Practical examples on traceability, measurement uncertainty and validation in chemistry Vol 2 [VOL 2] 9789279189982

Table of contents :
Introduction
Guest editorial
About the authors
Abbreviations and acronyms
Chapter 1
Simultaneous measurement of the concentrationof retinol and α‑tocopherol in human serumby HPLC with UV and fluorimetric detection
Antonella Semeraro, Ilaria Altieri, Elena Amico di Meane, Sabrina Barbizzi,Maria Belli, Antonio Menditto, Marina Patriarca, Giancarlo Pistone, Michela Sega
Chapter 2
Measurement of the concentration of cyclamateconcentration in soft drinks by a high‑performanceliquid chromatographic method
Gordana Horvat, Snježana Marinčić
Chapter 3
Measurement of the concentration of arsenicin groundwater by flame atomic absorptionspectrometry (hydride technique)
Nada L. Lazić, Jelena Bebić
Chapter 4
Measurement of the mass fraction of sodiumchloride in milk products by Volhard’s method
Tidža Muhić-Šarac, Munir Mehović, Mustafa Memić
Chapter 5
Measurement of the concentration of total organiccarbon (TOC) in waste water
Brigita Tepuš, Marjana Simonič
Appendix 1
How to use this book
Appendix 2
TrainMiC Exercises (white pages)
Appendix 3
Briefing of the trainees on the example session

Citation preview

Practical Examples on

Traceability, Measurement Uncertainty and Validation in Chemistry Volume 2 Edited by Nineta Majcen, Philip Taylor, Tomas Martišius Guest Editors: Antonio Menditto, Marina Patriarca

Authors: Ilaria Altieri Sabrina Barbizzi Jelena Bebić Maria Belli Elena Amico di Meane Gordana Horvat Nada L. Lazić Snježana Marinčić Munir Mehović

Mustafa Memić Antonio Menditto Tidža Muhić-Šarac Marina Patriarca Giancarlo Pistone Michela Sega Antonella Semeraro Marjana Simonič Brigita Tepuš

EUR24688 EN - 2011

Practical Examples on

Traceability, Measurement Uncertainty and Validation in Chemistry Volume 2

Edited by: Nineta Majcen, Philip Taylor Tomas Martišius Guest Editors: Antonio Menditto, Marina Patriarca Authors: Ilaria Altieri Sabrina Barbizzi Jelena Bebić Maria Belli Elena Amico di Meane Gordana Horvat Nada L. Lazić Snježana Marinčić Munir Mehović Mustafa Memić Antonio Menditto Tidža Muhić Šarac

Marina Patriarca Giancarlo Pistone Michela Sega Antonella Semeraro Marjana Simonič Brigita Tepuš

The mission of the JRC-IRMM is to promote a common and reliable European measurement system in support of EU policies. European Commission Joint Research Centre Institute for Reference Materials and Measurements Contact information Address: Institute for Reference Materials and Measurements, European Commission, Joint Research Centre, Retieseweg 111, 2440 Geel, BELGIUM E-mail: jrc irmm [email protected] Tel.: +32 14571608 Fax: +32 14571863 http://irmm.jrc.ec.europa.eu/ http://www.jrc.ec.europa.eu/ Legal Notice Neither the European Commission nor any person acting on behalf of the Commission is responsible for the use which might be made of this publication. Europe Direct is a service to help you find answers to your questions about the European Union Freephone number (*): 00 800 6 7 8 9 10 11 (*) Certain mobile telephone operators do not allow access to 00 800 numbers or these calls may be billed.

More information on the European Union is available on the Internet (http://europa.eu). Cataloguing data can be found at the end of this publication. Luxembourg: Publications Office of the European Union, 2011

JRC 65988 EUR 24688 ISBN 978-92-79-18998-2 ISSN 1018-5593 doi: 10.2787/36024 © European Union, 2011 Reproduction is authorised provided the source is acknowledged Printed in Belgium

Table oF ConTenTs InTrodUCTIon ..................................................................................................................5 GUesT edITorIal ...............................................................................................................7 aboUT The aUThors .....................................................................................................11 abbreVIaTIons and aCronyMs ...............................................................................21 ChapTer 1..........................................................................................................................23 simultaneous measurement of the concentration of retinol and α-tocopherol in human serum by hplC with UV and fluorimetric detection Antonella Semeraro, Ilaria Altieri, Elena Amico di Meane, Sabrina Barbizzi, Maria Belli, Antonio Menditto, Marina Patriarca, Giancarlo Pistone

ChapTer 2..........................................................................................................................57 Measurement of the concentration of cyclamate in soft drinks by a high-performance liquid chromatographic method Gordana Horvat, Snježana Marinčić

ChapTer 3..........................................................................................................................95 Measurement of the concentration of arsenic in groundwater by flame atomic absorption spectrometry (hydride technique) Jelena Bebić, Nada L. Lazić

ChapTer 4....................................................................................................................... 131 Measurement of the mass fraction of sodium chloride in milk products by Volhard’s method Tidža Muhić‑Šarac, Munir Mehović, Mustafa Memić

ChapTer 5....................................................................................................................... 167 Measurement of the concentration of total organic carbon (ToC) in waste water Brigita Tepuš, Marjana Simonič

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appendIx 1 ..................................................................................................................... 227 how to use this book appendIx 2 ..................................................................................................................... 233 TrainMiC exercises (white pages) appendIx 3 ..................................................................................................................... 249 briefing of the trainees on the example session

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Introduction 的汉语意思 Learning is like rowing upstream: not to advance is to drop back. (approximate translation of a Chinese proverb)

Chemical and bio‑analytical measurements are omnipresent and often very important in our society. Just think of the quality of the food we eat, the air we breathe … the role of these measurements in healthcare, in trade and in research. In all these cases, people strive to get reliable data. There is an international standard for assuring the quality of measurement data, namely ISO/IEC 17025:2005 (General requirements for the competence of testing and calibration laboratories). The standard contains particular management as well as technical requirements. These technical requirements are linked to the science behind these measurements, meaning that metrological issues such as traceability, uncertainty and validation are at the heart of this. In order to provide life long learning in this area, the TrainMiC® programme (http:// www.trainmic.org) was conceived in 2001 by the Institute for Reference Materials and Measurements of the European Commission’s Joint Research Centre. Firstly, it addressed the need arising in those countries wanting to become members of the European Union (EU) at that time. Rather than approaching such training in an anecdotal way and organising ad hoc events, a programme was set up — TrainMiC® — to create harmonised training material as well as to disseminate knowledge in the various countries via a network of authorised TrainMiC® trainers. Afterwards, the TrainMiC® programme spread to the rest of the EU and Europe’s largest Lifelong learning programme in this area was created. Up to the present, 20 national TrainMiC® teams have been set up and more than 6 000 experts had been trained all across Europe by the end of 2010. Trainers quickly realised the importance of having suitable examples for their training events and they soon realised that creating examples adapted to the various audiences is quite a labour intensive activity. For this reason, sharing such examples proved to be an attractive proposition. Today, examples are reviewed and then published primarily in an e‑collaboration environment only available to the authorised trainers. It was then decided to publish some of the examples in the format of a series of books: a first volume in 2010 with this book being the second volume.

