Quantification of mycophenolic acid and glucuronide metabolite in human serum by HPLC-tandem mass spectrometry.
BACKGROUND
The potent immunosuppressant mycophenolic acid (MPA) is metabolized to an inactive glucuronide (MPAG). The extent of metabolism varies among individuals, and the MPAG formed can be hydrolyzed to MPA and can displace MPA from serum albumin, creating a potential need to monitor both MPA and MPAG.


METHODS
After addition of the carboxybutoxy ether of MPA (MPAC) as internal standard, MPA and MPAG were isolated from serum by acidification followed by solid-phase extraction. Gradient chromatographic separation was performed on a Waters Atlantis reversed-phase liquid chromatography (HPLC) column, and the compounds were quantified by electrospray ionization tandem mass spectrometry (MS/MS) in the multiple-reaction monitoring mode. Results obtained by HPLC-MS/MS were compared with an HPLC assay using ultraviolet detection (HPLC-UV) performed at a reference laboratory.


RESULTS
MPAG, MPA, and MPAC were fully separated during a 7.0-min run time. Precision at both low and high concentrations of MPA ad MPAG met the suggested method validation criteria from a consensus panel report on MPA. The extraction efficiencies were 99% for MPA and MPAG. The assay was linear to 16 mg/L for MPA and 200 mg/L for MPAG. Limits of quantification were 0.1 mg/L for MPA and 1 mg/L for MPAG. Regression analysis gave the following results: HPLC-MS/MS = 1.03(HPLC-UV) - 0.03 mg/L (R2 = 0.982) for MPA; and HPLC-MS/MS = 0.93(HPLC-UV) + 0.89 mg/L (R2 = 0.967) for MPAG.


CONCLUSIONS
This HPLC-MS/MS assay can be used to reproducibly quantify MPA and MPAG across a large analytical range in serum from organ transplant patients.
Annesley
Thomas M
TM
Department of Pathology, University of Michigan Health Sciences Center, Ann Arbor, MI 48109-0054, USA. annesley@umich.edu
Clayton
Larry T
LT
D002851:Chromatography, High Pressure Liquid; D020719:Glucuronides / Q000097:blood*; D006801:Humans; D007166:Immunosuppressive Agents / Q000097:blood*; D009173:Mycophenolic Acid / Q000031:analogs & derivatives* / Q000097:blood*; D016377:Organ Transplantation; D044967:Serum; D021241:Spectrometry, Mass, Electrospray Ionization; D013056:Spectrophotometry, Ultraviolet
D020719:Glucuronides; D007166:Immunosuppressive Agents; D009173:Mycophenolic Acid