Analytical techniques for clinical chemistry methods and applications

Marı´a del Carmen Barciela Alonso, Trace Elements, Spectroscopy and SpeciationGroup, Department of Analytical Chemistry, Nutrition and Bromatology, Faculty of Chemistry, University of Sa

TECHNIQUES FOR CLINICAL CHEMISTRY

Copyright Ó 2012 by John Wiley & Sons, Inc All rights reserved

Published by John Wiley & Sons, Inc., Hoboken, NJ

Published simultaneously in Canada

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Library of Congress Cataloging-in-Publication Data:

Analytical techniques for clinical chemistry : methods and applications /

edited by Sergio Caroli, Gyula Za´ray.

ISBN: 9780470445273

In memory of Karoly Zimmer, our beloved friend Karcsi, who always stimulated us to

do well what we had to do, taught us that work can be a source of real fun, and lives

forever in our hearts

Sergio Caroli and Gyula Zaray

1 Good Clinical Practice Principles: Legal Background

Umberto Filibeck, Angela Del Vecchio, and Fabrizio Galliccia

1.2.2 WHO Guidelines for Good Clinical Practice

1.2.3 WHO Handbook for Good Clinical Research

1.4.1 EU 2007 Conference on the Implementation and

Applicability in the European Union of Legislation on

1.4.2 Directives 2001/20/EC, 2005/28/EC, and Good Clinical

1.5 Good Clinical Practice and Bioequivalence Trials: GCP

1.5.3 EMA Reflection Paper for Applicants Who Want

to Submit Bioequivalence Performed Outside the

1.5.4 Good Clinical Practice Bioequivalence Inspections 16

1.6 Good Clinical Practice for Clinical Trials with Advanced

1.7 Good Clinical Practice and Clinical Trials in Developing

viii CONTENTS

3.3.2 Reference Methods and Materials 69

4 The Role and Significance of Reference Values in the

Pietro Apostoli and Maria Cristina Ricossa

and Florabel G Mullick

6.3 Methodology for Trace Metal Ion Analysis in Clinical,

6.4 Case Studies of Relevance to Research and Diagnosis on

Clinical Chemistry, Forensic Toxicology, and Chemical

6.4.1 Case Study No 1: Copper Levels in Placental

6.4.2 Case Study No 2: Cd, Fe, Se, and Zn in Prostate

6.4.3 Case Study No 3: Measurement of Dental

Implant Corrosion Products and Histological

6.4.4 Case Study No 4: Skin Pigmentation after

6.4.5 Case Study No 5: Analysis of U Isotope Ratios by

using ICP-MS for the Assessment of Natural U or

8 The Role of Analytical Chemistry in

8.3 The Role of Analytical Chemistry in Drug Research,

8.3.1 QC of Drugs 200

8.3.4 Contribution of Analytical Chemistry to

11.3 Element Analysis in Environmental and

Marı´a del Carmen Barciela Alonso

CONTENTS xiii

12.3.1 Precautions During Sampling and Contamination

13.2 Advantages and Limitations of Inductively Coupled

13.5 Human Biomonitoring by Inductively Coupled

13.5.5 Precious Metals: Silver, Gold, Iridium, Palladium,

14 Molybdenum in Biological Samples and Clinical Significance

Munehiro Yoshida

14.2 Analysis of Molybdenum in Biological Samples by

15.2.5 Arsenic Speciation 41415.2.6 Application of Arsenic Speciation Techniques

15.3.4 High-Performance Liquid Chromatography and

15.4.4 Significance of Speciation Analysis of Organo-Tin

Daniela Deriu and Franco Mazzei

17 Bioimaging of Metals and Proteomic Studies of Clinical Samples

by Laser Ablation Inductively Coupled Plasma Mass

17.3 Experimental Aspects of Imaging Laser Ablation

17.3.4 Bioimaging of Metals in Biological Tissues by

Laser Ablation Inductively Coupled Plasma

Uta Ceglarek, Georg Martin Fiedler, and Joachim Thiery

18.1.2 Tandem Mass Spectrometry in the Clinical

