Quantification by liquid chromatography tandem mass spectrometry of mycophenolic acid and its phenol and acyl glucuronide metabolites.

BACKGROUND

We developed and validated a rapid and reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) procedure for the quantification of mycophenolic acid (MPA) and its phenol glucuronide (MPAG) and acyl glucuronide (AcMPAG) metabolites.

METHODS

We performed protein precipitation on all samples (calibrators, quality controls, and patient samples) and then subjected them to online solid-phase extraction followed by reversed-phase liquid chromatography for 4.0 min. The carboxybutoxy ether of MPA (MPAC) was used as the internal calibrator. The separated compounds (MPA, MPAG, AcMPAG, and MPAC) were detected by electrospray ionization-coupled MS/MS. We compared LC-MS/MS results with results for the same samples obtained with a validated HPLC procedure with an ultraviolet detector.

RESULTS

Comparison with the validated HPLC-ultraviolet procedure demonstrated good agreement. The Passing-Bablok regression was y = 0.968x - 0.058 for MPA, y = 1.08x - 1.697 for MPAG, and y = 0.952x + 0.076 for AcMPAG. Assay imprecision showed a CV <10% at 3 concentrations for each compound. The lower limit of quantification was 0.1 mg/L for MPA, 1.0 mg/L for MPAG, and 0.05 mg/L for AcMPAG. The mean analytical recovery was 90%-110%. The assay was linear from 0.1 to 50 mg/L for MPA (r = 0.9987), from 1 to 500 mg/L for MPAG (r = 0.9999), and from 0.05 to 10 mg/L for AcMPAG (r = 0.9988). Quantification of the compounds was not affected by in-source fragmentation or ion suppression.

CONCLUSION

The LC-MS/MS assay described here is valid and reliable for the quantification of total MPA, MPAG, and AcMPAG in serum.