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Practical examples of traceability, measurement uncertainty and validation in chemistry

Interestingly, one of the ways examples are generated is via a competition between the national teams which meet at the biannual TrainMiC® convention. In June 2006, this competition was won by the Bulgarian team (see the example in Chapter 1, Volume 1) and, in January 2009, it was the Italian team who won (this is the example in Chapter 1, Volume 2). Volume 1 contained five examples from the area of clinical, environmental, food, and material analysis. From the feedback we received, it seems that the book has inspired laboratory practitioners as to how to present their way of working during accreditation audits. It has also been used by teachers, for example within the Euromaster Measurement Science in Chemistry (http://www.msc‑euromaster.eu). This new volume contains five examples covering some new areas and produced by other authors. Nineta Majcen and Philip Taylor

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Guest editorial

Guest editorial Salute a TrainMiC®!

Most European regulations and directives require, for their implementation, results of analytical measurements. To ensure the uniform application of EU legislation across the Member States, the same quality of analytical results must be achieved. To this aim, education of practitioners is a key issue and, even more, the harmonisation across the EU of such education. In 2006, we came up with a novel idea: a harmonised platform across Europe for the interactive education of practitioners applying the concepts of metrology in analytical sciences to the tests they carry out every day. The TrainMiC® programme, born out of a project in support of the new accession countries, today provides the basis for a harmonised interpretation of the technical requirements of ISO/IEC 17025:2005 across the 27 EU Member States and beyond. The programme also provides an interactive platform through the implementation, by the European Commission’s JRC‑IRMM, of innovative IT (eRooms), allowing an ongoing interactive exchange of experiences, knowledge and discussion on emerging issues, of which the TrainMiC® authorised trainers are the key players. The Italian national team was, therefore, formed in 2006; public bodies, entrusted with responsibilities in the field of measurements in general (the National Institute for Research in Metrology, INRIM) joined forces with those in specific sectors, such as public health (the National Institute of Health, ISS), the environment (the National Institute for Environmental Research and Protection, ISPRA) and food control (the Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d’Aosta). At various levels, these parties were already carrying out activities aiming to improve the reliability of analytical results produced in Italy, each according to their area of competence. These activities included the promotion and dissemination of metrological concepts; production, certification and/or distribution of reference materials; organisation of inter‑laboratory comparisons; and training of laboratory staff, quality managers and managers. Coming together with the TrainMiC® idea provided the spark to bring our

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Practical examples of traceability, measurement uncertainty and validation in chemistry

competence and experience together, to fulfil our shared mission to promote further education in metrology applied to analytical sciences, within a European‑wide environment. Over four years (2006–10), nine TrainMiC® events were organised in Italy, all of them approved by the Italian Ministry of Health as part of the programme for the continuous education of staff providing services related to health. One of the key features of the TrainMiC® programme is to promote the use of national languages for training and, accordingly, TrainMiC® presentations and two examples were translated into Italian. Additional material was produced by the team to cover general aspects, such as the content of normative references for laboratories seeking accreditation to ISO/IEC 17025:2005 (ISO/IEC 17000:2004 (Conformity assessment — Vocabulary and general principles); ISO/IEC Guide 99:2007 (International vocabulary of metrology — Basic and general concepts and associated terms (VIM) Third edition)); the principles of metrological confirmation of equipment; and more specific issues such as the structure of the metrological system in Italy and alternative approaches to the estimate of measurement uncertainty. In total, 320 laboratory staff were trained including analysts, quality managers, metrology function managers and internal auditors, as well as end‑users of analytical data, spread across various areas of Italy (north, centre, south, islands). The training, which included learning evaluation on the request of the Italian Ministry of Health, was well received and successful for the vast majority of the participants. The main feedback from the participants was: ‘We need more!’ A key aspect of TrainMiC® is its focus on ‘learning by practicing’. The TrainMiC® examples are developed by the TrainMiC® national teams, based on real‑life experiences and according to a standardised model. Examples help the trainees, working together, to practice what they have learned and stimulate discussion and exchange of knowledge between trainees as well as between trainers and trainees. Our best achievement as a team is, therefore, the first of the five examples presented in this book, which shows, in a practical way and using real experimental data, how to plan a single‑laboratory validation of an analytical procedure in order to obtain all the necessary information to estimate the measurement uncertainty of the results. A lot of time and effort was put into this work by our team member Antonella Semeraro and we were pleased to see it acknowledged by the TrainMiC® community with the award of the TrainMiC® Cup for the best proposed example during the 2009 TrainMiC® Convention. The mascot in this book, who will guide you through the methodology and the five examples presented here, is Pinocchio, a well‑known Italian character, born out of the pen of the Italian writer Carlo Collodi in 1881. Pinocchio’s best‑known feature is that his nose grows when he tells lies. Analysts do not lie: but they may be mistaken, confused or contradicted if they do not have a strong basis to hold on to. So we chose Pinocchio’s story as a good example of how success is achieved by learning from one’s mistakes, with a little bit of advice from those who know (the talking crickets

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Guest editorial

— please don’t kill them straight away!), some help from the fairies (Always look around for help! Fairies? Well, they may also use the Internet nowadays!) and a lot of commitment to the professional code of conduct and practice. The pictures were created, on our suggestion, by Antoine Cesaroni, whose contribution is gratefully acknowledged. We hope you enjoy them! In 2011, at the same time as TrainMiC®’s 10th birthday, Italy celebrates 150 years of the birth of Italy as one country. The unification of the Italian territory under one flag was not an easy task to accomplish. Great effort was needed to bring together people of different origins, traditions, social organisations and even languages, but it was all worth it and, today, we are proud to celebrate the statesmen who led the change as well as the people that made it possible. In a somewhat similar way, TrainMiC® brings together people from different backgrounds, culture, history and languages, to develop a common understanding of metrology in chemistry across Europe. We would like to say: “Buon compleanno TrainMiC®!” Marina Patriarca and Antonio Menditto TrainMiC® National Team Leaders, Italy Acknowledgment The graphic illustrations in this book were created by Antoine Cesaroni on suggestions from the Italian Team Leaders at the Istituto Superiore di Sanità and his contribution is gratefully acknowledged.

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About the authors Introduction Philip Taylor Professor Philip Taylor completed his PhD in analytical chemistry at the University of Gent in 1986. He started his career as a research fellow for the Belgian Science Foundation, he then moved to the European R&D Centre of Proctor & Gamble in Brussels. Since 1990, he has worked at the Institute for Reference Materials and Measurements (Geel, Belgium), which is part of the European Commission’s Joint Research Centre. Professor Taylor has had many interests during his research career ranging from atomic spectrometry to mass spectrometry and isotopic measurements, with an emphasis on how to produce reliable results, and has produced some 200 research papers in areas ranging from fundamental constants to food, environmental and industrial reference materials. He enjoys making his activities in metrology relevant to the broader outside world, both within the Commission services as well as externally. He heads a unit specialising in technical assistance regarding quality infrastructure (metrology, accreditation) in support of EU legislation. He initiated and established the TrainMiC® programme as well as a joint university programme, the Euromaster Measurement Science in Chemistry. He also lectures on these topics at the University of Maribor in Slovenia and is the vice‑chair of the Eurachem working group on training and education. For his contributions, he got awarded by the Polish Chemical Society and University of Maribor.

