Urine hydrolysis: how did I choose which enzyme to use?

Urine hydrolysis is a part of sample preparation that we do to eliminate glucuronides. So first, what are glucuronides? And why do we want to get rid of them? Glucuronides are formed during metabolism. These glucuronide compounds attach to drugs to make them more water-soluble. This allows for easier excretion of the drugs in urine. However, this is not what we want to analyze when looking for our drugs of interest. We would rather look for just morphine or just oxazepam instead of morphine-3-β-D glucuronide or the oxazepam glucuronide. The process of removing these glucuronides from our analytes of interest is called hydrolysis.

In this post, I’ll tell you about my experience with urine hydrolysis and my success (or lack of success) when using different enzymes.

There are many enzymes on the market that we can use to cleave glucuronidated urinary metabolites. Choosing the best enzyme can depend on which metabolites I am trying to hydrolyze and the amount of time that can be devoted to hydrolysis. These different enzymes all hydrolyze our samples differently so it’s important to investigate which one allows for the most complete hydrolysis. When hydrolyzing, I want to remove most, if not all, of the glucuronide that is attached to my drug of interest in the sample. I looked at four different enzymes: BG100 (red abalone from Kura Biotech), Campbell (abalone from Campbell Scientific), IMCSzyme (recombinant from IMCS), and BGTurbo (recombinant from Kura Biotech).

I analyzed 4 opiates (codeine-6-β-D, morphine-3-β-D, hydromorphone-3-β-D, and oxymorphone-3-β-D), 2 benzodiazepines (lorazepam and oxazepam), norbuprenorphine, and THC-COOH glucuronides. I wanted a range of compounds, especially in the opiate and benzodiazepine classes, to determine if certain analytes were hydrolyzed better than others. There can be variations in hydrolysis, even in the same drug class! I used several incubation parameters: a 30 minute room temperature hydrolysis, a 10 minute 55°C hydrolysis, and a 30 minute 55°C hydrolysis. The samples were extracted using an EVOLUTE® EXPRESS CX 10 mg mixed mode extraction plate from Biotage®.

So how did I determine hydrolysis efficiency? I had my sample with glucuronidated compounds. I spiked these samples so that the final concentration of the sample if all of the glucuronides were cleaved would be 100 ng/mL. I also had a non-conjugated sample (a sample with “free” drug or metabolite and no glucuronides). This sample was also spiked at 100 ng/mL. To calculate hydrolysis efficiency, I used Equation 1.

Equation 1: Calculation of hydrolysis efficiency

This showed me what my hydrolysis efficiency was. Figure 1 shows the hydrolysis efficiency of the glucuronidated analytes when using a 55°C, 30 minute incubation time. As we can see from the graph, the BGTurbo enzyme seemed to have the best hydrolysis efficiency, while the Campbell enzyme had the worst. This was the case across the board, for all of my incubation times and temperatures. My opiate compounds were the most difficult to hydrolyze and I was never able to reach 100% hydrolysis efficiency.

Figure 1: Hydrolysis efficiency of glucuronide compounds when using a 55C, 30 minute incubation

I also wanted to take into consideration any recovery issues that could occur from the enzymes. I didn’t want the other compounds in my panel to have lower recovery when using one enzyme or another. (Dan talked about how to do recovery experiments in this blog post!) Figure 2 shows recovery for a range of analytes using the various enzymes.

Figure 2: Recovery of compounds in various drug classes for all enzymes using a 55C, 30 minute incubation

Based on the results, as far as recovery is concerned, it looks like I can use any of the enzymes and have similar recoveries across the board! When looking at both the hydrolysis efficiencies and recoveries of my analytes for my big 60 compound panel, I think I’m going to use the BGTurbo enzyme. It seemed to have the highest hydrolysis efficiency without sacrificing analyte recovery.

A lot of work goes into figuring out which enzyme to use, especially when you consider the amount of time you’re willing to hydrolyze for without sacrificing hydrolysis efficiency. Which enzymes have you had the most success with?

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