Determination of metrifonate enantiomers in blood and brain samples using liquid chromatography on a chiral stationary phase coupled to tandem mass spectrometry.

A novel enantioselective assay is described for the simultaneous determination of the metrifonate enantiomers BAY z 7216 and BAY z 7217 in extracts of whole blood samples obtained from rats, mice, rabbits and Beagle dogs as well as in rat brain tissue using liquid chromatography tandem mass spectrometry (LC/MS/MS) with thermally and pneumatically assisted electrospray ionization (TurboIonSpray(R)). Chromatographic separation is achieved on a chiral normal phase column with a mobile phase containing 0.25% water only. The total run time per sample is 11.0 min giving chromatographic base line separation of the enantiomers. Compared with previous methods this assay offers a higher sample throughput, excellent ruggedness and higher sensitivity. The limits of quantification for each enantiomer are 5.00 microg/L from 0.5 mL whole blood and 7.50 ng/g (ppb) using 0.333 g brain tissue, respectively. Similar assay specifications have been derived for the two enantiomers. The method has been validated for the analysis of blood samples from low and high dosed preclinical pharmacokinetic and toxicokinetic studies, corresponding to two analytical working ranges like e.g. 5.00 to 1000 microg/L and 200 to 40000 microg/L (0. 200 to 40.0 mg/L). For rat brain tissue the validated concentration range is 7.50 to 750 ng/g (ppb).

Determination of the enantiomers of nisoldipine in human plasma using high-performance liquid chromatography on a chiral stationary phase and gas chromatography with mass-selective detection.

A method is described that combines chiral HPLC and off-line GC with mass-selective detection for the quantitation of the enantiomers of nisoldipine [(+/-)-I] in human plasma. An isotope-labelled internal standard [nine-fold deuterated (+/-)-I] is used throughout the assay. The limit of quantification is 0.1 microgram/l for each enantiomer. Data on the precision, accuracy and selectivity of the method are presented. Enantioselective analysis was performed in subjects receiving the racemic drug in tablet form. In healthy volunteers the maximum concentration and the area under the curve of the pharmacologically more active (+)-enantiomer were greater by 9-fold and 13-fold, respectively, compared to those of the (-)-enantiomer. In elderly hypertensive patients plasma concentrations of (+)-I were ca. five times as high as those of the (-)-enantiomer. Stereoselectivity was not affected by hepatic impairment. After intravenous administration of (+/-)-I there were no relevant differences between the plasma concentrations of the enantiomers.

Enantioselective assay for the determination of nisoldipine in dog, rat and mouse plasma by chiral microbore high-performance liquid chromatography combined with gas chromatography-mass spectrometry.

A sensitive, selective and validated method for the enantioselective determination of (+)- and (-)-nisoldipine in rat, mouse and dog plasma following administration of nisoldipine racemate is described. The alkalized plasma samples containing [13C4]nisoldipine racemate as internal standard (ISTD) were extracted once with toluene. The enantiomers of nisoldipine were quantitatively separated by high-performance liquid chromatography on a 250 x 2 mm I.D. column containing tris(4-methylbenzoate)-modified cellulose on silica. The fractions containing either the (+) or (-)-enantiomer of the analyte and [13C4]ISTD were analysed by gas chromatography with mass-selective detection in the single-ion monitoring mode. The limits of determination and detection were 0.5 and 0.2 ng/ml, respectively, the total precision was better than 7% (R.S.D. at 5 and 50 ng/ml, n = 35) and the accuracy was better than 10% (0.5-100 ng/ml, n = 23). The sum of the concentrations of the enantiomers determined with this assay corresponds to the concentration of the racemate determined independently by capillary gas chromatography with electron-capture detection (accuracy better than 15%, 1-80 ng/ml). The method was used for the analysis of more than 500 plasma samples obtained from toxicokinetic studies.

Chromatographic resolution of chiral intermediates in beta-adrenergic blocker synthesis on chiral stationary phases.

The enantiomeric purities of optically active intermediates for beta-adrenergic blocking agents prepared via enzyme-assisted processes can be determined rapidly and with high accuracy using HPLC on commercially available columns with chiral supports [Chiralcel OD, OB; Chiralpak OT(+)]. The dependence of the resolution parameters on the substitution pattern of both hydroxy compounds and their esters is reported.