Yoshie Kobayashi

Fluorometric determination of guanidino compounds by new postcolumn derivatization system using reversed-phase ion-pair high-performance liquid chromatography.

A new postcolumn derivatization system using 1,2-naphthoquinone-4-sulfonate as fluorogenic reagent for the fluorometric determination of guanidino compounds is described. The guanidino compounds were separated by reversed-phase ion-pair high-performance liquid chromatography with an isocratic mobile phase containing the fluorogenic reagent and octane-sulfonate as the counterion. Fluorophors were derived from a condensation of guanidino compounds with the fluorogenic reagent in an alkaline solution. The chromatographic system using the mobile phase containing the fluorogenic reagent was simplified because only two pumps were required to deliver the mobile phase and the alkaline solution. Separation of guanidino compounds was completed within 25 min using a Nucleosil C8 column (5 microns, 15 cm X 4.6 mm i.d.). This method was applied to serum obtained from patients on hemodialysis therapy.

Micro-Scale Method for Determination of Tobramycin in Serum Using High-Performance Liquid Chromatography

Abstract A rapid, simple, accurate, and micro-scale method for the determination of tobramycin, sisomicin and netilmicin in serum using high-performance liquid chromatography has been developed. The method is sensitive to 0.3 pg/ml using only 20 μl of serum. The serum is deproteinized with methanol containing an internal standard: sisomicin for the tobramycin, netilmicin for the sisomicin, and sisomicin for the netilmicin. After centrifugation, a counter-ion reagent is added to the supernatant, then an aliquot of the solution is injected into the chromatograph. Tobramycin, sisomicin and netilmicin are separated by reversed-phase, ion-pair chromatography and detected by fluorescence using continuous-flow, post-column derivatization with o-phthalaldehyde. For the tobramycin, within-run and day-to-day variation was below 2.5%. Correlation of this method with microbiological assay and homogeneous enzyme immunoassay was good.

Post-column derivatization system for the fluorimetric determination of guanidino compounds with ninhydrin by reversed-phase ion-pair high-performance liquid chromatography

A post-column derivatization system for the fluorimetric determination of guanidino compounds by high-performance liquid chromatography was developed. A mobile phase containing ninhydrin was used as the fluorigenic reagent. Ten guanidino compounds were separated within 25 min on a Nucleosil C8 (5 microns) column (15 cm X 4.6 mm I.D.) by isocratic, reversed-phase ion-pair chromatography and detected as fluorophors derived from condensation with ninhydrin in an alkaline stream. In this simplified system only two pumps required to deliver the mobile phase and the alkaline solution. This method was applied to serum from patients on haemodialysis therapy.

Fluorometric determination of streptomycin in serum by high-performance liquid chromatography using mobile phase containing fluorogenic reagent

A new postcolumn derivatization method for the fluorometric determination of streptomycin in serum by high-performance liquid chromatography is described. The serum was treated with 3.5% perchloric acid to precipitate proteins and the supernatant was directly injected into the chromatograph. Streptomycin was separated by reversed-phase, ion-pair chromatography with a mobile phase containing ninhydrin as a fluorogenic reagent, octanesulfonate, and 1,2-ethanedisulfonate as counterions, and was detected by fluorescence using continuous-flow, postcolumn derivatization in an alkaline stream with ninhydrin in the mobile phase. This method is sensitive to 1.0 microgram/ml using only 100 microliter of serum. Comparison with a fluorescence polarization immunoassay gave a good correlation coefficient of 0.976.

Fluorometric determination of biguanides in serum by high-performance liquid chromatography with reagent-containing mobile phase

A simple post-column derivatization method for the fluorometric determination of biguanides (buformin and phenformin) in serum by high-performance liquid chromatography is described. The serum was treated with 4% perchloric acid to precipitate proteins, and the supernatant was directly injected into the column. Synthesized 9,10-phenanthrenequinonesulphonate (PSQ) was used as a fluorogenic reagent and added to the mobile phase. Biguanides were separated within 10 min on a Radial-Pak microBondapak C18 cartridge (10 microns, 10 cm x 8 mm I.D.) by reversed-phase ion-pair chromatography. They were then allowed to react with PQS in an alkaline stream and detected fluorometrically. This method was applied to the analysis of serum from patients with diabetes mellitus.

Improved Method for the Determination of Cimetidine in Human Serum by High - Performance Liquid Chromatography

Abstract A simple and reproducible method for the determination of cimetidine in serum is described. Separation and quantitation were performed by high-performance liquid chromatography using a Radial-Pak CN column with a mobile phase of 33 % acetonitrile solution containing 5 mM triethylamine (adjusted to pH 3.0 with phosphoric acid) at a flow rate of 2 ml/min and at a detection of 220 nm. Cimetidine and ranitidine as an internal standard were extracted from serum with ethyl acetate and then back-extracted into dilute acid. An aliquot of the dilute acid was analyzed in the chromatographic system. The limit of detection was as low as 0.02 μg/ml using 100 μl of serum at a signal-to-noise ration of 2. Recoveries of cimetidine and ranitidine from serum were both greater than 95%. Within-run and day-to-day reproducibility for 10.0 μg/ml samples were 2.6 % and 2.9 %, respectively. The method is applicable to pharmacokinetic studies.

Rapid Method for Determination of Disopyramide and Its Mono-N-Dealkylated Metabolite in Serum by High-Performance Liquid Chromatography Using Sep-Pak Silica Extraction

Abstract A rapid, simple and reproducible method for sample pretreatment and analysis of disopyramide and its mono-N-dealkylated metabolite in serum is described. Samples for analysis were prepared by using Sep-Pak silica cartridge treated with 1 N sodium hydroxide followed by elution in 8 ml ethyl acetate. The eluate was back-extracted with acidic solution and an aliquot of the solution was injected directly onto the column. Chromatographic separation using p-chlorodisopyramide as an internal standard was achieved on a radially compressed CN column, with a mobile phase of 0.01 M dibutylamine phosphate(pH 3.0)-acetonitrile (75:25, v/v), and at a flow rate of 2.0 ml/min. The, peaks were monitored by W at 210 nm. Replicate analysis of serum controls for drug and its metabolite resulted in a within-run coefficient of variation of ≦ 2.1 and ≦ 4.4% and a day-to-day coefficient of variation of ≦ 2.5 and ≦ 4.8%, respectively. Comparison with an EMIT assay gave a correlation coefficient of 0.950.