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Laboratory and Scientific Section,UNITED NATIONS OFFICE ON DRUGS AND CRIME. Guidelines for Testing Drugs,under International Control in Hair. Sweat and Oral Fluid,MANUAL FOR USE BY,NATIONAL DRUG ANALYSIS LABORATORIES. UNITED NATIONS,New York 2014, Operating and experimental conditions are reproduced from the original reference. materials including unpublished methods validated and used in selected national. laboratories as per the list of references A number of alternative conditions and. substitution of named commercial products may provide comparable results in many. cases but any modification has to be validated before it is integrated into laboratory. Mention of names of firms and commercial products does not imply the endorse. ment of the United Nations,ST NAR 30 Rev 3,Original language English. United Nations May 2014 All rights reserved worldwide. The designations employed and the presentation of material in this publication do. not imply the expression of any opinion whatsoever on the part of the Secretariat of. the United Nations concerning the legal status of any country territory city or area. or of its authorities or concerning the delimitation of its frontiers or boundaries. Mention of names of firms and commercial products does not imply the endorsement. of the United Nations,This publication has not been formally edited. Publishing production English Publishing and Library Section United Nations. Office at Vienna,Acknowledgements, UNODC s Laboratory and Scientific Section LSS headed by Dr Justice Tettey. wishes to express its appreciation and thanks to Professor Franco Tagliaro Uni. versity of Verona Professor Donata Favretto University of Padova and Mr Paolo. Fais of the University of Verona Italy for the preparation of the final draft of the. present Manual, The valuable comments and contribution of the following experts to the peer review. process is gratefully acknowledged, Mr Bill Corl of Omega Laboratories Inc United States Professor Olaf Drummer. of the Victorian Institute of Forensic Medicine Australia Dr Pirjo Lillsunde of the. National Institute for Health and Welfare Finland Dr Mee Jung Park of the Korea. National Forensic Service Republic of Korea and Dr Alain G Verstraete of Ghent. University Hospital Belgium, The preparation of the present Manual was coordinated by Dr Conor Crean staff. member of LSS The contribution of Dr Iphigenia Naidis staff member of LSS and. other UNODC staff members is gratefully acknowledged. Introduction 1,Background 1, Alternative specimens for analysing drugs of abuse 2. Purpose and use of the Manual 4,1 The analysis of drugs in hair 7. 1 1 Anatomy and physiology of hair 7,1 2 Specimen collection 10. 1 3 Sample preparation 13,1 4 Extraction and clean up 15. 1 5 Methods for the analysis of drugs in hair 19,1 6 Interpretation of results 30. 1 7 Applications of hair analysis in the forensic field 35. 2 The analysis of drugs in sweat 41,2 1 Anatomy and physiology 41. 2 2 Specimen collection 43,2 3 Drug extraction 45,2 4 Methods for the analysis of drugs in sweat 45. 2 5 Interpretation of results 47,2 6 Applications of sweat analysis 48. 3 The analysis of drugs in oral fluid 51,3 1 Production and composition 51. 3 2 Oral fluid as a biofluid for drug analysis 51,3 3 Advantages and disadvantages 52. 3 4 Oral fluid blood partition 53, 3 5 Methods for the analysis of drugs in oral fluid 54. 4 General considerations in the analysis of drugs in hair. sweat and oral fluid 67,References 73,Introduction. Background, The present Manual is a revision of the United Nations Office on Drugs and Crime. UNODC manual Guidelines for Testing Drugs Under International Control. ST NAR 30 Rev 2 This version has been prepared taking into account recent devel. opments in analytical technology to detect conventional and new unconventional. drugs and is based on up to date scientific knowledge of the physiology and phar. macology of the so called alternate biological specimens which today may offer. important information complementary to the analysis of the traditional biological. specimens blood and urine, In the intervening time period since the publication of the previous revision of this. Manual there have been significant advances in the analytical techniques used for. the analysis of drugs under international control in hair sweat and oral fluid Con. currently there has also been an increase in the number of substances that are. encountered in drug analysis laboratories which can vary considerably from country. to country and also from region to region within the same country 1 The drugs. appearing in markets in recent years include not only traditional drugs already under. international control but also new psychoactive substances NPS or combinations. of biologically active compounds distributed in the form of clandestine preparations. Concurrently it has been noted that there has been an expanding abuse of substances. and drugs used for medical purposes such as benzodiazepines antidepressants and. therapeutic substitutes for opioids, National institutions as well as clinical and forensic toxicology facilities are required. not only to analyse seized materials but also to detect and measure the abused. compounds and their metabolites in biological specimens In the clinical e nvironment. toxicologists are usually required to promptly identify drugs and drug metabolites. to support the physician in the diagnosis and treatment of acute intoxications Also. in non clinical settings forensic epidemiological etc the toxicologist is often the. cornerstone of policies and legislative provisions as well as of court decisions. As a result of the changes described above laboratories must be able to deal with. an ever increasing number of substances and use analytical methods coupling sen. sitivity and specificity with the widest analytical spectrum assuring both rapid. 