Background: Therapeutic Drug Monitoring (TDM) of immunosuppressant compounds has become a crucial practice to increasing the success of solid organ transplantation. Mycophenolate compounds are commonly deployed immunosuppressant drugs following a solid organ transplant and require accurate monitoring due to their narrow therapeutic range.
Mycophenolate Mofetil (MMF) is readily metabolized by most patients into MPA, the active agent in immunosuppression, which forms MPAG through glucuronidation. MPAG is the major metabolite of MPA and is tested to ensure normal metabolism of the drug.
The pharmacokinetics of mycophenolate compounds have proven to be highly complex, primarily due to enterohepatic circulation, comedication interactions, patient genetics, protein binding capacity, and the overall high variability these factors cause in patient metabolism.
Despite these factors, steady state analysis has become the most common practice for TDM of MPA post transplantation. Given the drastic variations in interpatient metabolisms of MPA and MPAG, it is important that more accurate and comprehensive methods of TDM are made available to transplant patients.
Methods: A quantitative method was developed using liquid chromatography with tandem mass spectrometry for therapeutic drug monitoring of MPA/MPAG. MPA/MPAG were measured using a Shimadzu LC-MS 8060. Serum samples (50uL) and internal standard solution (50uL 6ug/mL MPA d3/ 60ug/mL MPAG d3) were extracted with 300uL acetonitrile-methanol (1:1). 0.2uL of supernatant was injected into the LC. Chromatographic separation was acheived in 3.5 minutes with gradient elution of 0.1%acetic acid and 0.1%formic acid in DI H2O and 0.1% acetic acid and 0.1%formic acid in MeOH at 40ºC. The detection of MPA/MPA d3 were performed using ESI tandem mass spectrometry in the positive ion mode, while MPAG/MPAG d3 were analyzed using negative ESI with MRM. The precursor→product ion was monitored at 320.95→207.20m/z (MPA), 324.05→210.15m/z (MPA d3), 495.16→3.19.20m/z (MPAG), and 498.46→322.20m/z (MPAG d3).
Results: Extraction recovery was 98% and standard curve was linear (r2 = 0.998) from 0.1-30 ug/mL for MPA and 1-300 ug/mL for MPAG.
The method was validated according to the 2018 FDA Bioanalytical method validation guidance. The method is utilized for pharmacokinetic analysis by determination of the AUC for 7 samples collected over 12 hours. This provides an accurate and comprehensive understanding of each patient’s level of immunosuppression, as well as the individual nuances in their metabolism of MPA and MPAG.
Conclusions: Literature shows that by utilizing pharmacokinetic analysis an overall increase in solid organ transplant success rate can be achieved, alongside decreased rate of rejection, toxicity, and overall side effects. The above method has been successfully validated for immunosuppressive therapeutic drug monitoring of MPA/MPAG and has been implemented in a CAP CLIA certified clinical laboratory for AUC0-12 analysis to prevent organ rejection in solid organ transplant patients.