Effect of Enzyme Titer and pH on Hydrolysis Efficiency Using Recombinant Limpet β-Glucuronidase Enzyme

By: Jim Blasberg1, Danny Taglicht2, Ena Orzech2, Jeff Turner1, Ben Cutak1, and Kevin Ray1, 1 - MilliporeSigma, St. Louis, MO 2 – MilliporeSigma, Jerusalem, Israel

Overview

Urine drug testing has become an important tool to monitor compliance and detect the presence of illicit drugs. The primary metabolites of many such drugs are glucuronides, which offer analytical challenges due to their hydrophilic nature and poor ionization efficiencies. β-glucuronidase is often utilized for enzymatic hydrolysis of glucuronide metabolites back to their parent drug, simplifying the analytical workflow.

Here we evaluate enzyme titer and pH effect on hydrolysis efficiency of a recombinant limpet enzyme against a panel of glucuronide substrates. Traditionally difficult opioid substrate, Codeine-6-β-D-glucuronide; a quaternary amine substrate, Amitriptiyline-N-β-Dglucuronide; as well as benzodiazepine and steroid drugs are represented. We found the enzyme titer and solution pH effect on hydrolysis efficiency varied widely among tested substrates. Based on this data, experimental conditions can be optimized for minimal titer and optimal pH.

Enzyme Digestion

  • Glucuronide drug conjugates were spiked into a synthetic urine matrix along with isotopically labeled (deuterated) parent drug internal standards.
  • All hydrolysis experiments were conducted at 70 °C.
  • Enzyme titer experiments were performed at 0.5, 5.0, and 50 units/μL (final reaction mix) concentrations and incubation times 5, 10, 15, 20, 25, and 30 minutes.
  • Hydrolysis pH experiments were performed at pH 4.0, 4.5, 5.0, 5.5, 6.0 and enzyme titers 0.5, 5, and 50 units/μL (final reaction mix) concentrations.
  • Hydrolyzed samples were injected as-is with no additional cleanup prior to analysis by LC-MS/MS.

Results

  • Percent conversion of >85% in 15 minutes was achieved for all substrates (except Dihydrocodeine, requiring 30 minutes) a titer of 50 Units per μL and pH of 5.0, Figure 2 
  • The titer requirement at pH 5.0 (typical condition) to achieve complete hydrolysis within 15 minutes is substrate dependent.
  • Similarly, the optimal pH was found to be substrate dependent. 
  • Recovery of COOH-THC is problematic at lower pH due to apparent post-hydrolysis degradation.

Codeine-Gluc Hydrolysis Time Course 70 °C, pH 5.0,50 U/μL

Hydrolysis of Codeine-Glucuronide to Codeine over 30 minute time course

Figure 1. Hydrolysis of Codeine-Glucuronide to Codeine over 30 minute time course

 

16 Substrate Hydrolysis Efficiency 70 °C, pH 5.0, 15 min, 50 U/μL

Parent drug recovery of 16 glucuronide substrates

Figure 2. Parent drug recovery of 16 glucuronide substrates

 

Effect of pH on Amitriptyline Recovery 70 °C, 30 min, 5 U/μL

Increase in Amitriptyline recovery with corresponding drop in Amitriptyline-Glucuronide demonstrates pH effect on hydrolysis efficiency at low titer (5 U/μL).

Figure 3. Increase in Amitriptyline recovery with corresponding drop in Amitriptyline-Glucuronide demonstrates pH effect on hydrolysis efficiency at low titer (5 U/μL). Complete recovery of Amitriptyline is achieved at all pH conditions using the titer condition of 50 U/μL as shown in Figure 5.

 

Effect of pH on COOH-THC Recovery 70 °C, 30 min, 50 U/μL

Quantitative recovery of COOH-THC was problematic at lower pH

Figure 4. Quantitative recovery of COOH-THC was problematic at lower pH (data not shown). COOH-THC-Glucuronide was not detected in qualitative analysis by qTOF at any pH, indicating complete hydrolysis was achieved. Presence of an unidentified peak at lower pH suggests post-hydrolysis degradation of COOH-THC.

 

pH/Titer Heat Map – 16 Glucuronide Substrates

pH/Titer Heat Map – 16 Glucuronide Substrates

Figure 5. Recombinant Limpet enzyme was diluted in appropriate pH buffer and incubated with spiked synthetic urine; 1:1, v:v at 70 °C for 30 minutes under pH and titer conditions delineated above. Parent drug recovery is dependent upon substrate, enzyme titer, and solution pH. Preferred conditions can be selected for each substrate based on the heat map shown here.

Conclusions

We found that the effect of enzyme titer and solution pH on hydrolysis efficiency varied widely among tested substrates. Based on this data, optimal experimental conditions of titer, pH, and hydrolysis time can be recommended for a substrate of interest.