B-276 Evaluation of the Roche Benzodiazepines II Immunoassay for Urine Drug Testing in Clinical Specimens
Authors: Ge M , Alabi A , Kelner M , Fitzgerald R , Suhandynata R T 
Published: October 2024

Background

Benzodiazepines are one of the most commonly prescribed medications in the United States and are frequently linked to instances of abuse and overdose. Historically, FDA-cleared benzodiazepine urine immunoassays cross-react poorly with glucuronidated metabolites excreted in urine. False negative results are especially prevalent with lorazepam, which is almost exclusively excreted as lorazepam glucuronide. Some clinical laboratories have addressed this problem with the addition of beta-glucuronidase to enhance assay sensitivity as a laboratory developed test (LDT). Roche Diagnostics recently received FDA clearance to offer a benzodiazepine immunoassay that includes beta-glucuronidase.

Methods

Performance characteristics of two FDA-cleared benzodiazepine urine immunoassays (Benzodiazepines Plus, no glucuronidase and Benzodiazepines II, with glucuronidase; Roche Diagnostics) and a benzodiazepine immunoassay LDT (with glucuronidase) were evaluated using 258 urine specimens. These immunoassays were directly compared to an LC-MS/MS benzodiazepine LDT to determine clinical sensitivity and specificity. Cross-reactivity of all three immunoassays were compared and evaluated based on the measured benzodiazepine concentrations determined by the LC-MS/MS LDT. Cross-reactivity for 7-aminoclonazepam, lorazepam, and lorazepam glucuronide were assessed using drug-free urine spiked with reference materials (Cerilliant) at concentrations ranging from 100 to 1000 ng/mL. The cutoff for positive results was set at 1000 mAbs, corresponding to 200 ng/mL of the nordiazepam calibrator.

Results

The Benzodiazepines II and LDT immunoassays exhibited greater clinical sensitivity (100% and 95.2%) compared to the Benzodiazepines Plus assay (66.7%). Clinical specificity of 100% was observed for all three assays. Cross-reactivity of the Benzodiazepines II assay was greater across the range of benzodiazepine concentrations tested in comparison to the other immunoassays. In particular, the Benzodiazepines II assay was the most sensitive for 7-aminoclonazepam, as it was the only immunoassay out of the three that detected the presence of specimens containing this metabolite alone. Cross-reactivity analysis shows that both 7-aminoclonazepam and lorazepam yielded positive results when tested using the Benzodiazepines II assay at a concentration of 300 ng/ml. 7-aminoclonazepam tested positive at 800 ng/ml with the LDT and Benzodiazepines Plus assays, whereas lorazepam tested positive at 600 ng/ml with these assays. Additionally, the LDT and Benzodiazepines II assays detected lorazepam glucuronide at concentrations of 400 ng/ml and 250 ng/ml, respectively. However, the Benzodiazepines Plus assay yielded negative results for all concentrations of lorazepam glucuronide tested (up to 1000 ng/mL).

Conclusions

A comprehensive evaluation of these three immunoassays demonstrates that the Benzodiazepines II immunoassay has increased clinical and analytical sensitivity compared to the Benzodiazepines Plus and LDT immunoassays. The inclusion of a beta-glucuronidase greatly improved the sensitivity of the Benzodiazepines II and LDT immunoassays for lorazepam, which is primarily excreted as a glucuronide metabolite in urine.