Quantification of free mycophenolic acid and its glucuronide metabolite in human plasma by liquid-chromatography using mass spectrometric and ultraviolet absorbance detection

The immunosuppressant drug mycophenolic acid (MPA) and its major metabolite, mycophenolic acid glucuronide (MPAG), are highly bound to albumin. An HPLC-tandem-MS (HPLC/MS/MS) and an HPLC-UV assay were developed to measure free (unbound) concentrations of MPA and MPAG, respectively. Ultrafiltrate was prepared from plasma (500 mul ) by ultrafiltration at 3000 x g for 20 min (20degreesC). Both MPA and MPAG were isolated from ultrafiltrate (100 mul) by acidification and C-18 solid-phase extraction. Free MPA was measured by electrospray tandem mass spectrometry using selected reactant monitoring (MPA: m/z 338.2 --> 206.9) in positive ionisation mode. Chromatography was performed on a PFPP column (50 mm x 2 mm, 5 mum). Total analysis time was 7 min. The assay was linear over the range 1-200 mug/l with a limit of quantification of 1 mug/l. The inter-day accuracy and imprecision of quality controls (7.5, 40, 150 mug/l) were 94-99% an