc. makes fluorescence spectroscopic method much superior than other techniques [17].

In this work, we herein report a green, cost-effective and facile approach towards the synthesis of luminescent C.dots from mustard residue, which is a waste product usually discarded after removing the mustard oil from the mustard seeds. The ultra-sonication method which we adopted in this work is less time consuming, energy saving, without any passivating agent and also water is used as a solvent media makes this method more appreciable than other commonly used synthetic procedures. The highly fluorescent N-doped C.dots obtained were found to be aqueous soluble, photo stable and shows excitation tunable emission spectra. To find out fluorescence quenching efficiency of different metal ions towards the C.dots a series of metal ions were selected and fluorescence quenching was determined.

MATERIALS AND METHODS Chemicals

Mustard seeds were used as a molecular precursor for the synthesis of C.dots using ultra-sonication method. This was purchased from the local market and used for the study without any preservatives.

A.R. Grade Nickel Nitrate (Ni(NO3)2,Cupric Nitrate (Cu(NO3)2), Calcium Sulphate (CaSO4), Cobalt Nitrate (Co(NO3)2), Cadmium Nitrate Cd(NO3)2, Manganese Sulphate (MnSO4), Lead Nitrate Pb(NO3)2, Magnesium Sulphate (MgSO4), Ferrous Sulphate (FeSO4), Mercuric Chloride (HgCl2), Barium Nitrate Ba(NO3)2 and Zinc Sulphate (ZnSO4) were purchased from SDFCL and used without any further puri?cation. NaH2PO4.2H2O and Na2HPO4.7H2O were also purchased from SDFCL. These are used for preparing buffer solutions of different pH. All solutions were prepared using triply distilled water.

Instruments Ultra-Sonicator (BRANSON-1800) was used for the synthesis of C.dot from mustard seeds. A transmission electron microscope (TEM, Model TECNAI G2-20 U-Twin) with an operating voltage of 200 KV was used for determining the size of the synthesized C.dots. The absorbance and fluorescence measurements were recorded using UV-Vis spectrophotometer (Shimadzu) and spectrofluorimeter (Jasco FP-8300 with 150 W Xe lamp) respectively. The absorbance spectrum of the sample was recorded from 200 nm to 600 nm. Fluorescence spectra were obtained by exciting the solution at various wavelengths ranging from 300 nm – 400 nm. For fluorescence quenching studies excitation was performed at 360 nm with an emission range from 370 nm – 550 nm and a data interval of 1 nm. The slit width for excitation and emission were kept at 2.5 nm for all the measurements and scan rate was maintained at 1000 nm -1 min . The fluorescence life time measurements were carried out in FL-TCSPC fluorescent spectrometer (Horiba Jobin Yvon Fluorocube-01-NL, UK) with pico second laser diodes as excitation source. For decay fitting IBH-DAS6 software was used and the data were fitted using minimum number of exponential. The χ2 values and residual plot were used for assessing the goodness of the fitting. When χ2 value goes unfavourable (Ideally χ2~1) curve fitting was done using higher exponentials [18].

Environmentally friendly synthesis of C.dot

Figure 1. Digital image of the mustard seeds, and the schematic illustration of fluorescent C.dots with surface active groups 18

Roshni V. and O. Divya

Article The preparation of C.dots is as follows: 1 g of mustard seeds were taken in a beaker and subjected to ultra-sonication for about 30 min. During the course of synthesis the carbonisation of the mustard seeds resulted in black residue separated from the mustard oil. The residue is then crushed properly and dissolved in water. The insoluble carbonaceous particles were removed via centrifugation at 8000 rpm for 15 min. The solution was then lyophilized. When dispersed in water the lyophilized sample shows deep blue fluorescence on irradiation with UV light.

Details of metal ion sensing Detection of metal ions was performed at room temperature in aqueous solution. Stock solutions of all these metal ions were prepared from their respective salts. Aqueous solution of these metal ions were diluted to obtain a final concentration of 100 µM. Typically, 0.1 mL of these metal ion solution was added -1 2+ to C.dot solution (0.1 mg mL ) and stirred at room temperature. For Hg detection the similar steps were 2+ followed by changing the concentration of Hg from 1 µM to 10 µM. The PL measurements were recorded under excitation at 360 nm.

RESULTS AND DISCUSSION The mustard seeds which are rich in proteins and carbohydrates contain carbon, oxygen and nitrogen elements [19]. The pale yellow solution obtained after the ultra-sonication and the bright blue fluorescence indicate the complete carbonisation and the formation of C.dots from mustard seeds. The surface emissive traps formed by the various functional groups over the mustard surface were considered as the key factor which dominates emission. As reported, the structure of C.dot can be visualised as a carbon rich backbone with a lot of connected chemical groups on the surface [5].

Characterisation Methods Transmission Electron Microscopy (TEM) Figure 2 (Left) shows the TEM images of the nano sized C.dots obtained from mustard seeds having almost spherical morphology. The C.dots were having size distribution in the range of 3-6 nm. The small size and low contrast can be considered as the main reason for the non-clarity of HRTEM image. The chemical composition of these nanoparticles was further determined by collecting the corresponding energy-dispersed spectroscopy (EDS), as shown in Fig. 2 (Right). The peaks of carbon, nitrogen and oxygen elements are observed and the elemental composition of these C.dots are found to be 87.85% C, 9.86% N and 2.12% O.

Figure 2. (Left) TEM and (Right) EDS spectrum of C.dots produced by the ultra-sonication of mustard seeds

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Fluorescent N-doped carbon dots from mustard seeds: One step green synthesis and its application as an effective Hg (II) sensor

Article UV-Vis spectral analysis

The UV/Visible spectrum obtained in the case of C.dots from mustard seeds showed two peaks indicative of two types of transitions in the carbon core structure (Figure 3). The two peaks obtained are at 243 nm and 313 nm respectively. The peak at 243 nm is ascribed as π-π* transition of the respective aromatic groups, while an edge around 313 nm attributes to n-π* transition of connected groups present on the surface of the C.dots.

Figure 3. UV/Vis absorption spectra of C.dots obtained by the ultra-sonication of mustard seeds

Fluorescence spectroscopic analysis Similar to the C.dots obtained from other sources, the C.dots obtained from the mustard seeds also shows an excitation dependent emission spectra which is considered as the unique property of C.dots.

The broad emission spectra (Figure 4) of C.dots ranging from 352 nm to 423 nm were obtained when the excitation wavelength varied from 300 nm to 400 nm. The maximum emission peak intensity is observed at 440 nm when excited at 364 nm. This emission, however, is very sensitive to the excitation wavelength and with the increase in the excitation wavelength, the emission peaks shift to longer wavelengths with decreased intensity. The main reason for such a stokes shift can be due to the functional groups present on the C. dot surface which act as a series of emissive traps. When C.dots are illuminated by light of suitable wavelength the emissive trap on the surface state predominates the emission. Along with the above phenomena, a surface modification can also result in more surface defects, resulting in excitation dependant emission spectra [20]. The mustard seed solution is non emissive in the studied region, confirming the bright fluorescence is stemming from the synthesized C.dots.

Figure 4. Emission spectra of C.dots when excited with wavelengths ranging from 300 nm to 400 nm

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Roshni V. and O. Divya

Article Infrared spectral analysis For determining the exact chemical composition of C.dots infrared spectroscopic analysis was carried out and shown in Figure 5. The absorption bands of O–H vibrations were detected at 3285 cm−1, whereas peaks at 2920 cm−1 and 2361 cm−1 showed C–H stretching and C–N stretching peaks. Furthermore, peaks at 1709 cm−1 and 1665 cm−1 corresponded to C=O and C=C stretch of carbon backbone of C.dots. The −1 −1 − peaks appeared at 1590 cm and 1421 cm may be by the asymmetric stretching vibration of COO .

Notably, the peaks at 1375 cm−1, and 1090 cm−1 indicated asymmetric and symmetric vibrations of C–O–C, −1 while peaks at 1248 cm represented stretching vibrations of C–O bonds in carboxyl groups.

Figure 5. FTIR absorption spectra of C.dots

Factors influencing the fluorescence of C.dots: Photostability and pH study The obtained C.dots from mustard seeds possess good photostability and were verified by the continuous irradiation by a UV lamp at a wavelength of 365 nm for 5 hours. The C.dots were able to maintain almost the initial fluorescence intensity after irradiation proving their photo stability. The effect of pH on the fluorescence of C.dots was also studied. The fluorescence characteristic of the C.dots strongly depends upon the pH value as shown in Figure 6. In the acidic and basic pH the fluorescent intensity of C.dots got appreciably reduced whereas it was maximum at the neutral pH. In the strongly acidic and basic environments the protonation and deprotonation of the functional groups on the carbon core structure may happen, resulting in the quenching of fluorescence. Since there is no change in the emission intensity at the neutral pH, C.dots can be successfully applied as an optical sensor in various biological applications.

Figure 6. (Left) Photostability of C.dots with respect to time on continuous irradiation for 5 h (Right) Fluorescence spectra of C. dots in different pH conditions 21

Article

Fluorescent N-doped carbon dots from mustard seeds: One step green synthesis and its application as an effective Hg (II) sensor

Application of C.dot as a selective sensor for Hg (II) Many attempts were successfully carried out earlier for the selective and sensitive detection of Hg (II) using C.dots. Zhou and co-workers [21] were able to use the unmodified C.dots obtained by the pyrolysis of EDTA salts for the selective determination of Hg (II) and biothiols. They achieved good selectivity and sensitivity for mercury detection. In this present work we tried to synthesise C.dot through green strategy where the precursors used are natural and environment friendly. The synthetic method adopted is ultrasonication method which again is power and time saving.

To explore the feasibility of using such C.dots for the metal sensing application, we had selected a series of metal ions. The metal ions selected were Ba(II), Ca(II), Cu(II), Mg(II), Pb(II), Ni(II), Co(II), Cd(II), Hg(II), Zn(II), Mn(II) and Fe(II). The metal ion concentration in the solution was maintained at 1 µM. The sensing potential was evaluated by monitoring the initial fluorescent intensity of C.dot for possible intensity changes once the metal ion is added to it. From Figure 7 it is evident that the fluorescence of C.dots has been quenched in the presence of metal ions. Among the metal ions, Hg (II) shows appreciable quenching of fluorescence. This may apparently via electron or energy transfer from C.dot surface groups to Hg (II) which results in the quenching of fluorescence of C.dots. The hydroxyl and carboxyl or carbonyl groups present on C.dots surface can initiate this charge transfer process resulting in the strong binding interaction between Hg (II) and these functional groups [21]. The fact that without doing any surface modification these C.dots are selective towards Hg (II) makes this study interesting.

Figure 7. Effect of different metal ions (1 µM) on the emission intensity of the C.dots (excitation wavelength 364 nm)

Quantitative determination of C.dots fluorescence quenching by Hg (II) Figure 8 shows the influence of Hg (II) concentration on the fluorescence of C.dots. By varying the metal concentration from 1 µM to 10 µM a gradual decrease in fluorescence intensity was observed. Based on this, Stern-Volmer plot is generated and presented as shown in Figure 9. The photoluminescence quenching can be analyzed by Stern-Volmer equation.

Fo/F = 1 + KSV[Q] ( Eqn. 1) Where Fo and F are fluorescence intensities of the carbon dots before and after the addition of metal ions. [Q] is the concentration of metal ions and Ksv is the Stern-Volmer quenching constant.

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Roshni V. and O. Divya

Article

Figure 8. Fluorescence quenching of C.dots in the presence of Hg (II) of concentration varying from 1 µM to 10 µM

Figure 9. Stern-Volmer plot for fluorescence quenching of C.dots by H(II)

From the graph, which showed a linear correlation, correlation coefficient (r) and Ksv value (from slope 4 -1 of the line) were obtained and was found to be 0.98 and 1.3x10 M respectively. 3σ/s was used to calculate the Limit Of Detection (LOD); where, σ denote the standard deviation of C.dots corrected blank signals and s denote the slope of the linear curve derived from Stern-Volmer equation. LOD of the synthesised C. dots towards Hg (II) was found to be 2.03 µM. The bimolecular quenching constant, kq was found out using the lifetime value of C.dots using equation 2.

KSV = kqτ0 (Eqn. 2) Ksv is the Stern-Volmer quenching constant and τ0 is the fluorescence lifetime in the absence of metal ion. Fluorescence lifetime measurements were carried to obtain the τ0 value and were found to be 4.2 ns 12 −1 (Figure 10). The kq value is calculated using the above expression and was found out to be 3 x 10 M S−1. This value is much higher than a diffusion-controlled quenching in solution (about 1010 M−1 S−1). This demonstrates the presence of both diffusion as well as possible binding interaction between fluorophore and quencher.

Figure 10. Fluorescence decay profile (λex = 364 nm and λem = 440 nm) of C.dots

CONCLUSION We have demonstrated the green synthesis of C.dots from a bio-precursor using ultra-sonication method without any passivating or stabilizing agents. The C.dots produced were highly fluorescent and showed a blue fluorescence when excited at 364 nm with emission maximum at 440 nm. Due to their excellent photoluminescent property the synthesised C.dots were utilised for the metal sensing application where the intensity of fluorescence of C.dots were effectively quenched by Hg (II) ions.

23

Article

Fluorescent N-doped carbon dots from mustard seeds: One step green synthesis and its application as an effective Hg (II) sensor

ACKNOWLEDGEMENT This work is financially supported by the Science and Engineering Research Board (SERB) of Department of Science and Technology (DST), New Delhi, India (SB/FT/CS-109/2012) and the authors are thankful to IISc Bangalore and Physics Dept., SPPU for TCSPC and TEM facilities respectively.

Manuscript received Sept. 7, 2016; revised manuscript received Dec. 9, 2016; accepted Jan. 1, 2017.

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2. Xia, Y.H.; Zhou, Y.L.; Tang, Z.Y. Nanoscale, 2011, 3, p 1374.

3. Cao, L.; Harruy, A.; Veca, L.M.; Murray, D.; Xie, S.Y.; Sun, Y.P. J. Am. Chem. Soc., 2007, 129, p 11318.

4. Chandra, S.; Das, P.; Bag, S.; Laha, D.; Pramanik, P. Nanoscale, 2011, 3, p 1533.

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6. Hsu, P.C.; Chang, H.T. Chem. Commun., 2012, 48, p 3984.

7. Wang, L.; Zhou, H.S. Anal. Chem., 2014, 86, p 8902.

8. Baker, S.N.; Baker, G.A. Angew. Chem. Int. Ed., 2010, 49, p 6726.

9. Liu, Y.; Wu, P.Y. ACS Appl. Mater. Interfaces, 2013, 5, p 3362.

10. Lim, S.Y.; Shen, W.; Gao, Z. Chem. Soc. Rev., 2015, 44, p 362.

11. Wang, F; Chen, Y.H.; Liu, C.Y.; Ma, D.G. Chem. Commun., 2011, 47, p 3502.

12. Zhu, H.; Wang, X.L.; Li, Y.L.; Wang, Z.J.; Yang, F.; Yang, X.R. Chem. Commun., 2009, p 5118.

13. Kang, Z.; Li, H.; He, X.; Liu, Y.; Huang, H.; Lian, S.; Lee, S.T. Carbon, 2011, 49, p 605.

14. Yang, S.T.; Cao, L.P.; Luo, G.J.; Lu, F.S.; Wang, X.; Wang, H.F.; Meziani, M.J.; Liu,Y.F.; Qi, G.; Sun, Y.P. J. Am. Chem. Soc., 2009, 131, p 11308.

