Shunsuke Hata

Determination of simvastatin and its active metabolite in human plasma by column-switching high-performance liquid chromatography with fluorescence detection after derivatization with 1-bromoacetylpyrene

By using a fluorescent derivatization and column-switching technique, a highly sensitive and selective high-performance liquid chromatographic (HPLC) method has been developed for the determination of simvastatin (I, beta-hydroxy-delta-lactone form) and its active hydrolyzed metabolite (II, beta,delta-dihydroxy acid form of I) in human plasma. A plasma sample spiked with internal standards was applied to a C8 solid-phase extraction column. Compounds I and II were separately extracted from plasma into two fractions. Compound I in one of the fractions was hydrolyzed to II. A fluorescent derivative was prepared by esterification of II with 1-bromoacetylpyrene in the presence of 18-crown-6 for both fractions. The pyrenacyl ester of II thus obtained was purified on a phenylboronic acid (PBA) solid-phase extraction column, and was measured by column-switching HPLC with fluorescence detection. The calibration curves for both I and II were linear in the concentration range of 0.1-10 ng/ml. The intra-day coefficients of variation were less than 11.0%, and the accuracies were between 91.7% and 117% within the concentration range for both analytes. The limits of quantification (LOQ) for both analytes were set to 0.1 ng/ml. This assay method has adequate sensitivity and selectivity to measure the concentrations of I and II in human plasma from clinical studies.

High-performance liquid chromatographic determination of finasteride in human plasma using direct injection with column switching.

A fully automated column-switching high-performance liquid chromatographic (HPLC) method was developed for the quantification of finasteride [N-(1,1-dimethylethyl)-3-oxo-4-aza-5 alpha-androst-1-ene-17 beta- -carboxamide] in human plasma. Plasma samples were diluted with an equal volume of ethylene glycol-water (40:60, v/v), then the diluted sample (150 microliters) was injected into the HPLC system without clean-up. The analyte was retained on a pretreatment column, whereas plasma proteins and other endogenous components were washed out to waste. The analyte was transferred to the analytical column in the heart-cut mode and then detected at 210 nm. A quantification limit of 1 ng/ml was attained. There was a linear relationship between peak height and drug concentration in plasma in the range 1-50 ng/ml. This method was validated and applied to the assay of plasma samples to characterize pharmacokinetic parameters in clinical studies.

Simultaneous determination of a new anticancer agent (NB-506) and its active metabolite in human plasma and urine by high-performance liquid chromatography with ultraviolet detection

A high-performance liquid chromatographic method with ultraviolet detection has been developed to quantify NB-506 and its active metabolite in human plasma and urine. This method is based on solid-phase extraction, thereby allowing the simultaneous measurement of the drug and metabolite with the limit of quantification of 0.01 microgram/ml in plasma and 0.1 microgram/ml in urine. Standard curves for the compounds were linear in the concentration ranges investigated. The range for the drug in plasma was 0.01-2.5 micrograms/ml, and for the metabolite 0.01-1 microgram/ml. In urine, the range for both compounds was 0.1-10 micrograms/ml. The method was validated and applied to the assay of plasma and urinary samples from phase I studies.

Column-switching high-performance liquid chromatographic analysis of BO-2727, a new carbapenem antibiotic, in human plasma and urine by direct injection.

A column-switching high-performance liquid chromatographic method has been developed for the simple and sensitive analysis of BO-2727 (I) in human plasma and urine. Plasma samples were diluted with an equal volume of a stabilizer, and the mixture was directly injected onto the HPLC system. The analyte was enriched in a pre-treatment column, while endogenous components were eluted to waste. The analyte was then backflushed onto an analytical column and quantified with ultraviolet detection. Urinary concentrations were determined in a similar way except that the enriched analyte was eluted in the foreflush mode to a cation-exchange column used for chromatographic separation. The standard curves for the drug were linear in the range of 0.05-50 micrograms/ml in plasma and 0.5-100 micrograms/ml in urine. The limits of quantification for plasma and urine were found to be 0.5 micrograms/ml and 0.5 micrograms/ml, respectively. This method was used to support Phase I clinical pharmacokinetic studies.