Quantification of free mycophenolic acid by high-performance liquid chromatography–atmospheric pressure chemical ionisation tandem mass spectrometry

Abstract

To facilitate the investigation of free mycophenolic acid concentrations we developed a high-performance liquid chromatography tandem mass spectrometry method using indomethacin as an internal standard. Free drug was isolated from plasma samples (500 μl) using ultrafiltration. The analytes were extracted from the ultrafiltrate (200 μl) using C 18 solid-phase extraction. Detection was by selected reactant monitoring of mycophenolic acid ( m/z 318.9→190.9) and the internal standard ( m/z 356.0→297.1) with an atmospheric pressure chemical ionisation interface. The total chromatographic analysis time was 12 min. The method was found to be linear over the range investigated, 2.5–200 μg/l ( r>0.990, n=6). The relative recovery of the method for the control samples studied (7.5, 40.0 and 150 μg/l) ranged from 95 to 104%. The imprecision of the method, expressed in terms of intra- and inter-day coefficients of variation, was <8 and <9%, respectively. Further, analysis of pooled patient plasma produced an intra-day imprecision of 6.6%. The signal-to-noise ratio at the limit of quantification (2.5 μg/l) was approximately 5:1. The mean absolute recovery ( n=6) of mycophenolic acid and the internal standard were 76.0±13.5% and 86.0±9.1%, respectively. The method reported provides an accurate and precise quantification of free mycophenolic acid over a wide analytical range and thus can be used for routine monitoring and pharmacokinetic studies.