Free mycophenolic acid determination in human plasma ultrafiltrate by a validated liquid chromatography-tandem mass...

The aim of this study was to develop and validate fully the liquid chromatography-tandem mass spectrometry method for free mycophenolic acid (MPA) concentration measurements in plasma ultrafiltrate that will be reliable and simple in preparation with deuterated MPA (MPA-d3) chosen as an internal standard. The chromatographic separation was made with Zorbax Eclipse XDB-C18 column (4.6 × 150 mm) using a gradient of two solutions as a mobile phase: (A) water and (B) methanol, each containing 0.1% formic acid and 2.5 mm ammonium acetate. Satisfactory repeatability of retention times was achieved with average values of 7.54 ± 0.20 min and 7.50 ± 0.19 min for MPA and MPA-d3, respectively. The method was selective, with no carry-over or matrix effect observed. The analytical range was proven for MPA ultrafiltrate concentrations of 1-500 ng/mL. The accuracy and precision fell within the acceptance criteria for intraday (accuracy: 100.63-110.46%, imprecision: 6.23-7.76%), as well as interday assay (accuracy: 98.81-110.63%; imprecision: 5.36-10.22%). The method was used for free MPA determination in plasma samples from patients treated with mycophenolate mofetil. To the best of our knowledge this is the first liquid chromatography-tandem mass spectrometry method for free MPA monitoring using MPA-d3 that allows to measure plasma ultrafiltrate concentrations as low as 1 ng/mL.