Determination of PNU-248686A, a novel matrix metalloproteinase inhibitor, in human plasma by liquid chromatography–tandem mass spectrometry, following protein precipitation in the 96-well plate format

A sensitive, specific and high-throughput analytical method for the quantitation of PNU-248686A (I), in human plasma has been developed. I, sodium (2R)-3-{[(4′-chloro(1,1′-biphenyl)-4-yl]sulfonyl}-2-hydroxy-2-[(phenylsulfanyl)methyl] propanoate, is an orally active matrix metalloproteinase (MMP) inhibitor developed for the treatment of solid tumors over-expressing MMPs. Concentrations of I, as free acid, were determined in human plasma by LC–MS–MS after plasma protein precipitation in the 96-well plate format. Aliquots of plasma (50 μl) were placed into the plates and 0.2 ml of methanol was added. The plates were shaken for 5 min and centrifuged at 1500 g for 10 min. Aliquots of 10 μl of the supernatants were then directly injected into the LC–MS–MS system. A Symmetry Shield C8 column (50×2.1 mm, 3.5 μm) was used to perform the chromatographic analysis. The mobile phase was 5 mM ammonium formate buffer solution pH 5.0–acetonitrile (60:40, v/v) with a flow-rate of 0.3 ml/min. Retention time of I was about 1.2 min. Total cycle time was 2.5 min. MS detection used the Applied Biosystems–MDS Sciex API 3000 with TurboIonSpray interface and single reaction monitoring (461→251 m/z transition) operated in negative ion mode. Calibration curves were constructed by plotting the area of the compound (y) against its concentration (x). A weighed linear regression (weighting factor 1/x2) was used to calculate I concentrations in quality control and unknown samples. The method was fully validated over the range of 5.0–5000 ng/ml. The suitability and robustness of the method for in vivo samples was confirmed by analysis of plasma samples from a pilot clinical study.