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 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.

Validated HPLC/MS/MS assay for CI-1011 in rat plasma and a comparison with an HPLC/UV assay.

A liquid chromatographic/mass spectrometric (LC/MS/MS) method to quantitate CI-1011 in rat plasma has been validated and compared to an LC/UV assay. The analyte and internal standard were isolated from the plasma matrix by using liquid/liquid extraction with diethyl ether. The ether layer was evaporated to dryness and the residue reconstituted in acetonitrile-water (70:30, v/v). A 2.1 x 150 mm x 5 microns Zorbax RX-C18 column with a mobile phase of acetonitrile-ammonium acetate (pH 8.0; 5 mM)-triethylamine (70:30:0.03, v/v/v) delivered at a flow rate of 0.2 ml min-1 was used for chromatography. Analyte and internal standard ion chromatograms were obtained by operating the mass spectrometer in the negative ion multiple reaction monitoring mode to detect the presence of a precursor-product ion pair for both the analyte and the internal standard. Samples were introduced into the mass spectrometer using electrospray ionization. Retention times of CI-1011 and of the internal standard (IS), [13C6]CI-1011, were approximately 4.2 min. No peaks interfering with the quantitation of CI-1011 were observed throughout the validation process. Mean recoveries of CI-1011 from rat plasma ranged from 98.2 to 105%. The recovery of the IS was 100%. Assay precision for CI-1011, based on the percent relative standard deviation of replicate quality controls, was less than or equal to 5.60% with an accuracy of +/- 8.80%. The lower limit of quantitation for CI-1011 was 0.500 ng ml-1 for a 0.2-ml sample aliquot. CI-1011 is stable in rat plasma for 24 h at room temperature and for at least 34 days at -20 degrees C. This assay has been proven suitable for routine quantitation of CI-1011 in rat plasma at concentrations from 0.500 (100 pg on-column) to 500 ng ml-1. The applicability of this method to determine CI-1011 concentrations in rat plasma is reported in this manuscript. CI-1011 concentrations, in plasma samples from cholesterol- and chow-fed rats administered single daily oral doses of CI-1011 in a CMC/Tween suspension, obtained using a validated LC/UV assay were compared to concentrations obtained using the reported LC/MS/MS assay over the concentration range 0.0806-12.3 micrograms ml-1. The concordance correlation coefficient determined for this comparison was 0.9977, suggesting that the CI-1011 concentrations obtained by the two assays are in excellent agreement.

An HPLC assay utilizing solid-phase extraction for CI-1010, an alkylating radiosensitizer, in rat plasma.

CI-1010, a 2-nitroimidazole, is a chiral prodrug for the active moiety PD 146923 and is under development as an alkylating radiosensitizer to be used as an adjuvant to radiotherapy. Because CI-1010 has an estimated half-life < or = 2 min under physiological conditions its metabolites/degradation products PD 146415, an inactive moiety, and PD 146923 were assayed to support rat toxicology studies. The method involves the processing of plasma samples through phenyl solid-phase extraction cartridges followed by chromatography on CN columns with UV detection at 325 nm. The assay appears linear over the range 0.050-100 micrograms ml-1 for both PD 146415 and PD 146923. Interrun accuracy and precision estimates for PD 146415 and PD 146923 were within +/- 6.50 and < or = 3.27%, respectively, and +/- 12.8 and < or = 4.06%, respectively, for quality controls containing nominal concentrations of 0.400, 4.00 and 40.0 micrograms ml-1. The absolute recovery of CI-1010, PD 146415 and internal standard, PD 126675, were approximately 40, 96 and 95%, respectively. The recovery of PD 146923 appeared concentration dependent and ranged from 68 to 92%. PD 146415 and PD 146923 were both stable in rat plasma at 4 degrees C and -77 degrees C for at least 7 h and 154 days, respectively. CI-1010 was not stable in rat plasma at 4 degrees C. CI-1010, PD 146415 and PD 126675 were stable for at least 63 days in 10 mM phosphate buffer at pH 3.0 and 4 degrees C. Under identical conditions PD 146923 was stable for only 8 days. The applicability of this method to determine concentrations of PD 146415 and PD 146923 in rat plasma is reported in this paper.

High-performance liquid chromatographic assay for CI-980, a novel 1-deaza-7,8-dihydropteridine anticancer agent, in human plasma and urine.

CI-980, a 1-deaza-7,8-dihydropteridine, is a novel anticancer agent that is a potent mitotic inhibitor acting as a tubulin binder similar to the vinca alkaloids. CI-980 has shown equivalent or superior anticancer activity in vitro compared to vincristine and retains full activity against vincristine resistant tumors in vitro. A high-performance liquid chromatographic (HPLC) assay was developed and validated for human plasma and urine to support Phase 1 clinical trials. CI-980 and PD 080658, internal standard, were isolated from 2-ml samples of human plasma and urine by solid-phase extraction with Bond-Elut C18 cartridges. Urine samples must be pretreated with bovine serum albumin (BSA) to minimize the binding of CI-980 to glass and some plastics. The eluate from the cartridges for both matrices was evaporated to dryness and taken up in mobile phase. Zorbax RX C18 columns, mobile phase buffer of 10 mM ammonium dihydrogen phosphate at pH 7.5 and a flow-rate of 0.75 ml/min were used for both matrices. Column dimensions, column temperature and mobile phase acetonitrile-buffer ratio were 300 mm x 4.6 mm I.D., 30 degrees C and 38:62 (v/v), respectively, for the plasma assay and 250 mm x 4.6 mm I.D., 35 degrees C and 40:60 (v/v), respectively, for the urine assay. Column effluent was monitored fluorometrically for the plasma method using excitation and emission wavelengths of 388 nm and 473 nm, respectively. Ultraviolet detection at 380 nm was used for the urine method. Peak-area ratios were proportional to CI-980 concentrations from 0.2 to 25 ng/ml and 1 to 100 ng/ml for plasma and urine, respectively. CI-980 in water will bind to glass and plastics but not PTFE or stainless steel. Urine calibration standards were frozen prior to use in order to compensate for loss of CI-980 due to freezing in this matrix. The accuracy of the assay was within 4.7%, with a precision of 5.6% for both matrices. Recoveries ranged from 93.8 to 102% and 90.7 to 92.3% for plasma and urine, respectively. CI-980 was stable in plasma and urine for at least 275 and 217 days, respectively, when stored at -70 degrees C. The assay is suitable for studying the clinical pharmacokinetics of CI-980.

