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% and <7%, respectively. Free MPAG was chromatographed on a C-18 Nova-Pak column (150 mm x 3.9 mm, 5 mum) 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. (C) 2003 Elsevier B.V. All rights reserved.