Considerations When Performing Enzyme Hydrolysis for Drugs of Abuse Analysis by LC-MS/MS

Urine analysis can be relatively simple; however, metabolic processes result in the additional complication of transforming some drugs of abuse into glucuronides. Through this metabolic pathway, glucuronic acid links to the parent drug, which enhances its solubility in water and enables more efficient urinary excretion [1]. This results in the glucuronide metabolite being present in the urine. For example, morphine-3-glucuronide would be present in the urine of an individual who used morphine.

Since LC-MS/MS analysis primarily focuses on the analysis of the parent drug rather than the glucuronide, a hydrolysis step is required to release glucuronic acid from the analyte of interest [1]. One way of achieving this hydrolysis is through the use of the enzyme β-glucuronidase. This is the type of hydrolysis we will be discussing in this blog.  An example of this reaction can be found in Figure 1 below.

 

This is a simple reaction that involves the addition of enzyme to your sample followed by incubation under certain conditions. The addition of this enzyme releases the parent compound from the glucuronide. When performing this type of sample preparation, it is important to check the efficiency of the enzyme hydrolysis reaction. This is often done by running a hydrolysis control as part of the analysis.

When preparing these controls, it is important to keep a couple of factors in mind. For most quality control samples, it is standard to target somewhere in the middle of the linear range, but for hydrolysis controls, it is important to target the upper range. This is because as the concentration of glucuronide increases, the hydrolysis efficiency can decrease, which can result in underreporting analyte concentrations [2]. So, in order to make sure patient or test samples are being accurately hydrolyzed and quantitated, it is best to test near the upper limit of quantitation (ULOQ).

Additionally, it is important to consider that the hydrolysis control is prepared accurately. When reading literature regarding enzyme hydrolysis controls, the verbiage “when liberated” is often used. This means that the hydrolysis control sample was spiked to account for the loss of glucuronic acid post-hydrolysis. Typically, when preparing QCs, you simply spike the value of standard that you are hoping to see. For example, if you were targeting 250 ng/mL, you would dilute 250 µL of a 1000 ng/mL stock into 1 mL of final solution. However, when making the hydrolysis control, you need to look at the molecular weight for both the analyte of interest and the glucuronide and use a corrected calculation to prepare the QC. This can be done by using Formula 1 below.

 

It is important to use this equation in order to ensure accurate hydrolysis sample preparation. In the following experiment, two hydrolysis controls were prepared and analyzed by LC-MS/MS. The first control, labeled “Standard QC Calculation” in Table I and Table II below, was prepared using the standard calculation for making QCs. The second QC, labeled “Corrected QC Calculation” in Table I and Table II below, was prepared using Formula 1 above.  Table I includes results for a QC at 100 ng/mL and Table II includes results for 1000 ng/mL (linear range for the analytes in this study was 20–1000 ng/mL).

Table I: Comparison of Calculations at 100 ng/mL

Analyte	Preparation	Average	% Difference
Morphine	Standard QC Calculation	87.3	-12.6
	Corrected QC Calculation	114.7	14.7
Hydromorphone	Standard QC Calculation	85.9	-14.1
	Corrected QC Calculation	114.3	14.3
Amitriptyline	Standard QC Calculation	90.9	-9.1
	Corrected QC Calculation	103.3	3.3
Oxazepam	Standard QC Calculation	98.3	-1.7
	Corrected QC Calculation	100.0	0.0

 

Table II: Comparison of Calculations at 1000 ng/mL

Analyte	Preparation	Average	% Difference
Morphine	Standard QC Calculation	643.7	-35.6
	Corrected QC Calculation	991.7	-0.8
Hydromorphone	Standard QC Calculation	683.3	-31.7
	Corrected QC Calculation	1076.7	7.7
Amitriptyline	Standard QC Calculation	798.3	-20.2
	Corrected QC Calculation	1040.7	4.1
Oxazepam	Standard QC Calculation	635.3	-36.5
	Corrected QC Calculation	932.3	-6.8

 

In Table I, the target concentration of the hydrolysis control was 100 ng/mL, whereas the target concentration for Table II was 1000 ng/mL. Results in Table I are all within ±15% of the target value, but you can observe the low bias using the “Standard QC Calculation.” Table II shows how the low bias for the “Standard QC Calculation” impacts results as concentrations increase, while the “Corrected QC Calculation” values meet acceptance criteria.

In conclusion, it is important to properly prepare the hydrolysis control in order to assess that both the sample preparation and enzyme hydrolysis are working properly. It is also important to consider the concentration of your hydrolysis control to assess hydrolysis efficiency. Overall, utilizing these practices can have a substantial effect on the accuracy of drugs of abuse in urine testing by LC-MS/MS.

References and Further Reading

1. Neifeld, Jillian. Urine hydrolysis: how did I choose which enzyme to use? https://www.biotage.com/blog/urine-hydrolysis-how-did-i-choose-which-enzyme-to-use
2. Skov, Kathrine. et. al. Exploring Enzymatic Hydrolysis of Urine Samples for Investigation of Drugs Associated with Drug-Facilitated Sexual Assault. Pharmaceuticals. 2023. 21, 17.