Comparison of Immunoassay Screening Tests and LC–MS-MS for Urine Detection of Benzodiazepines and Their Metabolites: Results of a National Proficiency Test

Journal of Analytical Toxicology 2013;37:659 –664 doi:10.1093/jat/bkt063 Advance Access publication August 13, 2013 Article Comparison of Immunoassay Screening Tests and LC –MS-MS for Urine Detection of Benzodiazepines and Their Metabolites: Results of a National Proficiency Test Elisabetta Bertol1, Fabio Vaiano1, Maurizio Borsotti2, Massimo Quercioli2 and Francesco Mari1* 1 Forensic Toxicology Division, Department of Health Sciences, University of Florence, Florence, Italy, and 2Department of Security and Quality, Careggi Hospital of Florence, Florence, Italy *Author to whom correspondence should be addressed. Email: francesco.mari@unifi.it For most diverse purposes, different immunoassay (IA) screening methods are usually used to detect benzodiazepines and their metabolites in urine. In this study, we compared the main IAs used in forensic toxicology (Cloned Enzyme Donor Immunoassay, CEDIAw; EnzymeMultiplied Immunoassay Technique, EMITw; Fluorescent Polarization ImmunoAssay, FPIAw; Kinetic Interaction of Microparticles in Solution, KIMSw and Immunochromatographic Techniques, IMC) with liquid chromatography–tandem mass spectrometry (LC–MS-MS). Twelve urine specimens were analyzed by 178 laboratories in Italy that participated in a National Proficiency Test, providing both qualitative and semi-quantitative results. Each IA was evaluated by the parameters: true positive, true negative, false positive (FP), false negative (FN), sensitivity (SENS), specificity (SPEC), positive predictive value, negative predictive value (NPV) and accuracy. SPEC was affected by a high FP rate for all IAs. The lowest SENS and NPV were provided by FPIA due to a high number of FN cases. Comparing IA semi-quantitative data with LC–MS-MS results, an overestimation of benzodiazepine amount is noted. This paper draws attention to the problem of the careless use of IA tests for forensic purposes as they may provide FP and/or FN results that can lead to errors of great severity. Introduction Benzodiazepines are the drugs most frequently prescribed worldwide as tranquilizers, hypnotics, anesthetics, anticonvulsants or muscle relaxants to treat sleeplessness, depression, anxiety and epilepsy. Benzodiazepines are considered to be safer and more effective than barbiturates, but chronic use can produce dependence and abuse (1). Their intake, in combination with other central nervous system (CNS) depressants such as alcohol, may cause severe respiratory depression, which can lead to death (2– 4). Benzodiazepine misuse is increasingly associated with suicidal poisoning (5), driving under the influence of drugs (DUID) (6) and drug-facilitated sexual assault (DFSA) (7 –9). As such, benzodiazepines are among the most frequently encountered substances in clinical and forensic toxicological analyses, for which the simultaneous analysis of benzodiazepines and their metabolites in biological matrices is of great interest. The most representative difficulties in benzodiazepine analysis derive from the large number of these drugs (more than 50 different benzodiazepines are commercially available for clinical use) and from the possibility that many of them can be metabolized to multiple forms (many metabolites are drug substances in their own right) (10, 11). For these reasons, screening tests that can provide a rapid qualitative (or semi-quantitative) detection of benzodiazepines with high sensitivity (SENS) and specificity (SPEC) to include both abusive use and low-therapeutic doses are necessary, followed by instrumental techniques based on mass spectrometric detection. Immunoassays (IAs) are widely utilized for their rapidity, flexibility and semi-quantitative results. Urine is one of the preferred matrices for drug analysis, because the concentrations of benzodiazepines and their metabolites are higher in urine than in other biological specimens such as blood, oral fluid and hair. However, the capability of IAs to detect benzodiazepines is problematic due to the wide variation of the various representatives of this group of drugs in potency, structure, metabolism and elimination. The most critical limitation of IAs is related to the variable immunoreactivity of the antibodies to the diverse structural differences of the benzodiazepine class of drugs, leading to a large incidence of false positives (FPs) and false negatives (FNs) (12). In forensic laboratories, usually only positive results are investigated by instrumental techniques, while negative (and FN) ones are not. FNs can occur when benzodiazepine compounds with a low immunoreactivity rate are analyzed (i.e., Phase II metabolites, especially glucuronide forms) (13, 14). It is important to keep in mind these limitations, as in urine specimens benzodiazepine metabolites may be present and there is a risk of quantitative underestimation resulting in a false negativity (15). Analytical identification by IA is further complicated by the wide ranges of benzodiazepine therapeutic concentrations and metabolic pathways. Thus, these types of assays are not suitable to selectively identify the drug and to discriminate the parent drug from their metabolites (16). The present paper aims to evaluate the advantages and limitations of the use of IAs for forensic purposes on the basis of a National Proficiency Test (PT), for which we are the Reference Laboratory (RL). The study was conducted by comparing the liquid chromatography–tandem mass spectrometry (LC–MS-MS) method with the main IAs used in forensic toxicology (Cloned Enzyme Donor Immunoassay, CEDIAw; Enzyme-Multiplied Immunoassay Technique, EMITw; Fluorescent Polarization ImmunoAssay, FPIAw; Kinetic Interaction of Microparticles in Solution, KIMSw and Immunochromatographic Techniques, IMC) to determine benzodiazepines and their metabolites, with particular attention to false result cases. Methods Chemicals and urine specimens Drug standards 3-hydroxyflunitrazepam, 7-aminoclonazepam, 7-aminoflunitrazepam, 7-aminonitrazepam, a-hydroxyalprazolam, a-hydroxymidazolam, alprazolam, bromazepam, brotizolam, clonazepam, chlordiazepoxide, delorazepam, diazepam, flunitrazepam, halazepam (internal standard, IS), ketazolam, lorazepam, lormetazepam, midazolam, nitrazepam, nordiazepam, oxazepam, pinazepam, prazepam, temazepam and triazolam were purchased # The Author [2013]. Published by Oxford University Press. All rights reserved. For Permissions, please email: Table I Number and typology of IA techniques used in the quality control program for benzodiazepines and their metabolites detection IA technique Laboratories CEDIA EMIT-SYVA EMIT-I.L. EMIT-DIMENSION EMIT-BECKMAN EMIT-ARCHITECT FPIA-ABBOTT KIMS Immunochromatography 13 27 10 26 11 10 9 39 33 from Lipomed, Inc. (Cambridge, MA, USA). Dichloromethane was acquired from Panreac Quimica S.L.U. (Castellar del Vallès, Spain). Glacial acetic acid was obtained from J.T. Baker (Deventen, Holland). Sodium hydroxide, ammonium acetate and isopropanol were supplied by Carlo Er (...truncated)