Cultivation of fibroblast cells on keratin-coated substrata.

By means of a cell culture method, the attachment and growth of mouse L929 fibroblast cells were studied on matrices of the (-SDS)- and (+SDS)-keratins, which were extracted from wool in the absence and presence of sodium dodecyl sulfate, respectively. The (+SDS)-keratin showed some toxic effect on the cell growth, but upon washing with a pH 7/phosphate buffer, the protein behaved similarly to a substratum of the (-SDS)-keratin. The comparative culture assay on the keratins, collagen (type I), and glass revealed that the keratins were more adhesive to the cells and more supportive for cell proliferation than the collagen and glass. The results were explained by an enhanced initial adsorption of mediator proteins from fetal bovine serum onto the keratin substrata.

High-performance liquid chromatographic determination of 6-amidino-2-naphthyl [4-(4,5-dihydro-1H-imidazol-2-yl) amino]benzoate dimethanesulphonate and its metabolites in biological fluids.

6-Amidino-2-naphthyl [4-(4,5-dihydro-1H-imidazol-2-yl) amino]benzoate dimethanesulphonate has been developed for the therapy of pancreatitis. A reversed-phase high-performance liquid chromatographic assay of the levels of this drug and its metabolites in biological fluids was investigated. Fluorescence detection with post-column alkaline degradation was used for the determination of the intact drug and the amidinonaphthol moiety metabolite, and ultraviolet detection at 254 nm was used to determine the levels of the benzoic acid moiety metabolite. Satisfactory recoveries and variabilities of the intact drug and its metabolites from biological fluids were obtained. The detection limits for the intact drug and amidinonaphthol were 0.5 ng/ml at a signal-to-noise ratio of 12 in plasma and 10 ng/ml at a signal-to-noise ratio of 32 in urine and homogenized faeces, and those of benzoic acid were 5 ng/ml at a signal-to-noise ratio of 3 in plasma and 50 ng/ml at a signal-to-noise ratio of 7 in urine and homogenized faeces.

Gas chromatographic-negative-ion chemical ionization mass spectrometric determination of a new dihydropyridine calcium antagonist (MPC-1304) and its metabolites in human plasma and urine.

A gas chromatographic-negative-ion chemical ionization mass spectrometric method was developed for the determination of a new calcium antagonist, (+/-)-methyl 2-oxopropyl 1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridinedicarboxylate, and its metabolites in plasma and urine. The sample was extracted with n-hexane-diethyl ether. The dried organic layer was subjected to acetylation: the aqueous layer was acidified and extracted with ethyl acetate, and after the ethyl acetate extract was dried the resulting residue was subjected to methylation. Aliquots of each reactant solution were injected into the gas chromatograph-mass spectrometer, equipped with a chemical ionization source and negative-ion monitoring mode, and analysed by the selected-ion monitoring method using deuterium-labelled internal standards. Detection was limited to 0.02-0.05 ng/ml of plasma and urine for each metabolite. A precise and sensitive assay for the determination of a new dihydropyridine calcium antagonist and its metabolites in plasma and urine was thus established.

High-performance liquid chromatographic determination of a new beta-lactamase inhibitor and its metabolite in combination therapy with piperacillin in biological materials.

[2S-(2 alpha,3 beta,5 alpha)]-3-Methyl-7-oxo-3-(1H-1,2,3-triazol-1-yl- methyl)-4-thia-1-azabicyclo [3.2.0]-heptane-2-carboxylic acid 4,4-dioxide (YTR-830H) is a new beta-lactamase inhibitor and the combination therapy of this compound with piperacillin is now under study. For the determination of the beta-lactamase inhibitor and piperacillin in biological materials, plasma and visceral tissue homogenates were deproteinized, whereas diluted urine and filtered faeces homogenates were treated with a Sep-Pak C18 cartridge. In order to assay the inactive metabolite of beta-lactamase inhibitor, each sample was treated with a Sep-Pak C18 cartridge. Aliquots of each preparation were chromatographed using ion-pair and reversed-phase chromatographic techniques on a high-performance liquid chromatograph equipped with a UV detector, set at 220 nm. The detection limits of beta-lactamase inhibitor and piperacillin were 0.2 microgram/ml in plasma, 2.5-5.0 micrograms/ml in urine and 0.2-0.5 microgram/g in visceral tissue and faeces. Those of the metabolite were 1.0 microgram/ml in plasma, 2.5-5.0 micrograms/ml in urine and 1.0 microgram/g in visceral tissue and faeces. A precise and sensitive assay for the determination of the beta-lactamase inhibitor, its metabolite and piperacillin is described, and their stabilities in several media are reported.

