Method Description

Preliminary screen is performed by immunoassay.

The benzodiazepine assay is based on the kinetic interaction of microparticles in a solution as measured by changes in light transmission. In the absence of sample drug, soluble drug conjugates bind to antibody-bound microparticles causing the formation of particle aggregates. As the aggregation reaction proceeds in the absence of sample drug, the absorbance increases. When a urine sample contains the drug in question, this drug competes with the drug derivative conjugate for microparticle-bound antibody. Antibody bound to sample drug is no longer available to promote particle aggregation, and subsequent particle lattice formation is inhibited. The presence of sample drug diminishes the increasing absorbance in proportion to the concentration of drug in the sample. Sample drug content is determined relative to the value obtained for a known cutoff concentration of drug.(Package insert: BNZ2. Roche Diagnostics; V 2.0, 04/2024)

Benzodiazepines are extensively metabolized by the liver and subsequently exist in the urine primarily as conjugated esters (-glucuronides). The conjugated metabolites are cleaved during a mild hydrolysis utilizing the enzyme glucuronidase. Stable isotope forms of the compounds are added as internal standards to account for extraction losses. An aliquot of the hydrolyzed sample is diluted and the analytes are separated by liquid chromatography tandem mass spectroscopy and analyzed by multiple reaction monitoring.(Unpublished Mayo method)