Introduction:

Urine drug testing is one of the most common practices for monitoring the use of prescribed or illicit 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). β-glucuronidases hydrolyze glucuronidated compounds from phase II metabolites present in urine which simplifies parent analyte detection using LC-MS/MS. This study shows that endogenous chemicals in clinical samples can reduce enzyme performance compared to control samples.

Objectives:

Present data to show that endogenous chemicals in clinical samples could reduce enzyme hydrolysis compared to control samples. Not all commercially available recombinant enzymes are inhibited by these chemicals equally. Additionally, using an inadequate amount of enzyme could result in low hydrolysis for drugs of abuse which can potentially cause false negatives.

Methods:

Drug standards were from Cerilliant. All reagents were purchased from MilliporeSigma or Fisher Scientific. Opioid-positive urine specimens were obtained from a national testing laboratory and had no identifying information. Drug free human urine control was from UTAK and was fortified with glucuronidated drugs of abuse. Two commercially available β-glucuronidases from two different manufacturers are both advertised as room temperature hydrolysis. One is IMCSzyme RT from IMCS the other is “Premixed Enzyme”. Control and patient specimens were buffered and hydrolyzed with two commercial glucuronidases for 15-minutes at room temperature. The two β-glucuronidases were compared by two different methods. First method, ninety-six patent samples were hydrolyzed using an equivalent protein amounts of β-glucuronidases, measured by Bradford. Second method tested patient samples with a range of β-glucuronidase amounts that are below, at and over manufacturer recommendations.

Sample clean-up was performed by eluting hydrolyzed urine samples through β-Gone plus plates from Phenomenex and diluted with water. Sample were 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:

Ninety-six patient samples were hydrolyzed with equivalent protein amounts by two different β-glucuronidases. Samples were considered positive if opioid recovery was ≥ 25 ng/mL. Out of 96 patient samples analyzed, 50 results disagreed between IMCSzyme RT and Premixed Enzyme where they were reported above the cutoff specification when hydrolyzed with IMCSzyme RT and below the cutoff specification when hydrolyzed with Premixed Enzyme. Missing such thresholds resulted in 50 results being potentially reported as false negatives.

Four patient samples and a fortified control sample were hydrolyzed with a range of β-glucuronidase concentrations (0-100 µg of one β-glucuronidase per reaction and 0-200 µg of the second per reaction). Hydrolysis was reported as % hydrolysis and considered complete when hydrolysis reached ≥ 80%. Complete hydrolysis was confirmed based on ≤ 20% glucuronide remaining in the sample. Two out of four patient samples required more enzyme to complete hydrolysis than the control sample, indicating that these samples contain endogenous chemicals that reduced enzyme hydrolysis of morphine and oxymorphone glucuronide. Endogenous chemicals in urine that reduced enzyme activity include urea, ascorbic acid and flavonoids.

Discussion:

Some patient samples contain different amounts of endogenous chemicals that reduce enzyme activities compared to control samples and not all enzymes are affected equally by these chemicals. Using an inadequate amount of enzyme could result in low rates of hydrolysis for drugs of abuse, and ultimately, false negatives for patient samples.