Introduction:

Opiate and opioid metabolism primarily occurs in the liver, and these drugs are removed from the body by glucuronidation and sulfation to facilitate excretion in urine. Drug testing laboratories can quantify free opiates and opioids in urine by LC-MS/MS after hydrolysis with a β-glucuronidase (β-GUS) and/or sulfatase. We have shown in previous studies that glucuronidated substrates and glucuronidases have different pH preferences for optimal hydrolysis. Use of a single β-glucuronidase can constrain the recovery of glucuronide conjugates, potentially leading to incomplete detection of drug metabolites. Here, we show that individual enzymes exhibit substrate biases, and that combining multiple β-glucuronidases enhances hydrolysis efficiency across a diverse panel of glucuronidated compounds

Objectives:
Compare the activities, or hydrolysis efficiencies, of commercially available β-glucuronidase enzymes on various glucuronidated substrates by testing a range of enzyme concentrations at fixed pH and substrate amounts.

Methods:
Deuterated and reference standards for morphine, oxymorphone, codeine, O-desmethyl-tramadol, O-desmethyl-venlafaxine, amitriptyline, and nortriptyline were purchased from Cerilliant. All other reagents were purchased from MilliporeSigma or Fisher Scientific. Three β-glucuronidases were expressed and purified at IMCS: Aspergillus chimera β-GUS (AspGUS), Eubacterium eligens β-GUS (EelGUS), and Brachyspira pilosicoli β-GUS variant (BpiGUS). Samples were buffered with sodium acetate at pH 5.5. Reactions were stopped by transferring sample into methanol. Samples were further diluted and injected on a Thermo Scientific™ Vanquish™ UHPLC system coupled with a Thermo Scientific™ Endura™ Triple Quadrupole Mass Spectrometer. Analytes were separated using a Phenomenex Kinetex® 2.6 μm Biphenyl 100 Å, 50 x 4.6 mm column. Mobile phase A and B were 0.1% formic acid in water and 0.1% formic acid in acetonitrile, respectively

Results:
BpiGUS variant performed best on codeine, O-desmethyl-venlafaxine, trifluoperazine and haloperidol. Chimeric enzyme abbreviated as AspGUS performed best on morphine- and oxymorphone-glucuronides EeGUS performed best on glucuronidated O-desmethyl-tramadol, imipramine, amitriptyline and cotinine

Discussion:
Individual β-glucuronidases exhibit substrate bias. By combining enzymes, the mixture broadens substrate coverage and reduces the risk of incomplete hydrolysis