18.1.3 Pre-Analytical Aspects of Clinical Laboratory

18.1.4 Sample Preparation of Human Body Fluids for

Liquid Chromatography-Mass Spectrometry

18.2.3 Coupling of Liquid Chromatography with Mass

CONTENTS xvii

18.2.4 Development of Liquid Chromatography-Tandem

Mass Spectrometry Methods for Clinical Laboratory

18.3 Liquid Chromatography-Tandem Mass Spectrometry

18.3.1 Determination of Amino Acids and Acylcarnitines

19 Metabolomics Using UPLC/HPLC-Tandem Mass Spectrometry

20.2.2 Measurement of Ratios of Reduced/Oxidized

20.2.10 Ex Vivo Oxidizability of Low-Density Lipoproteins

21.3 Basic Equipment and Setup for X-Ray Fluorescence

21.3.5 Setup for Total Reflection X-Ray Fluorescence and

22 A New Tool Based on the Use of Stable Isotopes and Isotope

Pattern Deconvolution (IPD)-ICP-MS for

and Alfredo Sanz-Medel

22.5 Selenium Metabolism in Lactating Rats by Means of

22.6 Determination of Selenium in Urine, Faeces, Serum,

and Erythrocytes by Isotope Pattern Deconvolution

22.6.1 Determination of Endogenous and Exogenous

22.6.2 Determination of Endogenous and Exogenous

22.7 Quantitative Speciation of Selenium in Urine, Serum, and

Erythrocytes by High Performance Isotope Pattern Deconvolution

22.7.1 General 63722.7.2 High-Performance Isotope Pattern Deconvolution

Inductively Coupled Plasma Mass SpectrometryQuantification of Natural and Exogenous

22.7.3 Quantification of Endogenous (Natural) and

22.7.4 Quantification of Natural and Exogenous

Alessio Ceccarini, Fabio Di Francesco, Roger Fuoco,

Silvia Ghimenti, Massimo Onor, Sara Tabucchi,

and Maria Giovanna Trivella

23.7 Conclusions 677

Uwe Christians, Volker Schmitz, Jost Klawitter,

and Jelena Klawitter

24.4 Molecular Markers in Drug Development

xxii CONTENTS

The quality and reliability of data generated during the conduct of clinical trialsrepresent a very critical aspect in the development of pharmaceutical products Thelatter must meet all the regulatory and legislative requirements established at aninternational level in order to protect the health and well being of the patients exposed

to these new drugs Analytical techniques represent a very important aspect inproducing the supporting data required by clinical research protocols In this context,analytical work performed in research and control laboratories must comply withcurrent legislation and guidelines, especially with the requirements of the Interna-tional Conference on Harmonisation of Technical Requirements for Registration ofPharmaceuticals for Human Use This fact represents a real challenge in the conduct

of clinical investigations and, in particular, the development of appropriate analyticaltechniques, due to the fact that the actors in this field are faced with ever-changingregulations that attempt (but do not always succeed) to keep pace with the rapidtechnological advancements in developing instrumentation for use in research andcontrol laboratories

This multiauthored book aims at underlining the role played by analyticaltechniques in supporting and promoting research and control in the various fields

of clinical activity, starting from the very early stages of clinical research to theattainment of marketing authorizations as well as in practical applications The bookalso elicits the progress made in developing instrumentation that is fit-for-purpose

as well as to identify outstanding problems that deserve further investigation,investment, and improvement in both research and routine laboratories