15. Hatai, J.; Pal, S.; Jose, G.P.; Bandyopadhyay, S. Inorg. Chem., 2012, 51, p 10129.

16. Huang, X. Environ. Sci. Technol., 2011, 45, p 9442.

17. Yang, Z.; Li, Z.; Xu, M.; Ma, Y.; Zhang, J.; Su, Y.; Gao, F.; Wei, H.; Zhang, L. Nano-Micro Lett., 2013, 5, p 247.

18. Lakowicz, J.R. Principles of Fluorescence Spectroscopy, 3rd ed. Springer, 2006.

19. Banuelos, G.S.; Lin, Z.-Q. Development and Uses of Biofortified Agricultural Products. CRC Press, 2008.

20. Zhu, S.; Song, Y.; Zhao, X; Shao, J.; Zhang, J.; Yang, B. Nano Res., 2015, 8, p 355.

21. Zhou, L.; Lin, Y.; Huang, Z.; Ren, J; Qu, X. Chem. Commun., 2012, 48, p 1147.

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Br. J. Anal. Chem., 2017, 4 (14), pp 25 - 33

Article

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Determination of insecticides in different commercial formulations by Gradient HPLC Hadi Hassan Jasim1, Sarmad Bahjat Dikran2, Amer Saleh Mahdi1, Bahaa Malik Altahir3* 1

Chemistry Department, College of Science, University of Mustansiriyah, Baghdad, IRQ Chemistry Department, College of Education for Pure Science-Ibin Al- Haitham, University of Baghdad, IRQ 3 Biology Department, College of Science, University of Baghdad, IRQ

2

A simple, accurate and rapid method for separation and determination of most commonly used insecticides in Iraq [thiamethoxam (Thi), imidacloprid (Imi), indoxacarb (Ind), and abamectin (Aba)] is presented. The separation was performed by gradient reversed-phase high performance liquid chromatography on a C18 stationary phase column. The method was developed and validated. The -1 mobile phase was a mixture of acetonitrile and water using gradient flow. The flow rate was 1.0 mL min .

The optimum temperature of separation was 25 ºC. The detection was performed at multiple wavelengths.

The analysis time was up to 10.5 minutes with retention times of 3.221, 3.854, 6.385, and 9.452 min for -1 the studied insecticides. The linearity was in the range of 0.1-200 μg mL . The proposed method was applied for the quantitative determination of these insecticides in their pure form and in different commercial formulations with little interference of additives.

Keywords: High Performance Liquid Chromatography, Thiamethoxam, Imidacloprid, Indoxacarb, and Abamectin.

INTRODUCTION Pesticide residues in different environmental compartments, e.g. soil, water, fruits, and vegetables have adverse effects on the human health [1]. Pesticides are usually applied in the field to eliminate damage and infestation by insects and pathogens. To ensure safe consumption, herbs are routinely monitored for pesticide residues before putting them on the market [2]. Pesticide residue analysis in food and environmental samples has been performed in numerous government and private laboratories throughout the world for approximately 40 years [3]. Insecticides can be divided into inorganic compounds, organic compounds and naturally occurring chemicals. Insecticides impact the nervous system, by preventing nerve cells from communicating with each other. Normally, nerve cells in the brains or muscles of humans or insects send tiny electrical pulses down a tendril to the end of a cell where the pulse has to jump across a gap which is known as a synapse to another nerve cell [4].

Thiamethoxam, 3-[(2-chloro-5-thiazolyl)methyl]tetrahydro-5-methyl-N-nitro-4H-1,3,5-oxadiozin-4-imine belongs to a relatively new class of insecticides known as neonicotinoids, which act as agonists of the post synaptic nicotinic acetylcholine receptors [5]. Abamectin (Aba) is a highly pesticidal agent that contains a macrocyclic lactone derived from the soil bacterium Streptomyces avermitilis. Aba is a product developed by Merck Co. Inc. for use as an acaricide, insecticide and nematicides for crop protection [6].

The insecticides thiamethoxam [(EZ)-3-(2-chloro-1,3-thiazol-5-ylmethyl)-5-methyl-1,3,5-oxadiazinan-4ylidene(nitro)-amine], and imidacloprid [1-(6-chloro-3-pyridylmethyl)-N-nitro- imidazolidin-2-ylideneamine] are simultaneously applied during the growing season in different vegetable crops in India to control insects and fungal diseases [7]. Natural insecticides are based on chemicals occurring in nature, such as nicotine, pyrethrum and neem extracts [8]. Imidacloprid, thiacloprid, and thiamethoxam all belong to the neonicotinoid insecticide class [9]. Neonicotinoids, also known as chloronicotinyls, are a group of insecticides with a wide range of chemical and biological properties, for this reason they are used *Baha782004@gmail.com 25

Jasim, H.H.; Dikran, S.B.; Mahdi, A.S.; Altahir, B.M.

Article throughout the world for crop protection and in veterinary medicine [10]. Thiamethoxam, imidacloprid, indoxacarb, and abamectin (Figure 1) work to eliminate insects in various stages of life. They include ovicides, which are used against insect eggs, and larvicides, which are used against larvae and adults.

Insecticides can be divided into different classes based on how they affect the target insect. Some are "contact" insecticides that kill when they come into direct contact with the insect. Others are "systemic” insecticides that kill insects when they eat the plant and ingest the insecticide chemical [11].

Various analytical techniques are reported in the literature for the estimation of these pesticides. The chromatographic methods are one of the best choices. High-performance liquid chromatography (HPLC) is used by different researchers [12]. Gas chromatography is used as a good choice in determination of the studied compounds [13]. Different detectors have been used and combined with these chromatographic techniques such as mass spectrometry [14] and diode-array detection [15]. Pretreatment and extraction methods employed in the extraction of the pesticides include initial single-phase extraction [3], microwave-assisted extraction (MAE) [7], solid-phase extraction [16], dispersive solid-phase extraction [2], QuEChERS [17], dispersive liquid−liquid microextraction [10] and solid liquid extraction [13].

These pesticides have been detected in various samples such as fruits and vegetables [18], soil [5], water [14], bee samples [19], tea [20], tomato [16], and honey [10]. HPLC is the preferred technique for priority pollutant aromatic compounds separation, because it is simple, robust, reliable, accurate and highly selective [21].

Studies have been carried out to evaluate the amount of pesticides in environmental and agricultural samples, but few previous studies have been concerned with these compounds in commercial formulations.

In the current study, the separation of these compounds by HPLC was developed for the determination of thiamethoxam, imidacloprid, indoxacarb, and abamectin in a mixture of the four pesticides in their pure and commercial forms. The separation and determination of these four compounds together was achieved with short-time analysis using a new gradient flow program.

Figure 1. Chemical structures of the studied insecticides

MATERIALS AND METHODS Chemicals The HPLC grade acetonitrile, and pure insecticides standards (thiamethoxam, imidacloprid, indoxacarb and abamectin) were supplied from Sigma-Aldrich Company. The commercial insecticides were purchased from the local markets. Their names and specifications are given in Table I. Table I: List of commercial insecticides

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Name

Company

Active material

Concentration

Actara

Syngenta

Thiamethoxam

25% w/w

Avaunt

Dupont

Indoxacarb

150 g L

Confidor

Bayer

Imidacloprid

200 g L

Vertimec

Syngenta

Abamectin

18 g L

-1 -1

-1

Determination of insecticides in different commercial formulations by Gradient HPLC

Article Chromatographic system The HPLC system comprised two LC-20AD pumps, an SPD-20A UV-VIS detector, manual injector, DGU-20A5 degasser, CBM-20A system controller, and CTO-20A column oven (all from Shimadzu Kyoto Japan). The column used for separation was NUCLEODUR® 100-5 C18 ec (250 × 4.6 mm i.d.; particle size 5 μm, MACHEREY-NAGEL Germany) with EC guard column cartridges (4 mm × 3 mm i.d.). The guard column holder was REF 718966. The integrated data were recorded using Shimadzu's LC solution software operated on a computer system.

Separation conditions and mobile phase The separations were achieved by using mobile phase consisting of acetonitrile and deionized water (65:35 v/v). All solvents were filtered through a 0.45 μm nitrocellulose filter and de-gassed by ultra-1 sonication before use. The separation was accomplished with a flow rate of 1.0 mL min , temperature of 25 ºC and injection volume of 20 µL. A gradient of flow mode of flow was used and operated by the LC program. The detection was controlled at multiple wavelengths. Peak area and peak height were recorded and used in a quantitative study of the analytes.

Preparation of insecticide stock solution Stock solutions of insecticides were prepared by weighing the proper portion of a pure solid or measuring the proper volume of a pure liquid and diluting the solutions to a 50 mL volume in acetonitrile to get a 1000 µg mL-1 standard solution of each insecticide. These solutions were used to prepare a series of solutions at 100 ppm, 75 ppm, 50 ppm, and 25 ppm. Acetonitrile was used as solvent, diluent and standard blank. The quantities used in these preparations were 0.2 g of Actara insecticide (the active material (Thi) is 25% w/w), -1 0.333 mL of Avaunt insecticide (the active material (Ind) is 150 g L ), 0.25 mL of Confidor insecticide (the active material (Imi) is 200 g L-1), and 2.777 mL of Vertimec insecticide (the active material (Aba) is 18 g L-1).

All solutions were stored at 4 ºC and protected from light. All commercial formulations were prepared and injected on the HPLC of non-spiked samples with triplicate to study the recovery and relative standard deviation.

Method validation The proposed method was validated with respect to the suitability of the method. Linearity, precision, accuracy, limits of detection (LOD) and quantification (LOQ) were used in the validation study. The linearity of the method was expressed as the coefficient of determination (r square).The linearity was calculated by recording the peak area of different concentrations of the studied pesticides. The data were recorded and calculated using statistical analysis and a linear-regression method [19]. The accuracy of the method was estimated by the relative error percent (Er%). The procedure in calculating the Er% of the studied pesticides was achieved from three replicates of non-spiked solutions, each at three different levels: 25 μg mL-1, 50 μg mL-1, and 100 μg mL-1. Similarly, recovery percentages were calculated for the four insecticides (n=3) at three different insecticide concentrations [10]. The precision was calculated by preparing the solutions of insecticides at different concentrations [5]. The repeatability (intra-day) used in the precision calculations is the variation in assay obtained at different concentration levels for each day of the studied insecticides and expressed in terms of the relative standard deviation (RSD%) [9]. The LOD and LOQ were represented as the analytes concentrations where the observed signal-to-noise ratios were equal or greater than 3 for LOD and 10 for LOQ. LOD and LOQ were determined by measuring the analytical background, i.e., by injecting a series of blank solutions (n=3) and calculating the signal-to-noise ratio for each compound [22].

Mobile phase optimization protocol In order to achieve the full separation of the mixture of Thi, Imi, Ind, and Aba using RP-HPLC on C18 columns, some factors were adjusted to overcome issues such as the peaks tailing due to the compounds tendency to bind by hydrogen bonding with the residual silanols of the C18 materials. The optimization

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Jasim, H.H.; Dikran, S.B.; Mahdi, A.S.; Altahir, B.M.

Article study was accomplished by optimizing some separation factors such as the use of organic modifiers, flow rates, isocratic and gradient modes.

RESULTS AND DISCUSSION The present study aimed to develop a chromatographic method for the separation and determination of Thi, Imi, Ind, and Aba in their pure form and in different commercial formulations. The HPLC conditions were optimized by studying the effects of concentration of organic modifier, mobile phase flow rate and column temperature.

Effect of organic modifier The isocratic mode was used, and the RP-NUCLEODUR® 100-5 C18 ec (250 mm × 4.6 mm) column was maintained at 25 ºC for all organic modifier experiments. Acetonitrile, rather than methanol, was used in this study because of its ability to accept the weak hydrogen bond and its high dipole moment which enables it to participate in selective dipole-dipole interaction with certain solutes [23]. Moreover, the mobile phase becomes less viscous than the corresponding methanol/water mixtures. Thus it enables faster flow rates and develops faster separation methods [24]. Further studies were carried out to determine the effect of acetonitrile concentration on separation.

The organic modifier percentage was optimized using mixtures of acetonitrile and water with different organic solvent percentages (65, 70, 75, 80, 85, 90, 95 and 100%) as the mobile phase. The separation was followed by checking the values of the chromatographic parameters in addition to the shape of the chromatographic bands. It was found that increasing the acetonitrile percentage above 65% lead to a decrease in the analysis time as well as an increase in the peak heights, but insufficient resolution of peaks was observed. The study shows a large difference in the separation of the studied compounds when the mobile phase composition varied from 35:65 of water:acetonitrile (Table II).

Table II. The effect of organic modifier on efficiency and retention factors k'

N

Organic modifier (acetonitrile) (%) 65

Thi 1.10

Imi 1.32

Ind 11.26

Aba 24.68

Thi 6499

Imi 3464

Ind 2308

Aba 6135

70

1.03

1.21

7.24

16.67

3535

4156

5086

5720

75

0.99

1.12

4.87

11.74

4072

3177

6052

12824

80

0.96

1.06

3.44

8.61

2807

2391

4918

8264

85

0.97

0.97

2.52

6.57

1081

1081

6093

7052

90

0.98

0.98

1.90

5.10

1457

1457

4227

4625

95

1.00

1.00

1.51

4.08

2343

2343

4874

6819

100

1.01

1.01

1.29

3.78

2807

2807

3640

3833

Effect of Flow rate of the mobile phase Different flow rates of the mobile phase (1.0, 1.2, 1.4, 1.6, and 1.8 mL min-1) were tested to find the best flow rate to achieve a complete separation within a short analysis time with reasonable sensitivity and high column efficiency. The results in Figure 2 indicated that the optimum flow rate of the mobile phase was 1.0 mL min-1.

Figure 2. The effect of flow rate on retention factors (k').

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Determination of insecticides in different commercial formulations by Gradient HPLC

Article Temperature The column oven temperature was tested at different temperatures (25, 30, 35, and 40 ºC) using a thermostated oven. Generally, increasing column temperature in RP-chromatography caused decrease in the retention times of the separated bands and an increase in the column efficiency by decreasing the mobile phase viscosity [25] which in turn lowers the column back pressure. However, we observed that Aba exhibits the opposite behavior in the temperature range between 30-35 ºC. This may due to its high molecular weight that leads to a decreasing interaction with the stationary phase, which may have a positive retention enthalpy at that temperature ranges [26]. Table III shows the values of retention times, retention factors, selectivity, efficiency (N) and resolution (Rs) for each pesticide at different temperatures.

The optimum separation temperature was chosen to be 25 ºC. At this temperature, a good shape and resolution of the separated bands of the insecticides was obtained (Figure 3).

Table III. The effect of temperature on retention factors, selectivity and resolution in optimized conditions.

Figure 3: Chromatograms of four insecticides at 25 ºC.

Development of the gradient HPLC method Working in isocratic conditions is preferable, whereby the mobile phase composition remains constant.

The system and column stay equilibrated all the time and do not undergo fast chemical changes. However, as the needs of HPLC analysis have increased and the samples become complex in nature, HPLC systems have evolved into very robust and reliable machines, with columns manufactured to withstand thousands of injections. Therefore, most chromatographic separations are run based on gradient elution with a changing composition of the mobile phase. Different injections have been accomplished using gradient elution experiments to decrease the analysis time and improve the resolution, by increasing the efficiency of the column [27].

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Jasim, H.H.; Dikran, S.B.; Mahdi, A.S.; Altahir, B.M.

Article Figure 4 shows the high quality separations in terms of, resolution, reasonable run time, and peak symmetry, which are obtained using the gradient flow steps shown in Table IV.

Figure 4. Chromatogram of mixture of four insecticides (10 µg mL-1) in gradient elution system.

Table IV. Gradient elution program for separation of four insecticides using RP-HPLC. Time (min)

Module

Action

Value

0

Pumps

Acetonitrile %

50%

2.10

Pumps

Acetonitrile %

50%

2.11

Pumps

Acetonitrile %

100%

0

(DET. A)

Wavelength Ch1

258 nm

4.50

(DET. A)

Wavelength Ch1

370 nm

5.80

(DET. A)

Wavelength Ch1

307 nm

7.00

(DET. A)

Wavelength Ch1

370 nm

8.50

(DET. A)

Wavelength Ch1

245 nm

10.00

(DET. A)

Wavelength Ch1

370 nm

10.50

Controller

Stop

Insecticides

Thi, Imi

Ind

Aba

System suitability and HPLC parameters The column efficiency (retention factors) (k`), resolution (Rs), and selectivity (α) were calculated according to the observations from the chromatograms in Figure 4. All the chromatograms represent the pure standard compounds. The results of the HPLC parameters are expressed in Table V. The suitability of the system for analysis was clear from the ranges of data. This method has the ability to be used with environmental, commercial and clinical samples after being validated for these samples.

Table V. System suitability data and HPLC parameters of the studied pesticides.

30

Determination of insecticides in different commercial formulations by Gradient HPLC

Article Validation The calibration curve linearity was calculated using the linear-regression method. The linear regression equations and determination coefficients (r2) show good linearity as shown in Figure 5. The calibration curves were drawn by plotting peak areas vs. concentrations over the range of 0.1-200 µg mL-1 using an excel data sheet. Further statistical analysis of data was carried out using a linear-regression method to calculate the LOD and LOQ that were observed in Table VI. The results showed that the LOD and LOQ ranges were lower than the dynamic range.

