Sensitive method for the determination of KC 12291 in rat plasma and urine by high-performance liquid chromatography.

A sensitive and selective ion-pair reversed-phase HPLC method has been developed for the quantitative determination of KC 12291 and its major metabolite, KC 13194, in rat plasma and urine. An Ultrasphere ODS column constructed by using a mobile phase of 1 mM 1-octanesulfonic acid containing acetonitrile-0.1 M triethylamine phosphate buffer, pH 2.2 (29:71, v/v in plasma and 27:73, v/v in urine), an internal standard and a fluorescent detector (excitation 265 nm, emission 370 nm) were used for the separation and the quantitative determination, respectively. The plasma samples were made alkaline and both compounds were cleaned up by the use of liquid-liquid extraction. The limit of quantification was 10 ng/ml for KC 12291 in plasma and urine and for KC 13194 and 100 ng/ml in plasma, respectively. The assay has been validated with respect to system suitability, accuracy, precision, recovery, stability and ruggedness. All validated parameters were found to be within the necessary limits.

Simultaneous determination of nerisopam, a novel anxiolytic agent showing polymorphic metabolism, and its N-acetyl metabolite from human plasma by a validated high performance liquid chromatographic method.

A sensitive reversed-phase high-performance liquid chromatographic method with ultraviolet absorbance detection has been developed to simultaneously determine the concentrations of nerisopam (EGIS-6775) and its N-acetyl metabolite (EGIS-7649) from human plasma. The separation of the investigated compounds and internal standard was achieved on a Nucleosil 7 C(18) column with 2 mM heptanesulphonic acid containing 0.04 M phosphoric acid-acetonitrile-methanol (70:25:5, v/v), pH 2.7 mobile phase. The detection was performed at 385 nm. The compounds were isolated from plasma by Bakerbond C(18) solid-phase extraction. The limit of quantitation was 10 ng/ml plasma for each compound investigated. The assay has been validated with respect to accuracy, precision and system suitability. All validated parameters were found to be within the necessary limits. On the basis of the sensitivity, linearity and validation parameters, the developed analytical method was found to be suitable for the determination of nerisopam and its N-acetyl metabolite from human plasma and for application in pharmacokinetic studies and human drug monitoring. The pharmacokinetic parameters obtained from twelve human volunteers are reported. It was found that nerisopam acetylation is polymorphic: the volunteers with fast or slow acetylator phenotypes produced significantly different plasma concentrations. In slow acetylator phenotypes the concentration of nerisopam was considerably higher in plasma, while the level of its acetyl metabolite was higher in plasma of fast acetylators.

Pharmacokinetic and metabolism studies on girisopam by chromatographic and spectrometric methods in humans.

Girisopam possesses selective anxiolytic action without muscle relaxant and anticonvulsive activity. After a 100-mg oral dose of 14C-labelled girisopam to seven male subjects, the mean recovery of 14C radioactivity was 51% in urine and 33% in faeces. A high-performance liquid chromatographic method has been developed for studying girisopam in single-dose pharmacokinetic studies. The serum extract was chromatographed on a normal-phase column using a mobile phase of hexane-ethanol-diethyl ether (66:9:25, v/v) and ultraviolet detection at 235 nm. The recovery was 60% and the detection limit was 3 ng/ml, using 1 ml of serum. After a 20-min delay, girisopam is rapidly absorbed. After reaching a mean serum level of 178 ng/ml at a mean time of 2.0 h, the serum concentration of girisopam decreased with a mean elimination half-time of 22.2 h. The metabolites were separated by high-performance liquid chromatography, radio thin-layer chromatography and gas chromatography. Their structures were determined by liquid chromatography-mass spectrometry, mass spectrometry and gas chromatography-mass spectrometry. Their chemical structures were confirmed by comparison with synthesized reference compounds. The major urinary metabolites were 7-demethylgirisopam (I), 4'-hydroxygirisopam (II) and 4-hydroxymethyl-4-demethylgirisopam (III), which were in conjugated form, and 4-carboxy-4-demethylgirisopam (V), a compound with an open-chain structure (VII) and traces of 4-demethyl-4-oxogirisopam (VIII) and 4-hydroxymethyl-4-demethylgirisopam (III), which were in non-conjugated form. The metabolic profile in the serum consisted predominantly of the glucuronides of I, II and III. The non-conjugated metabolites were the metabolite with the open-chain structure (VII), III and V. Besides the parent compound, the faeces sample contained conjugates of I and II.

Highly active and selective anticoagulants: D-Phe-Pro-Arg-H, a free tripeptide aldehyde prone to spontaneous inactivation, and its stable N-methyl derivative, D-MePhe-Pro-Arg-H.

