Zhongzhou Shen

Pindolol is a non‐selective β ‐adrenergic antagonist ( β ‐blocker) for the treatment of cardiovascular diseases such as hypertension and angina pectoris. It has one chiral center, and, therefore, two optical isomers. It was essential to develop an enantioselective assay to measure each enantiomer in human plasma. However, separation of enantiomers using chiral chromatography usually requires relatively long retention times. This can pose a problem for rapid turnaround of a large number of samples (i.e., clinical studies). In the present study, a simple and sensitive chiral liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS) method was developed and validated for the determination of S ‐(−)‐ and R ‐(+)‐pindolol in human plasma. To increase throughput, staggered sample injection was employed using a CTC Trio Valve system on a CTC HTS PAL autosampler. The method exhibited good intra‐ and inter‐day accuracy and precision, and was linear over a dynamic range of 250 pg/mL to 250 ng/mL for each pindolol enantiomer. Intra‐ and inter‐day accuracy ranged between 90.0–106% and 91.6–104% for both quality control (QC) samples of S ‐(−)‐ and R ‐(+)‐pindolol, respectively. The respective intra‐ and inter‐day precision ranged between 4.24–7.86% and 4.98–10.4%. Copyright © 2005 John Wiley & Sons, Ltd.

MK‐0767, (±)‐5‐[(2,4‐dioxothiazolidin‐5‐yl)methyl]‐2‐methoxy‐ N ‐[[(4‐trifluoromethyl)phenyl]methyl]benzamide, is a thiazolidinedione‐containing dual peroxisome proliferator‐activated receptor (PPAR) α / γ agonist that has been studied as a potential treatment for patients with type 2 diabetes. MK‐0767 contains a chiral center at the C‐5 position of the thiazolidinedione ring and was being developed as the racemate, due to the rapid interconversion of its enantiomers in biological samples. In the present work the in vitro and in vivo concentration ratios of the (+)‐(R) to (−)‐(S) enantiomers of MK‐0767 were determined in plasma from humans ( in vitro only) and nonclinical species used in the toxicological evaluation of rac ‐MK‐0767, namely CD‐1 mice, Sprague‐Dawley rats, beagle dogs, New Zealand white rabbits, and rhesus monkeys. The R/S ratio was determined by chiral liquid chromatography/tandem mass spectrometry. Species differences were observed in the in vitro and in vivo enantiomeric ratios, as well as differences between in vitro and in vivo in some species. The in vitro R/S ratio was similar in dogs and humans (∼1.5–1.7). In rats and monkeys, the ratio was approximately unity, both in vitro and in vivo . In mice, the ratio was higher in vitro (∼1) than in vivo (∼0.6), while in rabbits it was higher in vivo (∼1) than in vitro (∼0.5). These results suggested that differential binding of the MK‐0767 enantiomers to plasma and tissue proteins and other macromolecules may be affecting the R/S ratio both in vitro and in vivo , since in protein‐free systems MK‐0767 exists as the racemate. Copyright © 2005 John Wiley & Sons, Ltd.

MK‐0767, 5‐[2,4‐dioxothiazolidin‐5‐yl)methyl]‐2‐methoxy‐ N ‐[[(4‐trifluoromethyl)phenyl]methyl]benzamide (I, Table ), is a dual peroxisome proliferator‐activated receptor (PPAR) α / γ agonist previously studied for the treatment of type 2 diabetes and dyslipidemia. To support further toxicological studies in one of the animal species used in chronic testing of I, a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the simultaneous quantification of I and seven metabolites in rat urine was developed and validated. In this method, urine samples were diluted with acetonitrile/methanol (50:50, v/v) and injected directly onto the column of an LC system. Detection was achieved by MS/MS using a turbo ion spray probe monitoring precursor → product ion combinations in selected reaction monitoring (SRM) mode. The linear range for I and three metabolites was 0.8–800 ng/mL, and 8–8000 ng/mL for four other metabolites found to be present in urine at higher concentrations than I. Intra‐day and inter‐day variation using this method were ≤13.0%. The method exhibited good linearity, reproducibility, specificity and sufficient sensitivity when used for the analysis of rat urine samples. Concentrations of I and its major metabolites in rat urine were determined in samples collected between 0–24 h after dosing on the last day of administration of nine daily oral doses to three male (1000 mg/kg/day) and three female (300 mg/kg/day) Sprague‐Dawley rats. The urinary concentrations of I and its metabolites were similar in male and female rats. The average concentrations of I were 0.51 and 0.33 μg/mL in male and female rats, respectively. Concentrations of four of the seven metabolites quantified were 6‐ to 45‐fold higher than those of I. The most abundant metabolite, with concentrations of 24.2 and 13.3 μg/mL in male and female rat urine, respectively, was a methyl sulfoxide derivative formed by oxidative cleavage of the thiazolidinedione ring, followed by S‐methylation and oxidation of the sulfide intermediate. Copyright © 2004 John Wiley & Sons, Ltd. Molecular weight (MW), SRM transitions and structures of MK‐0767 ( I ) and its metabolites: magnified image

1. Four new metabolites of pioglitazone were identified by liquid chromatography-mass spectrometry (LC-MS/MS) as being formed by hydroxylation (M-VII and M-VIII), opening of the thiazolidinedione ring (M-X) and by desaturation of the terminal ethyl side chain or tether ethoxy moiety (M-IX), respectively. The structure of one of the hydroxylated metabolites (M-VII) was confirmed by chemical modification using the Jones reaction. 2. Oxidative cleavage of the thiazolidinedione ring is a novel pathway not previously reported for pioglitazone. 3. The hydroxylated M-VII was detected in incubations with rat, dog and human liver and kidney microsomes, and in plasma from rats and dogs dosed orally with [(3)H]pioglitazone. 4. The carboxylic acid derivative of M-VII (M-V) and its taurine conjugate were the major radioactive components in dog bile.

Fluoxetine (Prozac®) is currently one of the widely prescribed selective serotonin reuptake inhibitors (SSRIs) for the treatment of depression. A high‐throughput sample preparation procedure using liquid‐liquid extraction (LLE) in a 96‐well plate format in conjunction with liquid chromatography/tandem mass spectrometry (LC/MS/MS) was developed and validated for quantification of fluoxetine enantiomers in human plasma. After addition of internal standard and ammonium hydroxide, samples were extracted with ethyl acetate. The organic extract was evaporated to dryness and reconstituted in methanol. Where possible, sample transfer and LLE steps were automated using a Tomtec Quadra 96 workstation. Adequate separation of fluoxetine enantiomeric pairs (resolution of 1.17) was achieved on a vancomycin column eluted with methanol containing 0.075% (by weight) ammonium trifluoroacetate. A triple quadrupole mass spectrometer, operated in the multiple reaction monitoring mode at m/z 310 → 44 for fluoxetine enantiomeric pairs and m/z 287 → 241 for oxazepam (internal standard), was used. Analysis was performed in the positive ion mode using atmospheric pressure chemical ionization (APCI). The standard curve range was 2.0–1000 ng/mL for each fluoxetine enantiomer. The intra‐ and inter‐day precision and accuracy of the quality control (QC) samples were <12.5% (CV) and 0.990. Method ruggedness was demonstrated by the reproducible performance of the assay during a 3‐day validation period. Copyright © 2002 John Wiley & Sons, Ltd.