Figure 5. Calibration graphs, concentration (µg mL-1) vs. peak area for Thi, Imi, Ind, and Aba.

Table VI. Calibration Data Insecticides

Linearity range -1 (μg mL )

Regression equation

r

Thi

0.1-200

y = 78845x - 9292.4

0.9998

78845

9292.4

9.9×10

Imi

0.1-200

y = 68849x - 40898

0.9994

68849

40898

1.2×10

Ind

0.1-200

y = 60767x - 85.43

0.9999

60767

85.43

1.53×10

Aba

0.5-200

y = 39161x - 1564.7

0.9999

39161

1564.7

2.7×10

2

Slope Intercept

LOD -1 (μg mL )

LOQ -1 (μg mL )

-6

3.28×10

-5

4.06×10

-5

-5

-5 -5 -5

5.04×10

-5

8.9×10

Analysis of bulk insecticides and commercial formulations The contents of Thi, Imi, Ind, and Aba were determined in commercial formulations. The results are shown in Table VII. The prepared commercial formulations chromatograms are shown in Figure 6. The recoveries ranged between 83.4 - 109.2% and RSDs ranged between 0.486 - 4.045%. The results showed good correlation and agreement between the declared and determined values of the studied insecticides for all analysed samples, which indicates that the method that was used is more specific, efficient and highly selective.

31

Jasim, H.H.; Dikran, S.B.; Mahdi, A.S.; Altahir, B.M.

Article

Figure 6. Typical overlaid chromatograms of four insecticides at three concentrations (100, 50 and 25 µg mL-1) in their commercial formulation.

Table VII. Application of the method for measurement of the insecticides concentration in different commercial formulation samples.

Actara (Thi) 25% w/w

Confidor -1 (Imi) 200 g L

Avaunt -1 (Ind) 150 g L

Vertimec -1 (Aba) 18 g L

Conc. Taken -1 (µg mL )

Conc.* found -1 (µg mL )

Recovery

RSD%

25

27.30

109.2

0.486

50

50.92

101.84

4.045

100

112.90

112.9

0.79

25

22.64

90.56

0.662

50

45.7

91.4

0.79

100

93.78

93.78

0.297

25

20.85

83.4

0.877

50

39.79

79.58

3.73

100

81.3

81.3

3.839

25

24.26

97.04

0.475

50

45.71

91.42

3.986

100

94.97

94.97

0.372

*Average of three determinations.

Conclusion A simple, sensitive, accurate and relatively fast analytical method for determination of thiamethoxam, imidacloprid, indoxacarb, and abamectin was developed. This validated method can be used in the determination of these insecticides in commercial preparations. Organic modifiers, flow rate, and temperature were effective factors to achieve a high column efficiency, resolution and purification of the separated bands. Although isocratic elution is the preferable elution mode, using gradient elution was necessary to decrease the analysis time and improve the resolution, since this mode of elution increase the efficiency of the column [6]. The low LOD and LOQ of the studied compounds enable the use of this method in the detection and determination in water at low concentrations [5]. The results showed good 32

Determination of insecticides in different commercial formulations by Gradient HPLC

Article correlation and agreement between the declared and determined values of the studied insecticides in all analysed commercial samples. The gradient programed method in separation of the studied compounds give the innovations over the previous studies because no any of these studies could determine each of the four compounds with short time [9].

Article

Manuscript received Sept. 17, 2016; revised manuscript received Dec. 3, 2016; accepted Dec. 15, 2016.

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2. Yang, R.Z.; Wang, J.H.; Wang, M.L.; Zhang, R.; Lu, X.Y.; Liu, W.H. J. Chromatogr. Sci., 2011, 49, pp 702-708.

3. Anastassiades, M.; Lehotay, S.J.; Stajnbaher, D.; Schenck, F.J. J. AOAC Int., 2003, 86, pp 412-431.

4. Zhang, W.; Jiang, F.; Ou, J. Proceed. Intern. Acad. Eco. Environ. Sci., 2011, 1, pp 125-144.

5. Da Rocha, C.G.; França, F.H.R.; Cardoso, C.A.L. Am. J. Anal. Chem., 2012, 3, pp 242-249.

6. Xie, W.; Ko, K.; Kim, S.; Chang, H.; Lee, K. Kor. J. Environ. Agri., 2006, 25, pp 359-364.

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8. Asogwa, E.U.; Ndubuaku, T.C.N.; Ugwu, J.A.; Awe, O.O. J. Med. Plants Res., 2010, 4, pp 1-6.

9. Ying, G.G.; Kookana, R.S. J. Environ. Sci. Health, 2004, 39, pp 737-746.

10. Campillo, N.; Vinas, P.; Férez-Melgarejo, G.; Hernández-Córdoba, M. J. Agric. Food. Chem., 2013, 61, pp 4799-4805.

11. Yeary, W.; Fulcher, A.; Klingeman, W.; Grant, J.; Xiaocun, S. J. Entomol. Sci., 2015, 50, pp 35-46.

12. Baig, S.A.; Akhter, N.A.; Ashfaq, M.; Asi, M.R.; Ashfaq, U. J. Agric. Techno., 2012, 8, pp 903-916.

13. Morais, E.H.; Rodrigues, A.A.Z.; Queiroz, M.E.L.R.; Neves, A.A.; Morais, P.H.D. Food Cont., 2014, 42, pp 9-17.

14. Hassan, J.; Ghafari, M.; Mozaffari, S.; Farahani, A. Aust. Chrom., 2014, 1, pp 1-3.

15. Abd-Alrahman, S.H. Food Chem., 2014, 159, pp 1-4.

16. Gong, Y.; Huang, W.; Zhang, Y.F.; Li, J.Z. Asian J. Chem, 2012, 24, pp 5289-5291.

17. Lee, S.W.; Choi, J.; Cho, S.; Yu, H.; Abd El-Aty, A.M. J. Chromatogr. A, 2011, 1218, pp 4366–4377.

18. Chai, M.K. Development and validation of a solid phase Microextraction method for simultaneous determination of pesticide residues in fruits and vegetables by gas chromatography. PhD thesis, 2008, University of Malaya, Kuala Lumpur, Malaysia.

19. Rancan, M.; Rossi, S.; Sabatini, A.G. J. Chromatogr. A, 2006, 1123, pp 60-65.

20. Juan, X.; Jie, C.; Hong-yi, Y.; Lan, W.; Linghui, S.; Zifeng, L. J. Instrum. Anal., 2011, 9, pp 1-6.

21. Jasim, H.H.; Altahir, B.M. Eur. J. Sci. Res., 2015, 135, pp 47-60.

22. Deshpande, D.; Srivastava, A.K.E. J. Forensic Sci., 2016, 3, pp 1-6.

23. Wysocki, J. LCGC, 2002, 19, pp 1150-1159.

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Br. J. Anal. Chem., 2017, 4 (14), pp 34 - 43

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A low-cost device for sample introduction and determination of mercury by Cold Vapour Atomic Absorption Spectrometry – application for irrigation water and paddy soil Marcelo Belluzzi-Muiños, Isabel Dol and Mariela Pistón* Analytical Chemistry, DEC, Faculty of Chemistry, Universidad de la República, Montevideo, Uruguay

Mercury (Hg) is a global pollutant that is released into the environment from geological and anthropogenic sources and its compounds are highly mobile and toxic at trace level. A simple method based on cold vapour atomic absorption spectrometry (CV AAS) for the determination of total mercury in irrigation waters and extractable mercury in paddy soil was validated, using a low-cost sample introduction device. The validated method presented a linear range from 0.2 to 2.0 µg L-1; detection limits were 0.03 µg L-1 and 0.012 mg kg-1 (dry basis) for irrigation water and paddy soil respectively. Trueness was evaluated using spiked samples; the mean recovery was 101% (n=8) and 99.4% (n=6) for irrigation water and paddy soil respectively. Precision, expressed as relative standard deviation (RSD), was 9.6% (n=10) and 24% (n=8) for irrigation water and paddy soil respectively. These figures of merit were adequate for the determination of total Hg levels in irrigation waters and extractable Hg in paddy soils established by the regulations. To assess analytical performance several samples from the Eastern zone of Uruguay were analysed. Sample preparation consisted of an acid digestion using an economical pressurized reactor. Hg levels in waters and paddy soils were all below the maximum allowed and therefore were apt for agricultural use. These results are very important not only locally but also for the world since Uruguay is currently among the eight-largest paddy rice and milled rice exporter in the world. This method can be an alternative instead of the more expensive techniques mostly not available in developing countries and very easy to implement in environmental laboratories.

Keywords: Cold vapour atomic absorption spectrometry, paddy soil, water, mercury, low cost sample introduction device.

INTRODUCTION The toxicity of mercury (Hg) is well known, affecting the environment (water, soil, air and biota), therefore, the scientific community has devoted efforts and research for the study of the damage that Hg causes to the environment in general. Historical and contemporary evidence suggests that Hg atmospheric levels increased between two and five times since the development of the industrial era. Due to its physicochemical properties, including high mobility and long period of residence in the atmosphere and the fact that the contamination of aquatic food chains is widespread geographically, Hg is considered as a global problem and a matter of concern [1, 2]. If waters and their sediments become contaminated from sources such as atmospheric deposition and discharges from industrial, municipal, or agricultural operations, toxic substances could concentrate in seafood and wildlife [3]. In 1992, under the Clean Water Act (CWA) the United States Environmental Protection Agency (USEPA) provided criterion concentrations for Hg as a priority toxic pollutant [4].

*mpiston@fq.edu.uy

34

A low-cost device for sample introduction and determination of mercury by Cold Vapour Atomic Absorption Spectrometry – application for irrigationwater and paddy soil

Article

The presence of Hg in aquatic systems is a matter of concern since it can participate in processes of bioaccumulation and biomagnification in the food chain. So many countries established the maximum admitted levels of total Hg as a parameter for the determination of the water quality [5-7].

In contaminated waters, such as industrial wastewaters, or water bodies near gold mines, Hg concentrations can reach the µg L-1 range [8]. For drinking water the maximum level of inorganic Hg recommended by the USEPA in accordance with the Safe Drinking Water Act (SDWA), is 0.002 mg L-1 [4].

The World Health Organization (WHO) established the maximum in 0.006 mg L-1 [8, 9]. In Uruguay, governmental authorities manage the water resources and provide drinking water to the population of the country. To prevent environmental contamination, water control is managed by local regulation. According to this regulation the maximum Hg concentration admitted is 0.001 mg L-1 [10].

The environmental mobility and toxicity of Hg in soils depend on its species. Soluble inorganic Hg species are more easily transported by natural processes and serve as the substrate for methylation process.

These extractable organomercury species and extractable inorganic species constitute the majority of potentially toxic Hg in soils. The "non-mobile" Hg species such as mercury sulfide are chemically stable in the soil for longer geologic time periods and thus are less toxic [11].

In addition to Hg that can be found naturally, an anthropogenic source contributing to the soil contamination exists since a wide variety of organic mercurials have been used in the past as disinfectants and pesticides. Primarily fungicides, insecticides and herbicides were used in agriculture as preservatives.

Some of these agrochemicals include organomercurials such as phenylmercuric acetate, phenylmercuric nitrate, nitromersol, thimerosol, mercurochrome and mercurobutol [12]. Currently none of them is registered in the USEPA, but because of its persistence in soil and environment, mercurial pesticides have been classified as permanent (half-alive more than 20 years) and hence monitoring them is relevant even though they are no longer applied [12]. According the Canadian Council of Ministers of the Environment the maximum amount of inorganic Hg in soils for agricultural use is 6.6 mg kg-1 [13].

In Uruguay surveillance of the quality of the water for agricultural use and of paddy soils is important not only for public health reasons but for economical reasons as well. According to the Food and Agricultural Organization for the United Nations (FAO) Uruguay is one of the top ten rice-exporting countries in the world. All major suppliers of rice belong to Asia but there are also a few countries, particularly Argentina, Uruguay, United States and Egypt, that consolidated themselves as reliable rice exporters [14]. Rice is planted once a year, mostly in the coastal plains (Eastern Zone), in the valley of Uruguay River, and in areas located along the border with Brazil and Argentina (Northern Zone).

Analytical determinations at trace levels of total Hg in environmental samples can be carried out by several techniques. The analyses must be reliable and fast, thus causing constant pressure on analytical laboratories which must fulfil an increasingly demand. This has triggered research for alternative rapid, simple and economical methods for the determination of contaminants at trace levels. Expensive techniques sometimes are not available mainly in developing countries.

The most popular detection technique for the determination of Hg in several matrices is cold vapour atomic absorption spectrometry (CVAAS) [15-19]. This technique also presents some important advantages, including the separation of the analyte from the matrix, which reduces the number of interferences that may occur. Some variations for the cold vapour method are commonly used, such as cold vapour atomic fluorescence spectrometry (CAFS) [19-21] and cold vapour inductively coupled plasma-optical emission spectrometry (CV-ICP-OES) [19, 22] all of them have also been successfully used for Hg determination.

These techniques require the use of a commercial module for sample introduction, typically flow injection manifolds. For this module, the estimate cost is USD 7.000 to USD 10.000. More recently, highly sensitive and selective detection methods include instrumental techniques, such as inductively coupled plasmamass spectrometry (ICP-MS) [20, 23], total reflection X-ray fluorescence (TXRF) [20, 24], and thermal desorption/atomic absorption technique [20, 25-26] have been used for mercury determination in environmental samples.

The aim of this work is to present a simple low-cost system for sample introduction and subsequent 35

Belluzzi-Muiños, M.; Dol, I.; Pistón M.

Article determination of Hg by CVAAS using an atomic absorption spectrometer. The system coupled to the spectrometer consists of a glass separation funnel and a piece of a commercial midget impinger, both devices commercially available for less than USD 300. The performance of the proposed system was evaluated in terms of figures of merit and once validated it was applied to environmental samples analysis.

In addition, the sample preparation of soils was carried out using a low-cost pressurized reactor.

MATERIALS AND METHODS Reagents All chemicals were of analytical reagent grade and all the solutions were prepared with ultrapure water of 18.2 MΩ cm resistivity (ASTM Type I). All glassware was soaked overnight in 10% (v/v) HNO3 before use. All reagents were with low content of Hg checked by blank analysis.

An Hg 1000 mg L-1 commercial standard solution (Merck) for atomic absorption spectrometry was used.

The reducing agent was stannous chloride (20% w/v SnCl2.2H2O). Nitrogen (dried and purified by a combined Drierite/molecular sieve trap) was used as carrier gas.

Acid digestion was made with a mixture of sulfuric acid (H2SO4) - nitric acid (HNO3) (1 + 1 v/v), potassium permanganate solution (KMnO4) 5% (w/v) and potassium persulfate solution (K2S2O8) 5% (w/v). The subsequent treatment was carried out with hydroxylamine hydrochloride solution (NH2OH.HCl) 12% (w/v).

For validation, a standard reference material (SRM) Inorganics in Marine Sediment was analysed (NIST 2702). The certified value for total Hg was 0.4474 ± 0.0069 mg kg-1.

Calibration solutions The commercial stock solution of 1000 mg L-1 was diluted to prepare an initial intermediate standard -1 -1 solution (10 mg L ) and from this, a second standard intermediate solution of 100 µg L . Calibration solutions in the range of 0.1 - 2.0 µg L-1 were prepared daily by stepwise dilutions from the second intermediate standard solution. Ultrapure water was used for all dilutions.

To evaluate possible matrix interferences, standard additions were made at two levels of concentration -1 spiking water and paddy soils so that the final concentration of Hg in the reactor was 1.0 and 1.8 µg L .

Method The analytical strategy for total Hg determination is based on a sample treatment that ensures that the metal is in its highest oxidation state (Hg2+) in inorganic form and a subsequent reduction to elemental Hg0 using a suitable reducing agent (SnCl2). The reduction step takes place in a reactor (separation funnel in this method) and an inert gas as N2 (or Ar) carries the Hg0 vapour formed in the reduction process from the reactor to the measuring system. This process will be detailed bellow for the proposed assembly.

