MSACL 2024 Abstract

Best Practices: Enzymatic Hydrolysis Protocols Are Not a "One Size Fits All" for Each Drug Class When Using B-One

Jose Luis Callejas (1), Nicholas Chestara (2), Janet Jones (1), Elias Villalobos (1), Camila Berner (1)
(1) Kura Biotech Inc, Atlanta, GA, USA. (2) DPX Technologies, Columbia, SC, USA

Elías Villalobos, Master in Chemistry (Presenter) Kura Biotech

Relevant Financial Disclosures (within past 24 months, reported on Jul 17, 2025) Conflict mitigation NOT REQUIRED.

INTRODUCTION: Glucuronidation plays a crucial role in the elimination of many drugs from the body. This metabolic process involves the conjugation of drugs with glucuronic acid, resulting in the formation of more polar compounds that can be easily excreted. Consequently, these conjugated drugs can complicate drug analysis by mass spectrometry for multiple reasons and to help resolve this issue, many laboratories utilize beta-glucuronidase enzymes to cleave the glucuronide conjugates to convert them back to their original, parent form. This approach allows for more accurate detection and quantification of these drugs and metabolites while streamlining the sample preparation workflow. However, it’s important to take into consideration when hydrolyzing different drug classes such as opiates, benzodiazepines, cannabinoids, and antidepressants, utilizing a universal approach may not be the most effective option. The reason for this is that each analyte has a unique interaction with beta-glucuronidase, leading to a customized hydrolysis method that is tailored to the specific class of drugs.

OBJECTIVE: Our main objective is to emphasize that a one-size-fits-all approach is not always ideal when hydrolyzing different drug classes and analytes, and to raise awareness about the importance of a customized hydrolysis protocol. This study also shows how to apply best practices while evaluating the hydrolysis performance using B-One® (Finden® by Kura Biotech®) recombinant beta-glucuronidase for the various drug classes for use in forensic and clinical toxicology laboratories.

MATERIALS AND METHODS: Common drug classes and analytes were quantitatively analyzed in different panels for hydrolysis efficiency using the following glucuronide standards fortified in drug-free urine: Opiates, Benzodiazepines, Naloxone/Buprenorphine/Norbuprenorphine, Carboxy-THC, and Amitriptyline. Quality control standards were prepared at low, medium, and high concentrations. The hydrolysis method was performed using B-One®, an “all-in-one” recombinant beta-glucuronidase stabilized in its reaction buffer for fast room-temperature hydrolysis. The hydrolysis was followed by a clean-up protocol using XTR™ tips 5 mg HLB (DPX Technologies) and then diluted with DI water for analysis by LC-MS/MS.

RESULTS: A quantitative method was used to determine the concentrations of free drugs for each analyte in quadruplicates and then the recoveries were calculated using a hydrolysis efficiency formula. Results demonstrated good recovery and precision with an optimized hydrolysis method for each drug class.

DISCUSSION AND CONCLUSION: The hydrolysis experiments conducted on various drug classes demonstrate the need for customized hydrolysis parameters for B-One,® including different hydrolysis times and enzyme amounts, by following the best practices suggested here. The addition of DPX XTR™ tips and NGX custom standard mixtures provides a streamlined protocol for ease of use as well.