A Fast Ultra-Performance Liquid Chromatography Method for Simultaneous Quantification of Mycophenolic Acid and Its Phenol- and Acyl-Glucuronides in Human Plasma

Abstract

Several studies have demonstrated a close relationship between mycophenolic acid (MPA) exposure and the risk for graft rejection or side effects. Measurements of MPA and its metabolites plasma levels are therefore recommended. A new chromatographic method has been developed using ultra-performance liquid chromatography (UPLC) to improve both analytical throughput and sensitivity. MPA and its phenol-glucuronide and acyl-glucuronide were extracted from plasma using Isolute C2 solid phase extraction (SPE) cartridges (100 mg, 3 mL). UPLC separations were performed with a Waters BEH C18 column (50 x 2.1 mm, 1.7 microm) maintained at 65 degrees C on a Waters Acquity instrument equipped with a photodiode array detector. The total UPLC run time was 3.5 minutes. The method was linear in the range of 0.1-40 microg/mL for MPA and acyl-glucuronide, and 1-400 microg/mL for phenol-glucuronide. Relative standard error and mean relative prediction error were <15% for all tested quality controls (in-house and external proficiency panels). UPLC performances are characterized by a dramatic reduction in retention times together with an improvement of the sensitivity without affecting peak resolution. Further validations have been obtained by analyzing routine and clinical trial patients' samples. Significant improvement of the analytical throughput (reduction of run time from >10 to 3.5 minutes) was obtained using UPLC for MPA analyses. This retention time reduction was accompanied by an improvement of other analytical performances such as sensitivity.