0 After generation of Hg vapour, the inert gas transports it to a closed measuring cell with quartz windows.

Detection takes place using a hollow cathode lamp or electrodeless discharge (EDL) and measurements are carried out at 253.65 nm.

Samples For evaluation of the analytical performance of this system, several samples of irrigation water and paddy soil were collected and analysed. The sampling plan was carried out following the International Organization for Standardization (ISO 5667-1:1980, ISO 5667-2:1991 and ISO 5667-3:2003) recommendations [30-33]. Nine sampling points in rivers and lagoons were selected on the East part of Uruguay (East zone in Figure 1); these are water sources used to irrigate rice crops.

36

A low-cost device for sample introduction and determination of mercury by Cold Vapour Atomic Absorption Spectrometry – application for irrigationwater and paddy soil

Article

Figure 1. Regions of rice crops in Uruguay.

From each location, the samples were taken in duplicate and were conserved in glass bottles (pH was adjusted to pH< 2 with HNO3) and stored at 4 °C during transportation. Twenty soil samples were collected from the three zones destined in Uruguay for rice crops (Figure 1). The sampling plan was done according to the recommendations of the USEPA standard operation procedure for soil sampling [34].

For each location, the sample was obtained by inserting a trowel (maximum depth: 15 cm) in 20 points within a surface of 1000 m2 to conform a bulk sample of approximately 1 kg. The bulk sample was placed inside a polyethylene zip-closed bag and transported to the laboratory. Before analysis, samples were dried in an oven at 70 °C (for maximum 48 hours) and sieved through a sieve DIN Nº 12 (106 µm).

Sample preparation For irrigation water, sample preparation was done according the standard methods for total Hg determination in environmental waters (USEPA 245.1 and 7470a) [27, 28]. Acid digestion was carried in a Kjeldahl digestion unit (VELP Scientifica) using glass tubes. 30 mL of each sample was heated for 1 hour at 80 °C with 1 mL of a mixture of HNO3:H2SO4 (1:1 v/v), 0.5 mL of K2S2O8 5% (w/v) and five drops of KMnO4 5% (w/v) solution. After digestion, the solution was let to cool until it reached room temperature, then 2 drops of NH2OH.HCl 12% (w/v) were added and was agitated until the colour disappeared and then completed to a volume of 40 mL with ultrapure water. Reagent blanks were also run.

For paddy soil, sample preparation was adapted from the USEPA 3200 Method [29] as follows: 0.2 g of paddy soil was accurately weighed in a 30 mL screw-capped Teflon® PFA vessel (Savillex, Minnetonka, MN, USA) and 7 mL of concentrated HNO3 and 1 mL of ultrapure water were added. The vessel was placed in a pressurized reactor (a domestic stainless steel pressure cooker with a capacity of up to 6 vessels) and heated in a hot plate for 1 hour. After cooling, the suspension obtained was centrifuged for 3 minutes at 3000 rpm and the supernatant was used for the analytical determinations after completed to a final volume of 40 mL with ultrapure water. Reagent blanks were measured alongside the samples.

Instrumentation All measurements were taken with a Perkin Elmer model AAnalyst 200® atomic absorption spectrometer ® coupled to a PC with the software WinLab 32 AA , fitted with a 15 cm absorption cell with quartz windows.

The operating conditions were continuous data acquisition mode (MHS); slit width 2.7 nm; integration time: 58 s; analytical signal: peak area; electrodeless discharge lamp (EDL) for Hg (Perkin Elmer®); intensity = 220 mA and λ = 253.65 nm.

The reactor, consisting of a separation funnel (borosilicate 3.3 glass, 125 mL) commonly used for liquidliquid separations with a PTFE stopcock in the lower part that allows the removal of the solution after each determination.

A commercial glass (borosilicate 3.3, Pyrex®) midget impinger, with fritted nozzle, was placed inside the separation funnel; the two pieces of glass were fitted together using a 20/40 ground joint. A complete impinger (commercially available as midget impinger for a 25 mL glass tube) costs approximately USD 37

Belluzzi-Muiños, M.; Dol, I.; Pistón M.

Article 150. The carrier gas (N2) passes through the porous glass membrane (pore size: 200 µm) located at the end and at the thinnest part of the reactor, into the solution. This allows both the input of the carrier gas and the released the vapour (Hg0) to move towards the detection system (Figure 1).

Figure 2 describes the complete system. This design permits the bubbling of the carrier gas into the reactor and therefore an efficient transportation of the analyte towards the detection system. Before the absorption cell, a tube containing silica gel was placed to prevent the passage of moisture to the detection system. The system was completed with silicone tubes and at the end of the absorption cell a flask with a KMnO4 solution (5% m/v) acts as a trap for the Hg vapour generated in the reactor thus avoiding contamination of the laboratory environment.

Figure 2. Scheme of the complete device used for Hg determination coupled to CVAAS. A: N2 inlet (1 L min-1); B: separation funnel with a piece that ends with a porous glass membrane; C: desiccant; D: absorption cell; E: trap for Hg vapour (the flask on the left side containing KMnO4, and the other one containing water to close the system); F: Waste; G: Hg lamp; H: transient signal.

The general procedure for the proposed system is the following: after sample preparation, the final volume was in all cases 40 mL completed with ultrapure water. Place the sample (previously digested) or standard in the reactor (separation funnel) and then add 2 mL of the reducing agent (SnCl2 20% w/v), and cover the reactor immediately.

Record the instrumental signal (peak area). After the measurement time (30 s for this system), discard the solution by opening the stopcock. Wash the reactor by placing ultrapure water in the separation funnel and discard by opening the stopcock at least 2 times. After this sequence, the system is ready for a new determination. Ultrapure water volume depends on the volume of the sample; in previous assays, it was demonstrated that for proper operation total volume in the reactor must not be less than 35 mL and not exceed 45 mL due to the geometry of the system.

RESULTS AND DISCUSSION Sample introduction system The use of a separation funnel with an impinger piece as a reactor coupled with the CVAAS detection system was successful in terms of analytical performance being a low-cost alternative to the expensive flow injection manifolds. The piriform separation funnel (reactor) was chosen due to its particular design since it has several advantages.

2+ 0 2+ 4+ Inside the reactor, the redox reactions that occur are: Hg + 2e → Hg and Sn → Sn + 2e the reaction is instantaneous. Elemental Hg is poorly soluble in water, but it can be detected when Hg is in the vapour state, so it is necessary to promote mass transfer from the liquid phase to the vapour phase. The angular design of the reactor allows less dead volume compared to a cylindrical one, enabling all Hg that is in the vapour state to migrate immediately into the carrier gas stream (N2). The entry of nitrogen as microbubbles generated by the porous glass membrane (pore size: 200 µm) favours two important aspects in the system: the generation of a vortex which acts as a mixer and simultaneously the exposure surface mass transfer is enhanced by the spherical shape thereof.

-1 A constant flow rate of 60 mL min ensures vigorous mixing and the pore size of the membrane ensures adequate mass transfer of Hg in the liquid phase to the gas phase, to reach the absorption cell quantitatively, generating a transient signal (Figure 3). The time required for each determination is about 3 minutes, whereby 38

A low-cost device for sample introduction and determination of mercury by Cold Vapour Atomic Absorption Spectrometry – application for irrigationwater and paddy soil

Article

the sampling frequency can be estimated at 20 samples/hour. This frequency is similar for FIAS commercial systems.

The amount of the reducing agent (SnCl2) in large excess was to ensure good reaction rate. Once the sample was added, the reactor was immediately closed allowing the passage of nitrogen. The curved shape at the top of the reactor also improves the breaking of the bubbles and decreases the possibility of generating a dead volume. Another advantage of this reactor is that is very easy to empty it using the PTFE stopcock of the bottom, facilitating its cleaning and reloading. The commercial flow analysers have very serious problems with carry over effects and tubing must be often removed because of cross contamination. This assembly did not present this inconvenient.

Figure 3. Transient signal obtained for Hg determination.

Figures of merit and method validation The figures of merit were obtained according the recommendations of the Eurachem Guide: The Fitness for Purpose of Analytical Methods [35]. For the evaluation of linearity, blank and 10 standard -1 solutions in the range 0.2 - 1.8 µg L were measured and the results (peak areas) were plotted versus concentration. Linearity in this range was confirmed by visual inspection of the plot (R2= 0.999) and analysis of residuals.

Detection (DL, 3s) and quantification (QL, 10s) limits were estimated by measuring (n=10) the dispersion of the blank signal and referring the measurements to the calibration curve. Blank signals were approximately 2.0% of the value of the signal (peak area) of the highest standard used for the calibration curve.

Slopes of external calibration curves and standard addition curves were determined for water and paddy soil. The slope values were 0.5502 L µg-1 for external calibration (n>30), 0.5628 L µg-1 for water samples -1 (n=9) and 0.5670 L µg for paddy soils (n=20). These results show no significant differences between slopes so no evidence of multiplicative interferences was found for either irrigation water or paddy soils (differences lower than 5% between slopes).

To establish the trueness of the method, nine irrigation waters and twenty paddy soils were analysed.

The trueness of the method was verified for both kinds of samples by a spike/recovery approach at two levels (40 and 70 ng Hg). Recoveries obtained from these analyses are shown in Table I. In addition, a standard reference material (SRM) Inorganics in Marine Sediment (NIST 2702) was analysed. Mean Hg concentration was 0.425 ± 0.053 mg kg-1 (n=15), this corresponds a recovery of 95% compared with the certified value.

Precision as relative standard deviation (RSD (%)) was estimated by both instrumental and analytical repetition. Table I summarizes the main figures of merit obtained for the validation.

39

Belluzzi-Muiños, M.; Dol, I.; Pistón M.

Article Table I. Figures of merit Parameter

Results Irrigation water -1

0.012 mg kg (dry basis)

-1

0.039 mg kg (dry basis)

Detection Limit (DL) (n = 10)

0.03 µg L

Quantification Limit (QL) (n = 10)

0.10 µg L

-1

Linearity (µg L ) R

Paddy soil -1

-1

0.2 – 2.0

2

> 0.997

Precision (Instrumental) RSD (%)

6.1 (n=15)

Intermediate Precision RSD (%)

9.6 (n=10)

24 (n=8)

Trueness (% R)

101.0 (n=8)

99.4 (n=6)

These figures of merit were adequate for the intended purpose since the quantification limit allows the methodology to be useful for water and soil surveillance and monitoring without a pre-concentration step or flow injection manifolds for sample introduction.

Almeida et al. postulate an alternative sample preparation method for the determination of extractable Hg in soils and sediments using CVAAS. The authors report a detection limit of 0.07 mg kg-1 using a flow injection manifold for sample introduction and our method provides a lower LD for paddy soils (0.012 mg kg-1) considering the above-mentioned advantages and the fit for purpose [37].

Precision was good, considering that is a manual method and it is in accordance with Horwitz theory about variability at trace levels (coefficients of variation less than 45%) [36]. Trueness resulted suitable, thus the method can be considered accurate (truthful and precise).

Real sample analysis For irrigation waters samples the concentration of total Hg was below the DL for eight of them, only one -1 -1 was < QL (0.10 µg L ). All irrigation waters analyzed were suitable for agricultural use (< 0.2 µg L ).

Table II shows the results of extractable Hg levels for paddy soil samples. All of them comply with the requirements established by the Canadian Council of Ministers of the Environment (< 6.6 mg kg-1).

USEPA 3200 standard method recommends a microwave assisted treatment for soils. An analytical microwave oven is an expensive instrument not always available in routine analysis laboratories. The use of a pressurized reactor as an alternative was successful in this work thus reducing the cost of analysis.

Table II. Mercury levels in paddy soils.

-1

-1

Sample

mg kg (dry basis)

Sample

mg kg (dry basis)

1 2 3 4 5 6 7 8 9 10

0.070 ± 0.003 0.094 ± 0.023 0.066 ± 0.002 0.063 ± 0.001 < 0.039 0.070 ± 0.003 0.155 ± 0.028 0.092 ± 0.002 < 0.039 0.051 ± 0.001

11 12 13 14 15 16 17 18 19 20

< 0.039 0.046 ± 0.002 < 0.039 0.041 ± 0.002 < 0.039 0.039 ± 0.004 0.066 ± 0.002 0.063 ± 0.001 0.113 ± 0.014 0.112 ± 0.002

Values expressed as mean ± dispersion between duplicates (QL = 0.039 mg kg-1) 40

A low-cost device for sample introduction and determination of mercury by Cold Vapour Atomic Absorption Spectrometry – application for irrigationwater and paddy soil

Article

CONCLUSIONS An economic analytical tool for sample introduction and subsequent determination of total Hg in irrigation waters and extractable Hg in paddy soil coupled to the CV-AAS technique was developed and validated.

This design can be postulated as a simple low-cost alternative to the commercial flow injection manifolds for sample introduction for total Hg determination in these samples. This method has proven to be an alternative to more expensive techniques such as CV-AFS not frequently available in developing countries and presented advantages compared with commercial flow systems used for sample introduction. Figures of merit were adequate for monitoring purposes according the regulations. Hg levels in all the analysed samples were far below the maximum admitted values.

ACKNOWLEDGEMENTS The ‘‘Programa de Ciencias Básicas’’ (PEDECIBA-Química) and the ‘‘Comisión Sectorial de Investigación Científica’’ (CSIC) supported this work. We are also grateful to Ph.D. Ana Acevedo for language assistance.

Disclosure Statement The authors declare that there is no conflict of interest regarding the publication of this article.

Manuscript received Sept. 17, 2016; revised manuscript received Jan. 27, 2017; accepted Mar. 2, 2017.

REFERENCES 1. World Health Organization. Preventing Disease through healthy environments. Exposure to mercury: A major public health concern. Geneva: WHO, 2007. http://www.who.int/phe/news/Mercury-flyer.pdf [Accessed 17 September, 2016].

2. de Azevedo, F.A.; Nascimento, E.S; Chasin, A.A.M. in: Metais, Gerenciamento da Toxicidade, edited

by Fausto Antonio de Azevedo and Alice A. da Matta Chasin, Atheneu, Brazil, 2003, p 299.

3. Abreu, S.; Pereira, E.; Valeand, C.; Duarte, A. Mar. Pollut. Bull., 2000, 40, p 293.

4. Agency for Toxic Substances and Disease Registry. Toxicological Profiles Toxicological profile for mercury. Atlanta, Georgia: ATSDR, 1999. http://www.atsdr.cdc.gov/toxprofiles/tp46-c7.pdf [Accessed 17 September, 2016].

5. Gray, J. Mar. Pollut. Bull., 2002, 45, p 46.

6. Lawrence, A.; Mason, R. Environ. Pollut., 2001, 111, p 217.

7. Boening, D. Chemosphere, 2000, 40, p 1335.

8. Huber, J.; Leopold, K. TrAC Trend. Anal. Chem., 2016, 80, p 280.

9. World Health Organization. Guidelines for Drinking-water Quality. WHO, Fourth Ed. http://www.who.int/water_sanitation_health/publications/2011/9789241548151_ch12.pdf [Accessed 17 September, 2016].

10. Instituto Uruguayo de Normas Técnicas. UNIT 833:2008. Agua potable. Requisitos. http://www.unit.org.uy/normalizacion/norma/100000158/ [Accessed 27 January, 2017].

11. U.S. Environmental Protection Agency. SW-846 Test Method 3200, 2007. Mercury species fractionation and quantification by microwave assisted extraction, selective solvent extraction and/or solid phase extraction.

12. James, R.; Roberts, M. P. H.; Reigart, J.R. Recognition and Management of Pesticide Poisoning, U.S. EPA, 2013. http://www2.epa.gov/pesticide-worker-safety [Accessed 17 September, 2016]

41

Belluzzi-Muiños, M.; Dol, I.; Pistón M.

Article 13. Canadian Council of Ministers of the Environment. Canadian Environment Quality Guidelines.

Canadian Soil Quality Guidelines for the Protection of Environmental and Human Health. Quebec, Canada: CCME; 2007. http://www.esdat.net/Environmental%20Standards/Canada/SOIL/rev_soil_summary_tbl_7.0_e.pdf [Accessed 17 September, 2016].

14. Calpe, C. Rice international commodity profile. Food and Agriculture Organization of the United Nations (FAO) markets and trade division, 2006. http://www.fao.org/fileadmin/templates/est/COMM_MARKETS_MONITORING/Rice/Documents/Rice _Profile_Dec-06.pdf [Accessed 17 September, 2016].