D-Phe-Pro-Arg-H sulfate (GYKI-14166) is a highly active and selective inhibitor of thrombin both in vitro and in vivo. Recent studies on the stability of D-Phe-Pro-Arg-H in neutral aqueous solution at higher temperature have revealed that it is transformed into inactive 5,6,8,9,10,10a-hexahydro-2-(3'- guanidinopropyl)-5-benzyl-6-oxo- imidazo[1,2-a]pyrrolo[2,1-c]pyrazine. No such inactivation could be observed with Boc-D-Phe-Pro-Arg-H (GYKI-14451), but this compound was far less specific than the free peptide as it inhibited thrombin and, for instance, plasmin equally well. Assuming that the transformation of free tripeptide aldehyde, mentioned above, can only be initiated by a primary amino terminus, the N-alkyl derivatives of D-Phe-Pro-Arg-H were prepared. Of the new analogues, D-MePhe-Pro-Arg-H (GYKI-14766) proved to be as highly active and selective anticoagulant as its parent compound and was not inactivated by transformation into a heterocyclic compound.

Sensitive method for the determination of 6-oxo-prostaglandin F1 alpha by gas chromatography with electron-capture and mass-fragmentographic detection.

A selective and sensitive quantitative method is described for the determination of the endogenous prostacyclin content of the aorta of rats, treated with GYKI-11679, a new hydrazone derivative with antihypertensive action. The thoracal aorta from three rats was excised and pooled, 1 h after treatment with 10 mg/kg of GY-KI-11679. The prostacyclin, transformed to 6-oxo-PGF1 alpha, was extracted. For gas chromatographic electron-capture detection (GC-ECD), 6-oxo-PGF1 alpha was converted into the O-methyloxime tris(trifluoroacetyl) hexafluoroisopropyl ester derivative. The GC-ECD properties, stability and mass-spectrometric characteristics of this new derivative allow the quantitative analysis of 6-oxo-PGF1 alpha by using nor-PGF2 alpha as internal standard.

Investigation of metabolites of tofizopam in man and animals.

The biotransformation of the labelled forms of 1-(3,4-dimethoxy-phenyl)-4-methyl-5-ethyl-7,8-dimethyoxy-5H-2,3-be nzodiazepine (Grandaxin, tofizopam) has been investigated after oral administration in animals and man. The major part of urinary metabolites was found to be conjugated with glucuronic acid. The metabolites were separated using TLC technique and analysed by GLC and GLC-MS. The chief way of the metabolic information of tofizopam is demethylation, however, the position of CH3 elimination as well as the rate of it was different in various species.

Investigation of the metabolites of tofizopam in man and animals by gas-liquid chromatography-mass spectrometry.

The metabolites of tofizopam [Grandaxin; 1-(3,4-dimethoxyphenyl)-4-methyl-5-ethyl-7,8-dimethoxy-5H-2,3-benzodiazepine] have been studied in patients and animals. The major pathway of the metabolic transformation of tofizopam was found to be demethylation. The position in which demethylation takes place and the rate of this process in various species were determined. Gas-liquid chromatography-mass spectrometry-mass chromatography was used for the identification of the metabolites.

Specific and sensitive method for the determination of tobanum using gas chromatography with electron-capture and mass fragmentographic detection.

Quantitative gas--liquid chromatographic (GLC) and GLC--mass spectrometric (MS) methods for the determination of Tobanum, a new beta-blocking agent, in human plasma have been developed. After solvent extraction, a bis-trifluoroacetyl derivative is formed which is measured by an electron-capture detector. The quantitation is controlled by using an internal standard, propranolol hydrochloride, which is added to all samples. The electron-capture detector response is linear in the concentration range used, 5--150 ng/ml. The identity and quantity of the GLC peaks is confirmed by GLC--MS. The minimum detectable concentration of Tobanum is 1 ng using 3-ml plasma samples.

Capillary gas chromatographic studies of cholesterol biosynthesis in rats treated with EGYT-1299.

Cholesterol and desmosterol concentrations were assayed by high-performance capillary gas chromatography. It was established that no toxic desmosterol accumulation occurred either in the serum or liver of rats receiving EGYT-1299 treatment. Desmosterol accumulation induced by triparanol was reduced by the agent at doses ten times lower than Atromid S; consequently its cholesterol lowering effect may be attributed to interference with cholesterol biosynthesis.

Quantitative analysis of corticosteroids in adrenal cell cultures by capillary column gas chromatography combined with mass spectrometry.

A quantitative method using glass capillary column gas-liquid chromatography (GLC) has been established for the analysis of corticosteroids in small biological samples such as a few milliliters of medium from steroidogenic cultured cells. The steroid extract is separated in three main thin-layer chromatographic fractions. The steroids assayed in these fractions are: less polar fraction (C21O2) steroids), 20 alpha-dihydroprogesterone, pregnenolone and 20 alpha-dihydro-pregnenolone; intermediate fraction (C21O3 steroids), deoxycorticosterone, 11-oxo- and several hydroxy-20 alpha-dihydroprogesterones; corticosteroid fraction (C21O4 steroids), corticosterone and 18-hydroxydeoxycorticosterone. The method has been assessed by characterizing the steroid structures and checking the purity of GLC effluents through direct coupling of the capillary column to the electron-impact or chemical-ionization source of a computerized mass spectrometer. Applications to the quantitative evaluation of the endocrinological status of newborn rat adrenocortical cell cultures under various conditions of development, hormonal stimulation or specific inhibition by drugs are described.