Evaluation of Buprenorphine CEDIA Assay versus GC-MS and ELISA using Urine Samples from Patients in Substitution Treatment

Journal of Analytical Toxicology, Vol. 29, November/December2005 Evaluation of BuprenorphineCEDIA Assayversus GC-MS and ELISAusing Urine Samplesfrom Patientsin SubstitutionTreatment Michael B6ttcher Arztpraxis f. Medizinische Mikrobiologie, Labordiagnostikund Hygiene, Dessau, Germany Olof Beck* Department of Medicine, Division of Clinical Pharmacology, Karolinska University Hospital, Stockholm, Sweden [ Abstract As buprenorphine becomes more clinically used in heroin substitution treatment, there is an increasing need for methods suitable for high-volume screening. In this study, a new immunochemical test based on CEDIA technology was evaluated for the use in clinical urine drug testing. The method was compared with an existing ELISA method and a gas chromatography--mass spectrometry (GC-MS) method on urine specimensfrom patients in heroin substitution treatment. The precision of the CEDIA assay was < 9% both within- and betweenday at levels at and above the cutoff limit of 5 pg/t. The concordance in qualitative results with an existing ELISA method was 96.8%. The CEDIA measuring range was extended by diluting urine samples100-fold with saline, and the results agreed well (slope of regression line was 1.09, r2 = 0.968) with GC-MS. The sensitivity of CEDIA in detecting authentic specimen containing buprenorphineat levels > 5 pg/L was 99.5%. Cross-reactivity causing false-positive response was discovered in patients receiving prescribed dihydrocodeine. The urine concentration of total buprenorphine in urine from patients prescribed daily doses between 0.2 and 24 mg ranged from 0.5 to 2900 pg/L. The concentration of the metabolite norbuprenorphine was usually higher, and the median ratio of buprenorphine to norbuprenorphinewas 0.23 (95% were below 1). We conclude that the CEDIA assay is suitable for application in high-volume screening of buprenorphine for urine drug testing. Introduction Buprenorphine (Subutex | is used as an analgesic drug and for detoxification and substitution therapy of opioid de* Author to whom correspondence should be addressed: Dr. Olof Beck, Department of Medicine, Division of Clinical Pharmacology, Karolinska University Hospital, SE-17176 Stockholm, Sweden. pendence (1,2). Since 1996, starting in France, the use of buprenorphine in substitution therapy has increased and spread to over 30 countries, many in Europe but also in Asia, Australia, and the Americas. Buprenorphine offersan attractive alternative to methadone, with benefits including increased safety for respiratory depression, suppressed heroin use, and possibility of longer dosing interval (2). However,in combination with the increased medical use, buprenorphine also occurs on the black market as an illicit drug (3,4), and fatalities due to poly-drug use have been reported (5,6). Buprenorphine is metabolized by dealkylation to norbuprenorphine primarily by the cytochrome P450 isozyme CYP 3A4 (7). Both buprenorphine and norbuprenorphine are conjugated with glucuronic acid and subsequently excreted in urine during the course of several days (8). In urine drug testing, parent buprenorphine has become the primary analytical target compound, and less focus has been devoted to the metabolite norbuprenorphine. The proportion of the two compounds in urine has not been well-documented in patient material. The activity of the CYP 3A4 isozyme is subjected to variability influenced by both genetic and environmental factors, which will determine the proportion of norbuprenorphine that is being formed and subsequently excreted in urine (9,10). As a consequence of increased legal and illicit use of buprenorphine, there is a need for an analytical method for toxicological monitoring of patients. In addition, an analytical method is necessary for compliance monitoring during substitution therapy, which has been integrated in many methadone programs. A number of methods for urinary determination of buprenorphine are available, including radioimmunoassay, enzyme-linked immunosorbent immunoassay (ELISA), thin-layer chromatography, high-pressure liquid chromatography, gas chromatography (GC), gas chromatography-mass spectrometry (GC-MS), and liquid Reproduction(photocopying)of editorialcontentof thisjournal is prohibitedwithoutpublisher'spermission. 769 Journal of Analytical Toxicology, Vol. 29, November/December 2005 chromatography-mass spectrometry (LC-MS) (11-20). However, automated high-volume screening tests for buprenorphine have not been available. The aim of this study was to evaluate whether a new buprenorphine CEDIAassay (cloned enzyme-donor immunoassay) could fulfill the need for a rapid and cost-effective urinary screening assay. Materials and Methods Chemicals Buprenorphine, morphine, morphine-3-glucuronide, codeine, norbuprenorphine, dihydrocodeine, hydrocodone, buprenorphine-d4, and norbuprenorphine-d3 were obtained as methanol solutions from Promochem GmbH (Wesel, Germany). Codeine-6-glucuronide,dihydrocodeine-6-glucuronide, dihydromorphine, dihydromorphine-3-glucuronide, dihydromorphine-6-glucuronide, dihydronormorphine, and dihydronorcodeine were obtained as solid material from Lipomed GmbH (Bad S~ickingen,Germany) and dissolved in pure water or methanol. Working solutions were prepared by dilutions with blank urine or saline (0.9%). The experiment for obtaining cross-reactivity data used typically 4-7 prepared solutions for each compound using linear regression analysis. For compounds with low cross-reactivity, one data point was used. Helix pomatia I~-glucuronidase/arylsulfatase (supplied in liquid form) was obtained from Roche GmbH (Mannheim, Germany); acetic anhydride was from Fluka (Buchs, Switzerland); methanol, ethyl acetate (suprasolve grade), sodium acetate, ammonia (32%), and acetic acid were from Merck GmbH (Darmstadt, Germany); acetonitrile was from Riedel-de Haen (Seelze, Germany); and 4-dimethylaminopyridine (crystalline grade) was from Sigma Chemical (Taufkirchen, Germany). All chemicals were of analytical grade unless otherwise stated. Urine samples Urine samples were collected from outpatients in heroin substitution treatment with methadone, buprenorphine, or dihydrocodeine assumed to be in steady-state. In total, 1552 samples were obtained from approximately 600 patients, consisting of 70% males and having an age range of 18-54 years. The samples were collected under supervision and sent to the laboratory for routine urine drug testing. Three different criteria were used for inclusion in various parts of the study: prescription of Subutex with known daily dose, prescription of other substitution medication (methadone or dihydrocodeine), or a specific request of urine buprenorphine testing. The ethics committee at the Karolinska Institute approved the study. CFDIA assay Reagents for the CEDIAbuprenorphine test were supplied by Microgenics (Fremont, CA). The test was performed on Hitachi 911 and 912 (Roche Diagnostics, Indianapolis, IN) instruments with a test (...truncated)