2 Guidelines for testing drugs under international control in hair sweat and oral fluid. response and robust operation at the same time Taking into account the analysis of. biological specimens additional challenges must be faced such as the need for high. sensitivity and for high selectivity towards numerous potential endogenous interfer. ences Furthermore the rapid decrease of drug concentrations in biological fluids. due to the metabolic changes of the parent compounds poses additional problems. to the toxicologist, Given the above considerations it is clear that an efficient exchange of information. between laboratories as well as between laboratories and regulatory agencies at the. national and international levels will offer a harmonization of methods which forms. the basis of an effective global control of the phenomenon of drug abuse In particular. the validation of analytical methods according to international standards e g ISO. IEC 17025 or 15189 and participation in proficiency testing p rogrammes are recom. mended to define the accuracy and the trueness of the laboratory s tests allowing an. efficient comparison between results obtained by different laboratories. Alternative specimens for analysing drugs, It is generally accepted that chemical testing of biological specimens is the only. objective means of diagnosis of exposure to therapeutic and non therapeutic drugs. including toxicants abused drugs doping compounds and other xenobiotics For. this purpose urine testing has been by far the most common toxicological approach. because relatively high concentrations of drugs and metabolites are generally present. in this biological matrix However urine analyses are essentially limited to testing. for and reporting on the presence or the absence of a drug or its metabolites over. a short retrospective period 2 Blood in which the presence of many compounds. is limited to a few hours is generally considered the biological sample of choice. to detect drugs in the actual phase of biological activity i e to test the drug related. impairment of a subject The relatively low concentrations and short half life of. exogenous compounds in the blood places important demands on the analytical. techniques which should be 10 to 100 fold more sensitive than for urine Since. the beginning of routine drug analysis in biofluids the standard approach has been. based on immunoassay screening in urine followed by confirmation with gas. chromatography mass spectrometry GC MS 3 Even if performed with the most. rigorous analytical procedure an intrinsic weak point of the analysis of drugs in. biofluids is the limited detection window from hours up to a few days and the. prevalence of metabolites versus the parent drug, In the late 1970s 4 hair was first reported to be suitable for retrospective toxico. logical analyses and since then numerous new applications of innovative analytical. techniques have been reported for the detection and measurement of many analytes. in hair including among others ketamine 9 tetrahydrocannabinol THC. Introduction 3, 11 nor 9 THC 9 carboxylic acid THCCOOH doping agents synthetic cannabi. noids and cathinones 5 Therefore hair analysis is now considered to be the most. efficient tool to investigate drug related histories particularly when the period of. use needs to be tested back to many days or even months before the sampling 6. On these grounds following recent suggestions from international associations such. as the Society of Hair Testing hair analysis can become not only a fundamental tool. in forensic toxicology and medicine but also a way to find traces of illicit drugs in. subjects claiming abstinence for months before sampling Following the success of. advances in hair analysis other alternative biological specimens such as sweat and. oral fluid have gained popularity as forensic specimens being able to provide infor. mation in specific circumstances As depicted in table 1 7 these alternate matrices. offer different detection windows In most instances they show significantly different. metabolic profiles when compared to traditional blood and urine testing. Table 1 Detection windows for drugs in various biological matrices 7. Specimen Detection window,Blood serum Several hours to 1 2 days. Urine Several hours to 3 days, Oral fluid Several hours to 1 2 days or more for basic drugs. Sweat patch Weeks,Hair Months years, In addition to differences in metabolism and pharmacokinetics the various biological. matrices show other peculiarities particularly relevant in the forensic environment. First of all there are issues with the possibility of urine substitution dilution and. adulteration during sample collection These problems are much less likely for hair. and the other alternate specimens compared to urine This advantage has promoted. the popularity and use of these specimens for drug testing 3 Also in comparison. to blood the alternate matrices have the undoubted advantage of a minimally invasive. collection procedure which can potentially be performed in a non medical setting. The analysis of drugs in hair was first reported outside the field of forensic toxico. logy in 1954 8 However only in 1979 4 was a radioimmunoassay for morphine. detection reported and used to document chronic opiate abuse histories To date a. literature search for the string of terms hair and analysis and toxicology results. in about 600 publications 160 of which were published in the last three years using. the PubMed search in MEDLINE for example The international interest in the. subject is also exhibited by the publication of a number of books 3 6 9 11 and. by the existence of an international society The Society of Hair Testing SoHT. founded in 1995 As mentioned above the major practical advantage of hair testing. compared to urine and blood testing for drugs is its larger detection window ranging. from weeks to several months depending on the length of hair shaft analysed In. practice by combining the detection windows offered by blood urine and hair a. 4 Guidelines for testing drugs under international control in hair sweat and oral fluid. toxicologist can gather objective information on drug use exposure within an. extended time frame, While the great majority of publications concern the National Institute on Drug. Abuse NIDA five marijuana opioids cocaine amphetamines and phencyclidine. 3 6 10 11 the number of drugs metabolites whose detection in hair has been. published is increasing constantly including ethyl glucuronide and fatty acid ethyl. esters alcohol metabolites lysergic acid diethylamide 12 13 ketamine 14. antidepressants 12 15 17 mephedrone 18 gamma hydroxybutyric acid 19 20. synthetic cannabinoids 21 25 and other new psychoactive substances 26 Hair. analysis has also been used for the d etermination of a large number of p harmaceutical. drugs 11 and chemical compounds 27,Purpose and use of the Manual. The present Manual is focused on the application of up to date techniques of ana. lytical toxicology to the biological specimens hair sweat and oral fluid These. biological matrices having a different composition to more traditional biofluids i e. urine and blood require robust analytical methodologies based on unequivocal deter. mination of the analytes of interest obtained by accurate qualitative and quantitative. techniques The major issues that are still open regarding the interpretation of the. qualitative and quantitative results will be discussed.