15. Welz, B.; Sperling, M. Atomic Absorption Spectrometry, 3rd ed. Willey-VCH, Weinheim, 1999.

16. Ma, X.; Huang, B.; Cheng, M. Rare Metals, 2007, 26, p 541.

17. Puanngam, M.; Dasgupta, P.K.; Unob, F. Talanta, 2012, 99, p 1040.

18. Chahid, A.; Hilali, M.; Benlhachimiand, A.; Bouzid, T. Food Chem., 2014, 147, p 357.

19. Ferreira, S.L.F.; Lemos, V.A.; Silva, L.O.B.; Queiroz, A.F.S.; Souza, A.F.; da Silva E.G.P; dos Santos, W.N.L.; das Virgens, L.F. Microchem. J., 2015, 121, p 227.

20. Kallithrakas-Kontos, N.; Foteinis, S. Curr. Anal. Chem., 2016, 12, p 22.

21. Saniewska, D.; Beldowska, M.; Beldowski, J.; Saniewski, M.; Romanowski, A.; Falkowska, L. Environ. Monit. Assess., 2014, 186, p 7593.

22. Hellings, J.; Adeloju, S.B.; Verheyen, T.V. Microchem. J., 2013, 111, p 62.

23. Ma, S.; He, M.; Chen, B.; Deng, W.; Zheng, Q.; Hu, B. Talanta, 2016, 146, p 93.

24. Marguí, E.; Kregsamer, P.;Hidalgo, M.; Tapias, J.; Queralt, I.; Streli, C. Talanta, 2010, 82, p 821.

25. Palmieri, H.E.L.; Nalini Jr, H.A.L.; Leonel, V.; Windmöller, C.C.; Santos, R.C.; de Brito, W. Sci. Total Environ., 2006, 368, p 69.

26. Fernández-Martínez, R.; Rucandio, I. Anal. Method., 2013, 5, p 4131.

27. U.S. Environmental Protection Agency. Method 245.1, 1994. Determination of Mercury in Water by Cold Vapour Atomic Absorption Spectrometry (CVAA). http://www.epa.gov/homeland-securityresearch/ epa-method-2451-determination-mercury-water-cold-vapor-atomic-absorption [Accessed 17 September, 2016].

28. U.S. Environmental Protection Agency. SW-846 Test Method 7470A, 1994. Mercury in Liquid Waste (Manual Cold-Vapor Technique). https://www.epa.gov/sites/production/files/201512/documents/7470a.pdf [Accessed 17 September, 2016].

29. Breckenridge, R.P.; Crockett, A.B. Determination of background concentrations of inorganics in soils and sediments at hazardous waste sites. Engineering Forum Issue, ed. U.S. EPA, 1995. http://www.epa.gov/sites/production/files/201506/documents/determine_background_concentrations.pdf [Accessed 17 September, 2016].

30. International Organization for Standardization. ISO 5667-1, 1990. Water quality - Sampling Part 1: Guidance on the design of sampling programs.

31. International Organization for Standardization. ISO 5667-2, 1991. Water quality - Sampling Part 2: Guidance on sampling techniques.

42

A low-cost device for sample introduction and determination of mercury by Cold Vapour Atomic Absorption Spectrometry – application for irrigationwater and paddy soil

Article

32. International Organization for Standardization. ISO 5667-3, 2003. Water quality - Sampling Part 3: Guidance on the preservation and handling of water samples.

33. Hall, P.; Selinger, B. Anal. Chem., 1989, 61, p 1465.

34. U.S. Environmental Protection Agency. Standard operating procedure. Soil Sampling, 1997. http://archive.epa.gov/region9/toxic/web/pdf/ee-soilsampling-sop-env-3-13.pdf [Accessed 17 September, 2016].

35. Magnusson, B.; Örnemark, U. (eds.) Eurachem Guide: The Fitness for Purpose of Analytical Methods – A Laboratory Guide to Method Validation and Related Topics, 2nd ed., 2014. www.eurachem.org [Accessed 17 September, 2016].

36. Horwitz, W.; Albert, R.J. Assoc. Off. Anal. Chem., 2006, 89, p 1095.

37. Almeida, I. L. S.; Oliveira, M. D. R.; Silva, J. B. B.; Coelho, N. M. M. Microchem. J., 2016, 124, p 326.

43

Br. J. Anal. Chem., 2017, 4 (14), pp 44 - 47

Technical Note

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Determination of inorganic contaminants in meat by ICP OES: a simple method to comply with Brazilian and Chinese market demands Bruno Menezes Siqueira*, Diego de Moura Leite, Mariana Ortega Garcia, Raquel Rainone Nova Analítica Importação e Exportação, São Paulo, SP, BR A sample preparation method and elemental determination of As, Cd, Cr, Hg, and Pb in meat samples was developed in order to comply with Chinese and Brazilian food contamination regulations. The very low limits allowed for those elements make the analysis by ICP OES challenging, forcing many laboratories to use ICP-MS, a much more expensive technique. This work showed a very simple method of combining digestion by dilute acid in a microwave system, with optimization of ICP OES parameters in order to achieve the necessary limits of quantification of those toxic elements in meat. The digestion temperature, acid volume, and sample mass were optimized to achieve the best digestion with a low dilution of the sample.

The ICP OES parameters were optimized for better sensitivity. Determination of Hg was performed using a hydride generation method with 0.5% NaBH4 solution (m/v) in 0.05% NaOH. The samples were digested with 10 mL of 10% dilute nitric acid (HNO3) at a maximum temperature of 230 °C. The limits of detection -1 obtained in ICP OES for As, Cd, Cr, Hg, and Pb were 0.63, 0.06, 0.88, 0.58, 0.89 μg kg respectively.

Keywords: Food safety, sample preparation, microwave digestion, elemental determination, ICP OES INTRODUCTION The area of food safety has become increasingly prominent in recent years once chemical and microbiological contaminants have become increasingly worrying. In response to this, regulatory agencies around the world have increased their demands regarding the presence of residues in this type of matrix.

The main elements monitored by the legislations of several countries are: Arsenic (As), Cadmium (Cd), Lead (Pb) and Mercury (Hg). Such elements, when ingested, in general may cause cardiovascular, renal, neurological, and respiratory disorders.

In Brazil, the National Agency of Sanitary Surveillance (ANVISA), in resolution RDC No. 42, dated August 29, 2013, provides for the MERCOSUR technical regulations regarding the maximum permissible limits of inorganic contaminants in foods. In addition, the Ministry of Agriculture and Livestock (MAPA) has a National Plan for the Control of Residues and Contaminants (PNCRC) in food. This plan was divided into Vegetable PNCRC and Animal PNCRC and also provides maximum limits of inorganic contaminants in food [1,3]. Brazil is one of the biggest exporters of soybeans and meat in the world; such a position makes the requirements of the legislation of importing countries a matter of importance and also of suitability for those who are going to export their food. China is one of the major importers of Brazilian meat, so we take Chinese legislation as an example, for comparison with Brazilian legislation. The maximum limits allowed for toxic metals in food in China are described in the National Food Safety Standard of Maximum Levels of Contaminants in Foods (GB 2762-2012) [4].

Many works use hot plates for food digestions, using HNO 3 and HClO4 [5,6]. Other works use H2SO4 to digest heart, kidney and meat of beef, mutton and chicken. Digestions performed in open systems allow the use of a larger amount of sample, which helps the method to reach the concentrations necessary to meet the regulations. However, this kind of methodology requires higher acid volumes and long digestion times [7].

In this work we report the use of microwave assisted digestion with dilute acids, in order to achieve low levels of dilution, without the disadvantages of open-vessel digestions. The less diluted the sample is, the higher can be the limits of detection of the method, allowing the use of inductively coupled plasma optical *bruno.menezes@novanalitica.com.br

44

Determination of inorganic contaminants in meat by ICP OES:a simple method to comply with Brazilian and Chinese market demands

Technical Note

emission spectrometry (ICP OES), instead of inductively coupled plasma mass spectrometry (ICP-MS).

Table I shows the maximum permitted limits of As, Cd, Hg, Pb and Cr in meat in Brazil and China.

Table I. Metals maximum limits permitted in meat samples by Brazilian and Chinese legislation [1,3,4] ANVISA -1

PNCRC

GB2762-2012

-1

-1

(mg kg )

(mg kg )

(mg kg )

As

0.5

1

0.5

Cd

0.05

1

0.1

Hg

***

0.03

0.05

Pb

0.1

0.5

0.2

Cr

***

***

1

***This element is not required for the related legislation

MATERIALS AND METHODS Sample Preparation The sample preparation was performed by microwave-assisted acid digestion. Three samples of each type of meat (beef, pork, and chicken) were digested in triplicate. The microwave oven system used to perform these experiments was SpeedWave 4, Berghof with DAP-60 vessels. The samples were cut to sizes of about 0.5 cm. The pieces, of around 500 mg, were weighed and 10 mL of 10% dilute nitric acid (HNO3) added. The nitric acid was purified using Distillacid (Berghof), a sub-boiling distillation apparatus.

The samples were then submitted to ramp heating in the microwave system following the temperature program shown in Table II. The final dilution factor of each sample was 20x. For addition and recovery tests, spikes of 10 μg kg-1 were performed before the digestion for all elements studied.

Table II. Temperature ramp parameters for meat acid digestion Temperature o ( C)

Ramp Time (min)

Plateau Time (min)

Power (%)

140

8

5

60

190

8

5

65

230

8

20

75

Sample digestion by the microwave system using dilute acid was efficient. In order to improve the digestion (lower residual carbon) 2 mL of H2O2 may be added. The hydrogen peroxide must be ultrapure because it can act as a source of contamination.

In this work we did not perform the analysis of residual carbon; however Gouveia et al. [8] obtained 11.0 ± 0.4 wt% of residual carbon content in a digestion of 250 mg of lyophilized bovine liver with 2 mL of HNO3 + 1 mL of H2O2 in a microwave oven. Residual carbon around this value is considered satisfactory for determinations by ICP OES [8]. Visually, the samples were low in residual carbon, as to be expected since the method was based on dilute acid, as in the work of Bizzi et al. [9].

Elemental determination After preparation of the samples, the elemental determination was performed ICP OES, using iCAP 7400 Duo (Thermo Scientific) equipment. The analysis parameters are shown in Table III.

45

Siqueira, B.M.; Leite, M.D.; Garcia, M.O.; Rainone, R.

Technical Note Table III. ICP OES method parameters Radiofrequency power (W)

1250

Pump rotation (rpm)

65 -1

Nebulizer gas (L min )

0.45

-1

Auxiliary gas (L min )

0.5

-1

Plasma gas (L min )

12

Emission lines (nm)

As (189.042); Cd (214.488); Cr (267.716); Hg (194.277); Pb (220.353)

To evaluate the accuracy of the method, standard additions of 10 μg kg-1 were performed on samples.

For Hg analysis, hydride generation was required. A 6M HCl solution and a 0.5% NaBH4 solution (m/v) in 0.05% NaOH was used. A hydride generation accessory with a gas/liquid separator was used to separate the hydrides from the sample matrix.

RESULTS AND DISCUSSION The detection limits for As, Cd, Cr, Hg, and Pb in meat samples were calculated according to the indication by Thomsen et al., which incorporates the background equivalent concentration (BEC) signals and relative standard deviations (RSD) of 10 analytical blank measurements in the calculation of detection and quantification limits [1]. It is possible to see in Table IV that ICP OES, using iCAP 7400 equipment, presents sufficiently sensitive limits of detection to meet the requirements of the regulatory agencies in Brazil and China.

Table IV. Limits of detection and quantification obtained for meat elemental determination by ICP OES As

Cd

Cr

Hg

Pb

-1

0.63

0.06

0.88

0.58

0.89

-1

2.11

0.2

2.94

1.95

2.97

LOD (μg kg ) LOQ (μg kg )

The concentrations obtained in the three types of meat are shown in Table V, and it can be concluded that all values are below the maximum limit allowed by GB 2762-2012, ANVISA and PNRCR. Addition and recovery tests were performed to evaluate the accuracy of the method. Spikes of 10 μg kg-1 were added to the samples, and in the sequence the recoveries were evaluated in terms of percentage. It can be observed that the recoveries varied around 100 ± 20%, showing that the method has satisfactory accuracy.

Table V. Obtained concentrations in meat samples and maximum permitted limits (MPL) for each legislation (µg kg-1).

As

Cd

Cr

Hg

Pb

Beef

13.78 + 2.89

1.98 + 0.59

129.98 + 10.35

< LD

< LD

Pork

27.34 + 3.11

1.72 + 0.48

84.26 + 9.89

< LD

10.02 + 2.79

Chicken

16.74 + 3.59

2.26 + 0.61

188 + 11.67

< LD

7.9 + 2.52

500

100

1000

50

200

MPL-ANVISA

500

50

***

***

100

MPL-PNCR

1000

1000

***

30

500

MPL-GB 2762-2012

***This element is not required for the related legislation 46

Determination of inorganic contaminants in meat by ICP OES:a simple method to comply with Brazilian and Chinese market demands

Technical Note

Table VI. Blank matrix, spiked concentrations (µg kg-1), and percentage of recovery for spiked samples (%).

Beef

Pork

Chicken

Analyte

Blank Matrix

Spiked

Recovery

Blank

Spiked

Recovery

Blank

Spiked

Recovery

Arsenic

<LD

26.06

109.6

LD

38.05

101.9

<LD

31.05

116.1

Cadmium

0,07

12.24

102.2

LD

11.97

102.1

0,06

12.68

103.4

Chromium

<LD

177.07

126.05

<LD

86.44

91.7

<LD

162.56

82.1

Mercury

<LD

10.22

102.2

<LD

9.38

93.8

<LD

10.54

105.4

Lead

<LD

9.57

95.7

<LD

16.34

81.6

<LD

16.09

89.9

CONCLUSIONS Sample digestion by a microwave system using dilute acid was efficient. In order to improve the digestion (lower residual carbon) 2 mL of H2O2 may be added. But hydrogen peroxide must be ultrapure because it can act as a source of contamination.

The ICP OES technique has been proved capable of detecting the maximum permitted limits of metals in meat required to comply with the requirements of Brazilian and Chinese legislation.

It is important to remember that most studies use ICP-MS for the determinations of these elements in these concentrations [10,11]. However, with this method of sample preparation and sensitive limits of detection, the low concentrations of these elements demanded by legislation in Brazil and China can be determined by ICP OES Addition and recovery tests indicated that the proposed method, from sample preparation to elemental determination by ICP OES, is of satisfactory accuracy.

Manuscript received Feb. 14, 2017; revised manuscript received Apr. 11, 2017; accepted Apr. 18, 2017.

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REFERENCES

1. http://www.agricultura.gov.br/portal/page/portal/Internet-MAPA/pagina-inicial/pncrc [Accessed 11 January 2016].

2. Thomsen, V.; Roberts, G.; Burgess, K.; Spectroscopy, 2000, 15, p 33.

3. http://www.anvisa.gov.br/alimentos/legis/especifica/contaminantes.htm [Accessed 11 January 2016] 4. http://www.seafish.org/media/publications/China_Max_levels_of_contaminants_in_food.pdf [Accessed 11 January 2016] 5. Gonçalves, J.R. Determinação de Pb, Cd, Fe, Zn, e Cu em carnes de bovinos e Pb, Cd e P em suplementos minerais no Estado de Goiás. Master thesis, 1999, Escola de Veterinária, Universidade Federal de Goiana, Goiás, Brazil.

6. Chowdhury, M.Z.A.; Siddique, Z.A.; Hossain, S.M.A.; Kazi, A.I.; Ahsan, M.A.; Ahmed, S.; Zaman, M.M. J. Bangladesh Chem. Soc., 2011, 24 (2), pp 165–172.

7. Al-Zuhairi, W.S.; Farhan, M.A.; Ahemd, M.A. Int. J. Recent. Sci. Res., 2015, 6 (8), pp 5965–5967.

8. Gouveia, S.T.; Silva, F.V.; Costa, L.M.; Nogueira, A.R.A.; Nobrega, J.A. Anal. Chim. Acta, 2001, 445 (2), pp 269–275.

9. Bizzi, C.A; Flores, E.L.M; Nobrega, J.A.; Oliveira, J.S.S.; Schmidt, L.; Mortari, S.R. J. Anal. At. Spectrom., 2014, 29, pp 203–203.