The 25 chapters of this book have been written by prominent scientists and coverprimary issues which include three main parts Fundamentals, Selected Applications,and Future Trends The first area provides a survey of the current legal framework (inparticular the EC Directive 2005/28 of April 2005 on the principles of GoodLaboratory Practice), the major challenges of clinical investigations and the avail-ability of analytical techniques for research and routine work In this section the readerwill encounter topics such as uncertainty in clinical chemistry measurements, the roleand significance of reference values in the identification of trace elements from diet,sample collection, storage and pretreatment in clinical chemistry, metal toxicology inclinical, forensic and chemical pathology, elemental speciation in clinical sciences,and detection of drugs in biological fluids for antidoping control, which are discussed

in detail The book then goes on to illustrate the applicability of the most popular andsuccessful analytical techniques as well as the relevant quality systems and theirimplementation Here the reader can find information such as the applicability of

plasma-based techniques to biological monitoring, atomic spectrometry, tallic speciation, the clinical meaning of molybdenum, bioimaging of metals andproteomic studies of clinical samples by laser ablation inductively coupled plasmamass spectrometry, application of liquid chromatography combined with tandemmass spectrometry in clinical laboratory diagnostics, metabolomics using high-performance liquid chromatography-tandem mass spectrometry, biomarkers of stress

organome-in plasma and urorganome-ine, X-ray techniques organome-in medical research, and analytical examorganome-ina-tion of drugs in the forensic science laboratory The third part gives the reader a look atpromising innovative approaches and their possible exploitation, e.g., for breathanalysis, development of proteo-metabolic strategies and optimization of laboratorymedicine

examina-This book greatly benefits from the enthusiastic participation and support of allauthors who greatly collaborated with the editors and to whom the editors expresstheir sincere gratitude

VALENTINEANTHONYSFORZA

The first idea of a multiauthored book devoted to the role played by analyticalchemistry in fostering clinical research was conceived by the Editors some four yearsago during a lively conversation had in the aftermath of the publication by Wiley of

was well received by the readers, or perhaps the exciting atmosphere of the Hungariantavern where we were dining (not to speak of a bottle of excellent red wine which made

us rather loquacious), or perhaps—and most likely—the synergistic action of thesefactors altogether, that fertilized our minds and led us to plan a new book in the beliefthat it would meet the needs of the scientific community Greatly inspired by optimismand self-confidence (never out of place under such circumstances), the more wedebated this issue, the more the project became a fascinating challenge Deliberatelyminimizing all the difficulties that we knew by personal experience would thwart theprogress of the work and make our professional lives uneasy for quite a long period oftime, a list of key topics was promptly drafted and a tentative list of potential

Now that the book has finally reached completion in spite of an endless number oftechnical problems, delays, withdrawal of manuscripts, and all kinds of unexpectedevents, we wish to express our sincere gratitude to all contributing authors for theirvaluable competence, their willingness to cooperate at all stages of preparation oftheir chapters, and their infinite patience in tackling all our often complex, alwaystime-consuming, and certainly tedious requests Needless to say, we do hope that theywill be happy with the outcome of their unremitting efforts

The sponsorship of PerkinElmer, Inc to the making of this book is gratefullyacknowledged Without their generosity, constant support, and firm trust in ourproject, it would never be possible for us to accomplish it If the result of ourcommitment pleases them, this will also significantly add to our satisfaction

SERGIOCAROLI

GYULAZARAY

Mass Spectrometry, John Wiley & Sons, Inc., Hoboken, NJ, USA, 2007.

Marı´a del Carmen Barciela Alonso, Trace Elements, Spectroscopy and SpeciationGroup, Department of Analytical Chemistry, Nutrition and Bromatology, Faculty

of Chemistry, University of Santiago de Compostela, Spain

Pietro Apostoli, Department of Experimental and Applied Medicine, Section ofOccupational Health and Industrial Hygiene, University of Brescia, Brescia, ItalyPilar Bermejo Barrera, Trace Elements, Spectroscopy and Speciation Group,Department of Analytical Chemistry, Nutrition and Bromatology, Faculty ofChemistry, University of Santiago de Compostela, Spain

J Sabine Becker, Central Division of Analytical Chemistry, Forschungszentrum

J Susanne Becker, Aeropharm, Francois-Mitterrand-Allee 1, Rudolstadt, GermanyBjørn J Bolann, Laboratory of Clinical Biochemistry, Haukeland UniversityHospital, Helse Bergen HF, Bergen, Norway; and Institute of Medicine, University

of Bergen, Bergen, Norway

Alessio Ceccarini, Department of Chemistry and Industrial Chemistry, University ofPisa, Pisa, Italy

