title: Urine benzodiazepine screening using Roche Online KIMS immunoassay with beta-glucuronidase hydrolysis and confirmation by gas chromatography- mass spectrometry.
abstract: Performance of the Roche Online KIMS (kinetic interaction of microparticles in solution) benzodiazepine (BZD) immunoassay (IA) with and without beta-glucuronidase treatment was evaluated on a Hitachi Modular automated IA analyzer calibrated using nordiazepam at 100 ng/mL. Reproducibility, linearity, accuracy, sensitivity, and interferences were evaluated. Precision of the assay (percent coefficient of variation (%CV)) with and without addition of the enzyme was less than 6% and 9%, respectively, with linearity (r(2) value of 0.9578 and 0.9746), respectively. Between-run precision of a 125 ng/mL nordiazepam control (n = 287) over 67 days, produced a %CV of 13.6% for the hydrolytic assay. Modification of the BZD assay to include automated hydrolysis of urinary BZD glucuronide conjugates was evaluated using three glucuronidated BZD standards prepared at concentrations ranging from 250 to 10,000 ng/mL. With hydrolysis, temazepam, oxazepam, and lorazepam glucuronides, produced cross-reactivities of 25%, 15%, and 20%, respectively. Without hydrolysis, the glucuronidated BZD standards produced less than 1% cross-reactivity in the assay. The ability of the assay to differentiate between positive and negative samples was evaluated by assaying 20 negative urine samples and serial dilutions of certified drug-free urine fortified with 28 different BZDs. All of the negative and positive urine samples produced the appropriate screening result. Cross-reactivities of 27 different BZDs, calculated as the normalized IA response divided by the BZD concentration that produced a response approximately equivalent to the response of a 100 ng/mL nordiazepam standard and multiplied by 100, ranged from 15% to 149%. Human urine samples (n = 28) that were previously found to contain BZDs by gas chromatography-mass spectrometry (GC-MS) also produced a positive BZD IA result. The IA was challenged with 78 potentially interfering compounds, and none produced a positive BZD response. As a part of the validation, a large number of human urine samples (29,500) were assayed using the modified Online BZD IA method to evaluate the performance of the method in production. Of the 29,500 samples tested, 80 produced a positive IA result. Analysis by GC-MS confirmed the presence of at least 1 BZD compound in 61 of the samples corresponding to a confirmation rate of 76%. The Online BZD IA modified by the automatic addition of beta-glucuronidase appears well adapted for the rapid detection of BZDs and their metabolites in human urine.
authors.affiliation: Navy Drug Screening Laboratory, 320 B Street, Great Lakes, Illinois 60088-2815, USA. klklette@nhgl.med.navy.mil
authors.forename: Kevin L
authors.initials: KL
authors.lastname: Klette
authors.forename: Russell F
authors.initials: RF
authors.lastname: Wiegand
authors.forename: Carl K
authors.initials: CK
authors.lastname: Horn
authors.forename: Peter R
authors.initials: PR
authors.lastname: Stout
authors.forename: Joseph
authors.initials: J
authors.lastname: Magluilo
mesh_terms: D001569:Benzodiazepines / Q000652:urine*; D002138:Calibration; D008401:Gas Chromatography-Mass Spectrometry; D005966:Glucuronidase / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D007118:Immunoassay / Q000379:methods; D009862:Online Systems; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D015813:Substance Abuse Detection / Q000379:methods*
chemical_list: D001569:Benzodiazepines; D005966:Glucuronidase