Nineta Majcen Nineta Majcen started her career as a researcher at the University of Ljubljana (Slovenia) where she gained her PhD on the validation of newly developed methods and chemometrics. She continued her analytical work in quality control laboratories in industry before stepping into metrology activities at the national and European level. In metrology, she has mainly been involved in topics related to metrology in chemistry, issues related to metrological infrastructure and knowledge transfer activities. She also collaborates closely with accreditation and standardisation bodies and lectures as a guest lecturer at universities, postgraduate summer schools and other knowledge transfer events. She is the author of more than 200 bibliographic publications in both research and expert areas. Several international conferences, workshops, seminars and high‑level events have been organised under Nineta Majcen’s leadership: for example, the Eurachem workshop on proficiency testing (2006), EURAMET’s European metrology research programme launch event (2008), Quality for south‑eastern European countries (2008), the TrainMiC® Convention (2009), and the Measurement science in chemistry summer school (2009). Proactively contributing to the TrainMiC® programme since the beginning of the initiative in 2001, Nineta Majcen received special recognition in 2005 from EC JRC‑IRMM for

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Practical examples of traceability, measurement uncertainty and validation in chemistry

her contribution to the TrainMiC® programme. She is the Slovenian TrainMiC® team leader, a member of the TrainMiC® Management Board and chairs the TrainMiC® Editorial Board. She is the Slovenian representative at Eurachem and is a member of the Eurachem working group on training and education. She is currently working as the Secretary General of the European Association for Chemical and Molecular Sciences (EuCheMS), managing also policy issues in these areas.

Guest Editorial Antonio Menditto Antonio Menditto gained his degree in medicine and surgery at the University of Rome La Sapienza (Italy). He is a senior scientist at the Department of Public Veterinary Health and Food Safety of the Istituto Superiore di Sanità (Italian National Institute of Health) and is active mainly in the food and medical analysis fields. He is the author of more than 100 scientific publications and has been involved in the organisation and administration of more than 100 courses in the fields of laboratory accreditation, quality assurance, method validation, uncertainty of measurement and metrological verification of measuring equipment. Antonio Menditto has been involved in the organisation and development of external quality assessment schemes in clinical, environmental and occupational laboratory medicine and is a member of the Eurachem working group on proficiency testing. He is also a lead assessor for ISS ORL, the body involved in the accreditation of Italian laboratories in charge of official control of food products. Since 2006, Antonio Menditto has been an authorised TrainMiC® trainer and, jointly with Marina Patriarca, coordinated the TrainMiC® activities in Italy as national TrainMiC® team leader.

Marina Patriarca Marina Patriarca gained her PhD in chemistry from the University of Rome La Sapienza (Italy) and her MSc in medical sciences from the University of Glasgow (United Kingdom). She joined the Italian National Institute of Health (Istituto Superiore di Sanità) in 1981, where she still currently works as a senior research scientist. Her research activity has mainly been devoted to the application of atomic spectrometry and has involved the development and validation of analytical methods including the estimation of uncertainty of measurement; population surveys for risk factors, including environmental exposure to metals; studies on the metabolism of copper and nickel in humans; the development and organisation of external quality assessment schemes and the assessment and certification of reference materials. She is author of more than 80 papers and a member of the Atomic Spectrometry Updates Editorial Board.

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About the authors

Currently, she supports the quality system at her home institution by providing advice on metrology issues related to the implementation of the technical requirements of ISO/ IEC 17025:2005. Together with Enzo Ferrara, she represents Italy at Eurachem. Marina Patriarca has gained considerable experience in training practitioners by lecturing at more than 50 courses and seminars for the staff of public and private laboratories devoted to aspects of quality assurance, implementation of ISO standards in testing laboratories and the uncertainty of measurement. Recently, she has been involved in training activities for laboratory staff in developing countries. Since 2006, Marina Patriarca has been an authorised TrainMiC® trainer and, jointly with Antonio Menditto, coordinated the TrainMiC® activities in Italy as TrainMiC® team leader: she is also a member of the TrainMiC® Editorial Board.

Chapter 1 Antonella Semeraro Antonella Semeraro gained her masters degree in chemistry at the University of Rome La Sapienza (Italy). She continued her analytical work on biological markers at the Istituto Superiore di Sanità (Italian National Institute of Health) for three years. Then she spent six months at the Isotope Measurement Unit of the Institute for Reference Materials and Measurements as a member of the International Measurement Evaluation Programme (IMEP). As a trainee under supervision, she organised the inter‑laboratory comparison IMEP‑27 (levels of Cd, As and Pb in a mineral feedstuff). Antonella Semeraro is currently working as a research scientist at the Department of Public Veterinary Health and Food Safety of the Istituto Superiore di Sanità. Her scientific work is mainly in the field of analysis of trace elements in biological fluids by Inductively coupled plasma mass spectrometry (ICP‑MS). She is also involved in the organisation of programmes for external quality assessment in preventive medicine and food safety. The author of several scientific papers, Antonella Semeraro has also been involved in the organisation and administration of seven courses in the field of laboratory accreditation, quality assurance, method validation and metrological confirmation for measuring equipment. Since 2007, Antonella Semeraro has been an authorised TrainMiC® trainer and member of the Italian TrainMiC® team.

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Practical examples of traceability, measurement uncertainty and validation in chemistry

Ilaria Altieri Ilaria Altieri gained her masters degree in biology at the University of Rome La Sapienza in 1991: she is a specialist in biotechnology applications. Since 1990, Ilaria Altieri has worked at the Istituto Superiore di Sanità (Italian National Institute of Health) as a researcher in charge of developing analytical methods (chromatographic and radioimmulogical) to determine drugs and their metabolites in biological fluids to detect and monitor the activity and toxicity of anti‑epileptic, anti‑tumor and antipsycotic drugs in pharmacokinetic studies. Ilaria Altieri worked for five years on the batch release of blood derivatives following the implementation in Italy of Legislative Decree (22/4/1996). Since 2004, she has been working in the Food Safety and Veterinary Public Health Department where she is developing and applying new methodologies for the measurement of concentration of biomarkers (biochemical and molecular), indicators of risk and protective factors for the study of the effects of dietary components on human health, and effects of contaminants such as xenobiotics, residues and additives. The author of more than 40 scientific papers, Ilaria Altieri has been involved in the organisation of more than 20 courses in the field of laboratory accreditation, quality assurance, method validation and the metrological verification of measuring equipment and has also collaborated on the translation of some of the TrainMiC® material in to Italian.

