Author(s)

Ashraf Mina^1,2*, Leah McNeice¹, Shanmugam Banukumar¹, Santiago Vazquez¹

¹NSW Health Pathology, Forensic & Analytical Science Service (FASS), Toxicology Unit, Macquarie Hospital, Sydney, Australia.
²Affiliated Senior Clinical Lecturer, Faculty of Medicine and Health, Sydney University, Sydney, Australia.

Background: Hydrolysis improves the sensitivity of drug detection for drug classes such as opiates/opioids and benzodiazepines, which are highly metabolized by glucuronidation and sulfation and should be implemented in analytical procedures to convert conjugated metabolites into the free or unbound form. This study was aimed to compare different enzymes to make an informed decision. Methods: In this study, the CEDIA Benzodiazepine assay was compared with the LC-MS-MS method using 150 positive urine samples and 50 negative urine samples. The samples were analysed without adding any enzyme and then by adding different enzymes to compare their performance. Results: The Kura Escherichia coli enzyme performed better than the Roche Escherichia coli enzyme which had 20% false-positive results. Kura BG-100 enzyme performed well but Kura B-One enzyme performed better The Kura B-One enzyme had only 11.5% false-positive results. When double the volume of Kura B-One enzyme was used to test to see if it will have any impact on reducing the number of false negatives, it performed worse. Kura Turbo enzyme behaved similarly to Kura BG-100. Conclusions: The β-glucuronidase enzymes comparison allowed us to identify the Kura B-One enzyme as the enzyme of choice for our operation because it reduces the false positives from 20% to 11.5% when compared with the Roche enzyme. It also improved the detection of oxazepam. The Kura B-One enzyme has a short incubation time for hydrolysis when used with the LC-MS-MS method. As a result, we improved the overall turn-around time and reduced the number of false positives that needed confirmation.

β-Glucuronidase Enzyme, CEDIA, Benzodiazepine, 7-Amino Clonazepam, 7-Amino Clonazepam

Mina, A. , McNeice, L. , Banukumar, S. and Vazquez, S. (2022) Optimisation of Benzodiazepine Immunoassay Using β-Glucuronidase Enzymatic Hydrolysis: A Comparison of Five Different β-Glucuronidase Enzymes. Journal of Biosciences and Medicines, 10, 7-15. doi: 10.4236/jbm.2022.101002.