Validated LC–MS/MS method for quantitation of total and free mycophenolic acid concentration and its application to a pharmacokinetic study in pediatric renal transplant recipients

Abstract

A simple and sensitive LC–MS/MS method was established to quantify total and free mycophenolic acid (MPA) plasma concentrations during immunosuppressive medication for pediatric renal transplantation. The chromatographic separation was performed with the Hypersil GOLD C18 column, using a mobile phase consisting of 0.1% formic acid in water and acetonitrile (60:40, v/v) at an isocratic flow rate of 0.4 ml/min. An Agilent 6420 triple quadrupole mass spectrometer was operated via a positive electrospray ionization interface using the transitions m/z 321.14 → 206.9 for MPA and m/z 324.15 → 209.9 for MPA‐d3 (internal standard). The linearity was 0.1–50 μg/ml for total MPA and 0.0025–0.5 μg/ml for free MPA. The within‐run and between‐run precisions were all <5% and accuracy was within 96.23–107.63%. The validated method was successfully aspplied to a pharmacokinetic study in 28 pediatric renal recipients. The mean free fraction of MPA in our patients was 0.89% (ranging from 0.62 to 1.25%) and albumin level played a major role in the variability of free fraction of MPA, thus, in pediatric patients with hypoproteinemia, close free drug monitoring and dose adjustments should be considered to prevent toxicity.

Quantitation of Mycophenolic Acid and Mycophenolic Acid Glucuronide in Serum or Plasma by LC‐MS/MS

“…There are various methods for the quantification of MPA and MPAG levels. These include immunassays, HPLC linked to an ultraviolet detector (HPLC/UV) and HPLC linked to a tandem mass spectrometer (HPLC‐MS/MS) (Danafar & Hamidi, 2015; Glahn‐Martinez et al., 2018; Kikuchi et al., 2018; Klepacki et al., 2012; Kunicki et al., 2015; Liu et al., 2021; Seyfinejad & Jouyban, 2021; Zhou et al., 2021). LC‐MS/MS is the most commonly used method for the quantification of MPA followed by immunoassays (College of American Pathologists Surveys, 2022).…”

“…This could be a serious problem in renal failure, in which MPA metabolites accumulate to significantly high concentrations. Total MPA concentrations are generally measured, but an assay of free MPA levels may be desirable in patients with renal failure and hypoalbuminemia (Liu et al., 2021; Luszczynska et al., 2017). However, measurement of free MPA may be challenging, as only ∼2% of the drug is in the free form.…”

Pharmacokinetics of free and total mycophenolic acid in paediatric and adult renal transplant recipients: Exploratory analysis of the effects of clinical factors and gene variants

“…tMPA and fMPA concentrations in plasma were determined by a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method previously published by our laboratory. 30 The plasma samples were treated by protein precipitation using acetonitrile as a precipitant, and free forms were isolated using ultrafiltration. Ultrafiltrates were prepared with 400 μl aliquots of plasma in Centrifree ultrafiltration device under centrifugation (4 C) at 2000 Â g for 30 min.…”

Recent Advances in Therapeutic Drug Monitoring of Voriconazole, Mycophenolic Acid, and Vancomycin: A Literature Review of Pediatric Studies

“…Although unbound, free MPA (fMPA) is the pharmacologically active form of the drug, relatively small number of studies focus on fMPA analysis [116]. Smits et al [76] tried to answer the question whether fMPA TDM might have any advantages over current practice of monitoring total MPA.…”

“…In a recent study of Liu et al [116], who developed a simple and sensitive ultrafiltration and LC-MS/MS method for total and fMPA determination, the authors recommended close free drug monitoring and dose adjustments in pediatric patients with hypoproteinemia to prevent toxicity. The mean fMPA in pediatric renal transplant recipients was 0.89% (ranging from 0.62 to 1.25%), and the authors indicated that the fMPA was small and concluded that plasma albumin level plays a major role in the variability of fMPA.…”