Elena Amico di Meane Elena Amico di Meane gained her PhD in ‘Metrology: Science and Technique of Measurements’ at the Politecnico of Turin (Italy) in 2002, with an experimental thesis, on the ‘Study of methods for measurement of concentration of gaseous substances in conditions traceable to the measurement units of the International System’. The research was carried out at the Institute of Metrology ‘G. Colonnetti’ of the Italian National Council of Research (IMGC‑CNR). Afterwards, she continued to work, as a research scientist, at IMGC‑CNR and at the National Electrotechnical Institute ‘G. Ferraris’ (IEN). In 2006, both institutes merged into the National Institute of Metrological Research (INRIM) where she continues to be employed. Her field of interest is focused on metrology in chemistry applied in gas analysis. She has expertise in analytical techniques and the preparation of reference gas mixtures by the gravimetric method. Elena Amico di Meane is the author of more than 30 scientific papers and has contributed to the organisation of several training courses: she is also taking part in the activities of CCQM. Elena Amico di Meane joined the TrainMiC® programme in 2007 as a member of the Italian team of authorised TrainMiC® trainers.

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About the authors

Sabrina Barbizzi Sabrina Barbizzi gained her PhD in physics at the University of Bologna (Italy) and started her career at the Italian National Agency for New Technologies, Energy and the Environment. Since 2000, she has worked at ISPRA — Istituto Superiore per la Protezione e la Ricerca Ambientale in Rome (Italy) — in the field of environmental metrology. Sabrina Barbizzi is the author of some 40 scientific papers and reports. Her research interests include the sampling and laboratory analysis by energy dispersive X‑ray fluorescence of environmental matrices; production and characterisation of reference materials; evaluation of data of inter‑laboratory comparisons; evaluation of uncertainties associated with analytical methods and sampling phases; statistical and geostatistical processing of environmental data for the validation of methods and strategies of soil sampling. Sabrina Barbizzi joined the TrainMiC® programme in 2008 as a member of Italian team of authorised TrainMiC® trainers.

Maria Belli Maria Belli started her career in 1976 working on neutron activation analysis applied to environmental science. She is a physicist and until 2000 worked on environmental radioactivity and radio‑ecology. Since 2000, she has been the head of the Environmental Metrology Unit of the National Environmental Protection Institute of Italy (ISPRA). The unit she heads produces environmental reference materials and organises inter‑laboratory comparisons of environmental pollutants. Since 2009, the Environmental Metrology Unit has been accredited according to ISO/IEC 17025:2005 and ISO Guide 34:2000 (General requirements for the competence of reference material producers). Maria Belli is the author of about 150 scientific papers and joined the TrainMiC® programme in 2007 as a member of the Italian team of authorised TrainMiC® trainers.

Antonio Menditto (refer guest editorial)

Marina Patriarca (refer guest editorial)

Giancarlo Pistone Giancarlo Pistone gained his degree in veterinary medicine at the University of Turin (Italy). He is the head of the Quality Assurance Department, Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d’Aosta, and is also the Director of the

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Practical examples of traceability, measurement uncertainty and validation in chemistry

Cuneo Section of the Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d’Aosta. Giancarlo Pistone has more than 20 years experience in laboratory management and the training of staff members and veterinarians concerning validation and quality assurance in analytical chemistry. He is also a lead assessor for the Italian accreditation body in charge of accrediting laboratories for the official control of food products. He is the author of some 40 scientific publications and has been involved in the organisation of more than 50 courses in the field of laboratory accreditation, quality assurance, method validation and metrological verification of measuring equipment. Since 2007, Giancarlo Pistone has been an authorised TrainMiC® trainer and member of the Italian TrainMiC® team.

Michela Sega Michela Sega gained her masters degree in chemistry and PhD in analytical chemistry at the University of Turin (Italy). She has been working at the Italian National Institute of Metrological Research (INRIM) in Turin (Italy) since 1998. Michela Sega works in the field of metrology in chemistry, particularly in gas analysis and organic analysis. Her main activity is devoted to the realisation of primary gas mixtures with the gravimetric method and to the analysis of organic micro‑pollutants by means of Gas chromatography‑mass spectrometry (GC‑MS). Michela Sega closely collaborates with the Italian accreditation body for calibration laboratories (SIT) for amount of substance. She has active collaborations with Italian national bodies, such as the National Institute of Health and the Italian Environmental Protection Agency. She is the Italian contact person for the EURAMET Technical Committee of Metrology in Chemistry (TC‑MC) and is involved in the activities of the Consultative Committee for Amount of Substance — Metrology in Chemistry (CCQM). The author of more than 30 scientific papers, Michela Sega has been involved in the organisation of more than 20 courses in the field of quality assurance, method validation and metrological verification of measuring equipment. Since 2008, Michela Sega has been an authorised TrainMiC® trainer and member of the Italian TrainMiC® team. Currently, she is chairing the EURAMET Technical Committee on Metrology in Chemistry (TC‑MC).

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About the authors

Chapter 2 Gordana Horvat Gordana Horvat has been involved in analytical chemistry since she started working at the Institute of Public Health ‘Dr Andrija Štampar’ in Zagreb, Croatia, in 1975. Currently, she works as a head of laboratory for food additives. Gordana Horvat has a lot of experience in analytical chemistry gained while working in the field of water and food analysis using GC and HPLC techniques. Her area of expertise is the development and implementation of new analytical methods in the field of food testing using liquid chromatography. Currently, Gordana Horvat is engaged in the implementation of new analytical methods for the testing of food additives as a part of the harmonisation of Croatian national legislation to European Union legislation. Gordana Horvat lectures on food additives at the Faculty of Applied Health Studies.

Snježana Marinčić Snježana Marinčić works at the Institute of Public Health ‘Dr Andrija Štampar’ in Zagreb, Croatia, and holds the position of quality manager. As a quality manager, she is involved in QA/QC of measurements in the field of testing samples from environmental, food and commonly used objects. Snježana Marinčić’s long experience in analytical chemistry includes testing in the field of water examination, mainly wet chemistry, and the application of liquid chromatographic methods in the field of environmental and food analysis. Snježana Marinčić lectures and is a trainer on issues related to laboratory accreditation and metrology in chemistry such as measurement uncertainty evaluation and QA/QC measures. She actively collaborates with the Croatian Accreditation Agency, where she is a member of the working group in inter‑laboratory comparisons, and the Croatian Metrology Society, where she is a member of Management Board. Snježana Marinčić is a member of the TrainMiC® Editorial Board and is the national TrainMiC® team leader for Croatia.

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Practical examples of traceability, measurement uncertainty and validation in chemistry

Chapter 3 Jelena Bebić Jelena Bebić works at the Directorate of Measures and Precious Metals, Serbia, in the field of physico‑chemical measurements and reference materials. She gained her diploma in 2000 at the Faculty of Technology and Metallurgy, University of Belgrade (Serbia): her area of study was the antimicrobial activity of essential oils related to their composition. At present, she is finishing her PhD, studying natural antioxidants in plants. Jelena Bebić’s main interests are measurement methods and techniques in biotechnology (GC‑MS, UV‑VIS, GC‑FID, GC‑ECD) and measurement standards in the field of physico‑chemical quantities (density of liquids, refractometry). An important part of her work is knowledge transfer in QA/QC for analytical laboratories and the dissemination of knowledge in metrology in chemistry with a focus on measurement uncertainty. In 2007, Jelena Bebić joined the TrainMiC® as a member of Serbian team of authorised TrainMiC® trainers.

