Development and validation of a sensitive liquid‐chromatography tandem mass spectrometry assay for mycophenolic acid and metabolites in HepaRG cell culture: Characterization of metabolism interactions between p p ‐cresol and mycophenolic acid

摘要

Abstract Mycophenolic acid (MPA), a frequently used immunosuppressant, exhibits large inter‐patient pharmacokinetic variability. This study (a) developed and validated a sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) assay for MPA and metabolites [MPA glucuronide (MPAG) and acyl‐glucuronide (AcMPAG)] in the culture medium of HepaRG cells; and (b) characterized the metabolism interaction between MPA and p ‐cresol (a common uremic toxin) in this in vitro model as a potential mechanism of pharmacokinetic variability. Chromatographic separation was achieved with a C 18 column (4.6?×?250?mm,5?μm) using a gradient elution with water and methanol (with 0.1% formic acid and 2?m m ammonium acetate). A dual ion source ionization mode with positive multiple reaction monitoring was utilized. Multiple reaction monitoring mass transitions ( m / z ) were: MPA (320.95?→?207.05), MPAG (514.10?→?303.20) and AcMPAG (514.10?→?207.05). MPA‐d 3 (323.95?→?210.15) and MPAG‐d 3 (517.00?→?306.10) were utilized as internal standards. The calibration curves were linear from 0.00467 to 3.2?μg/mL for MPA/MPAG and from 0.00467 to 0.1?μg/mL for AcMPAG. The assay was validated based on industry standards. p ‐Cresol inhibited MPA glucuronidation (IC 50 ≈ 55?μ m ) and increased MPA concentration (up to 2‐fold) at physiologically relevant substrate‐inhibitor concentrations ( n ?=?3). Our findings suggested that fluctuations in p ‐cresol concentrations might be in part responsible for the large pharmacokinetic variability observed for MPA in the clinic.