Urine Benzodiazepine Screening using Roche Online(R) KIMS Immunoassay with  -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.

Liquid Chromatography–Tandem Mass Spectrometry for Detection of Low Concentrations of 21 Benzodiazepines, Metabolites, and Analogs in Urine: Method with Forensic Applications

“…Compared with other reports on analysis of several BZPs (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35), in the screening method described in this study, the LLOQs for 19 BZPs or their major metabolites and 2 related compounds (zopliclone and zolpidem) are lower and seem to be appropriate for the detection and quantification of these compounds in urine samples from DFSA cases.…”

Evaluation of KIMS immunoassays on a cobas c 501 analyzer for drugs of abuse and ethyl glucuronide testing in urine for forensic abstinence control

“…For the EDDP assay, two samples (EDDP and methadone in sample 1: 37 ng/mL and 55 ng/mL, and in sample 2: 54 ng/mL and 52 ng/mL) yielded ng/mL-immunoassay (IA) values of 0 leading to a sensitivity of only 92% even at cut-offs lower than 16. [12] The specificity of the ethyl glucuronide assay reached 100% only at a cut-off value of 103 ng/mL immunoassay units. A possible explanation may be an adulteration of the samples with substances interfering with the enzymatic reaction.…”

Review and Recommendations for Drug Testing in Substance Use Treatment Contexts

“…In the US alone, sedative use has reached upwards of 20% prevalence [71,74]. Testing for sedatives can be conducted using hair, nail, urine, saliva and blood collection methods [75][76][77][78].…”