Uta Ceglarek, University Hospital Leipzig, Institute of Laboratory Medicine,Clinical Chemistry and Molecular Diagnostics, Leipzig, Germany

Stephen Ave., Silver Spring, MD, USA

Marcello Chiarotti, Institute of Forensic Medicine, Catholic University of theSacred Heart, Rome, Italy

Uwe Christians, iC42 Clinical Research & Development, Department of siology, University of Colorado Denver, Bioscience East, Aurora, CO, USA

Budapest Metropolis, Budapest, Hungary

Daniela Deriu, Department of Chemistry and Drug Technologies, La SapienzaUniversity, Piazzale Aldo Moro Roma, Italy

Fabio Di Francesco, Department of Chemistry and Industrial Chemistry, University

of Pisa, Pisa, Italy

Angela Del Vecchio, Italian Medicine Agency (Agenzia Italiana del Farmaco,AIFA), Rome, Italy

Maria Luisa Fernandez-Sanchez, Department of Physical and AnalyticalChemistry, University of Oviedo, Oviedo, Spain

Georg Martin Fiedler, Institute of Laboratory Medicine, Clinical Chemistry andMolecular Diagnostics, University Hospital Leipzig, Leipzig, Germany

Umberto Filibeck, Italian Medicine Agency (Agenzia Italiana del Farmaco, AIFA),Rome, Italy

Rossella Fioravanti, Department of Chemistry and Drug Technologies, Faculty ofPharmacy, La Sapienza University, Rome, Italy

Roger Fuoco, Department of Chemistry and Industrial Chemistry, University ofPisa, Pisa, Italy

Fabrizio Galliccia, Italian Medicine Agency (Agenzia Italiana del Farmaco, AIFA),Rome, Italy

Silvia Ghimenti, Department of Chemistry and Industrial Chemistry, University ofPisa, Pisa, Italy

Sandor G€or€og, Gedeon Richter Plc., Budapest, Hungary

Bin He, State Key Laboratory of Environmental Chemistry and Ecotoxicology,Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences,Beijing, P.R China

Peter Heitland, Medical Laboratory Bremen, Bremen, Germany

Hector Gonzalez Iglesias, Department of Physical and Analytical Chemistry,University of Oviedo, Oviedo, Spain

Guibin Jiang, State Key Laboratory of Environmental Chemistry and ogy, Research Center for Eco-Environmental Sciences, Chinese Academy ofSciences, Beijing, P.R China

Ecotoxicol-Jelena Klawitter, iC42 Clinical Research & Development, Department ofAnesthesiology, University of Colorado Denver, Bioscience East, Aurora, CO,USA

Jost Klawitter, iC42 Clinical Research & Development, Department of siology, University of Colorado Denver, Bioscience East, Aurora, CO, USA

Metropolis, Budapest, Hungary

Helmut D Ko¨ster, Medical Laboratory Bremen, Bremen, Germany

Willem Kulik, Academic Medical Center, University of Amsterdam, Laboratory

of Genetic Metabolic Diseases, Department of Clinical Chemistry, Amsterdam,The Netherlands

xxviii CONTRIBUTORS

Fedele Manna, Department of Chemistry and Drug Technologies, Faculty ofPharmacy, La Sapienza University, Rome, Italy

Franco Mazzei, La Sapienza University, Department of Chemistry and DrugTechnologies, Roma, Italy

Florabel G Mullick, Biophysical Toxicology, The Joint Pathology Center, 606Sitter Stephen Ave., Silver Spring, MD, USA

Sandor Nagy, Monash Medical Centre, Southern Cross Pathology Australia,Biochemistry Clayton, Victoria, Australia

Massimo Onor, Institute of Chemistry of Organometallic Compounds, UOS Pisa,CNR, Pisa, Italy

Department of Analytical Chemistry, Nutrition and Bromatology, Faculty ofChemistry, University of Santiago de Compostela, Spain

Maria Cristina Ricossa, Department of Experimental and Applied Medicine,Section of Occupational Health and Industrial Hygiene, University of Brescia,Brescia, Italy

