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 μl) by ultrafiltration at 3000×g for 20 min (20 °C). Both MPA and MPAG were isolated from ultrafiltrate (100 μl) by acidification and C18 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×2 mm, 5 μm). Total analysis time was 7 min. The assay was linear over the range 1–200 μg/l with a limit of quantification of 1 μg/l. The inter-day accuracy and imprecision of quality controls (7.5, 40, 150 μg/l) were 94–99% and <7%, respectively. Free MPAG was chromatographed on a C18 Nova-Pak column (150 mm×3.9 mm, 5 μm) using a binary gradient over 20 min. The eluent was monitored at 254 nm. The assay was linear over the range 1–50 mg/l with the limit of quantification at 2.5 mg/l. The inter-day accuracy and imprecision of quality controls (5, 20, 45 mg/l) was 101–107% and <8% (n=4), respectively. For both methods no interfering substances were found in ultrafiltrate from patients not receiving MPA. The methods described have a suitable dynamic linear range to facilitate the investigation of free MPA and MPAG pharmacokinetics in transplant patients. Further, this is the first reported HPLC-UV method to determine free MPAG concentrations.