Liquid chromatography-tandem mass spectrometry (LC-MS-MS) offers specific advantages over gas chromatography-mass spectrometry (GC-MS) such as the ability to identify and measure a broader range of compounds with minimal sample preparation. Comparative analysis of LC-MS-MS versus GC-MS was performed for urinalysis detection of five benzodiazepine compounds currently part of the Department of Defense (DoD) Drug Demand Reduction Program (DDRP) testing panel; alpha-hydroxyalprazolam, oxazepam, lorazepam, nordiazepam and temazepam. In the analyses of internally prepared control urine samples at concentrations around the DDRP administrative decision point for benzodiazepines (100 ng/mL), both technologies produced comparable results with average accuracies between 99.7 and 107.3% and average coefficients of variation (%CV) <9%. Analysis of service member specimens that screened positive for benzodiazepines using both technologies produced comparable results for all analytes. Different degrees of matrix effect were observed for all analytes in the LC-MS-MS analysis. However, the effects were controlled by using deuterated internal standards (ISTDs). Additionally, there was a 39% increase in nordiazepam mean concentration analyzed by LC-MS-MS due to suppression of the ISTD ion by the flurazepam metabolite 2-hydroxyethylflurazepam. The ease and speed of sample extraction, the broader range of compounds that can be analyzed and shorter run time make the LC-MS-MS technology a suitable and expedient alternative confirmation technology for benzodiazepine testing.

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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.

2-Oxo-3-hydroxy lysergic acid diethylamide (O-H-LSD), a major LSD metabolite, has previously been demonstrated to be a superior marker for identifying LSD use compared with the parent drug, LSD. Specifically, O-H-LSD analyzed using liquid chromatography-mass spectrometry has been reported to be present in urine at concentrations 16 to 43 times greater than LSD. To further support forensic application of this procedure, the specificity of the assay was assessed using compounds that have structural and chemical properties similar to O-H-LSD, common over-the-counter products, prescription drugs and some of their metabolites, and other drugs of abuse. Of the wide range of compounds studied, none were found to interfere with the detection of O-H-LSD or the internal standard 2-oxo-3-hydroxy lysergic acid methyl propylamide. The stability of O-H-LSD was investigated from 0 to 9 days at various temperatures, pH conditions, and exposures to fluorescent light. Additionally, the effect of long-term frozen storage and pH was investigated from 0 to 60 days. There was no significant loss of O-H-LSD under both refrigerated and frozen conditions within the normal human physiological pH range of urine (4.6-8.4). However, significant loss of O-H-LSD was observed in samples prepared at pH 4.6-8.4 and stored at room temperature or higher (24-50 degrees C).

The purpose of this study was to evaluate the ability of two amphetamine class screening reagents to exclude ephedrine (EPH), pseudoephedrine (PSEPH), and phenylpropanolamine (PPA) from falsely producing positive immunoassay screening results. The study also sought to characterize the prevalence and concentration distributions of EPH, PSEPH, and PPA in samples that produced positive amphetamine screening results. Approximately 27,400 randomly collected human urine samples from Navy and Marine Corps members were simultaneously screened for amphetamines using the DRI® and Abuscreen® online immunoassays at a cutoff concentration of 500 ng/mL. All samples that screened positive were confirmed for amphetamine (AMP), methamphetamine (MTH), 3,4-Methylenedioxyamphetamine (MDA), 3,4-Methylenedioxymethamphetamine (MDMA), EPH, PSEPH, and PPA by gas chromatography/mass spectrometry (GC/MS). The DRI AMP immunoassay identified 1,104 presumptive amphetamine positive samples, of which only 1.99% confirmed positive for the presence of AMP, MTH, MDA, or MDMA. In contrast, the online AMP reagent identified 317 presumptive amphetamine positives with a confirmation rate for AMP, MTH, MDA, or MDMA of 7.94%. The presence of EPH, PSEPH, or PPA was confirmed in 833 of the 1,104 samples that failed to confirm positive for AMP, MTH, MDA, or MDMA; all of the 833 samples contained PSEPH. When compared to the entire screened sample set, PSEPH was present in approximately 3%, EPH in 0.9%, and PPA in 0.8% of the samples. The results indicate that cross reactivities for EPH, PSEPH, and PPA are greater than reported by the manufacturer of these reagents. The distribution of concentrations indicates that very large concentrations of EPH, PSEPH, and PPA are common.