10. Julshamn, K.; Maage, A.; Norli, H.S.; Grobecker, K.H.; Jorhem, L.; Fecher, P.; Dowell, D.J.; J. AOAC Int. 2013, 96 (5), pp 1101–1102.

11. Jackson, B.P.; Punshon, T. Curr. Environ. Health Rep. 2015, 2 (1), pp15–24.

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Br. J. Anal. Chem., 2017, 4 (14), pp 48 - 53

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Determination of Multiclass Veterinary Drug Residues in Meat, Plasma, and Milk on a Quadrupole-Orbitrap™ LC-MS System Olaf Scheibner and Maciej Bromirski Thermo Fisher Scientific, Bremen, Germany This technical note describes a screening method for veterinary drug analysis, showing how the variable data-independent acquisition (vDIA) workflow achieves high sensitivity and selectivity, providing a complete high-quality data record of the measured sample. Quantitative analysis of the acquired data in combination with non-targeted and screening is shown.

Keywords: Orbitrap, veterinary drugs, HRAM quantitation, HRAM screening, vDIA, screening, retrospective data analysis.

INTRODUCTION The analysis of veterinary drugs in animal products is usually a time-consuming process with respect to both sample preparation and mass spectrometric analysis [1, 2]. The quantitative analysis of multiclass veterinary drug residues from animal products - including meat, milk, and plasma - often requires multiple sample injections in order to achieve optimal conditions for individual classes of compounds. This includes multiple chromatographic methods for different compound classes, as well as separate mass spectrometric methods, each specifically directed to small groups of compounds. Even using conventional multi-residue methods [3, 4], the data obtained only contains information on the targeted compounds and is not suitable for any retrospective analysis on additional analytes of interest.

Here, an improved method utilizing ultrafast chromatography with a benchtop quadrupole-Orbitrap mass spectrometer is described. It consists of a short generic chromatographic method and a mass spectrometric method called variable data-independent acquisition (vDIA). The advantages of this approach are short overall analysis time, superior selectivity, and high sensitivity. This robust method provides data with suitable options for additional targeted and non-targeted screening. The vDIA approach has been developed and is utilized here for generation of calibration curves and analyses of samples for targeted and nonargeted compounds. vDIA can use multiple MS/MS isolation windows with widths from 50 Da up to 800 Da. Typically, smaller windows are used for lower mass regions to increase dynamic range and therefore sensitivity, while larger windows cover higher mass regions to improve the duty cycle.

In a typical acquisition setup, shown here, five MS/MS isolation windows were set to cover the entire mass range of the preceding full scan while maintaining speed of MS/MS analysis for the fast chromatography. Forty-four multi-class veterinary drug residues listed in Table I, were analyzed in extracts of muscle, kidney, milk, and plasma using a single standardized chromatographic and mass spectrometric method. For absolute quantification, standard samples with known concentrations of all 44 drug residues -1 -1 covering eight calibration points (from 100 pg mL to 500 ng mL ) were prepared. For evaluation of the method, spiked matrix samples (muscle and kidney for antibiotics, milk for avermectins and plasma for nitroimidazoles) were analyzed by high-resolution, accurate-mass (HRAM) LC-MS/MS.

48

Olaf Scheibner and Maciej Bromirski

Sponsor Report Table I. List of components used for analysis with limits of quantitation (LOQ), defined here as the lowest concentration level on which a component could be confirmed with at least one fragment ion.

Compound Abamectin Amoxicillin Ampicillin Cefalexin Cefalonium Cefaperazone Cefapirim Cefquinome Chlorotetracycline Ciprofloxacin Cloxacillin Danofl oxacin Dapsone Difloxacin Dimetridazol Doramectin Doxycyclin Enrofloxacin Eprinomectin Erythromycine Flumequine Ipronidazol-OH

LOQ -1 (ng mL ) 5.0 1.0 0.5 0.5 0.5 1.0 0.1 5.0 1.0 0.5 0.1 5.0 0.5 0.5 5.0 10.0 0.5 1.0 5.0 1.0 1.0 0.5

Compound Marbofloxacine Metronidazole Metronidazole-OH Moxidectin Nafcillin Oxacillin Penicillin G Penicillin V Ronidazol Sarafloxacine Sulfadiazine Sulfadimethoxin Sulfadimidin/Sulfamethazine Sulfadoxin Sulfamerazin Sulfamethoxazole Sulfamethoxypyridazine Sulfathiazole Tetracycline Thiamphenicol Trimethoprim Tylosine

LOQ -1 (ng mL ) 5.0 0.5 0.5 0.5 0.5 0.1 0.5 0.5 0.5 0.5 0.1 0.5 0.1 0.5 0.1 0.5 0.1 0.5 0.5 0.5 0.1 1.0

MATERIALS AND METHODS A generic LC method was used for all samples. Instrumentation: Thermo Scientific™ Dionex™ UltiMate™ 3000 Rapid Separation LC (RSLC), column Thermo Scientific™ Accucore™ C18 aQ 100 x 2.1 mm, 2.6 µm particle size; Mobile phase A water + 0.1% formic acid; Mobile phase B acetonitrile + 0.1% formic acid; 6 -1 min gradient from 5% B to 95% B. Flow rate 300 µL min ; total chromatographic cycle 15 min.

Mass Spectrometry Method: A generic full-scan method with wide-isolation variable data-independent acquisition (FS-vDIA) was used for all samples. Instrumentation: Q Exactive™ Focus MS system from Thermo Scientific™; Full Scan Resolution setting 70,000 (FWHM) at m/z 200; mass range (m/z) 100– 1000. vDIA, resolution setting 17,500 (FWHM) at m/z 200, isolation windows (m/z) 100–205, 195–305, 295–405, 395–505, 495–1000. Spray voltage 4.4 kV; sheath gas 30.0 (arbitrary units); aux gas 5.0 (arbitrary units); capillary temperature 250 °C; heater temperature 300 °C; RF-lens level 50; HCD collision energy 35 eV. Data processing was performed using Thermo Scientific™ TraceFinder™ software version 3.2. For generation of extracted ion chromatograms, an extraction window of 5 mg kg-1 was used. For non-targeted screening, a built-in component and fragment m/z values database was used, consisting of 450 components. Analytes were quantified based on full scan information (protonated ions). Additionally, one to five fragment ions were used for identity confirmation, according to EU regulatory requirements (EC/657/2002), achieving linear calibration curves over the ranges described above.

49

Determination of Multiclass Veterinary Drug Residues in Meat, Plasma, and Milk on a Quadrupole-Orbitrap™ LC-MS System

Sponsor Report RESULTS AND DISCUSSION

2

2

The vDIA approach described bridges the gap between full-scan data-dependent MS (FS-ddMS ) experiments and full range fragmentation scan modes such as all-ion fragmentation (AIF). As Figure 1 shows, this is a combination of a full scan with several wide range isolation MS2 scans. In this setup, the 2 isolation widths of the MS windows vary between 100 Da and 500 Da and together cover the entire mass range of the preceding full scan.

Figure 1. Setup of a typical FS-vDIA experiment.

FS-ddMS2 experiments, where MS2 scans are performed on targets of interest (present on an inclusion list) upon their detection in the full scan, are known to be very selective and sensitive with respect to the 2 fragment ion information obtained [5]. Retrospective FS-ddMS data analysis for additional compounds of interest, however, is limited to full-scan quantitation by accurate mass without confirmation of identity by MS/MS. Full-range fragmentation experiments like AIF [6], where fragments from all species present in the full 2 2 scan are detected in a single MS scan, have the advantage of collecting all possible full scan and MS information for the sample. Thus, they are fully suitable for retrospective data analysis. Dynamic range, selectivity, and achieved detection limits, however, are limited as the number of ions fragmented per species is lower due to the combined nature of the analyses.

The vDIA approach, where fragments from wide isolation windows covering the entire mass range are detected in multiple MS2 scans, maintains very high levels of sensitivity and selectivity while keeping a full digital record of the sample. Therefore, it is fully suitable for retrospective data analysis. Table I shows the observed limits of detection (LOQs) in a dilution series for full scan quantitation with fragment ion confirmation. LOQ is defined here as the lowest concentration level on which a component could be confirmed with at least one fragment ion.

For accurate quantification and confirmation of identity, it is crucial that the fragment ions used for confirmation are clearly resolved in time and mass, so that the confirmation is not affected by elevated background or interfering peaks. Figure 2 shows examples of how the overlay of the extracted ion chromatograms of the confirming fragment ions (right panel) match with the quantifier precursor ion (left panel), acquired in vDIA scan mode and automatically processed in TraceFinder software. All confirming ions were free of any interference and co-eluted with the quantifier, providing unambiguous confirmation that is essential, particularly for complex matrices. Since the vDIA mode generates full elution profiles for all fragment ions, the chromatographic peaks of the fragments can be integrated and this way the fragment ion ratio can be calculated with high confidence and used for confirmation as well.

50

Olaf Scheibner and Maciej Bromirski

Sponsor Report

Figure 2. Selectivity of selected components in matrix; A: ampicillin in pig muscle at 5 µg kg-1; B: sulfadiazin in pig kidney at 5 µg kg-1; C: ronidazol in pig plasma at 1 µg kg-1; D: moxidectin in cow milk at 1 µg kg-1.

Figure 3 shows an example of linear dynamic range for selected compounds using the vDIA approach.

The calibration range spanned from 0.5 µg kg-1 to 500 µg kg-1 and linearity in all cases was better than R2 = 0.99. In the cases of abamectin and doramectin, limited stability of the protonated ions was observed and the sensitivity in the full scan detection was therefore limited. This instability may be due to the use of average parameters for this generic method, suitable for a wide panel of drugs. In these few cases, the vDIA approach allows quantitation using several fragment ions of abamectin and doramectin, permitting quantitation and identity confirmation down to much lower levels than the protonated ions in the full scan.

After quantitation curves were established, all components were quantified in the spiked matrices at low levels. Antibiotics were spiked into muscle and kidney matrix at levels of 5 µg kg-1, while abamectins were spiked into milk at a level of 1.0 µg L-1 and nitroimidazoles were spiked into plasma at a level of 1.0 µg L-1.

Figure 3. Linearity of selected compounds.

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Sponsor Report

Determination of Multiclass Veterinary Drug Residues in Meat, Plasma, and Milk on a Quadrupole-Orbitrap™ LC-MS System

The processing software provides component detection with high selectivity (by means of the narrow extraction window of 5 mg kg-1) in the full scan with identification according to retention time. In addition to this, it offers three automated options for confirmation of identity. The first uses isotopic information of the precursor detected in the full scan. Figure 4 shows an overlay of the detected spectrum (in red) of ciprofloxacin and the theoretical isotopic distribution (in blue). With a resolution setting of 70,000, even in the complex matrix of muscle extract at a concentration of 5 µg kg-1, the isotopic distribution match is free from interference and gives an unambiguous confirmation.

Figure 4. Isotope pattern match for the example of ciprofloxacin. The detected spectrum is shown in red, and the theoretical isotopic distribution is shown in blue.

Confirmation of identity can be performed using the MS/MS data by detecting known fragment ions.

-1 The following results were achieved for a muscle tissue sample at a spike level of 0.5 µg kg . Figure 5 shows the result of the fragment ion confirmation in vDIA mode. As the vDIA spectra are generated using a wide isolation window, fragment ions from multiple precursors can be detected. The high resolution, accurate-mass nature of Orbitrap MS/MS detection allows selective identification of the fragment ions combined with RT profile matching giving confident identification.

Figure 5. Fragment match for the example of ciprofloxacin. The experimental spectrum is shown in red, the confirmation masses are shown in blue.

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Olaf Scheibner and Maciej Bromirski

Sponsor Report Most routine targeted methods only cover a limited number of analytes. The ability to reanalyze the sample for a suspected drug post-MS analysis becomes very beneficial for saving instrument/lab time and sample. To demonstrate retrospective analysis of the data files, a wide-range screening approach was performed on vDIA data files of analytes spiked in muscle tissue samples. A non-targeted screen with a 1500 component built-in database was conducted, providing several strong matches to additional components present in the sample. Figure 6 shows the example of cortisol (hydrocortisone) confirmed by isotopic pattern match, fragment search, and library match.

Figure 6. Non-targeted screening result.

CONCLUSION The Orbitrap QExactive™ Focus was used to create a variable data-independent acquisition general method for the detection and quantification of 44 multiclass veterinary drug residues in different matrices.

This method proved to have the required LOD sensitivity, and ability to confirm identity using retention time, accurate m/z, isotopic ratio, and fragment ions to exceed the EU regulatory requirements (EC/657/ 2002). The vDIA approach extends confirmation options for non-targeted screening approaches, while maintaining a high level of sensitivity and selectivity. All data processing was accomplished with the easyto-use proprietary Trace Finder™ software both for identification and all stages of identity confirmation. vDIA accomplishes the goal of being an accurate and sensitive analysis for the detection of targeted compounds (i.e. those from the 44 compound panel) and also for the screening of unexpected or suspected compounds post-analysis.

Obs.: vDIA method is not available in the United States of America.

This sponsor report is the responsibility of Thermo Fisher Scientific.

w9 C9 w9 b / 9 { 1. Masiá, A.; Suarez-Varela, M.M.; Llopis-Gonzalez, A.; Picó, Y. Anal. Chim. Acta, 2016, 936, pp 40-61.

2. Xie, J.; Peng, T.; Zhu, A.; He, J.; Chang, Q.; Hu, X.; Chen, H.; Fan, C.; Jiang, W.; Chen, M.; Li, J.; Ding, S.; Jiang, H. J. Chromatog. B, 2015, 1002, pp 19-29.

3. Stubbings, G.; Bigwood, T. Anal. Chim. Acta, 2009, 637 (1–2), pp 68-78.

4. Dasenaki, M.E.; Thomaidis, N.S. Anal. Chim. Acta, 2015, 880, pp 103-121.

5. Jia, W.; Chu, X.; Ling, Y.; Huang, J.; Chang, J. J. Chromatog. A, 2014, 1347, pp 122-128.

6. Gómez-Pérez, M.L.; Plaza-Bolaños, P.; Romero-González, R.; Martínez-Vidal, J.L.; Garrido-Frenich, A. J. Chromatog. A, 2012, 1248, pp 130-138.

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Br. J. Anal. Chem., 2017, 4 (14), pp 55-57

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1 Ibero-American & 6 BrMass Conference consolidated as the largest Mass Spectrometry congress in Latin America Prof. Dr. Marcos Eberlin, at the Opening Conference of the 6th BrMASS - 1st IbMASS

Last December the 6th Conference of the Brazilian Mass Spectrometry Society - organized in conjunction st th th with the 1 Ibero American Conference on Mass Spectrometry - occurred between the 10 and 14 at Rio de Janeiro, Brazil.

For the president and founder of the Brazilian Mass Spectrometry Society, Prof. Marcos Eberlin, also a professor at the Chemistry Institute, University of Campinas, the congress accomplished its roll despite a year of crisis. “We simply carried through the largest and best mass spectrometry congress in Latin America and established BrMASS as the third largest Mass Spectrometry congress in the world. It had a total of 1410 participants and it was a show of science and sociability” he accounted. In total, five successful BrMASS conferences have been held, the first of which happened in 2005 at the Royal Palm Plaza Resort in Campinas, SP, Brazil.

Prof. Dr. Alexander Alexeyevich Makarov, Director of Global Research for Life Sciences Mass Spectrometry at Thermo Fisher Scientific, was invited for the second time to deliver the BrMass opening conference. As leader of the team that developed the Orbitrap, a type of mass spectrometer, he also highlighted the presence of a larger number of international scientists in this edition of the event. “I think this current meeting brought more international scientists than the one held in 2011, and I witnessed a stronger presence of instrument suppliers as well. Therefore, I think that 6th BrMASS & 1st IbMASS indeed fulfilled the intention of becoming the first Opening lecture by Alexandre Makarov, Thermo Ibero-American Conference of Mass Spectrometry” Prof.

Orbitrap Inventor Dr. Alexander said.

The participants in this edition had the opportunity to attend 105 lectures, in addition to a wide and diversified technical and scientific program, in which pre-congress courses were also held. Moreover, in parallel to the scientific congress, sponsoring companies had organized an instrument exhibition. Among these, included: Agilent, Allcrom, Analitica, Bruker, LECO, Merck, Peak, Perkin Elmer, Sciex, Shimadzu, Thermo Fisher Scientific and Waters.

