A high-throughput U-HPLC–MS/MS assay for the quantification of mycophenolic acid and its major metabolites mycophenolic acid glucuronide and mycophenolic acid acyl-glucuronide in human plasma and urine

Abstract

Mycophenolic acid (MPA) is used as an immunosuppressant after organ transplantation and for the treatment of immune diseases. There is increasing evidence that therapeutic drug monitoring and plasma concentration-guided dose adjustments are beneficial for patients to maintain immunosuppressive efficacy and to avoid toxicity. The major MPA metabolite that can be found in high concentrations in plasma is MPA glucuronide (MPAG). A metabolite usually present at lower concentrations, MPA acyl-glucuronide (AcMPAG), has been implicated in some of the adverse effects of MPA. We developed and validated an automated high-throughput ultra-high performance chromatography-tandem mass spectrometry (U-HPLC-MS/MS) assay using liquid-handling robotic extraction for the quantification of MPA, MPAG, and AcMPAG in human EDTA plasma and urine. The ranges of reliable response were 0.097 (lower limit of quantitation) to 200 μg/mL for MPA and MPAG and 0.156– 10 μg/mL for AcMPAG in human urine and plasma. The inter-day accuracies were 94.3–104.4%, 93.8–105.0% and 94.4–104.7% for MPA, MPAG and AcMPAG, respectively. Inter-day precisions were 0.7–7.8%, 0.9–6.9% and 1.6–8.6% for MPA, MPAG and AcMPAG. No matrix interferences, ion suppression/enhancement and carry-over were detected. The total assay run time was 2.3 min. The assay met all predefined acceptance criteria and the quantification of MPA was successfully cross-validated with an LC-MS/MS assay routinely used for clinical therapeutic drug monitoring. The assay has proven to be robust and reliable during the measurement of samples from several pharmacokinetics trials.

A comprehensive review of the published assays for the quantitation of the immunosuppressant drug mycophenolic acid and its glucuronidated metabolites in biological fluids

“…Also, it was shown that MPAG-free after storage beyond 6 months at À80°C would have a tendency to degrade as a function of time (Figurski et al, 2009). The urinary stability of MPAG and AcMPAG has also been raised as a key issue during the validation work of Klepacki et al (2012. During the first 24 h of storage both MPAG and AcMPAG were stable in urine; however, beyond 24 h there was degradation evident for MPAG and AcMPAG in urine such that by 7 days of storage there was a complete degradation of AcMPAG in urine, while there was a 70% degradation of MPAG in urine (Klepacki et al, 2012). In this report, instability of both analytes also occurred in plasma samples during storage, although the extent of degradation did not appear to be severe (Klepacki et al, 2012).…”

Simultaneous quantification of mycophenolic acid and its glucuronide metabolites in human plasma by an UPLC–MS/MS assay

“…Several assays have been reported for the analysis of MPA and its metabolites by LC-MS/MS in different matrices (Benech et al, 2007;Brandhorst et al, 2006;Delavenne et al, 2011;Heinig et al, 2010;Kawanishi et al, 2015;Klepacki et al, 2012;Kuhn et al, 2009;Laverdiere et al, 2012;Md Dom et al, 2014;Shen et al, 2009;Upadhyay et al, 2014;Wiesen et al, 2012). However, relatively few assays are available for simultaneous quantification of MPA and its glucuronide metabolites with sufficient sensitivity for AcMPAG, which has a relatively low abundance in comparison with MPAG.…”

Bioanalytical challenges and strategies for accurately measuring acyl glucuronide metabolites in biological fluids

“…The incorporation of a chip‐based high‐field asymmetric waveform ion mobility spectrometry (FAIMS) separation in the ultra (high)‐performance liquid chromatography–high resolution mass spectrometry (UHPLC–HRMS) for determination of the ( R / S ) ibuprofen 1‐ O ‐acyl glucuronide metabolite in urine has also been reported (Smith et al, ). Analytical methods for acyl glucuronide metabolites quantitation are listed in Table (Klepacki et al, ; Surendradoss et al, ; Liu & Smith, ; Luigi et al, ; Mano et al, ; Merrigan, Kish‐Trier, Seegmiller, & Johnson‐Davis, ; Qiang & Zhang, ; Schwartz, Desai, Bi, Miller, & Matuszewski, ; Smith et al, ; Zhou et al, ).…”