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Urine Variability Could Compromise Efficiency of β-Glucuronidase Hydrolysis

Urine Hydrolysis Using IMCSzyme® RT

INTRODUCTION

Are urine samples destroying your β-glucuronidase enzyme? Why do some enzymes work with reference materials and eventually fail when challenged with real human samples?

Does the enzyme you choose matter?

Learn how you can prevent your difficult urine samples from compromising your confirmatory tests and prevent the likelihood of false negatives.

BACKGROUND

What is the role of β-glucuronidase in urine hydrolysis?

Glucuronidation is a typical, well-characterized metabolic pathway for a variety of chemicals such as drugs and endogenous substances.

β-glucuronidase plays an important role in the analysis of biological fluids for the presence of metabolites for drug screening and drug metabolism studies. Hydrolysis using β-glucuronidase is often necessary to separate the bonds, especially when extensive drug metabolism complicates drug detection in biological samples.

CHALLENGES

Why are some urine samples difficult to hydrolyze? Urine samples are difficult to hydrolyze due to β-glucuronidase enzyme inhibition and heterogeneity in urine samples.

Sample specific urine properties compromise β-glucuronidases to varying levels, more pronounced for some enzymes, and thereby lower the recovery of some drug analytes in an enzyme-specific manner. In fact, clinical urine specimen pH ranges from 4.5 to 8.0, and a shift of 0.5 pH unit can alter enzyme performance by 20% or more.

Enzyme B
Enzyme C

Enzyme B is a wild-type glucuronidase. Enzyme C is limpet (Patella vulgata).

Enzyme B does not effectively hydrolyze codeine-6-β-glucuronide while Enzyme C fails to complete the hydrolysis of most glucuronidated drugs.

Additionally, human urine samples come in a wide range of physical characteristics. A person’s diet, hormonal levels, lifestyle, medications, and overall health greatly influence the properties of their urine samples. These samples may contain inhibitors or inactivators that may interfere with enzyme activity (exact mechanism is still being widely studied). Examples include flavonoids, thiosulfinates, iminosugars, natural acids, and lactones.

SOLUTION

How can we prevent urine samples from compromising confirmatory tests?
IMCSzyme RT is highly resistant to naturally occurring inhibitors found in human urine. It is a next-generation genetically modified β-glucuronidase enzyme designed to hydrolyze glucuronidated drugs of abuse in 15 minutes or less at room temperature. After rapid hydrolysis with IMCSzyme RT, samples can be analyzed by immunoassay, mass spectrometry, and high-performance liquid chromatography.

IMCSzyme

Only IMCSzyme RT consistently outperforms other enzymes across multiple glucuronidated substrates and across different urine types​.

Our product has been optimized to provide results that are:

Fast. IMCSzyme RT is perfectly developed for fully automated, high throughput testing. It is the only enzyme in the market capable of completing greater than 95% room temperature hydrolysis of 16 glucuronidated drugs of abuse in 15 minutes or less. It hydrolyzes in the time it takes to pipette samples into a 96-well plate and eliminates any need for an incubator or a water bath.

Clean. IMCSzyme RT is a clear solution with no interference, which minimizes matrix effects. It includes room temperature buffer (RTB) prepared with mass spectrometry-friendly volatile salts and increases LC column life.

Accurate. IMCSzyme RT is resistance to naturally occurring inhibitors found in urine. Its broad-working pH range ensures accuracy even with the most difficult urine samples. Additionally, there is no conversion of 6-MAM to morphine.

REFERENCES

  1. Lee LA, McGee AC, Sitasuwan P, Tomashek JJ, Riley C, Muñoz-Muñoz AC, Andrade L. (2022). Factors Compromising Glucuronidase Performance in Urine Drug Testing Potentially Resulting in False Negatives. Journal of Analytical Toxicology. 46: 689–696.
  2. McGee AC, Sitasuwan PN, Tomashek JJ, Munoz-Munoz AC, Andrade L, Lee LA. (2021). Overcoming Heterogeneity in Urine Specimens: Avoiding False Negatives caused by Endogenous Inhibitors. Annual Meeting, Society of Forensic Toxicologists, Nashville, TN. September 26 - October 1, 2021.
  3. Grenier AC. (2020). Comparing Glucuronidase Hydrolysis Efficiencies in Patient Urine Samples. SOFTember Virtual Program, Society of Forensic Toxicologists, [Online]. September 9-30, 2020.
  4. McGee AC, Sitasuwan PN, Tomashek JJ, Schlachter CR, Grenier AC, Andrade L, Lee LA. (2020). Urine Variability Compromises β-Glucuronidase Performance Causing Inaccurate Drug Quantitation. SOFTember Virtual Program, Society of Forensic Toxicologists, [Online]. September 9-30, 2020.
  5. Sasaki TA. (2019). Simplifying Urine Drug Testing with “Flash Hydrolysis” Using New Recombinant β-Glucuronidase Enzymes. Annual Meeting, Society of Forensic Toxicologists, San Antonio, TX. October 13-18 2019.
  6. IMCSzyme RT Master Mix Stability. (2021). [Online]. Accessed March 29, 2022.
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