Published: Journal of Analytical Toxicology, Volume 20, Number 6, October 1996, pp. 416-424.

Identification of Urinary Benzodiazepines and their Metabolites: Comparison of Automated HPLC and GC–MS after Immunoassay Screening of Clinical Specimens
Jimmie L. Valentine, Rosalyn Middleton, and Carolyn Sparks

An automated high-performance liquid chromatographic method, benzodiazepines by REMEDi HS, was used to analyze benzodiazepines and their metabolites after b-glucuronidase hydrolysis of 1-mL urine specimens from the following: 924 clinic and hospital patients whose specimens had previously been found to be presumptively positive using either EMIT® or Triage® immunoassay methodologies and 128 individuals whose specimens had screened negative by EMIT d.a.u.™. REMEDi analyses did not correlate with the immunoassay results in 136 of the positive and three of the negative urine specimens. Gas chromatographic–mass spectrometric (GC–MS) confirmatory analyses were performed on these discordant specimens using 3 mL b-glucuronidase-hydrolyzed urine followed by extraction with chloroform–isopropanol (9:1) and derivatization with N,O-bis(trimethylsilyl)trifluoroacetamide. Two benzodiazepines, flunitrazepam and clonazepam, and their 7-amino metabolites were analyzed without prior derivatization. The analyses established 87% concordance between REMEDi and GC–MS versus 13% concordance with immunoassay for the subset. GC–MS analysis of these 142 specimens demonstrated two reasons for the nonconcurrence between REMEDi and EMIT: EMIT had given either false-negative or false-positive results and EMIT had given a positive result even though the determined metabolites were below the 200-ng/mL cutoff for the immunoassay and the
80-ng/mL cutoff for REMEDi. A total of 23 specimens were found to contain only lorazepam by REMEDi and GC–MS, 15 of which had been screened by Triage. A reevaluation of these 23 specimens by EMIT d.a.u. demonstrated that 11 were positive. This finding was in contrast to previous reports that EMIT will not detect lorazepam glucuronide in urine. An unexpected finding was the REMEDi identification and subsequent GC–MS confirmation of 7-aminoflunitrazepam, a urinary metabolite of flunitrazepam that is not available in the United States and that represented illicit use by four patients. A distinct advantage of REMEDi proved to be its capability in identifying demoxepam, a major metabolite of chlordiazepoxide; GC–MS analysis could not detect this metabolite because of its thermal decomposition to nordiazepam. To further evaluate the specificity of REMEDi, we conducted GC–MS analyses in a random fashion on 55 additional nondiscordant urine specimens that were identified as either positive or negative, as well as 22 specimens identified as containing 7-aminoclonazepam by REMEDi. Concurrence was observed between the two methods for all specimens, with the exception of one apparent false positive for a-hydroxyalprazolam by REMEDi. The reproducibility of the REMEDi method was found to be excellent; it was assessed by comparing results of 266 specimens that were reprocessed in different batches and for known calibrators and controls also processed with each batch. Study results demonstrated that the automated REMEDi assay for urinary benzodiazepines and their metabolites was comparable with GC–MS but had distinct advantages over GC–MS because of the following reasons: simplicity of the assay, less time required for analyses, and provision of additional information concerning the parent benzodiazepine.

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