Optimal enzymatic hydrolysis of urinary benzodiazepine conjugates

Conditions for the enzymatic hydrolysis of benzodiazepine conjugates were systematically examined. Optimal recovery of the free drugs occurs when 1 mL of urine buffered to pH 4.5 is incubated with 5000 U of Helix pomatia beta-glucuronidase at 56 degrees C for 2 h. Urine specimens containing conjugates of temazepam, oxazepam, lorazepam, alpha-hydroxyalprazolam, 2-hydroxyethylflurazepam, and N-desalkyl-3-hydroxyflurazepam as targeted benzodiazepines were used. The freed drugs were quantitated by gas chromatography-mass spectrometry.