Quantitation of the acid and lactone forms of atorvastatin and its biotransformation products in human serum by high-performance liquid chromatography with electrospray tandem mass spectrometry

A method for simultaneous quantitation of both the acid and lactone forms o f atorvastatin, a new synthetic inhibitor of HMG-CoA reductase that is bein g marketed for the treatment of high serum cholesterol, and both the acid a nd lactone forms of its two biotransformation products, 2-hydroxyatorvastat in and 4-hydroxyatorvastatin, in human serum (a total of six analytes) by h igh-performance liquid chromatography with electrospray tandem mass spectro metry was developed and validated. A deuterium labeled analog was used as i nternal standard for each of the six analytes. Each point of the calibratio n standard curve, which ranged from 0.5 to 200 ng/mL, contained the six ana lytes at equal concentrations. Three groups of quality control (QC) samples were used. In the first group, combination QC samples contained all six an alytes at equal concentrations. In the second group, acid-only QC samples c ontained only the acid forms (i.e. three analytes) at equal concentrations. In the third group, lactone-only QC samples contained only the lactone for ms (i.e. three analytes) at equal concentrations. After adding the internal standards to 0.5 mt of each standard and the QC sample kept at 4 degrees C , the samples were acidified with sodium acetate buffer (pH 5.0) and then e xtracted with methyl tert-butyl ether. Detection was by positive ion electr ospray tandem mass spectrometry using eight selected reaction monitoring ch annels. The acid compounds were stable in human serum at room temperature b ut the lactone compounds were unstable as they hydrolyzed rapidly to their respective acid forms. The conversion of the lactone compounds in both QC a nd post-dose human serum samples was nearly complete after 24 h at room tem perature. The lactone compounds in serum could be stabilized by lowering th e working temperature to 4 degrees C or lowering the serum pH to 6.0. The a cid-only and the lactone-only QC samples showed that, under the sample proc essing conditions used, the degree of the hydrolysis of the lactone compoun ds or the lactonization of the acid compounds during the assay procedure wa s minimal (<5%). The intra-day C.V., inter-day C.V. and the deviations from the nominal concentrations for all six analytes were within 15%, demonstra ting good precision and accuracy. The required lower limit of quantitation (LLQ) of 0.5 ng/mL was achieved for each analyte. Copyright (C) 1999 John W iley & Sons, Ltd.