The CEDIA dau Benzodiazepine assay has been reformulated to include online hydrolysis of urinary benzodiazepine glucuronide conjugates. The new antibody possesses enhanced cross-reactivities toward the low-close benzodiazepines, which are excreted at low urinary drug-metabolite concentrations. The screening method was evaluated using Iorazepam as the probe benzodiazepine. Four subjects each consumed a l-rag Iorazepam tablet. Sequential urine voids over the same time intervals were collected for

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... the next 48 h. Twelve postdose urine samples were collected from each subject. Positive results were obtained from 5-24 h to 2-35 h using a 200-ng/mt nitrazepam calibration cutoff. There was no practical difference between hydrolyzing online with the supplied E. coil ~-glucuronidase or offline with Helix pomatia 13-glucuronidase purchased separately. Without hydrolysis, all urine samples tested negative. The cross-reactivities of Iorazepam in terms of nitrazepam calibration equivalents, varied from 108 to 178% for Iorazepam concentrations between 50 and 2500 ng/mL. Lorazepam glucuronide gave cross-reactivities (expressed as Iorazepam base) between 72 and 136% using the online hydrolysis procedure with E. coli ~-glucuronidase. Offline hydrolysis with Helix pomatia gave cross-reactivities between 84 and 134%. Without hydrolysis, Iorazepam glucuronide gave less than 4% cross-reactivity in the assay.