Francesca Rossi, Department of Chemistry and Drug Technologies, Faculty ofPharmacy, La Sapienza University, Rome, Italy

Sabina Strano Rossi, Institute of Forensic Medicine, Catholic University of theSacred Heart, Rome, Italy

Alfredo Sanz-Medel, Department of Physical and Analytical Chemistry, University

of Oviedo, Oviedo, Spain

Volker Schmitz, iC42 Clinical Research & Development, Department of siology, University of Colorado Denver, Aurora, CO, USA

Anesthe-Valentine Anthony Sforza, Quality Management Associates, Anesthe-Valentine A Sforza &

C S.a.s., Via Volturno, 69-00042 Anzio, Rome, Italy

Ilse Steffan, Department of Analytical and Food Chemistry, University of Vienna,Vienna, Austria

Papasani V Subbaiah, Department of Medicine, University of Illinois at Chicago,Chicago, IL, USA

Jing Sun, State Key Laboratory of Environmental Chemistry and Ecotoxicology,Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences,Beijing, P.R China

Marit Sverresdotter Sylte, Laboratory of Clinical Biochemistry, HaukelandUniversity Hospital, Helse Bergen HF, Bergen, Norway

Imre Szaloki, Institute of Nuclear Techniques, Budapest University of Technologyand Economics, Budapest, Hungary

CONTRIBUTORS xxix

Norbert Szoboszlai, Department of Analytical Chemistry, E€otv€os Lorand sity, Budapest, Hungary

Univer-Sara Tabucchi, Department of Chemistry and Industrial Chemistry, University ofPisa, Pisa, Italy

Andrew Taylor, Department of Clinical Biochemistry, Royal Surrey CountyHospital, Guildford, UK; and Faculty of Health and Medical Sciences, University

of Surrey, Guildford, UK

Maria Giovanna Trivella, Institute of Clinical Physiology, CNR, Pisa, ItalyDouglas M Templeton, University of Toronto, Department of Laboratory Medicineand Pathobiology, 1 Kings College Circle, Toronto, ON, Canada

Joachim Thiery, University Hospital Leipzig, Institute of Laboratory Medicine,Clinical Chemistry and Molecular Diagnostics, Leipzig, Germany

Todor I Todorov, Crustal Geophysics and Geochemistry Science Center, USGeological Survey, Denver, CO, USA

Gijsbert B van der Voet, Gezondheidsraad Parnassusplein 5 - 2511 VX Den Haag,The Netherlands

Andre B P van Kuilenburg, Academic Medical Center, Laboratory of GeneticMetabolic Diseases, Department of Clinical Chemistry, University of Amsterdam,Amsterdam, The Netherlands

Goran Vujicic, Institute for Testing of Materials (IWM), Glattbrugg, SwitzerlandTore Wentzel-Larsen, Centre for Clinical Research, Haukeland University Hospi-tal, Bergen, Norway

Munehiro Yoshida, Department of Life Science and Biotechnology, Faculty ofChemistry, Materials and Bioengineering, Kansai University, Osaka, JapanChungang Yuan, State Key Laboratory of Environmental Chemistry and Ecotox-icology, Research Center for Eco-Environmental Sciences, Chinese Academy ofSciences, Beijing, P.R China; and School of Environmental Science & Engineering,North China Electric Power University, Hebei, P.R China

Budapest, Hungary; Cooperative Research Centre of Environmental Sciences,

Trace Element for UNESCO, Budapest, Hungary

xxx CONTRIBUTORS

FIGURE 2.2 Overall scheme of a GLP inspection performed at a TF by a National GLP MA.

FIGURE 2.3 Simplified cross-sectional view of a clean room for GMP activities 1, Air inlets;

2, decontaminated air; and 3, exhausts

Analytical Techniques for Clinical Chemistry: Methods and Applications, First Edition.

Edited by Sergio Caroli and Gyula Za´ray.

Ó 2012 John Wiley & Sons, Inc Published 2012 by John Wiley & Sons, Inc.