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Feature The companies took full advantage of the exhibition space, building their booths in such a way as to enrich the event even more. They also hosted live music and cocktails at hospitality suites. The social events were a novelty to the conference this year. Including the 'Beach Break', a collective swim in the sea in front of the host hotel and the opening show with the samba school 'União da Ilha'.

Participants enjoying of Hospitalities Suites

The lectures were given by important names in the mass spectrometry field such as Alexander Makarov of Thermo Fisher Scientific, Livia Eberlin of the University of Texas at Austin, Gary M. Hieftje of Indiana University and others. The opening lecture, 'Orbitrap mass spectrometry: a road to ultra-high resolution in every lab', was lead by the Prof. Dr. Alexander Makarov. “I was honored by the invitation of Prof. M. Eberlin and I really wanted to support his enthusiasm and belief in the future of mass spectrometry in Brazil. This was also reflected in my talk, in which I devoted a lot of time to the long and difficult history of Orbitrap development, with universal lessons about perseverance and thinking outside the box at any circumstance. Another aspect of my talk was the ongoing effort to expand the applications of Orbitrap technology, making it more accessible and indispensable for every analytical laboratory. I hope to see this technology not only in leading proteomics or metabolomics research centers, but also in institutions and companies that works in the environmental, toxicological and clinical fields” Prof. Dr. Alexander Makarov explained.

In the poster exhibition area, the congress members presented their work related to the diverse areas and applications of Mass Spectrometry.

Some congress participants presenting their posters

BrMass usually honors professionals who have contributed to the development of mass spectrometry with the BrMASS Medal. The board of directors of BrMASS nominates candidates, followed by voting, to choose three medal winners. This year, the tribute was made to the following three professionals who have worked or still work in companies that supply equipment, providing all the support for the implementation of advanced technologies in mass spectrometry:

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Delivery of the BrMass Medal held at gala dinner

Feature Juarez Araújo Silveira, founding partner of Sinc Brazil, a company that has worked for 30 years in the area of scientific instrumentation. He currently serves as commercial director of Brazil's Shimadzu in the analytical division.

Larry L. Burchfield - in memoriam. Mr. Burchfield was a highly accomplished professional with 40 years of professional experience. He was well known throughout professional circles for his knowledge of mass spectrometry, chromatography, analytical chemistry and computer science. Attended Texas A & M University where he earned a Bachelor of Science in Physics and Chemistry, and Master of Science in Physical Chemistry and Computer Science. He was a faculty member of the chemistry department at Texas A & M University, a sales account manager at Finnigan Instruments and Southwest region sales and service manager at Finnigan MAT. Before retiring, he worked for Thermo Fisher Scientific.

Luiz Bravo, founding partner of “Nova Analítica”, which, since 1992, supplies scientific equipment manufactured by companies of international renown. In the area of mass spectrometry this Brazilian company is a distributor of Thermo Fisher Scientific.

The medals were handed out during an elegant gala dinner offered to provide for the participants a relaxed atmosphere in which everyone could interact and have a pleasant time.

Photo credits: All photos are from brmass.com/galerias.php

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18 ENQA - Analytical Chemistry Integrated into Society th

18th ENQA Opening Ceremony

The 18 Brazilian Meeting of Analytical Chemistry (ENQA) had the theme 'Analytical Chemistry integrated into society' th and took place in Florianópolis, SC, BR, between 18 and st 21 of September 2016. The main purpose of the event was to promote a forum to discuss the advances of Analytical Chemistry. Therefore, different kinds of activities were performed, such as conferences, lectures, coordinated sessions, technical lectures, workshops, mini-courses and poster presentations. The ENQA also enabled the direct interaction between participants and companies involved with the Research & Development and Quality Control sectors.

ENQA is a biannual and itinerant event of the Analytical Chemistry Division of the Brazilian Chemical Society (SBQ). The first edition of the event took place in the early 1980s, which gives ENQA a tradition of more than 30 years as the largest and most important scientific event of Analytical Chemistry in Brazil, and one of the leading events of chemistry in Latin America. The number of participants has grown with every edition. An average of 1100 participants in the last three editions was obtained, which demonstrates the success of the event and the expansion of Brazilian Analytical Chemistry. th The opening ceremony of the 18 ENQA occurred on Sérgio L. C. Ferreira (left) and Bernhard Welz (right) who received the 'Adilson José Curtis' medal Sunday (09/18), in which important names of Analytical Chemistry were honored.

Prof. Dr. Fabio Augusto, professor of the Institute of Chemistry, University of Campinas (UNICAMP), SP, BR, received the medal 'Dr. Janusz Pawliszyn' for the contribution given to the sample preparation area.

Prof. Dr. Bernhard Welz (photo), a volunteer professor at the Federal University of Santa Catarina, Florianópolis, SC, BR, and author of several publications in the area of atomic absorption spectrometry (AAS), received the 'Adilson José Curtis' medal.

Others honored participants at ENQA for their contributions to the Analytical Chemistry field were: Prof. Dr. Mauro Bertotti (photo), professor at the Institute of Chemistry of the University of São Paulo (USP) SP, BR, who works mainly in research on microelectrodes, modified electrodes and electrochemical microscopy.

Thiago Paixão (left) and Mauro Bertotti (right) who was honored at ENQA 58

Prof. Dr. Maria das Graças Andrade Korn, professor of the Institute of Chemistry of the Federal University of Bahia, Salvador, BA, BR, who works mainly in atomic spectroscopy and preparation of samples applied to environmental samples, medicines, fuels and food.

Feature Dr. Lídio Kazuo Takayama, president of FEMTO Industry and Commerce of Instruments Ltd. Degree in Physics from the Institute of Physics, USP, SP, BR, and postgraduate degree from the Technological Institute of Aeronautics, São José dos Campos, SP, BR. Dr. Takayama was honored for his pioneering efforts in founding FEMTO in 1989. The company produces a wide range of spectrophotometers and has an important record in the history of Brazilian analytical instrumentation.

Prof. Dr. José Luís Costa Lima, professor and ex-director of the Faculty of Pharmacy of the University of Porto, Porto, PT, joined as an Assistant Professor at the Faculty of Pharmacy in 1986. His aim was to establish a group dedicated to Analytical and Applied Chemistry, with research compatible with the teaching of Chemistry in Pharmaceutical Sciences.

After all the tributes were honored, the event carried on with the opening lecture given by Prof. Dr. Jailson Bittencourt de Andrade, from the Federal University of Bahia (UFBA), entitled as 'What is the role of (analytical) chemistry in the 21 st century?'. Dr. Bittencourt has been a professor of the Department of General and Inorganic Chemistry of the Institute of Chemistry of UFBA since 1976. He is currently professor of UFBA and also Secretary of Policies and Programs of Research and Development of the Brazilian Ministry of Science Technology and Innovation.

Prof. Dr. Jailson Bittencourt de Andrade held the opening lecture

Following the event, as a tradition, cocktails were held, in which all the participants had the opportunity to interact in a relaxed atmosphere.

On Monday (09/19) the scientific programming began with the first four of eight Coordinated Sessions.

The Coordinated Sessions are a set of oral presentations that interrelate or complement each other around the sub-themes or thematic axes of the event. At the end of each session, the responsible for that session coordinates a discussion around the presented works.

The first four Coordinate Sessions topics of the day were: Electrochemical and Electroanalytical, Sample Preparation (Organic Analytes), Mass Spectrometry and Analytical Instrumentation. Afterwards, participants attended the Plenary Conference 'Engineering Nucleic Acid Aptamers for Molecular Analysis', given by Prof. Dr. Anthony E. G. Cass, currently Professor of Chemical Biology in the Department of Chemistry and Institute of Biomedical Engineering at Imperial College London, London, UK.

Parallel to the scientific congress, the ExpoCenter was held, which is a space used by companies sponsoring the event to exhibit their new equipment and technologies in the area of Analytical Chemistry.

The posters accepted at the 18th ENQA were also discussed in the ExpoCenter area.

ExpoCenter

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Feature The participants then attended the Plenary Conference Vapor Generation - 2016: 'The Ideal Sample for an ICP would be in the Gas Phase' by Dr. Ralph E. Sturgeon (National Research Council Canada, Ottawa, CA). Dr. Sturgeon has published more than 280 articles and several book chapters in the field of Analytical Chemistry. Furthermore, he participates in the consulting board of eight international Analytical Chemistry journals.

Poster session

During the afternoon the coordinated sessions continued, with the topics: Chemometrics, Separation Methods, Atomic Spectrometry and Other Topics. To close the agenda of the second day of the meeting, four corporate sponsor conferences were presented.

On Tuesday (09/20), the Coordinated Sessions listed below opened another day of lectures and presentations: Analytical Chemistry and Energy, Environmental Chemistry, Separation Methods and Atomic Spectrometry. Participants then attended the Plenary Conference 'Advances and Applications of TwoDimensional Gas Chromatography in Bioanalytical Chemistry and Organic Geochemistry', given by Prof.

Dr. Fabio Augusto, professor at the Institute of Chemistry, UNICAMP, who works in the area of analytical separations.

Participants also welcomed the book launch ‘‘Métodos de Preparo de Amostras para Análise Elementar’’ edited by professors Dr. Francisco José Krug and Dr. Fabio Rodrigo Piovezani Rocha, both from the Center for Nuclear Energy in Agriculture (CENA-USP), Piracicaba, SP, BR. The book brings together texts from 28 authors regarding preparation of samples for elemental analysis. Prof. Piovezanni commented “Despite being a very important subject in Analytical Chemistry, there were no such complete The editors Francisco José Krug and Fabio compilations in this area, even in English”. Additionally, Rodrigo Piovezani Rocha, signing the book.

Prof. Krug stated “The book brings scientific literature up to the beginning of 2016”.

Subsequently, a Plenary Conference entitled 'Chemometrics from the Laboratory to the Industry.

Practical implementation, benefits and drawbacks' was held by Prof. Dr. José Manuel Amigo Rubio, Associate Professor of the Department of Food Science - Quality and Technology at the University of Copenhagen, Copenhagen, DK. In the afternoon, three symposiums were offered: Bioanalysis, Microsystems of Analysis, and Analysis of Fuel and Biofuels.

On Wednesday (09/21), the participants attended the closing of the following Coordinated Sessions: Environmental Chemistry, Electrochemistry and Electroanalytical, Sample Preparation and Chemometrics.

After the coffee break, the scientific schedule continued with the last Plenary Conferences: 'Analyzes in Flow: from Paradigm Shifts to the Future (Uncertain?)', given by Dr. Fabio Rodrigo Piovezani Rocha, professor at CENA-USP; 'Microextraction by Gas Diffusion' by Dr. Luís Moreira Gonçalves, from the University of Porto, Porto, PT; 'Panorama of Scientific Production of Analytical Chemistry in Brazil' by Dr. Maria Valnice Boldrine Zanoni, professor at the Institute Coffee break 60

Feature of Chemistry, São Paulo State University (UNESP), Araraquara, SP, BR; and for last 'Magneto-Mips: From Selective Extraction to Highly Sensitive Quantification' by Dr. Maria Del Pilar Taboada Sotomayor, who is currently Coordinator of the Chemistry Undergraduate Program of the Institute of Chemistry of UNESP, Araraquara. After lunch, Prof. Dr. Marco Tadeu Grassi, from the Federal University of Paraná, Curitiba, PR, BR, coordinated a round table with the theme 'The Performance of Analytical Chemistry in Environmental th Crimes'. In conclusion of the scientific program of the 18 ENQA, a symposium was held on the theme: 'Use of Alternative Energies for Intensification of Chemical Processes'.

Lastly, there was the Closing Session with awards for the best posters and a recapitulation of the activities at the 18th ENQA, which was attended by 1044 people, including professors and senior researchers, postdocs, graduate and undergraduate students.

Photo credits: All photos are from the organization of ENQA 2016

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Instituto GAIA de Espectrometria (IGE) offers courses to strengthen ties between academia and the private sector In 2016, seeking to strengthen ties between academia and professionals of the private sector, the Instituto GAIA de Espectrometria (IGE) - in English, GAIA Institute of Spectrometry - was created as a division of the Group for Applied Instrumental Analysis (GAIA).

“The IGE purpose is to establish a link between academia and professionals of the private sector.

The courses are aimed at those interested in deepening and updating knowledge, mainly professionals involved with analytical instrumentation and resolution of analytical demands in multiple industrial sectors” explained Prof. Dr. Joaquim de Araújo Nóbrega, from the Department of Chemistry of the Federal University of São Carlos (UFSCar), SP, Brazil.

Practical class on ICP OES. Course offered in June 2016 - Photo: IGE

The IGE offers short courses on various topics aimed at the application of atomic spectrometry and inorganic mass spectrometry in solving contemporary demands. "The courses are divided into two parts, one theoretical and another practical. In the morning the theoretical part is presented, and in the afternoon the knowledge is put into practice. The students are divided into groups and take turns with the instruments.

Collective discussion sessions and the exchange of experiences are also carried out”, explained Prof. Dr. Edenir Rodrigues Pereira Filho, from the Department of Chemistry of UFSCar, who joined GAIA in 2006.

The offered courses seek to update knowledge in the instrumental techniques of spectrochemical analysis that help solve various analytical problems and also demands imposed by the legislation.

For instance, according to Dr. Nóbrega, electronic waste, such as old cell phones, screens and computers, has become an important environmental and legislation issue: "Today, there is a growing trend, including in Brazil, in which the company that sold the product is also responsible for its collection and recycling. Therefore, it is important to develop analytical procedures for the characterization of these products”, explained Nóbrega.

Practical class on ICP-MS. Course offered in June 2016 - Photo: IGE

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Members of GAIA / December 2016 Photo: IGE

The courses offered include; inductively coupled plasma optical emission spectrometry (ICP OES), inductively coupled plasma mass spectrometry (ICP-MS), sample preparation assisted by microwave radiation, chemical data processing using factorial design, statistical treatment of analytical data and new pharmaceutical standards for elemental impurities in drugs. These courses are periodically given and may also be offered in-company, depending on specific demands.

In addition to these topics, IGE also develops activities in X-ray fluorescence spectrometry and direct analysis of solid samples using laser techniques.

Additional information can be obtained by contacting Prof. Dr. Edenir (erpf@ufscar.br) or Prof. Dr. Joaquim (djan@terra.com.br).

About GAIA The GAIA was started in 1994 by Dr. Ana Rita de Araujo Nogueira (Embrapa Pecuária Sudeste) and Prof. Dr. Joaquim de Araújo Nóbrega. Later, Prof Dr. Edenir Rodrigues Pereira Filho and Dr. Lucimar Lopes Fialho, both from the Department of Chemistry of UFSCar, joined GAIA. The group acts for the development and evaluation of procedures for the decomposition of organic and inorganic samples using microwave ovens with open and closed flasks for subsequent quantification of the inorganic constituents by spectroscopic techniques. Techniques such as ICP-MS, ICP OES, microwave induced plasma optical emission spectrometry (MIP OES), graphite furnace electrothermal atomic absorption spectrometry (GFAAS) and flame atomic absorption spectrometry (FAAS), laser-induced breakdown spectroscopy (LIBS), and X-ray fluorescence spectrometry (XRF) are used.

Below is the schedule of IGE courses for this year: · March, 15 to 17: Sample Preparation and Elemental Analysis by ICP OES · June, 21 to 23: Elemental analysis of pharmaceuticals · September, 13 to 15: ICP-MS · November, 8 to 10: XRF Follow the IGE news along the year on the website: www.espectrometria.com.br

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Releases Thermo Scientific iCAP TQ ICP-MS Redefining triple quadrupole ICP-MS

With ever decreasing limit of detection requirements, interference elimination on specific analytes is becoming more important. Removal of most of the common interferences can be efficiently achieved with high resolution ICP-MS; however, budget limitations means this technology is not accessible to all laboratories. Quadrupole ICP-MS systems have both speed and fairly comprehensive interference removal capability. Systems fitted with collision/reaction cells (CRCs) are able to reduce polyatomic interferences, however, this mode is not effective for isobaric interferences. Triple quadrupole (TQ) technology adds a third quadrupole in front of the CRC to pre-filter the ion beam and limit the masses entering the CRC. If the mass shift reaction creates a product ion at the same mass as an existing matrix component, the matrix component is filtered out so that the analyte can be measured interference free.

