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

BACKGROUND AND

OBJECTIVES

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).

METHODS

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.

RESULTS

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.

CONCLUSION

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.