FIGURE 6.1 Case number 4 Photomicrograph demonstrating fine brown-black granularpigment both intracellular and scattered along the epidermal basement membrane (indicated bygreen arrows) Hemotoxylin & eosin-stained section (100 magnification).

FIGURE 17.2 Workflow of imaging LA-ICP-MS starting from sample preparation cutting of tissue sections of 20mm), MS measurements, data acquisition, evaluation, andquantification using laboratory prepared matrix-matched standards

(cryo-FIGURE 17.4 Rat brain experiments to study tumor growth Copper and P distribution in ratbrain tissues with a tumoral region as measured by LA-ICP-MS in comparison to autoradio-graph (on the top).

FIGURE 17.5 Images of the selected elements (C, Cu, Fe, K, Mg, Mn, P, S, and Zn) in a thinslice of rat brain tissue as obtained by LA-ICP-MS The LA-ICP-MS measurements wereperformed using the ICP-MS (Agilent 7500 CE coupled with the laser ablation systemNew Wave UP 266)

FIGURE 17.6 Images of selected metals (Cu, Fe, K, Mg, Mn, Ni, and Zn,) and two metals (C and P) in a slice from a rat brain with photothrombosis (on the upper left edge)measured by LA-ICP-QMS (Agilent 7500 CE coupled with the laser ablation system NewWave UP 266) (77).

non-FIGURE 17.7 Images of metals (Cu, Fe, and Zn) and the non-metal C in two different sections

of Parkinson’s mouse brain as measured by LA-ICP-MS (Agilent 7500 CE coupled with the laserablation system New Wave UP 266)

FIGURE 17.8 Imaging LA-ICP-MS of brain tissue combined with proteome studies viaprotein separation by 2D gel electrophoresis (See text for full caption.)

FIGURE 17.9 LA-ICP-QMS measurement on 2D native gel from bovine serum samplestained with Coomassie Distribution of63Cuþ,34S , and64Znþmeasured in 5 line scans of gel

is shown

FIGURE 17.10 Detection of Cu-, Pb-, and Zn-containing proteins on a 2D BN-PAGE gel (ratkidney water extract) by imaging of gel section using LA-ICP-MS.

Si(Li), PIN, SDD detector

Si reflector

Sample Reflector

Reflected beam Primary beam

X-ray fluorescent radiation

X-ray tube or

synchrotron beamline

FIGURE 21.7 Setup for TXRF and SR-TXRF measurements

Synchrotron storage ring

Polycapillary or mono capillary X-ray lens

Monochromators: Si(111) or Ni/C multilayer

FIGURE 21.8 Setup of synchrotron-based X-ray fluorescence scanning and tomography

Metabolites Proteins RNA DNA are released into urine

Histological Changes

Pathophysiological

Changes (GFR)

Apoptosis / Necrosis/ Repair/ Inflammation/

Fibrosis and Other Secondary Processes

Normal Epithelium

RECOVERY

RECOVERY INCOMPLETE

INJURY

RECOVERY INJURY

Functional impairment

Metabolite pattern changes in urine

Protein pattern changes in urine

Damage, cell death and repair

Genetic

Symptomatic phase

FIGURE 24.1 Time-dependency of kidney tubular epithelium injury and molecular markers

in urine (modified, based on references [11, 12]) After and during an injury, such as drugtoxicity, a disease process, or ischemia/reperfusion injury, cell function will be affected first.This may include absorption from and excretion into urine as well as cell metabolism Theresulting extent of urine metabolite pattern changes will depend on the intensity of the injuryand how many cells/tubuli are affected Depending on the type of injury (acute or chronic),sooner or later damage to the cells will lead to changes in protein patterns in urine So-calledrepair proteins will be formed and also the pattern of proteins excreted into urine may change(please see also Fig 17.5 and Table 24.7) While changes in metabolite patterns may be almostimmediate and occur within minutes, changes of intracellular protein expression and proteinconcentrations typically are slower and may take hours and even days The biochemical phase

of injury will progress toward the symptomatic phase, as increasing numbers of cells die bynecrosis and/or apoptosis These cells will release at least some of their contents such asmetabolites, proteins, RNA, and DNA into the urine (See text for full caption.)