Nada L. Lazić Nada L. Lazić started her professional career at the research and development institute of the rubber shoe and tyre company Borovo Gumitrade D.O.O., Croatia, where she worked until 1987. Her main research topics were related to polymer engineering, the reinforcement of rubbers and methods of rubber testing. From 1987 to 1991, she worked within the same company as a head of the control quality department laboratory. In 1991, Nada Lazić joined the Institute of General and Physical Chemistry, Belgrade, Serbia, and is currently working in polymer science and the quality issues related to methods of water and industrial waste testing, as a research fellow and quality manager. In 2004, Nada Lazić gained her MSc at the Faculty of Technology and Metallurgy, University of Belgrade, Serbia, with work on the influence of silica filler structure and surface characteristics on the properties of rubber properties. Nada Lazić has published over 20 scientific papers and presented at numerous international and national conferences and since 2008 she has been an authorised TrainMiC® trainer and a member of the Serbian TrainMiC® team.

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About the authors

Chapter 4 Tidža Muhić‑Šarac Tidža Muhić‑Šarac started work as a researcher at the motor factory Famos, Sarajevo (Bosnia and Herzegovina). She finished her PhD study in 1999 at the Faculty of Science, Department of Chemistry, University of Sarajevo (Bosnia and Herzegovina) with her thesis ‘Extractability of amounts of metals (Fe, Mn, Cu and Zn) from soils in Bosnia and Herzegovina’. She has continued her analytical work in the quality control of metals, alloys, water, soils and food. Currently, Tidža Muhić‑Šarac is employed at the Faculty of Science, Department of Chemistry, University of Sarajevo (Bosnia and Herzegovina), as an assistant professor. A major part of her work is teaching analytical chemistry, environmental chemistry, quality in analytical chemistry and metrology in chemistry. Her research interests include experimental and theoretical studies of species metals from water and soil. Tidža Muhić‑Šarac is the TrainMiC® team leader for Bosnia and Herzegovina and since 2000 she has also been associated to BATA, the Institute of Accreditation BiH, as technical assessor and technical expert for measurement uncertainty issues.

Munir Mehović Munir Mehović finished his masters degree in chemical science in 2009 at the Faculty of Science, Department of Chemistry, the University of Sarajevo (Bosnia and Herzegovina). His masters thesis was related to inter‑laboratory collaboration and proficiency testing. He works at the University Džemal Bijedić, Mostar, in the Department of Chemistry at the Faculty of Education as a senior assistant in analytical chemistry. His main interest is quality management and quality assurance in testing analytical laboratories. He has published several papers in the field of environmental chemistry engineering and is about to start working on his doctoral dissertation. Munir Mehović joined the TrainMiC® programme in 2006.

Mustafa Memić Mustafa Memić began his professional career as a chemist at the Institute of the Centre of Development and Research of New Materials — Energoinvest — in Sarajevo (Bosnia and Herzegovina) in 1989. Since 1990, he has worked in the Department of Analytical Chemistry, Faculty of Science at the University of Sarajevo. He finished his PhD in 2007, with his thesis ‘Study of biodegradation levels of polycyclic aromatic hydrocarbons (PAHS) and chlorinated phenols by ligninilytic fungi using gas chromatography with mass spectrometry detection (GC/MS)’. His main research interests are in the area of trace metals and analysis with FAAS, ET‑AAS, HG‑AAS and UV/VIS spectrometry.

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Practical examples of traceability, measurement uncertainty and validation in chemistry

As assistant professor, he lectures on analytical chemistry, electro‑analytical chemistry, spectroscopic methods of analysis, analytical methods in forensic chemistry, and sensors and analysis. He has supervised more than 10 MSc and more than 20 BSc students. He has published results of his research work in over 20 scientific papers and presented them at numerous international conferences. He was actively involved in the project SIMCA — a scientific cooperation between research institutions for the study of airborne particles in important cities of the Adriatic area. Mustafa Memić is a member of the working group of BAS Technical Committees of the Institute for Standardisation of Bosnia and Herzegovina and is a member of the TrainMiC® team for Bosnia and Herzegovina.

Chapter 5 Brigita Tepuš Brigita Tepuš gained her PhD ‘Hybrid removal of atrazine using catalytic ozonisation and nitrate using ion exchange from drinking water’ at the University of Maribor (Slovenia) at the Faculty of Chemistry and Chemical Engineering and started her career at the Komunalno podjetje Ptuj d.d. in the area of domestic water treatment and analytical chemistry in 2001. Brigita Tepuš became the head of the laboratory for analytical chemistry in 2003 and now works in the field of validation and measurement uncertainty in analytical chemistry.

Marjana Simonič Marjana Simonič gained her PhD at the University of Maribor (Slovenia) and started her career at the same Faculty of Chemistry and Chemical Engineering, in the Laboratory for water treatment in 1992. Her main research interests are in the area of water treatment, membrane separation and water analyses using UV/VIS spectrometry, atomic spectrometry, HPLC, and other advanced instrumental techniques. She is the author and co‑author of more than 30 scientific papers. Marjana Simonič has been the head of the laboratory for water treatment since 2003. Her research group has accomplished a number of research and applied projects in the field of water treatment, wastewater treatment and analytical chemistry. As assistant professor, Marjana Simonič lectures at the University of Maribor, Faculty of Chemistry and Chemical Engineering in water treatment and chemistry and water analysis.

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About the authors

abbreviations and acronyms Eurachem

Network of organisations in Europe having the objective of establishing a system for the international traceability of chemical measurements and the promotion of good quality practices.

EURAMET

European Association of National Metrology Institutes

ILC

Inter‑laboratory comparisons

PT

Proficiency testing

QC

Quality control

RM

Reference materials

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Chapter 1

simultaneous measurement of the concentration of retinol and α-tocopherol in human serum by hplC with UV and fluorimetric detection Antonella Semeraro, Ilaria Altieri, Elena Amico di Meane, Sabrina Barbizzi, Maria Belli, Antonio Menditto, Marina Patriarca, Giancarlo Pistone, Michela Sega • • • •

TrainMiC® example summary form (‘blue page’) A short introduction to the analytical procedure (‘slides’) All the input needed to do the three exercises (‘yellow pages’) The solved exercises (‘green pages’) 23

Practical examples of traceability, measurement uncertainty and validation in chemistry

TrainMiC® example summary form

General information about the example

24

Measurand

Simultaneous measurement of concentration of retinol and α‑tocopherol in human serum by HPLC with UV and fluorimetric detection

Example No

Ex‑22

Authors of the example

Antonella Semeraro, Ilaria Altieri, Elena Amico di Meane, Sabrina Barbizzi, Maria Belli, Antonio Menditto, Marina Patriarca, Giancarlo Pistone, Michela Sega

