Quantitative determination of verapamil and metabolites in human serum by high-performance liquid chromatography and its application to biopharmaceutic investigations.

A specific and sensitive high-performance liquid chromatographic method for the quantitative analysis of verapamil and N-desmethylverapamil in human serum is described. The analytes were extracted from serum using diethylether under alkaline conditions, followed by back extraction into dilute hydrochlorid acid for chromatographic analysis on a reversed-phase column with a mobile phase consisting of acetonitrile, water and perchloric acid at a flow rate of 1 l/min. The analytes were detected by fluorescence detection, the influence of temperature on retention is discussed. The method is linear, quantitative and reproducible for two calibration ranges in serum (2.5 ng/ml-100 ng/ml and 12.5 ng/ml-500 ng/ml) using peak area ratios analyte/internal standard for quantification. At ultimate sensitivity, concentrations down to 250 pg/ml could be assayed. The method was selective to 6 other metabolites of verapamil and common exogenous interferences. It was applicated to the serum samples of a comparative 120 mg - verapamil hydrochloride tablet single dose two-way cross-over study comprising 18 volunteers. The pharmacokinetic data for both formulations are presented.

Determination of nitrates in plasma.

Because of the high efficacy of existing nitrates, alternative dosage formulations have been developed rather than new compounds. These new formulations challenge the analyst to develop analytical methods with lower sensitivity and higher precision. Isosorbide dinitrate is analysed in the presence of its major metabolites isosorbide 2-mononitrate and isosorbide 5-mononitrate. The present method is an improvement over previously published methods. It is not possible to analyse glyceryl trinitrate and its major metabolites 1,2-glyceryl dinitrate and 1,3-glyceryl dinitrate with the necessary sensitivity in one chromatogram. A 2-step procedure is therefore applied. The extraction procedures, the gas chromatographic conditions, imprecision and inaccuracy of the methods, the lower limits of quantitation, sample chromatograms and applications of all methods are described in detail. Thus, the possibilities of modern analytical methods in the determination of nitrates in the lower picogram region are demonstrated.

[Comparative studies on the tolerance of bioequivalent doses of intravenous methylxanthine preparations in healthy subjects]. / Vergleichende Untersuchungen zur Verträglichkeit bioäquivalenter Dosen intravenöser Methylxanthin-Zubereitungen bei gesunden Probanden.

In a comparative tolerance study with two different intravenous methylxanthine preparations, a theophylline-ethylendiamine preparation (TE-reference preparation) was tested against a combination of theophylline, proxyphylline (7-(2-hydroxypropyl)-theophylline) and diprophylline (7-(2,3-dihydroxypropyl)-theophylline) (Neobiphyllin; TPD = test preparation) in 10 healthy volunteers by a single blind cross-over design. Both preparations were infused under continuous control of vital parameters (blood pressure, pulse, respiration frequency, heart rhythm) as infusions (1 ampoule with 800 mg TPD or 1 short-infusion with 480 mg of TE for 20 min, each) up to the individual tolerance limit or the pre-defined limit of 3 ampoules/short infusions, respectively. The maximum tolerated infusion time and the serum levels at which the first side-effects appeared, were compared. These maximum doses could be administered to 6 volunteers under TPD, but only to two under medication with the reference preparation. Side-effects under TPD occurred in 5, after infusion of the reference preparation in 9 volunteers. Serum levels of theophylline at the end of the infusion period reached (14.6 +/- 4.21 (TPD) and 23.01 +/- 6.02 mg/l (TE), respectively. The average infusion time for the test preparation was 54.8, for the reference preparation 46.2 min. The average serum theophylline levels of the 5 volunteers with side-effects under TPD reached--when these side-effects occurred --11.26 +/- 4.52 mg/l; the same volunteers showed after administration of TE levels of 14.94 +/- 7.49 mg/l. Our results showed an approx. additive effect of the side-effects together with an--according to literature--over-additive pharmacological effect of the single components of TPD.(ABSTRACT TRUNCATED AT 250 WORDS)

[Studies of the effect of effectors on proteinases from rat liver lysosomes in vitro]. / Untersuchungen zum Einfluss von Effektoren auf die Proteinasen aus Rattenleberlysosomen in vitro.

New test systems using azocasein or double-labelled cytosol proteins from rat livers were developed to test the influence of a lot of possible effectors on lysosomal proteinases from rat liver. All as yet tested inhibitors of serine proteinases did not show substantial inhibition of the sum of all proteinases activity in the soluble part of rat liver lysosomes. Moreover at least 200 different substances were found to be without significant effects against these lysosomal proteinases. These proteinases are only effected by peptide aldehydes and other compounds which influence thiolproteinases. This provides further evidence that mainly the thiolproteinases (cathepsin L, H and B) are responsible for the proteolytic activity in rat liver lysosomes.