Mycophenolic acid quantification in human peripheral blood mononuclear cells using liquid chromatography-tandem mass...

Mycophenolic acid (MPA) is an immunosuppressant widely used to prevent organ rejection after organ transplantation. MPA therapeutic drug monitoring (TDM) is currently recommended by plasma C0 - or even better - by AUC determinations, but little is known about the potential interest of measuring the intra-lymphocyte MPA concentrations, representing the target site of action. The aim of this study is to develop and validate a selective and sensitive analytical method for quantification of MPA levels in human peripheral blood mononuclear cells (PBMCs).

PBMCs were extracted from heparin blood samples by Leucosep®. Methanol and MPA-d3 were used as extraction solvent and internal standard, respectively. Chromatographic separation was obtained on a XBridge BEH C18 column (2.1mm×75mm, 2.5μm) maintained at 50°C on a Waters Alliance 2795 coupled to a QuattroMicro tandem mass-spectrometer. The multiple reaction monitoring transitions used for quantification were m/z 320.97→207.0 for MPA, and 324.2→210.1 for MPA-d3, in positive ESI mode.

The total HPLC run time was 6min. The retention times for MPA-d3 and MPA were 2.55 for both compounds. The method was linear from 0.1 to 50ng/mL MPA. The coefficient r(2) ranged from 0.996 to 0.998. Intra-assay and inter-assay imprecisions were <10% in the whole range of concentrations, and <20% at the LLOQ, and accuracy level was >90%. The matrix and ion suppression effects were <6%. The MPA limit of quantification was 0.1ng/mL. No interference was identified in the assay. From the preliminary results, intra-cellular MPA concentrations in kidney transplant patients ranged from 0.69 to 3.39ng/10(7) cells.

We described a robust, rapid and simple method suitable for the determination of MPA concentrations in PBMCs. This method is currently used in pharmacokinetics-pharmacodynamics (PK-PD) clinical trials, in comparison to standard plasma TDM.