Analytical procedure

Developed in‑house

Customer’s requirements

Working range, linearity, LoQ (from scientific reference)

Simultaneous measurement of the concentration of retinol and α‑tocopherol in human serum…

Attached files

3 — Green

2 — Yellow

1 — I

File No, type and name Ex‑22‑1‑I‑vitamines‑ serum‑HPLC‑2009‑ Ver1.ppt

Ex‑22‑2‑Y‑vitamines‑ serum‑HPLC‑2009‑ Ver1.doc

Ex‑22‑3‑G‑ vitamines‑serum‑ HPLC2009‑Ver1.doc

File attached Yes No

Content of the file About the analytical procedure: short introduction Description of the analytical procedure

ü

PART II

The customer’s requirements concerning the quality of the measurement result

ü

PART III

Validation of the measurement procedure — relevant equations and measurement data

ü

PART IV

Measurement uncertainty of the result — relevant equations and measurement data

ü

PART I

Establishing traceability in analytical chemistry

ü

PART II

Single laboratory validation of measurement procedures

ü

Building an uncertainty budget

ü

PART III

Addendum 1: By spreadsheet approach Addendum 2: By dedicated software

Given by the lecturer

ü

PART I

Remarks

Each participant receives their own copy to keep

You may also want to include the relevant certificates here.

ü ü

History of the example Version

Uploaded on

1

17 September 2009

Short description of the change

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Practical examples of traceability, measurement uncertainty and validation in chemistry

a short introduction to the analytical procedure

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Simultaneousmeasurement Simultaneous measurement oftheconcentration of the concentration ofretinolandα‑tocopherol of retinolinand human α‑tocopherol serumbyHPLCwith in human UVandfluorimetric serum…detection

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Practical examples of traceability, measurement uncertainty and validation in chemistry

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Simultaneousmeasurement Simultaneous measurement oftheconcentration of the concentration ofretinolandα‑tocopherol of retinolinand human α‑tocopherol serumbyHPLCwith in human UVandfluorimetric serum…detection

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Practical examples of traceability, measurement uncertainty and validation in chemistry

all the input needed to do the three exercises (yellow pages)

analytical procedure simultaneous measurement of the concentration of retinol and α-tocopherol in human serum by hplC with UV and fluorimetric detection

parT I ...................................................................................................................................31 description of the analytical procedure parT II .................................................................................................................................34 The customer’s requirements concerning the quality of the measurement result parT III ................................................................................................................................35 Validation of the measurement procedure — relevant equations and measurement data parT IV ................................................................................................................................38 Measurement uncertainty of the result — relevant equations and measurement data

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Simultaneous measurement of the concentration of retinol and α‑tocopherol in human serum…

PART I. Description of the analytical procedure

Aim Oxidative damage caused by free radicals plays an important role in the development of several degenerative pathologies. The vitamins retinol and α‑tocopherol are major components of the antioxidant system in humans, protecting cell membranes against peroxidation. Serum concentrations of retinol and α‑tocopherol are the best available biomarkers of their respective levels in the body. The aim of the analytical procedure is to quantify vitamins in serum to allow the investigation of the antioxidant status in relation to the development of degenerative pathologies.

Measurement principle Serum samples are extracted using a liquid‑liquid technique. Since retinol and α‑tocopherol are affected by the oxidising nature of air, ultraviolet light, high temperature and oxidant agents, t‑butyl‑hydroxytoluene (BHT) is added as a protective factor during the extraction step. Retinol and α‑tocopherol are separated on LC‑18 pre‑column and column by isocratic elution with methanol as the mobile phase at a flow rate of 1 mL min– 1. The effluent is monitored by measuring its absorbance at 292 nm, to quantify α‑tocopherol, and its fluorescence, at 340 nm in excitation and 520 nm in emission, to quantify retinol. To compensate for possible losses of analyte, two internal standards are used: retinyl acetate for retinol and α‑tocopheryl acetate for α‑tocopherol.

Equipment — — — — —

HPLC equipped with UV and fluorimetric detectors UV‑IS spectrophotometer Analytical balance Automatic pipettes Centrifuge

Reference materials Retinol alcohol (≥ 99.0 %), α‑tocopherol (≥ 98.0 %), retinyl acetate (~ 100 %), α‑tocopheryl acetate (≥ 97.0 %) were purchased from Fluka (Buchs/Schweiz, Switzerland). The last two compounds are used as internal standards. Stock solutions, at approximate concentrations of 100 mg L– 1 (retinol), 600 mg L– 1 (α‑tocopherol and retinyl acetate) and 1 000 mg L– 1 (α‑tocopheryl acetate) are prepared

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Practical examples of traceability, measurement uncertainty and validation in chemistry

in acetonitrile from weighed amounts of vitamins. Tocopherol is a slightly viscous pale yellow oil and for this reason it is difficult to weigh accurately; to overcome this problem, concentrations of retinol and α‑tocopherol in the stock solutions are estimated spectrophotometrically after dilution in ethanol on the basis of their molar absorption coefficient values. In addition, internal standard solutions (retinyl acetate (5.6 mg L– 1) and α‑tocopheryl acetate (210 mg L– 1)), to be added to the control and human serum samples, are prepared by dilution of the respective stock solution in acetonitrile. Stock solutions are stored in plastic containers covered with aluminium foil and kept at – 20 °C for up to three months. Three calibration materials are prepared to assess linearity by diluting the stock solutions in acetonitrile to the final concentrations of 0.2, 0.6 and 1.0 mg L– 1 for retinol, 5.0, 15.0 and 25.0 mg L– 1 for α‑tocopherol, 0.80 and 30.0 mg L– 1 for retinyl acetate and α‑tocopheryl acetate respectively. All working standard solutions are protected from light and kept at + 4 °C for not more than a week.

Reference materials for quality control Matrix‑matched control materials (CM) were prepared from a pool of human sera obtained from healthy subjects. Two portions (25 g each) of the pool, one unspiked and the other spiked with known amounts of the stock solutions of retinol and α‑tocopherol to the final added concentration of 0.34 mg L–1 and 12.6 mg L–1, respectively, were aliquoted into amber tubes (1.5 mL) and stored at – 80 °C.

Certified reference materials (CRM) The two‑level CRM ‘Fat‑soluble vitamins, carotenoids and cholesterol in human serum’ (Standard reference material SRM 968c) was purchased from the NIST (Gaithersburg, MD, USA).

Reagents All reagents are at least PA grade: — Phosphate saline buffer (PBS) — BHT — Butanol — Ethyl acetate — K2HPO4 — Methanol HPLC grade

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Simultaneous measurement of the concentration of retinol and α‑tocopherol in human serum…

Sample pretreatment Dilute 200 µL of serum with 300 µL of PBS into an amber tube. Add 50 µL of each internal standard solution followed after vortexing for 15 s by 250 µL of the butanol and ethyl acetate solution containing 5 g L– 1 of BHT and further vortexing for 60 s. Finally add 150 µL of the K2HPO4 saturated solution, vortex for 30 s, then centrifuge at 10 000 rpm for 10 min. The organic upper layer has to be transferred to a 1.5 mL amber tube and centrifuged again at 8 000 rpm for 5 min, then placed into an amber auto‑sampler vial and injected (20 µL) into the HPLC apparatus. The auto‑sampler is cooled to 8 °C.

