KEY TAKEAWAYS:
- IMCS pioneered the development of recombinant β-glucuronidases, replacing animal-derived enzymes.
- Continuous formulation advances (IMCSzyme®, IMCSzyme 3S, 3P, and IMCSzyme® RT) improved enzyme stability, temperature tolerance, and hydrolysis efficiency.
- Independent studies confirmed that IMCSzyme® β-glucuronidases provide faster, more consistent hydrolysis and higher resistance to inhibitors than competing enzymes.
Over the past decade, researchers at Integrated Micro-Chromatography Systems (IMCS) have made significant advancements in β-glucuronidases (BGUS), as reflected in the number of issued US patents and peer-reviewed publications. These advances address everyday challenges faced by clinical, forensic, and research laboratories where incomplete hydrolysis can delay results or lead to inaccurate drug detection. BGUS is an enzyme that breaks down, or hydrolyzes, the glucuronic acid linked to an analyte of interest (Figure 1). This process enables the analysis of the aglycone by various laboratory techniques such as immunoassay, gas chromatography, or liquid chromatography. Direct analysis of the glucuronidated target may be possible with highly sensitive mass spectrometers, but the presence of glucuronic acid often suppresses signals or broadens peak shapes.
The team’s innovations focused on developing genetic variants of β-glucuronidases under the IMCSzyme® brand with enhanced properties compared to their original counterparts. These improvements included higher enzymatic activities, broader substrate ranges, wider pH ranges, higher resistance to inhibitors, and greater enzyme stability across storage temperatures.
Prior to IMCSzyme’s release, many laboratories relied on natural sources from snail or abalone entrails, assuming that a mixture of these natural enzymes was necessary to hydrolyze a range of different analytes. IMCS was first to demonstrate in 2013 that a genetically engineered, recombinant enzyme can outperform those natural enzymes, reducing the hydrolysis time from hours to minutes [1]. Furthermore, sourcing a recombinant enzyme eliminated the need for harvesting snails, limpets or abalone, providing a more reliable supply chain and greater consistency in the final product.
The original IMCSzyme® (Cat. No. 04-E1F-010) was designed to hydrolyze at neutral pH and at elevated temperatures of 45-60 °C. The enzyme’s compatibility with neutral pH conditions enables users to test plasma, blood, oral fluids, or cell cultures using mild buffer conditions, such as phosphate-buffered saline or cell culture media.
To improve stability during shipping, IMCS developed IMCSzyme 3S (Cat. No. 04-INT3S-005), a thermostable liquid formulation that can tolerate temperatures of 40 °C for 160 hours and 37 °C for four weeks and can undergo multiple freeze-thaw cycles without loss of activity. This robustness to both elevated and freezing temperatures makes the product ideal for shipping overseas, where cold-chain logistics can be challenging. The product is also available as a dried film or lyophilized format, IMCSzyme 3P (Cat. No. 04-ESF3P-002), reducing the transportation mass. The original IMCSzyme, 3S and 3P contain the same genetically modified β-glucuronidase in different formats to accommodate the different logistical needs across the globe (Figure 2).
In recent years, the IMCS R&D team blended two distinct BGUS variants to provide a product with enhanced activity at room temperature (20-25°C), allowing laboratories to process a large number of samples without the need for an incubator. The blending of the two enzymes also resulted in the added benefit of expanding its substrate profile and pH range that an individual enzyme alone could not achieve. This blended product is called IMCSzyme® RT (Cat. No. 04-RT-005) and has been designed to function at slightly acidic pH levels of 5-6 using a volatile buffer compatible with mass spectrometers. This product represents the most advanced formulation by requiring the least amount of enzyme for compliance testing using urine as the primary biological matrix and liquid chromatography tandem mass spectrometry as the major downstream analytical instrument [2].
