Extraction of Mycophenolic Acid (MPA) and Mycophenolic Acid Glucuronide (MPAG) from Serum Using ISOLUTE® SLE+ prior to LC-MS/MS

Introduction

This application note describes the extraction of the immunosuppressant drug mycophenolic acid and its metabolite mycophenolic acid glucuronide from fortified serum using ISOLUTE SLE+ in a 96-well plate format.

Application Note AN810

Immunosuppressant drugs are instrumental in preventing organ and tissue rejection in patients undergoing transplant surgery. Mycophenolic Acid is a common immunosuppressant drug used in patient transplant therapy. The ability to monitor the trough levels in patients to evaluate dosing is important for the administration of the drug. The free drug and its glucuronidated metabolite can be found in patient serum. The ability to quantitate the amount of free drug and metabolite in patient serum is supported by using a fast and efficient supported liquid extraction sample preparation method using ISOLUTE^® SLE+plates. The free drug and metabolite can be recovered from serum using ISOLUTE SLE+ with high enough efficiency to allow for quantitation at target trough levels.

Figure 1. Structure of Mycophenolic Acid (MPA) and Mycophenolic Acid Glucuronide (MPAG)

Analytes Mycophenolic Acid (MPA) and Mycophenolic Acid Glucuronide (MPAG)

Sample Preparation Procedure

Format: ISOLUTE SLE+ 200 μL Supported Liquid Extraction Plate, part number 820-0200-P01 Matrix: Pooled human serum Sample Pre-treatment: Fortify 100 μL of negative serum with target analytes as needed to prepare target concentrations ranging from 0.1 μg/mL to 10 μg/mL (up to 10 μL of working standard). Add 90 μL of 20% aqueous formic acid to the samples then gently vortex the solutions. NOTE: Total sample load volume should not exceed the recommended load capacity (200 μL) for each well. For larger sample volumes, the method can be scaled up for use with higher capacity ISOLUTE SLE+ plates or columns. Sample Loading: Load pre-treated samples onto wells. Apply a short pulse of vacuum (VacMaster-96 Sample Processing Manifold) or positive pressure (PRESSURE+ 96 Positive Pressure Manifold) to initiate flow and then allow sample to absorb on column for 5 minutes. Analyte Elution: Apply ethyl acetate (2 x 500 μL) to each well and allow solvent to gravity flow. Apply positive pressure or pull slight vacuum as needed during collection process to facilitate a flow rate of 1 mL per minute. Post Extraction: Evaporate sample and reconstitute in water:acetonitrile (50:50, v/v, 500 μL). Additional Information: Working standards were prepared in 100% acetonitrile

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