IMCS at SOFT 2022

Background/Introduction:

Urine drug testing is one of the most common practices for monitoring the use of prescribed opioid medications. Testing is typically performed by a preliminary screening assay, such as immunoassay, followed by a confirmatory assay such as liquid chromatography coupled with mass spectrometry (LC-MS/MS). Screening and confirmatory assays benefit from the use of enzymes that hydrolyze, or deconjugate, glucuronidated analytes. New generation of beta-glucuronidases can now effectively cleave glucuronides in urine at room temperature, however during the studies we have identified additional challenges in urine drug testing across biologically relevant pH extremes and patient urine specimens.

Objectives:

Screening and characterizations of new generation of beta-glucuronidases against varying concentrations of chemicals present in urine.

Methods:

IMCSzyme RT was provided by Integrated Micro-Chromatography Systems (IMCS). BpGUS is a recombinant enzyme was expressed in E. coli with gene sequence sourced from Brachyspira pilosicoli. The enzyme contained poly-his tag for immobilized metal affinity chromatography (IMAC) followed by buffer exchange. Both enzymes are approximately 2 mg/mL of protein.

Enzyme activities were measured using a substrate-metabolite cross titration assay where substrate and metabolite concentrations varied while enzyme concentration remained constant. Hydrolysis rates were calculated and plotted as a function of substrate concentration and hydrolysis rate at each metabolite concentration. 4-methylumbelliferyl glucuronide was utilized as the substrate with 4-methylumbelliferyl measured with fluorescence. Substrate and metabolites were mixed, and hydrolysis was initiated with BpGUS addition to substrate-metabolite mixture. Reaction was quenched in 20 second intervals for 120 seconds.

Opioid-positive urine specimens were obtained from a national testing laboratory. Negative controls and patient specimens were buffered and hydrolyzed with IMCSzyme RT or BpGUS. Hydrolyzed urine samples were cleaned with solid phase extraction, solvent evaporated, reconstructed in methanol and diluted with mobile phase A (0.1% formic acid in water). 10 μL of sample was injected on a Thermo Scientific™ Vanquish™ UHPLC system coupled with a Thermo Scientific™ Endura™ Triple Quadrupole Mass Spectrometer using a Phenomenex Kinetex® 2.6 μm Phenyl-Hexyl 100 Å, 50 x 4.6 mm column. Mobile phase B was 0.1% formic acid in acetonitrile.

Results:

BpGUS activity drops approximately 15% at urea level of 25 mM or 70 mg/dL and drops nearly 40% when urea concentration is 100 mM or 350 mg/dL while the activity of IMCSzyme RT activity drops approximately 10% and 30% at the same urea concentrations. Well-known inhibitor D-saccharic acid 1,4-lactone monohydrate has a Ki value of 0.001 mM towards BpGUS, which is one of the lowest inhibition concentrations reported thus far for a glucuronidase. Other natural metabolites inhibiting this enzyme show higher Ki values ranging between 40-50 μM, and other common metabolites from coffee, vitamin C, and glucuronic acid did not have a significant impact on either enzyme. The potential negative impact is highlighted with six patient samples that exhibit varying degrees of inhibition. In one patient out of six, oxymorphone recovery is incomplete despite the addition of a large quantity of enzyme.

Conclusion/Discussion:

Not all enzymes have the same tolerances toward inhibitors in urine. One approach to avoid the complexity of natural inhibitors or urea reducing the hydrolysis efficiency is to dilute down the urine to lower the urea concentration and the concentration of the natural inhibitors. Such dilution is also beneficial in achieving the target pH for optimal hydrolysis, whereas limited dilution or buffer addition can result in reduced pH control. Heterogeneity of urine is a common fact, even if sourced from a single patient, the hydration levels and food metabolites will significantly vary. Although further dilution of urine will dilute target analytes, and possibly require a higher instrument sensitivity, there will always be economical and practical balancing act of efficient enzymatic hydrolysis.