Distal tubule Proximal tubule

PART I

EXPLORING FUNDAMENTALS

EC (GCP and Clinical Trials, CTs), and Directive 2005/28/EC (Detailed Guidance

on GCP)

European Directive 2001/20 on GCP and CTs has been widely criticized by a largeportion of the scientific community more directly involved in the promotion andmanagement of noncommercial academic CTs Since 2003 several scientists fromacademia highlighted through the international scientific literature the difficultiesinherent in the new EU regulation, in particular as regards GCP compliance andquality monitoring problems Such difficulties have also been acknowledged byDirective 2005/28 where, among others, it is stated that for academic CTs theapplication of certain GCP aspects may be unnecessary or guaranteed by other means.None of these documents oblige CTs to be in compliance with the GCP ICHGuideline (GCP-ICH) full text and details Rather, they prescribe that CTs be incompliance only with GCP principles and with GCP Guidelines laid down inDirective 2005/28 EC, this being less binding when compared to the GCP-ICHGuideline At the national level, EU Member States (MS) adopted different legislation

to implement the GCP obligations MS GCP Inspectorates generally act as if all GCPaspects were mandatory to verify the reliability of data reported by the CTs audited

At the international level, in particular in developing countries, where severalbioequivalence (BE) studies are conducted and the number of CTs is increasing, often

Analytical Techniques for Clinical Chemistry: Methods and Applications, First Edition.

Edited by Sergio Caroli and Gyula Za´ray.

Ó 2012 John Wiley & Sons, Inc Published 2012 by John Wiley & Sons, Inc.

3

neither specific aspects nor the principles of GCP are complied with In the case ofdata submitted to EU MS Regulatory Authorities (RAs) for a Marketing Authori-zation (MA) of a medicinal product, the CTs of which were performed outside the EU,Directive 2001/83 allows the MA to be granted only if the CTs are in compliance withthe ethical principles of GCP, although in practice only a few RAs assess that thisactually has been done.

Specific examples are given to illustrate the above issues and a number of keyaspects related to laboratory activities are reported and discussed in the frame ofdifferent international normative and guidelines on GCP

1.1 INTRODUCTION

Guidelines of the International Conference on Harmonization (ICH) on Good ClinicalPractice (GCP) In this regard, one European Union (EU) Guideline and three EUDirectives are in force at present, that is, the E6/CPMP/ICH/135/95 GCP Guide-line [1], Directive 2001/83/EC (Community Codex on Pharmaceuticals) [2], Direc-tive 2001/20/EC (GCP and Clinical Trials, CTs) [3], and Directive 2005/28/EC(Detailed Guidance on GCP) [4]

The introduction of GCP in EU is linked with Good Manufacturing Practice(GMP), particularly Annex 1, 13, and 16 (The rules governing medicinal products inthe European Union, EudraLex, Vol 4) [5] regarding directly or indirectly theproduction of Investigational Medicinal Product (IMP) and Directive 2003/94/EC

on GMP for Medicinal Products and IMP [6] At global level, besides ICH-GCP,WHO has also issued WHO GCP

The GCP legal background and applicability are discussed hereafter along with thedescription of related documents and implementation problems This legal frame-work is of paramount importance to attach credibility to the experimental informationobtained when carrying out clinical investigations, thus substantially contributing topreserving and improving human health

1.2 GOOD CLINICAL PRACTICE

1.2.1 ICH E6: Guidelines for Good Clinical Practice

ICH Guidelines for GCP [1] have been prepared by the ICH of Technical ments for Registration of Pharmaceuticals for Human Use, which is composed bythe Medicine Regulatory Agencies and members of the pharmaceutical industry of the

Require-EU, Japan, and the USA The WHO, Canada, and European ParliamentaryTechnology Assessment (EPTA) have observer status Since its creation in 1990,the ICH has issued 58 Tripartite Guidelines on issues related to its four main areas ofwork, namely quality, safety, efficacy, and multidisciplinary topics The process to

to EMA.

