Direct-injection LC-MS-MS method for high-throughput simultaneous quantitation of simvastatin and simvastatin acid in human plasma

A direct-injection liquid chromatography -mass spectrometry--mass spectrome try (LC-RIS-MS) method was developed and validated for the simultaneous qua ntitation in human plasma of the widely used cholesterol-lowering prodrug s imvastatin and its in vivo generated active drug, simvastatin acid. The pla sma samples were injected into the LC-MS-MS system after simply adding the internal standard solution in an aqueous buffer and centrifuging. The analy tes in the buffered plasma samples were found to be stable For at least 24 h at 4 degrees C. The method was successfully validated under the challengi ng condition of using a large number of quality control (QC) samples includ ing those in which the ratio of the simvastatin concentration to the simvas tatin acid concentration was different from the concentration ratio in the calibration curve standards. Under the dual stabilizing conditions of lower temperature (4 degrees C) and lower plasma pl-l of 4.9, the in-process hyd rolysis of simvastatin to simvastatin acid or the lactonization of simvasta tin acid to simvastatin was minimized to less than or equal to 1.0%. Althou gh the entire run time for on-line cleanup and analysis was only 2.5 min, c hromatographic base-line separation of simvastatin from simvastatin acid, w hich was required to avoid the interference by simvastatin acid with the si mvastatin selected reaction monitoring channel, was achieved. The desired l ower limit of quantitation of 0.5 ng/ml was achieved by injecting only an e quivalent of 8.0 mu l of the plasma sample. The extraction column lasted fo r at least 500 injections. (C) 2000 Elsevier Science B.V. All rights reserv ed.