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Evaluation on the Metabolic Activity of Two Carboxylesterase Isozymes in Mouse Liver Microsomes by a LC-MS/MS Method
Lulu Lan1, Min Li2, Yanjiao Xu2
1Department of Clinical Research, Guangxi Medical University Cancer Hospital, 71 Hedi Road, Qingxiu District, Nanning, Guangxi 530021, China.
Abstract:
An applicable method for the precise measurement of major carboxylesterase (CESs) activity in liver still limited. Clopidogrel and irinotecan are specific substrates for CES1 and CES2, respectively. Clopidogrel is metabolized to the inactive metabolite clopidogrel carboxylate (CCAM) by CES1. Irinotecan is metabolized to the active metabolite 7-ethyl-10-hydroxycamptothecin (SN-38) by CES2. In the present study, the LC-MS/MS method for the determination of CCAM and SN-38 were separately developed to characterize the metabolic activities of CES1 and CES2 in mouse liver microsomal. CCAM was separated on a Ecosil ODS column with an isocratic mobile phase consisted of 5 mmol/L ammonium formate and 0.1% formic acid in water and acetonitrile (15:85, V:V) at a flow rate of 0.4mL/min. SN-38 was separated on a Waters symmetry C18 column with an gradient mobile phase consisted of 5 mmol/L ammonium formate and 0.1% formic acid in water and acetonitrile at a flow rate of 0.3 mL/min. Calibration curves were linear within the concentration range of 100-20,000 ng/mL for CCAM and 1-200 ng/mL for SN-38. The results of method showed excellent accuracy and precision. The recovery rate, matrix effect and stability inspection results were within the acceptance criteria. The optimized incubation conditions were as follows: protein concentration of microsomes were all 0.1 mg/mL, incubation time was 60 min for clopidogrel and 30 min for irinotecan, respectively. This method was sensitive and applicable for the determination of the activity of CESs in the mouse liver microsomes.
Insights
This study developed a precise LC-MS/MS method to measure carboxylesterase (CES) activity in mouse liver microsomes, using clopidogrel for CES1 and irinotecan for CES2 activity determination.
Area of Science:
- Pharmacology
- Biochemistry
- Analytical Chemistry
Background:
- Precise measurement of carboxylesterase (CES) activity in liver is crucial for drug metabolism studies.
- Clopidogrel and irinotecan are specific substrates for CES1 and CES2, respectively, making them valuable tools for activity assessment.
Purpose of the Study:
- To develop and validate a sensitive LC-MS/MS method for the simultaneous determination of clopidogrel carboxylate (CCAM) and SN-38.
- To characterize the metabolic activities of CES1 and CES2 in mouse liver microsomes using the developed method.
Main Methods:
- Development of separate LC-MS/MS methods for CCAM (CES1 activity) and SN-38 (CES2 activity) in mouse liver microsomes.
- Optimization of chromatographic separation using ODS and C18 columns with specific mobile phases.
- Validation of calibration curves, accuracy, precision, recovery, matrix effect, and stability.
Main Results:
- Linear calibration curves were established for CCAM (100-20,000 ng/mL) and SN-38 (1-200 ng/mL).
- The developed methods demonstrated excellent accuracy, precision, and stability within acceptance criteria.
- Optimized incubation conditions included 0.1 mg/mL protein concentration, 60 min for clopidogrel, and 30 min for irinotecan.
Conclusions:
- The validated LC-MS/MS method is sensitive and applicable for determining CES1 and CES2 activity in mouse liver microsomes.
- This method provides a reliable tool for pharmacokinetic and pharmacodynamic studies involving CES enzymes.
