Comparison of four immunoassays for the detection of lorazepam in urine

Four healthy patient subjects were each given a single, 1-mg lorazepam tablet. Urine samples from all patient subjects were collected at 12 intervals (0-2, 2-5, 5-8, 8-11, 11-14, 14-24, 24-26, 26-29, 29-32, 32-35, 35-38, and 38-48 hours). An aliquot from each urine collection was screened using cloned enzyme donor immunoassay (CEDIA), enzyme-multiplied immunoassay technique (EMIT) II, EMIT dau, and fluorescence polarization immunoassay (FPIA) without and with hydrolysis using beta-glucuronidase. Using a 200 ng/mL calibrator cut-off, none of the four immunoassays gave a positive response before hydrolyzation of the urine samples. For offline hydrolysis using Helix pomatia beta-glucuronidase, 35, 3, 0, and 4 of 48 urine samples gave positive responses on the previously listed immunoassays. The CEDIA method also gave 32 of 48 positive responses for online hydrolysis using Escherichia coli beta-glucuronidase. Online hydrolysis can be conveniently automated by including the beta-glucuronidase in the first of the two reagents combined with the urine sample.