A high-performance liquid chromatographic assay for CI-973, a new anticancer platinum diamine complex, in human plasma and urine ultrafiltrates.

CI-973 is a new platinum compound with antitumor properties that is currently undergoing phase II clinical trials. A high-performance liquid chromatographic (HPLC) assay was developed and validated for ultrafiltrates of human plasma and urine to support phase I clinical trials. Plasma ultrafiltrate (0.5 ml) was extracted using C18 solid-phase cartridges. Urine was diluted 10-fold and extracted first with SAX solid-phase cartridges and then with C18 cartridges. For both matrices, the eluate from the C18 cartridges was injected directly. A Whatman PAC 10 column (4.6 x 250 mm, 10-microns particle size) and ultraviolet detection at 205 nm were used for both analyses. The mobile-phase buffer was 0.05 M sodium perchlorate (pH 2.3). The mobile-phase acetonitrile:buffer ratio, column temperature, and flow rate were 89:11 (v/v), 40 degrees C, and 2.0 ml/min, respectively, for the plasma ultrafiltrate assay and 85:15 (v/v), 50 degrees C, and 1.0 ml/min, respectively, for the urine ultrafiltrate assay. Standard curves were linear from 0.25 to 500 micrograms/ml and from 1.0 to 250 micrograms/ml for the plasma and urine assays, respectively. The accuracy of the assay lay within 4.5% of the nominal values, and the precision was 6.2%; the recovery of CI-973 varied from 79.2% to 105%. CI-973 remains stable in plasma for at least 6 h, at room temperature, in ultrafiltrates of both matrices for at least 15 days at -72 degrees C, and in water for at least 6 months at -72 degrees C.

Quantitative analysis of 1-(2-pyrimidinyl)piperazine in plasma by capillary gas chromatography-mass spectrometry.

A sensitive method for detecting and quantifying 1-(2-pyrimidinyl)piperazine, an important metabolite of buspirone, in human plasma was developed and validated. The range of the method is 0.2-15 ng/ml. The analyte was removed from buffered, sodium chloride-saturated plasma with benzene, extracted into aqueous acid, washed with diethyl ether and reextracted into benzene. The processed extract was derivatized with pentafluorobenzoyl chloride. Instrumental analysis involved on-column injection into a fused-silica capillary gas chromatography column and detection by selected-ion monitoring mass spectrometry.

Capillary gas chromatographic-mass spectrometric determination of buspirone in plasma.

Methods for determining buspirone plus a deuterated analogue and for buspirone alone in plasma samples are described. Analytes are prepared from plasma by liquid extraction into n-butyl chloride and subsequent back-extraction clean-up steps. Instrumental analysis involves selected-ion monitoring gas chromatography-mass spectrometry with fused-silica capillary chromatography. Quantification is in the range 0.05-10 ng/ml with acceptable accuracy and precision.

Gas chromatographic-mass spectrometric method for trazodone and a deuterated analogue in plasma.

A plasma assay method for trazodone and a 2H4 analogue is described which uses gas chromatography--electron-impact selected-ion monitoring mass spectrometry. Etoperidone is used as an internal standard. The analytes are extracted from basic medium into n-butyl chloride, then back extracted into aqueous 0.1 M hydrochloric acid. The aqueous layer is made basic and re-extracted with n-butyl chloride. The solvent is reduced under nitrogen at 35 degrees C and the residue is redissolved in toluene for gas chromatographic--mass spectrometric analysis. The ions monitored are m/z 231, 235, and 225 for trazodone, [2H4] trazodone and etoperidone, respectively. Quantitation is in the range 40-1000 ng/ml with acceptable precision and accuracy. The method is suitable for biopharmaceutical studies.

Human glycerol kinase: comparison of properties from fibroblasts and liver.

Kinetic differences in the glycerol kinase activities from human liver and fibroblasts have been previously demonstrated by this laboratory. The present study was undertaken to characterize these enzymatic activities more completely. The pH profiles were similar for the hepatic and fibroblast activities, but the stability of hepatic glycerol kinase varied much more markedly with pH than the fibroblast activity. Subcellular distribution of this enzymatic activity was quite different for liver and fibroblasts, as was the electrophoretic mobility of these activities on polyacrylamide gels. While these data do not distinguish between tissue specific isozymic forms or post-transcriptional modification, hepatic and fibroblast activities are altered similarly in patients with an inherited deficiency of glycerol kinase.

Human glycerol kinase deficiency: enzyme kinetics and fibroblast hybridization.

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.

Glycerol kinase deficiency with neuromuscular, skeletal, and adrenal abnormalities.

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.