Calibration In routine work, only one calibration standard (retinol 0.6 mg L– 1; α‑tocopherol 15 mg L– 1) is run at the beginning of the day. The daily measurement of the calibration standard is used to calculate sample concentrations from the following formula:

C = CSTD ×

where: CSTD

Areasample sample AreaSTDi

×

Std AreaSTDi × DF AreaStd

= Working standard concentration (0.6 mg L– 1 for retinol and 15 mg L– 1 for α‑tocopherol)

Areasample

= Peak area of sample

AreaStd

= Peak area of standard

sample AreaSTDi

= Peak area of internal standard for sample

Std AreaSTDi

= Peak area of internal standard for standard

DF (dilution factor) = 1.75

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Practical examples of traceability, measurement uncertainty and validation in chemistry

PART II. The customer’s requirements concerning quality of the measurement result The requirements for linearity, working range and limit of quantification (LoQ) are defined considering the ranges of values reported for healthy subjects in the scientific literature as follows: — working range and linearity:

—

0.19–0.92 mg L– 1 for retinol 3.1–22.4 mg L– 1 for α‑tocopherol, LoQ: 0.19 mg L– 1 for retinol 3.1 mg L– 1 for α‑tocopherol.

Desirable quality specifications for intermediate precision (I %), bias (B %) and total allowable error (TE %) can be defined based on the components of biological variability, namely, within and between subjects variation 1. A database of such information is available online (http://www.westgard.com). Performance targets defined in this way were: I % < 6.8 %, B % < 5.8 %, TE % = 17.1 % for retinol I % < 6.9 %, B % < 5.1 %, TE % = 16.5 % for α‑tocopherol. The requirement for TE % is intended as a requirement for expended measurement uncertainty.

1

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Fraser, C. G., Scandanavian Journal of Clinical Laboratory Investigation, 59 (1999), 487.

Simultaneous measurement of the concentration of retinol and α‑tocopherol in human serum…

parT III. Validation of the measurement procedure — relevant equations and measurement data Linearity Calibration curves were studied in the following concentration ranges: 0-1.0 mg L– 1 for retinol and 0-20.0 mg L– 1 for α‑tocopherol. Twelve standard curves were analysed and the determination coefficients were always greater than 0.998 for retinol and 0.996 for α‑tocopherol. In addition, linearity was verified by visual inspection and residual analysis.

Limit of detection and quantification Since retinol and α‑tocopherol are always present in serum, a matrix blank was simulated by a fourfold diluted serum from normal subjects and analysed 11 times under repeatability conditions. The results of the 11 measurements under repeatability conditions are shown in Table 1. Table 1: Results under repeatability conditions Measurement

Retinol mg L– 1

α‑tocopherol mg L– 1

1 2 3 4 5 6 7 8 9 10 11

0.154 0.140 0.141 0.140 0.146 0.152 0.147 0.153 0.147 0.145 0.144

1.651 1.691 1.731 1.609 1.545 1.645 1.581 1.715 1.725 1.427 1.652

Precision Repeatability and intermediate precision Two different CMs near the extremes of the physiological range were analysed 10 times in the same analytical session. The same samples were reanalysed in different analytical sessions, over a period of three months to evaluate intermediate precision. The means, the standard deviations and the relative standard deviation (RSD %) are shown in Table 2.

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Practical examples of traceability, measurement uncertainty and validation in chemistry

Table 2: Repeatability and intermediate precision data α‑tocopherol (mg L– 1)

Retinol (mg L– 1) CM 1

CM 2

CM 1

CM 2

Mean

0.53

0.88

4.0

14.9

SD

0.01

0.03

0.1

0.3

RSD %

2.7 %

3.3 %

2.9 %

2.2 %

n

10

10

10

10

Intermediate

Mean

0.53

0.85

4.2

15.7

precision

SD

0.03

0.03

0.1

0.4

RSD %

4.6 %

3.6 %

3.2 %

2.4 %

n

24

26

25

26

Repeatability

Dependency of repeatability and intermediate precision on concentration was tested by means of test F, with critic F value of F9.9 = 3.18, F23.25 = 2.02 and F24.25 = 1.96. Trueness Trueness was studied by analysing, under repeatability conditions, the NIST SRM 968c (n = 5) consisting of two vials at different levels of concentration. In order to verify the absence of systematic errors, a significance test was applied (with t = 2.78 (n - 1 = 5 - 1; p = 0.05)) using the ratios shown in the following formula: 1 − Rm ≤t u '( Rm)

where: Rm is the ratio between the mean measured value and the certified value and u(Rm) is the uncertainty associated with the bias estimate, calculated as follows:

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Simultaneous measurement of the concentration of retinol and α‑tocopherol in human serum…

where: u(Cr)

= Standard uncertainty calculated from the expanded uncertainty associated to the certified value divided by the stated coverage factor k (k = 2);

u '( Rm) = Cr RSD n

  u(C )  2  RSD  2  r     +  n     Cr 

= Certified value = Relative standard deviation obtained for replicate measurements of the CRM = Number of repeated measurements of the CRM

Compound

α‑tocopherol (mg L– 1)

Retinol (mg L– 1) CRM 1

CRM 2

CRM 1

CRM 2

Cr ± U (Cr)

0.841 ± 0.027

0.484 ± 0.012

7.47 ± 0.47

16.79 ± 0.76

Mean

0.83

0.49

7.5

16.9

SD

0.01

0.02

0.2

0.3

RSD %

1.2

4.0

2.1

1.9

N

5

5

5

5

Rm

0.987

1.012

1.004

1.006

u’(Rm)

0.017

0.022

0.033

0.024

|1‑Rm| /u(Rm)

0.764

0.545

0.121

0.250

U(Cr) = expanded uncertainty (coverage factor = 2 for a confidence level of 95 %).

