Development and validation of a high-performance liquid chromatography tandem mass spectrometry assay for atorvastatin, ortho-hydroxy atorvastatin, and para-hydroxy atorvastatin in human, dog, and rat plasma

A liquid chromatographic/mass spectrometric method to quantitate atorvastat in (AT) and its active metabolites ortho-hydroxy (o-AT) and para-hydroxy (p -AT) atorvastatin in human, dog, and rat plasma was validated. The method c onsisted of washing plasma samples at high pH with diethyl ether and subseq uently extracting the analytes and two internal standards, [d(5)]-atorvasta tin ([d(5)]-AT) and [d(5)]-ortho-hydroxy atorvastatin ([d(5)]-o-AT), from a cidified plasma by using diethyl ether. The ether layer was evaporated to d ryness and the residue reconstituted in ammonium acetate (20 mM, pH 4.0)-ac etonitrile-isopropanol (60:40:1, v/v/v). Chromatographic separation of anal ytes was achieved by using a YMC J'Sphere H80 (C-18) 150 x 2 mm, 4 mu m par ticle size, column with a mobile phase consisting of acetonitrile-0.1% acet ic acid, (70:30, v/v). Analytes were detected by using MS/MS. Sample introd uction and ionization was by electrospray ionization in the positive ion mo de. The method proved suitable for routine quantitation of AT, o-AT, and p- AT over the concentration range of 0.250 to 25.0 ng/mL. Approximate retenti on time ranges of p-AT, o-AT, [d(5)]-o-AT, AT, and [d(5)]-AT were 2.27 +/- 0.21, 3.36 +/- 0.23, 3.54 +/- 0.46, 4.12 +/- 0.61, and 4.65 +/- 0.65 min, r espectively. No peaks interfering with quantitation were observed throughou t the validation processes. Mean recoveries of AT, o-AT, and p-AT from plas ma ranged 100%-107%, 70.6%-104%, and 47.6%-85.6%, respectively. Mean recove ries of the [d(5)]-AT and [d(5)]-o-AT internal standards ranged 98.0%-99.9% and 97.3%-97.9%, respectively. Interassay precision, based on the percent relative deviation for replicate quality controls for AT, o-AT, and p-AT, w as less than or equal to 7.19%, 8.28%, and 12.7%, respectively. Interassay accuracy for AT, o-AT, and p-AT was +/-10.6%, 5.86%, and 15.8%, respectivel y. AT, o-AT, and p-AT in human, dog, and rat plasma quality controls were s table to three freeze-thaw cycles. AT, o-AT, and p-AT were stable frozen fo r 127, 30 and 270 days in human, dog, and rat plasma quality control sample s, respectively. Human plasma quality control samples containing AT, o-AT, and p-AT were stable for at least 4 days at ambient room temperature and 37 degrees C. The lower Limit of quantitation for all analytes was 0.250 ng/m L for a 1.0-mL sample aliquot. (J Am Soc Mass Spectrom 1999, 10, 55-66) (C) 1999 American Society for Mass Spectrometry.