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 by hydrolysis with a β-glucuronidase (β-GUS) and/or sulfatase. The activity of the β-glucuronidases will be affected both by the pH of the solution and the aglycone moiety of the drug glucuronide. Furthermore, for a single β-GUS, different drug glucuronides may display different pH optimums which is a consequence of aglycone functional groups and glucuronide linkage.

Objectives:

Compare the activities of commercially available β-glucuronidase enzymes on O-linked and N-linked drug glucuronides in different pH buffers.

Methods:

Deuterated and reference standards for morphine, oxymorphone, codeine, O-desmethyltramadol, O-desmethylvenlafaxine, amitriptyline, and nortriptyline were purchased from Cerilliant. All other reagents were purchased from MilliporeSigma or Fisher Scientific. Four β-glucuronidases were expressed and purified at IMCS: modified Escherichia coli β-GUS (E1F), Aspergillus chimera β-GUS (R3), Eubacterium eligens β-GUS (EeGUS), and Brachyspira pilosicoli (B. pi.) β-GUS variant (BpiGUS). A triple buffer system of Tris, Acetate, and MES was used from pH 4-9. Calibration curves include parent and glucuronide standards. Reactions were stopped by transferring sample into methanol (40%). 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:

The pH optimum was dependent on both the enzyme and the linkage (O- or N-) of the sugar to the drug aglycone. For O-linked drug glucuronides, E1F and BpiGUS favored neutral to basic pH while β-GUS Variant R3 and EeGUS had preference in acidic buffers. However, when N-linked drug glucuronides were hydrolyzed by the same enzymes, the pH optima of all enzymes shifted towards a more basic pH.

Discussion:

The β-GUS enzymes characterized here display variable pH optimums on drug glucuronides. Additionally, the pH optimum changes per enzyme based on whether the drug glucuronide is O-linked or N-linked, and the O-linked drug glucuronides are generally hydrolyzed at lower pH than N-linked drug glucuronides relative to each enzyme tested. The change in pH optimum based on sugar linkage may be related to the pKa of the amine in the N-linked drug glucuronides. When analyzing and comparing different β-GUS enzymes, the pH of the reaction buffer and sugar linkage of the target substrate(s) should be acknowledged in order to achieve optimal hydrolysis.