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Practical examples of traceability, measurement uncertainty and validation in chemistry

PART IV. Measurement uncertainty of the result: relevant equations and measurement data Retinol Input quantity

Value

Units

Remarks

CSTD

0.60

mg L

Calibration standard concentration

AreaStd

859 007.8

AU

Peak area of standard

Areasample

468 383.53

AU

Peak area of sample

Area

522 950.67

AU

Peak area of internal standard in the calibration standard

sample AreaSTDi

535 940.5

AU

Peak area of internal standard in the sample

DF

1.75

—

Dilution factor

Input quantity

Value

Units

Remarks

CSTD

14.83

mg L

Calibration standard concentration

AreaStd

102 256.8

AU

Peak area of standard

Areasample

23 652.8

AU

Peak area of sample

Std AreaSTDi

75 853.2

AU

Peak area of internal standard in the calibration standard

sample AreaSTDi

72 136.4

AU

Peak area of internal standard in the sample

DF

1.75

//

Dilution factor

Std STDi

– 1

α‑tocopherol

– 1

Relevant equations

1. Combined relative standard uncertainty of the result

u(C ) = C

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u 'c ( P ) + u 'c ( Rm ) + u ' ( x ) 2

2

2

where: u 'c (P)

= The best estimate of precision

u 'c (Rm)

= The uncertainty contribution associated with the bias estimate

u 'c (x)

= Any other relevant contribution to uncertainty (e.g. method robustness, sample variability)

Simultaneous measurement of the concentration of retinol and α‑tocopherol in human serum…

2. Best estimate of precision contribution, expressed as relative SD u 'c ( P ) =

( n1 − 1)( RSD1 )2 + ( n1 − 1) +

+ ( ni − 1)( RSDi )2 + ( ni − 1)

This equation is valid when precision is independent on concentration.

3. Uncertainty of the bias estimate u 'c ( Rm) =

u 'i ( Rm) =

where: u(Cr) Cr RSD n i

u '1 ( Rm)2 +

+ u 'i ( Rm)2 i

  u(C )  2  RSD  2  r     +  n     Cr 

= Standard uncertainty calculated from the expanded uncertainty associated to the certified value divided by the stated coverage factor k (k = 2) = Certified value = Relative standard deviation obtained for replicate measurements of the CRM = Number of repeated measurements of the CRM = Number of analysed CRM

4.  Other contributions to uncertainty, u ' ( x ) — relative standard uncertainty of this contribution Other contributions such as sample variability or robustness were not taken into account at this stage.

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Practical examples of traceability, measurement uncertainty and validation in chemistry

The solved exercises (green pages)

TrainMiC® exercises Analytical procedure Simultaneous measurement of the concentration of retinol and a‑tocopherol in human serum by HPLC with UV and fluorimetric detection

exerCIse 1: establishing traceability in analytical chemistry exerCIse 2: single laboratory validation of measurement procedures Part I: General issues Part II: Parameters to be validated Part III: Some calculations and conclusions exerCIse 3: building an uncertainty budget

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Simultaneous measurement of the concentration of retinol and α‑tocopherol in human serum…

ESTABLISHING TRACEABILITY IN ANALYTICAL CHEMISTRY

1. Specifying the analyte and measurand Analyte

Retinol and α‑tocopherol

Measurand

Concentration of retinol and α‑tocopherol in human serum in mg L– 1

Units

mg L– 1

2. Choosing a suitable measurement procedure with associated model equation Measurement procedure

The analytes are isolated from the serum samples by extraction with a mixture of butanol and ethyl acetate 1:1 (v/v). The extract is analysed by HPLC with UV and fluorescence detection. Separation is accomplished using LC‑18 pre‑column and column by isocratic elution with methanol at a flow rate of 1 mL min– 1.

Type of calibration

Standard curve

Standard addition

Internal standard

Model equation: C = CSTD ×

where: CSTD

Areasample sample AreaSTDi

×

Std AreaSTDi × DF AreaStd

Areasample AreaStd

= Calibration standard concentration (0.6 mg L– 1 for retinol, 15 mg L– 1 for α‑tocopherol) = Peak area of sample = Peak area of standard

sample AreaSTDi

= Peak area of internal standard for sample

Std AreaSTDi

= Peak area of internal standard for standard

DF

= Dilution factor

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Practical examples of traceability, measurement uncertainty and validation in chemistry

3. List the input quantities according to their influence on the uncertainty of the result of the measurement (first the most important ones): at this point, your judgement should be based on your previous experience only. Area sample

1

STD : extraction efficiency, separation efficiency, UV and fluorimetric Areasample and quantification, repeatability, vitamins degradation

2

Std Areastandard and AreaSTDi : separation efficiency, UV and fluorimetric quantification, repeatability, vitamins degradation

3 4 5

CSTD: Absorbance (spectroscopic measurement of standard concentrations), Volume (necessary dilutions to obtain calibration standards) DF (Vf /Vi): pipette calibration, repeatability, temperature

4. List the reference standards needed and state the information regarding traceability of the reference value For the analyte Name/chemical formula/producer

Name/chemical formula/producer

Retinol alcohol (≥ 99.0 %), retinyl acetate (~ 100 %), Fluka (Buchs/Schweiz, Switzerland) The standard content is evaluated considering the UV absorbance of the standard solution. Traceability is based on the calibration of the UV spectrophotometer and the molar absorption coefficient value, α‑tocopherol (≥ 98.0 %), α‑tocopheryl acetate (≥ 97.0 %) Fluka (Buch/Schweiz, Switzerland) The standard content is evaluated considering the UV absorbance of the standard solution. Traceability is based on the calibration of the UV spectrophotometer and the molar absorption coefficient value,

Name/chemical formula/producer

For the other input quantities

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1

Quantity/equipment/calibration (e.g. mass/balance/calibrated by NMI, U  = xx (k = 2))

Volume/automated pipettes/calibrated by an accredited centre

2

Quantity/equipment/calibration

Volume/volumetric flasks/Class A

3

Quantity/equipment/calibration

Absorbance/UV‑VIS spectrophotometer/certified filters and solutions

4

Quantity/equipment/calibration

UV detection‑relative measurement. Fl intensity/detector/ relative measurement, not directly part of the traceability chain.

Simultaneous measurement of the concentration of retinol and α‑tocopherol in human serum…

5. Estimating uncertainty associated with the measurement Are all important parameters included in the model equation?

Yes

No 

Other important parameters:

6. How would you prove traceability of your result? 1

Analysing certified reference materials: CRM: ‘Fat‑soluble vitamins, carotenoids and cholesterol in human serum’ (Standard reference material SRM 968c), NIST (Gaithersburg, MD, USA).

2

Using traceable calibrated glassware and instrumentation

3

7. Any other comments, questions …

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Practical examples of traceability, measurement uncertainty and validation in chemistry

SINGLE LABORATORY VALIDATION OF MEASUREMENT PROCEDURES parT I: General IssUes 1. Specify the measurement procedure, analyte, measurand and units The measurement procedure

The analytes are isolated from the serum samples by extraction with a mixture of butanol and ethyl acetate 1:1 (v/v). The extract is analysed by HPLC with UV and fluorescence detection. Separation is accomplished using LC‑18 pre‑column and column by isoctratic elution with methanol at a flow rate of 1 mL min– 1.

Analyte

Retinol and α‑tocopherol

The measurand

Concentration of retinol and α‑tocopherol in human serum in mg L– 1

Units

mg L– 1

2. Specify the scope Matrix

Serum

Measuring range

Retinol: 0.05–1.0 mg L– 1 α‑tocopherol: 0.95–23.0 mg L– 1

3. Requirements of the measurement procedure Intended use of the results

Assessment of vitamin status in population groups Parameters to be validated

Value requested by the customer

LOD LoQ Mark the customer’s requirements and give their values

Repeatability Within‑lab reproducibility Trueness Measurement uncertainty Other — state

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Retinol: