Comparison of Two β-Glucuronidases
β-glucuronidases recover drug compounds by hydrolyzing glucuronides on metabolites formed by normal metabolism. In this poster, Dr. Nguyen Nguyen, Clinical Chemist/Toxicologist and Safety Officer at Baylor Scott & White, presented his group's evaluation of two novel β-glucuronidase products, IMCSzyme RT and "CE" (their "current enzyme", an All-in-One β-glucuronidase), for recovery of common opioids and benzodiazepines at room temperature.
Study Summary
Urine samples (100 µL) were pooled from patients previously confirmed positive for benzodiazepines and opioids. Two experiments were conducted to compare recoveries from samples hydrolyzed with IMCSzyme RT or an "all-in-one" β-glucuronidase (CE, Current Enzyme). The first experiment compared recoveries from a range of enzyme volumes (0 – 100 µL) for samples incubated for 15 minutes at room temperature. The second experiment determined whether incubation times (0-30 minutes at room temperature) improved recoveries when using a fixed enzyme volume (100 µL).
The first experiment (Figure 2) tested various volumes of enzyme (0 – 100 μL) to determine the amount needed for complete hydrolysis at 15 minutes room temperature incubation.
IMCSzyme RT required only 20 μL enzyme volume while the all-in-one β-glucuronidase (CE, Current Enzyme) needed more. For instance, codeine did not hydrolyze fully even at 100 μL CE (1:1 ratio), which opens up the potential risk of false negatives. Hydromorphone needed >50 μL CE, while morphine, oxymorphone, o-desmethyltramadol all needed 65 μL CE.
A fixed enzyme amount of 100 µL for IMCSzyme RT and CE and varying incubation times did not improve recoveries for CE (Figure 3). A possible explanation for this may be attributed to endogenous compounds in urine that reduce the efficiency of enzymes to varying degrees, resulting in lower recoveries of certain analytes (Lee et al. 2021).
Recoveries from pooled urine samples hydrolyzed at 5 or 30 minutes with either IMCSzyme RT or CE were compared. There is a dramatic difference in codeine recovery between IMCSzyme RT and CE for both the 5-minute (A) and 30-minute incubations (B): IMCSzyme RT completely hydrolyzed codeine in only 5 minutes, while CE did not even get there after 30 minutes. Lower concentration targets (hydromorphone, morphine, o-desmethyltramadol, and oxymorphone) show recovery differences for both enzymes and incubation times.
Summary
Even with 5x less enzyme, IMCSzyme RT enabled complete hydrolysis in a shorter amount of time.