High-performance of liquid chromatographic determination of [3,4-di-(4-methoxyphenyl)-5-isoxazolyl]acetic acid and its metabolites in human plasma and urine.

A high-performance liquid chromatographic method is described for the simultaneous determination of the non-steroidal analgesic and anti-inflammatory agent [3,4-di-(4-methoxyphenyl)-5-isoxazolyl]acetic acid and its three metabolites in plasma and urine. Deproteinized plasma (with acetonitrile) or urine was applied to a Sep-Pak C18 cartridge, washed with distilled water and then eluted with methanol. The methanol eluate was reduced to dryness. The resulting residues from the plasma and urine were redissolved in methanol aqueous solution, respectively. Aliquots of each solution were chromatographed on a reversed-phase column using a mobile phase of methanol-20 mM potassium dihydrogenphosphate (pH 6.4) (linear gradient from 0 to 100% methanol at 3%/min with a flow-rate of 1.5 ml/min) on a liquid chromatograph equipped with an ultraviolet absorbance detector (254 nm). Detection was limited to 10 ng/ml in plasma and 100 ng/ml in urine for each compound. An accurate and sensitive assay for the determination of [3,4-di-(4-methoxyphenyl)-5-isoxazolyl]acetic acid and its metabolites was established.

Determination of cefodizime in biological materials by high-performance liquid chromatography.

Cefodizime (THR-221) is a new semi-synthetic cephalosporin. A high-performance liquid chromatographic method has been developed for the determination of cefodizime in biological materials. A plasma or serum sample was deproteinized with methanol and the resulting methanol eluate was concentrated to a volume of 0.5 ml. Urine and bile samples were diluted with buffer and each diluted sample was filtered. Faeces samples were homogenized and the supernate obtained after centrifugation was filtered. Visceral tissue samples were homogenized, the centrifuged supernate was deproteinized with methanol, and the methanol eluate was concentrated to a volume of 0.5 ml. Aliquots of each preparation were chromatographed on a reversed-phase column with an ion-pair chromatographic technique on a high-performance liquid chromatograph equipped with an UV detector set at 264 nm. The detection limits for cefodizime were 0.1 microgram/ml in plasma or serum, 0.3 microgram/ml in bile, and 0.5 microgram/ml in urine, 0.5 microgram/g in faeces and visceral tissue. This precise and sensitive assay for the determination of cefodizime is described, and its stability in several media is reported.

Gas chromatographic-mass fragmentographic determination of propiverine and its metabolites in plasma and urine.

1-Methyl-4-piperidyl diphenylpropoxyacetate hydrochloride has been developed clinically for the therapy of urinary bladder dysfunction. A gas chromatographic-mass fragmentographic method was developed for the determination of this drug and its seven metabolites in plasma and urine. The sample was first treated with a Sep-Pak C18 cartridge, the methanol eluate was evaporated to dryness, and the resulting residue was redissolved in distilled water. This solution was then extracted with chloroform and adjusted to pH 9.0 with 0.1 M sodium borate solution. The acidified aqueous layers were extracted with ethyl acetate. The chloroform layer, which contained non-polar metabolites, was concentrated to dryness, then subjected to trifluoroacetylation, decomposition and methylation. The extract from the plasma sample was trimethylsilylated. The dried residue of the ethyl acetate layer, which contained polar metabolites, was subjected to methylation, trifluoroacetylation and decomposition. Aliquots of each reactant solution were injected into the gas chromatograph-mass spectrometer and analysed by the selected-ion monitoring method using an internal standard. Detection was limited to 1-2 ng/ml of plasma and urine for each metabolite. A precise and sensitive assay for the determination of 1-methyl-4-piperidyl diphenylpropoxyacetate hydrochloride and its metabolites in plasma and urine was thus established, and it should prove useful in basic and clinical pharmacological studies.