This blend of β-glucuronidases exhibited 10x fold higher activity compared to a competing product, as presented in the Society of Forensic Toxicologists (SOFT) meeting in 2019 [3]. The competing enzyme is premixed with its own buffer (Table 1, Premixed Enzyme) but still required 100 μg of enzyme per reaction, whereas IMCSzyme RT only needed 10 μg. Despite this larger quantity of enzyme from the competitor, the competing product could not recover the target analyte even with extended incubation time of 50 minutes whereas IMCSzyme RT achieved over 90% hydrolysis in 15 minutes (Figure 3).
| Table 1. Sample preparation | ||
|---|---|---|
| IMCSzyme® RT | Premixed Enzyme | |
| Urine | 50 µL | 50 µL |
| Enzyme |
5 µL IMCSzyme RT (10 μg of enzyme – 2 mg/mL concentration) |
100 µL Premixed Enzyme (100 μg of enzyme – 1 mg/mL concentration; requires 10x more) |
| Buffer | 150 µL 1x RTB buffer | No buffer to add |
| Internal standard | 10 µL | 10 µL |
| Water | 295 µL | 350 µL |
Further studies presented by Dr. Nguyen Nguyen showed pooled human urine samples treated with IMCSzyme RT recovered analytes in 5 minutes with the lowest amount of product (20 µL) (Figure 4), whereas the competing enzyme (Premixed Enzyme) under-hydrolyzed even after 30 minutes of incubation with five times more enzyme [4].
The underperformance of the premixed enzyme is more noticeable when using challenging human urine samples that contain a mixture of different chemicals that reduce enzyme performance [5]. Common chemicals in urine like urea or vitamin C can negatively affect the premixed enzyme, reducing their performance, resulting in lower recoveries of analytes (Figure 5). Not only is IMCSzyme RT a superior product with higher activities towards glucuronidated substrates, its tolerance to natural inhibitors in urine provides more consistent performance over other BGUS products in the market.
Based on the historical results from independent third parties, the benefits of IMCSzyme RT in compliance testing are multi-fold.
- Less enzyme used per sample, meaning less protein per sample for removal and helps reduce clogging the liquid chromatography tubing[6].
- Less enzyme per sample also means more tests per volume of the same amount of product, which reduces the cost per test.
- Greater tolerance towards natural inhibitors in urine like urea and vitamin C means performance is maintained and reduces the risk of under hydrolyzing or reporting false negatives[5]
- Blend of different β-glucuronidases to provide synergistic benefits as a single enzyme has weaknesses[7].
REFERENCES
- Morris AA, Chester SA, Strickland EC, McIntire GL. (2014). Rapid Enzymatic Hydrolysis Using a Novel Recombinant β-Glucuronidase in Benzodiazepine Urinalysis. Journal of Analytical Toxicology; 38(8):610-614. doi: 10.1093/jat/bku083.
- Lee LA, McGee AC, Sitasuwan P, Tomashek JJ, Riley C, Muñoz-Muñoz AC, Andrade L. (2021). Factors Compromising Glucuronidase Performance in Urine Drug Testing Potentially Resulting in False Negatives. Journal of Analytical Toxicology. doi: 10.1093/jat/bkab090.
- 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.
- Abddelgader A, Karamooz S, Nguyen N. (2022). Evaluation of new beta-glucuronidase for urine drug testing. 12th Annual Conference and Exhibits, The Association for Mass Spectrometry & Advances in the Clinical Lab, Monterey, CA. April 5-8, 2022.
- McGee AC, Schlachter CR, Collins C, Lee LA. (2022). Natural inhibitors in urine can reduce glucuronidase performance and result in lowered recoveries. Annual Meeting, Society of Forensic Toxicologists, Cleveland, OH. October 31 - November 4, 2022.
- McGee AC, Lee LA, Schlachter CR. (2023). Balancing Column Deterioration with Enzyme Performance for Drug of Abuse Hydrolysis. IACFT Spring 2023. https://imcstips.com/news/balancing-column-deterioration-with-enzyme-performance-for-drug-of-abuse-hydrolysis/
- McGee AC, Chaparala A, Lee LA, Schlachter CR. (2025). Hydrolysis of Drug Glucuronides by β-Glucuronidase is Contingent on Both pH and N- or O-Glucuronide Linkage. Annual Meeting, Society of Forensic Toxicologists, Portland, OR. October 26 - November 31, 2025.
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