4 GOOD CLINICAL PRACTICE PRINCIPLES: LEGAL BACKGROUND AND APPLICABILITY

reach harmonization of technical requirements resulting from scientific progress goesalong with the process of keeping up-to-date the current guidelines, in order to ensurethat the harmonization process, so far achieved, is not lost Guidelines are adopted bythe Steering Committee and signed by the three regulatory parties to ICH However,guidelines become binding only when the regulatory bodies in the three regionsimplement them The objective of this guidance is to provide “international ethicaland scientific quality standards for designing, conducting, recording and reportingtrials that involve the participation of human subjects Compliance with this standardprovides public assurance that the rights, safety and well-being of trial subjects areprotected, consistent with the principles that have their origin in the Declaration ofHelsinki, and that the clinical trial data are credible.”

ICH E6 GCP Guideline is designed to set a unified standard for the ICH countries inorder to facilitate the mutual acceptance of clinical data by RAs in these jurisdictionsand speed up registration for market authorization of medicines Topics coveredinclude the composition of Ethics Committees/review boards, the responsibilities ofinvestigators and sponsors, provisions regarding trial protocols and protocol amend-ments, including treatment of data, informed consent, payment of subjects, insurance

in case of harm This guideline has been adopted by the EU in 1995 (updated version) [1]and largely transposed into their legislation by the United States [7] and Japan in 1997

ICH Good Clinical Practice Principles

The ICH-GCP Principles can be divided into three different categories:

(a) Principles to guarantee the “ethical aspects” of the CT, as follows

their origin in the Declaration of Helsinki and that are consistent with GCPand the applicable regulatory requirements

considerations and should prevail over interests of science and society A trialshould be initiated and continued only if the anticipated benefits justify therisks

CT participation

(b) Principles to guarantee the “technical-scientific aspects” of CT, namely

product should be adequate to support the proposed CT

should always be the responsibility of a qualified physician or, whenappropriate, of a qualified dentist

education, training, and experience to perform his/her respective task(s)

accordance with applicable Principles of GMP They should be used inaccordance with the approved protocol

GOOD CLINICAL PRACTICE 5

(c) Principles to guarantee the “quality and procedural aspects” of CT.

allows for its accurate reporting, interpretation, and verification

pro-tected, respecting the privacy and confidentiality rules in accordance with theapplicable regulatory requirements

prior Institutional Review Board (IRB)/Independent Ethics Committee (IEC)approval/favorable opinion

implemented

GCP Details in the Field of the Quality

ICH-GCP Guidelines describe in detail how the principles can be implemented.Principles related to the quality of the CT are crucial for all clinical investigations.Many paragraphs of GCP are related to quality principles Among others, thefollowing should be noted

“Essential Documents for the Conduct of a Clinical Trial” and as required bythe applicable regulatory requirement(s) The investigator/institution shouldtake measures to prevent accidental or premature destruction of these docu-ments (par 4.9.4)

(QA) and Quality Control (QC) systems with written Standard OperatingProcedures (SOPs) to ensure that trials are conducted and data are generated,documented (recorded), and reported in compliance with the protocol, GCP, andthe applicable regulatory requirement(s) (par 5.1.1)

reliable and have been processed correctly (par 5.1.3)

product(s) during the course of clinical development, the results of anyadditional studies of the formulated product(s) (e.g., stability, dissolution rate,bioavailability) needed to assess whether these changes would significantlyalter the pharmacokinetic profile of the product should be available prior to theuse of the new formulation in clinical trials (par 5.13.5)

(a) take steps to ensure that the investigational product(s) are stable over theperiod of use;

(b) maintain sufficient quantities of the investigational product(s) used in thetrials to reconfirm specifications, should this become necessary, and main-tain records of batch sample analyses and characteristics To the extentstability permits, samples should be retained either until the analyses of the

6 GOOD CLINICAL PRACTICE PRINCIPLES: LEGAL BACKGROUND AND APPLICABILITY