© Michael Court
Created December 2002
-Glucuronidase/sulfatase deconjugation of drug metabolites in urine
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To 1.5 mL polypropylene Eppendorf tubes add 250 uL of urine and 250 uL of 1 M
KH2PO4 buffer (pH = 4.0) to adjust urine pH to ~ 5.0. (or 200 mM sodium acetate
buffer, pH 5.0)
a. Optional – for quantitation – add drug standards (dissolved in methanol) and dry
down then add known blank urine.
Add 10 uL of -glucuronidase liquid (1000 units).
a. Use Helix pomatia -glucuronidase liquid 100 U / uL
i. Sigma G-0762
ii. **Note that this also has sulfatase (and probably other) activities
Vortex and incubate for at least 2 hours (preferably overnight) at 370C.
Add either
a. 250 uL acetonitrile/5% acetic acid (for most compounds) OR
b. 250 uL of 0.1 N HCL (for highly polar compounds)
Optional – for quantitation - add internal standard (up to 50 uL dissolved in methanol)
may be combined with stop solution
Vortex and centrifuge for 5 minutes.
Transfer to HPLC tube and perform HPLC assay – inject up to 100 uL.
*** Should also include NEGATIVE control for comparison –
a. Add stop solution (HCl or acetonitrile as in step 4) before -glucuronidase (step 2).