A liquid chromatographic/mass spectrometric method to quantitate atorvastatin (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 consisted of washing plasma samples at high pH with diethyl ether and subsequently extracting the analytes and two internal standards, [d5]-atorvastatin ([d5]-AT) and [d5]-ortho-hydroxy atorvastatin ([d5]-o-AT), from acidified plasma by using diethyl ether. The ether layer was evaporated to dryness and the residue reconstituted in ammonium acetate (20 mM, pH 4.0)-acetonitrile-isopropanol (60:40:1, v/v/v). Chromatographic separation of analytes was achieved by using a YMC J'Sphere H80 (C-18) 150 x 2 mm, 4 microns particle size, column with a mobile phase consisting of acetonitrile-0.1% acetic acid, (70:30, v/v). Analytes were detected by using MS/MS. Sample introduction and ionization was by electrospray ionization in the positive ion mode. 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 retention time ranges of p-AT, o-AT, [d5]-o-AT, AT, and [d5]-AT were 2.27 +/- 0.21, 3.36 +/- 0.23, 3.54 +/- 0.46, 4.12 +/- 0.61, and 4.65 +/- 0.65 min, respectively. No peaks interfering with quantitation were observed throughout the validation processes. Mean recoveries of AT, o-AT, and p-AT from plasma ranged 100%-107%, 70.6%-104%, and 47.6%-85.6%, respectively. Mean recoveries of the [d5]-AT and [d5]-o-AT internal standards ranged 98.0%-99.9% and 97.3%, respectively. Interassay precision, based on the percent relative deviation for replicate quality controls for AT, o-AT, and p-AT, was < or = 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%, respectively. AT, o-AT, and p-AT in human, dog, and rat plasma quality controls were stable to three freeze-thaw cycles. AT, o-AT, and p-AT were stable frozen for 127, 30 and 270 days in human, dog, and rat plasma quality control samples, 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/mL for a 1.0-mL sample aliquot.

Two brothers with a recently described inborn error of metabolism characterized by glyceroluria, hyperglycerolemia, and generalized glycerol kinase deficiency had moderate psychomotor retardation, spasticity, growth failure, a nonspecific myopathy, osteoporosis, and adrenal insufficiency. Glycerol kinase activity in leukocytes and cultured fibroblasts was less than 5% of control values. Hepatic and renal tissue obtained at autopsy in one patient had similarly low enzyme activity. Thus the deficiency of glycerol kinase in these patients appears to be generalized and heritable, though the relationship of the clinical phenotype to the enzymatic defect is not yet established.

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Glycerol kinase deficiency has been associated with neuromuscular, skeletal and adrenal abnormalities and has also been seen in individuals without these clinical findings. Examination of residual enzyme activity in patients' liver, kidney, leukocytes and fibroblasts showed a generalized, heritable defect: the apparent Km for glycerol was increased 5-200-fold over control values, whereas the apparent Km for ATP was not significantly altered. This kinetic defect was similar in fibroblasts from clinically different individuals with this inborn error of metabolism. Hybridization of fibroblasts from these individuals showed no evidence of complementation for glycerol kinase activity.