The Thermo Scientific™ iCAP™ TQ ICP-MS is the solution to future-proofing your laboratory: explore developing markets, push the boundaries of your research and meet the requirements of evolving legislation.

'Right first time' analysis with powerful interference removal, even in the toughest matrices. Highly effective interference removal for accurate and reliable results: Utilizes up to four different collision and reaction gases and the unique Reaction Finder simplifies method development. There is no requirement for advance knowledge of complex reaction chemistry: Reaction Finder does it all for you! Integrated reaction gas handling features ensure safe and reliable instrument operation.

Reduced re-runs in the daily workload with assured accuracy and repeatability. Maintains the flexibility of single quadrupole (SQ) mode for less challenging analyses.

For ultimate flexibility, the iCAP TQ ICP-MS enables you to easily switch from SQ to TQ mode in a single sample analysis, providing the best results for your complete suite of elements.

Qtegra Intelligent Scientific Data Solution Software: intuitive software enables plasma ignition to data reporting in just five clicks, enhancing your productivity for rapid results. Beside this, automated sample handling, managed within the simple interface of Qtegra ISDS Software can be used. Routinely tackle advanced applications with hyphenated techniques to expand your application capabilities, like speciation, nanoparticles and laser ablation analysis.

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Releases

Thermo Scientific Q Exactive Focus New hybrid quadrupole-Orbitrap Mass Spectrometer The Q Exactive Focus mass spectrometer combines quadrupole precursor selection with high-resolution and accurate-mass detection to produce sensitivity that rivals triple quadrupole mass spectrometers, and resolution that surpasses Q-TOF instruments. · Exceptional sensitivity thanks to superior selectivity that eliminates interferences allowing lower limits of detection and quantitation · Fast polarity switching so more classes of compounds can be processed in a single analysis · High scan speeds for compatibility with fast chromatography and high-throughput screening · Resolution superior to Q-TOF instruments for more confident identification and confirmation · Exceptionally wide linear dynamic range Exceptional flexibility The unique hybrid quadrupole-Orbitrap configuration provides exceptional flexibility, allowing Q Exactive Focus instruments to perform both targeted and untargeted screening with confident identification and confirmation. Q Exactive Focus instruments can take advantage of high-resolution, accurate-mass in a number of quantification approaches, including: · Full-scan – data-dependent MS/MS (FS-ddMS2) acquisition for ultimate scan speed and quantitative performance · Variable data-independent acquisition (vDIA) for qualitative coverage for screening unknowns without compromising quantitative performance · Selected-ion monitoring (SIM) for simple setup and highest sensitivity · Parallel-reaction monitoring (PRM) for high selectivity and high throughput with confident confirmation NB Application-specific software is available to make full use of the power of the Q Exactive Focus instrument.

For component identification in untargeted and unknown workflows, Thermo Scientific™ TraceFinder™ software includes libraries of high-resolution accurate-mass (HRAM) spectra. Combined with TraceFinder data analysis software, the Q Exactive Focus offers a powerful, yet simple-to-use solution for food safety, environmental, clinical research, forensic, and toxicological screening.

Compound Discoverer software enable fast compound identification and differential quantitation.

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Events 2017 5-9 March PITTCON Conference and Expo 2017 McCormick Place, Chicago, Illinois, USA http://pittcon.org/pittcon-2017/ 2-7 April 14th Rio Symposium on Atomic Spectrometry Vitória, ES, Brazil http://www.riosymposium.com/ 17-21 April XXI Brazilian Symposium of Electrochemistry and Electroanalytics Natal, RN, Brazil http://www.sibee.com.br/ 14-17 May th 6 Latin-American Pesticide Residue Workshop: Food and Environment (LAPRW 2017) Wyndham Herradura Hotel, San Jose, Costa Rica https://laprw2017.fundacionucr.ac.cr/index.php/en-us/ 11-16 June Colloquium Spectroscopicum Internationale XL Congress Palace, Pisa, Italy http://www.csi-conference.org/ 9-14 July th 46 IUPAC World Chemistry Congress (IUPAC-2017) & th 40 Annual Meeting of the Brazilian Chemical Society WTC Sheraton, São Paulo, SP, Brazil http://www.iupac2017.org/ 31 July - 5 August IMEKO TC1-TC7-TC13 Joint Symposium Rio Othon Palace, Rio de Janeiro, RJ, Brazil http://imeko-tc7-rio.org.br/ 26-28 September 14th Analitica Latin America & 5th Analitica Latin America Congress São Paulo Expo, São Paulo, SP, Brazil http://analiticanet.com.br/ 3-8 December 3rd BrMASS School of Mass Spectrometry SERHS Hotel, Natal, RN, Brazil http://www.escola.brmass.com 10-13 December 5th Brazilian Meeting on Chemical Speciation Águas de Lindóia, SP, Brazil http://www.unesp.br/portal#!/cea/home/espeqbrasil2017 67

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Notices of Books Applications in High Resolution Mass Spectrometry: Food Safety and Pesticide Residue Analysis Roberto Romero-González and Antonia Garrido Frenich (Editors) March 2017, Elsevier This is the first book to offer complete coverage of all aspects of high resolution mass spectrometry (HRMS) used for the analysis of pesticide residue in food. Aimed at researchers and graduate students in food safety, toxicology, and analytical chemistry, the book equips readers with foundational knowledge of HRMS, including established and state-of-the-art principles and analysis strategies. Additionally, it provides a roadmap for implementation, including discussions of the latest instrumentation and software available. The book also discusses extraction procedures and the challenges of sample preparation, and the application of UHPLC- & GC- & flow injection-HRMS, ambient ionization, and identification of pesticide transformation products in food. Read more…

Food Authentication: Management, Analysis and Regulation Constantinos A. Georgiou & Georgios P. Danezis (Editors) May 2017, Wiley-Blackwell This book covers the most advanced techniques used for the authentication of a vast number of products around the world. The reader will be informed about: the latest pertinent analytical techniques; concepts of food authentication; analytical techniques for the detection of fraud relating to geographical, botanical, species and processing origin; consumer attitudes towards food authenticity, the application of bioinformatics to this field, and the Editor's conclusions and future outlook. Read more…

Analysis of Food Toxins and Toxicants Yiu-Chung Wong & Richard J. Lewis (Editors) August 2017, Wiley-Blackwell This book provides up-to-date descriptions of the analytical approaches used to detect a range of food toxins. Part I reviews the recent developments in analytical technology including sample pre-treatment and food additives. Part II covers the novel analysis of microbial and plant toxins. Part III focuses on marine toxins in fish and shellfish. Part IV discusses biogenic amines and common food toxicants. Part V summarizes quality assurance and the recent developments in regulatory limits for toxins, toxicants and allergens, including... Read more…

Olives and Olive Oil as Functional Foods: Bioactivity, Chemistry and Processing Paul Kiritsakis, Fereidoon Shahidi August 2017, Wiley-Blackwell

This book will provide thorough information about olives and olive oil, including on composition, analysis, fruit processing, quality control, utilisation of by-products, and more.

Readers will learn about the importance of olive oil's role in reducing oxidative stress - a serious danger factor for human health. The book will provide very significant information about the new trends in olive oil and lipids. 30 chapters are planned, to be written by distinguished authors and industry leaders in the field covering… Read more…

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Notices of Books Food Safety: Innovative Analytical Tools for Safety Assessment Umile Gianfranco Spizzirri & Giuseppe Cirillo (Editors) December 2016, John Wiley & Sons The book aims to cover all the analytical aspects of the food quality and safety assessment.

For this purpose, the volume describes the most relevant techniques employed for the determination of the major food components (e.g. protein, polysaccharides, lipids, vitamins, etc.), with peculiar attention to the recent development in the field. Furthermore, the evaluation of the risk associated with food consumption is performed by exploring the recent advances in the detection of the key food contaminants (biogenic amines, pesticides, toxins, etc.). Read more…

Chemical Analysis of Non-antimicrobial Veterinary Drug Residues in Food Jack F. Kay, James D. MacNeil, Jian Wang (Editors) November 2016, John Wiley & Sons Provides a single-source reference for readers interested in the development of analytical methods for analyzing non-antimicrobial veterinary drug residues in food; comprehensive set of information in the area of consumer food safety and international trade; analytical quality control and quality assurance, measurement uncertainty, screening and confirmatory methods; nanotechnology and aptamer-based assays covering current and potential applications for non-antimicrobial veterinary drugs; guidance for analysis of banned drugs including... Read more…

Handbook of Food Analysis Instruments Semih Otles (Editor) CRC Press, 2016

This book discusses how to apply proper methods and use increasingly sophisticated instruments. It begins with information relevant to all techniques, including calibration, standard addition, internal standards, selectivity, accuracy, precision, detection limit, quantification limit, range, robustness, speed, and convenience. Each subsequent chapter focuses on a specific type of instrument and includes a description of the information the technique can provide, a simple explanation of how it works, and examples of its application. Read more…

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Author's Guidelines

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The Brazilian Journal of Analytical Chemistry (BrJAC) is a peer-reviewed scientific journal intended for professionals and institutions acting mainly in all branches of Analytical Chemistry. BrJAC is an open access journal which does not charge authors an article processing fee.

Scope BrJAC is dedicated to professionals involved in science, technology and innovation projects in the area of analytical chemistry at universities, research centers and in industry.

About this journal BrJAC publishes original, unpublished scientific articles and technical notes that are peer reviewed in the double-blind way. In addition, it publishes reviews, interviews, points of view, letters, sponsor reports, and features related to analytical chemistry.

BrJAC's review process begins with an initial screening of the manuscripts by the editor-in-chief, who evaluates the adequacy of the study to the journal scope. Manuscripts accepted in this screening are then forwarded to at least two referees indicated by the editors. As evaluation criteria, the referees will employ originality, scientific quality and contribution to knowledge in the field of Analytical Chemistry, the theoretical foundation and bibliography, the presentation of relevant and consistent results, compliance to the journal's guidelines, and the clarity of writing and presentation.

Brief description of the BrJAC sections · Articles: Full descriptions of an original research finding in Analytical Chemistry. Manuscripts submitted for publication as articles, either from universities, research centers, industry or any other public or private institution, cannot have been previously published or be currently submitted for publication in another journal. Articles undergo double-blind full peer review. · Reviews: Articles on well-established subjects, including a critical analysis of the bibliographic references and conclusions. Manuscripts submitted for publication as reviews must be original and unpublished, and undergo double-blind full peer review. · Technical Notes: Concise descriptions of a development in analytical method, new technique, procedure or equipment falling within the scope of BrJAC. Technical notes also undergo double-blind full peer review.

The title of the manuscript submitted for technical note must be preceded by the words "Technical note". · Sponsor Reports: Concise descriptions of technical studies not submitted for review by referees. Sponsor responsibility documents. · Letters: Discussions, comments, suggestions on issues related to Analytical Chemistry, and consultations to authors. Letters are welcome and will be published at the discretion of the editor-in-chief. · Points of view: The expression of a personal opinion on some relevant subject in Analytical Chemistry. · Interviews: Renowned chemist researchers are invited to talk with BrJAC about their expertise and experience in Analytical Chemistry. · Features: A feature article gives to the reader a more in-depth view of a topic, a person or opinion of acknowledged interest for Analytical Chemistry.

Manuscript preparation (download a template on the BrJAC website) The manuscript submitted to BrJAC must be written in English and should be as clear and succinct as possible. It must include a title, an abstract, a graphical abstract, keywords, and the following sections: Introduction, Methods, Results and Discussion, Conclusion, and References. Because the manuscripts are subjected to double-blind review, they must NOT contain the authors' names, affiliations, or acknowledgments. The manuscript must be typed in Arial font size 11 pt., and the lines numbered consecutively and double-spaced throughout the text, except in the figure captions, titles of tables and references.

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Author's Guidelines The manuscript title should be short, clear and succinct, and a subtitle may be used, if needed. The abstract should include the objective of the study, essential information about the methods, the main results and conclusions. Then, three to five keywords must be indicated. The section titles should be typed in bold and subsections in italics. Graphics and tables must be numbered according to their citation in the text, and should appear close to the discussion about them. For figures use Arabic numbers, and for tables use Roman numbers. The captions for the figures must appear below the graphic; for the tables, above. The same result should not be presented by more than one illustration. For figures, graphs, diagrams, tables, etc. identical to others previously published in the literature, the author must ask for permission for publication from the company or scientific society holding the copyrights, and send this permission to the BrJAC editor-in-chief with the final version of the manuscript.

The chemical nomenclature should conform to the rules of the International Union of Pure and Applied Chemistry (IUPAC) and Chemical Abstracts Service. It is recommended that, whenever possible, authors follow the International System of Units, the International Vocabulary of Metrology (VIM) and the NIST General Table of Units of Measurement. Abbreviations are not recommended except those recognized by the International Bureau of Weights and Measures or those recorded and established in scientific publications. If the abbreviations are numerous and relevant, place their definitions in a separate section (Glossary).

The manuscript must include only the consulted references, numbered according to their citation in the text, with numbers in square brackets. It is not recommended to mention several references with identical statements - select the author who demonstrated them. It is recommended that references older than 5 (five) years be avoided, except in relevant cases. Include references that are accessible to readers.

References should be thoroughly checked for errors before submission.

Examples of reference formatting Journals 1. Cochran, R.E.; Dongari, N.; Jeong, H.; Beránek, J.; Haddadi, S.; Shipp, J.; Kubátová, A. Anal.

Chim. Acta, 2012, 740, pp 93-103.

The titles of journals must be abbreviated as defined by the Chemical Abstracts Service Source Index (CASSI). 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-drug-substanceheadspace-gc-flame-ionization-detection [Accessed 10 November 2015].

Books 3. Burgot, J.-L. Ionic Equilibria in Analytical Chemistry. Springer Science & Business Media, New York, 2012, Chapter 11, p 181.

4. Griffiths, W.J.; Ogundare, M.; Meljon, A.; Wang, Y. Mass Spectrometry for Steroid Analysis. In: Mike, S.L. (Ed.). Mass Spectrometry Handbook, v. 7 of Wiley Series on Pharmaceutical Science and Biotechnology: Practices, Applications and Methods. John Wiley & Sons, Hoboken, N.J., 2012, pp 297-338.

Standard methods 5. International Organization for Standardization. ISO 26603. Plastics — Aromatic isocyanates for use in the production of polyurethanes — Determination of total chlorine. Geneva, CH: ISO, 2017.

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. 71

Author's Guidelines 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 Two different PDF files, as described below, must be sent online through the website www.brjac.com.br I. A cover letter addressed to the editor-in-chief with the full manuscript title, the full names of the authors and their affiliations, the complete contact information of the corresponding author, including the ORCID iD, and the manuscript abstract. This letter must present why the manuscript is appropriate for publication in BrJAC, and contain a statement that the article has not been previously published and is not under consideration for publication elsewhere. The corresponding author must declare on behalf of all the authors of the manuscript any financial conflicts of interest or lack thereof. This statement should include all potential sources of bias such as affiliations, funding sources and financial or management relationships which may constitute a conflict of interest. When the manuscript belongs to more than one author, the corresponding author must also declare that all authors agree with publication in BrJAC. II.The manuscript file that must NOT mention the names of the authors or the place where the work was performed, but must include the title, abstract, keywords, and all sections of the work, including tables and figures, but excluding acknowledgments that will be included in the final paper upon completion of the review process.

A sponsor report should be sent as a Word file attached to a message to the email brjac@brjac.com.br Revised manuscript submission Based on the comments and suggestions of the reviewers and editors a revision of the manuscript may be requested to the authors. The revised manuscript submitted by the authors must contain the changes made in the manuscript clearly highlighted. A letter without any author's information must also be sent with each reviewer's comment items and a response to each item.

Copyright When submitting their manuscript for publication, the authors agree that the copyright will become the property of the Brazilian Journal of Analytical Chemistry, if and when accepted for publication. The copyright comprises exclusive rights of reproduction and distribution of the articles, including reprints, photographic reproductions, microfilms or any other reproductions similar in nature, including translations.

Final Considerations Whatever the nature of the submitted manuscript, it must be original in terms of methodology, information, interpretation or criticism.

As to the contents of published articles, the sole responsibility belongs to the authors, and Br. J. Anal.

Chem. and its editors, editorial board, employees and collaborators are fully 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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Jan. - Mar., 2017 Volume 4 Number 14