A single method for confirmation and quantitation of a panel of commonly prescribed benzodiazepines and metabolites, alpha-hydroxyalprazolam, alpha-hydroxyethylflurazepam, alpha-hydroxytriazolam, alprazolam, desalkylflurazepam, diazepam, lorazepam, midazolam, nordiazepam, oxazepam, temazepam, clonazepam, and 7-aminoclonazepam, was developed for three specimen types, urine, serum/plasma, and meconium. Quantitation was by liquid chromatography tandem-mass spectrometry (LC-MS-MS) using a Waters Alliance-Quattro Micro system. The instrument was operated in multiple reaction monitoring mode with an electrospray ionization source in positive ionization mode. The method was evaluated for recovery, imprecision, linearity, analytical measurement range, specificity, and carryover. Average recovery and imprecision (within-run, between-run, and total % CV) were within +/- 15% of the target concentrations for urine (10 to 5000 ng/mL) and serum/plasma (10 to 2500 ng/mL) and within +/- 20% for meconium (10 to 5000 ng/g). In all, 205 patient specimens were analyzed, and the results compared to a previous in-house gas chromatography-MS method or LC-MS-MS results from an outside laboratory. Oxazepam glucuronide was evaluated as a hydrolysis control for the urine and meconium specimens.

The results of meconium specimens and fortified samples screened for drugs of abuse by both enzyme multiplied immunoassay technique (EMIT((R) )II) and enzyme-linked immunosorbent assay (ELISA) methods were compared. The sample preparation for the ELISA screen was a simple buffer extraction versus a lengthy and more laborious sample preparation procedure for the EMIT II screen. The ELISA method was automated using a TECAN Genesis. The EMIT II analysis was automated with an Olympus AU400e. The opioid screen was calibrated with hydromorphone and the benzodiazepine screen was calibrated with clonazepam to maximize detection for these analytes. Previously validated gas chromatography-mass spectrometry (GC-MS), two-dimensional GC-MS, or liquid chromatography-tandem MS methods were used for confirmation. Results from the two techniques compared well. Agreement of the ELISA assay was greater than 90% when compared to EMIT II for all drug classes except barbiturates and benzodiazepines. ELISA appears to be more sensitive than EMIT II for the detection of amphetamines, methadone, propoxyphene, and cocaine. ELISA compared well to EMIT II for cannabinoids, opioids, and PCP. Specificity of the ELISA assay was slightly better for PCP and opioids. EMIT II appears to be more sensitive for the detection of barbiturates and benzodiazepines. The ELISA method reduced turnaround time by 50% compared to the EMIT II method.

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The results of meconium specimens and fortified samples screened for drugs of abuse by both enzyme-linked immunosorbent assay (ELISA, Immunalysis) and biochip microarray (Randox) methods were compared. The ELISA method was semi-automated using a TECAN Genesis. The Randox assay used the Randox Evidence Investigator system. Previously validated gas chromatography-mass spectrometry (GC-MS), GC-GC-MS, or liquid chromatography-MS-MS methods were used for confirmation and quantitation. Results from the two techniques compared well. Agreement of the Randox assay was greater than 90% when compared to the ELISA assay for all drug classes except cannabinoids (88%). Specificity of the biochip assay was slightly better for amphetamines and cocaine.

Two versions of enzyme-linked immunosorbent assay (ELISA) kits designed for the detection of benzodiazepine drugs and metabolites (Immunalysis) were evaluated for use with meconium specimens. One was an older kit, and one was a new replacement kit developed for better detection of several commonly prescribed benzodiazepines and metabolites. The kits were evaluated by analyzing 68 patient specimens previously analyzed by liquid chromatography-tandem mass spectrometry and eight quality control samples. In addition to the recommended calibrator (oxazepam), clonazepam was evaluated as an alternate calibrator for the new kit. Detection and sensitivity for some analytes was improved using the new ELISA kit, but was reduced for others. The new kit using clonazepam as the calibrator provided the most sensitive assay for detection of the 11 benzodiazepines and metabolites reported here.