Michio Motohashi

High-performance liquid chromatographic determination of pioglitazone and its metabolites in human serum and urine

A high-performance liquid chromatographic (HPLC) method for the simultaneous determination of pioglitazone and its metabolites (M-I to M-V) in human serum and urine was developed. The method for serum involved the solid-phase and liquid-liquid extraction. Urine with and without enzymatic hydrolysis using beta-glucuronidase was treated with liquid-liquid extraction. The compounds in the extract were analyzed using HPLC with UV detection at 269 nm. The detection limits of pioglitazone, M-I, M-II, M-III, M-IV, and M-V in serum were 0.01-0.05 micrograms/ml, those in urine were 0.1-0.5 micrograms/ml, and those in urine after enzymatic hydrolysis were 0.3-0.5 micrograms/ml, respectively. The method was applied to the clinical trials of pioglitazone.

Quantitation of a new potent angiotensin II receptor antagonist, TCV-116, and its metabolites in human serum and urine

A sensitive high-performance liquid chromatographic (HPLC) method is described for the determination of a new potent antihypertensive agent, TCV-116, and its two metabolites (M-I and M-II) in human serum or urine. After pre-treatment of the specimens, the analytes were determined using a column switching technique, except for the metabolites in urine which were determined by gradient elution mode HPLC. The quantitation limits for TCV-116, M-I and M-II were all 0.5 ng/ml in serum, and 0.5, 10 and 110 ng/ml in urine, respectively. The methods were applied to clinical trials of TCV-116.

Selective β-adrenoceptor activities of tetrahydronaphthalene derivatives

Abstract Alpha - and beta -adrenoceptor activities of a group of tetralin derivatives prepared as fixed analogs of isoproterenol, adrenaline and noradrenaline were evaluated in vitro assays. All of the present compounds showed strong direct β-stimulating activity fairly selective for tracheal muscle. Activity of a trans isomer about the C(1)-C(2) bond was ten times higher than that of the cis isomer. None of the compounds tested had any practical α-adrenoceptor activity. Structure-activity interrelationships between the present series of compounds and some known adrenergic agonists are discussed.

Development and validation of column-switching high-performance liquid chromatographic methods for the determination of a potent AII receptor antagonist, TCV-116, and its metabolites in human serum and urine

Column-switching HPLC methods have been developed and validated for the determination of a new antihypertensive prodrug, TCV-116 (I), and its metabolites, CV-11974 (II) and CV-15959 (III), in human serum and urine. Initial sample cleanup was achieved by extracting the analytes into an organic solvent. After chromatographing on an ODS column with a mobile phase consisting of acetonitrile and an acidic phosphate buffer, the zone of the analytes retention was heart-cut onto a second ODS column with a mobile phase of acetonitrile and a phosphate buffer at a higher pH. Complete separation of the analytes and the endogenous peaks was accomplished by the two-dimensional chromatography. Good precision and linearity of the calibration standards, as well as the inter-day and intra-day precision and accuracy of quality control samples, were achieved. The limit of quantitation (LOQ), using 0.5 ml of serum, was 2 ng/ml for I, 0.8 ng/ml for II, and 0.5 ng/ml for III. The LOQ for urine sample was 10 ng/ml for II and III. Stability of the analytes during storage, extraction, and chromatography processes was established. The results illustrate the versatile application of column switching to method development of multiple analytes in various biological matrices. The methods have been successfully used for the analyses of I and its metabolites in thousands of clinical samples to provide pharmacokinetic data.

Column-switching techniques for high-performance liquid chromatography of ibuprofen and mefenamic acid in human serum with short-wavelength ultraviolet detection

Column-switching techniques for high-performance liquid chromatography of two acidic drugs, ibuprofen and mefenamic acid, in human serum with short-wavelength ultraviolet detection are described. The method involved extraction of the analyte from acidified serum followed by the chromatographic analysis using column switching. Three ODS columns were used each with different mobile phase, utilizing the difference of ion-pair formation or of ionization caused by pH change. The method offered high sensitivity and selectivity, with short-wavelength ultraviolet detection at 221 nm for ibuprofen and at 219 nm for mefenamic acid. The detection limits were 0.5 ng/ml (2.4 pmol/ml) for ibuprofen and 0.1 ng/ml (0.4 pmol/ml) for mefenamic acid using 1 ml of serum, both at a signal-to-noise ratio of 3. With some modifications, the principle of the method would be applicable to other acidic compounds in biological fluids.

High-performance liquid chromatography using on-line solid-phase extraction: determination of furosemide in human serum

An on-line solid-phase extraction technique based on column switching (heart-cutting) was developed for direct injection analysis of furosemide in human serum. In order to minimize the influence of deterioration in pre-treatment column efficiency, which was caused by protein precipitation with repeated injections of serum, furosemide was completely enriched at the top of the analytical column by ion-pair formation with tetra-n-butylammonium ion during heart-cutting. The robustness of the established on-line solid-phase extraction system was confirmed under routine conditions. As a result, almost comparable chromatograms could be obtained even though 50 repeated injections of a 100-microliter volume of serum were carried out using one pre-treatment column. The linearity of the calibration curves was demonstrated by the correlation coefficient which was greater than 0.99999 (5-1000 ng/ml). The relative errors and C.V. of quality control samples were within 4.00 and 5.88%, respectively (furosemide concentrations: 5, 100 and 1000 ng/ml).

Determination of manidipine and its pyridine metabolite in human serum by high-performance liquid chromatography with ultraviolet detection and column switching

A highly sensitive and selective high-performance liquid chromatographic method using column switching is described for the determination of the dihydropyridine calcium antagonist manidipine (I),2-[4-(diphenylmethyl)-1-piperazinyl]ethyl methyl (+/- )-1,4-dihydro-2,6-dimethyl-4-(m-nitrophenyl)-3,5- pyridinedicarboxylate, and its pyridine metabolite (II), 2-[4-(diphenylmethyl)-1-piperazinyl]ethyl methyl 2,6-dimethyl-4-(m-nitrophenyl)-3,5-pyridinedicarboxylate, in human serum. The method is based on the combination of the column-switching technique and ion-pair chromatography. In the first ODS column, I and II are preseparated from endogenous substances in serum with a mobile phase containing sodium nonane sulphonate as an ion-pair reagent. After column switching, in the second ODS column, the heart-cut fraction containing I and II is further separated from the co-eluted substances through the first column with a mobile phase containing no ion-pair reagent. By using microbore columns with a diameter of 2.1 mm, the sensitivity is almost double that given by conventional bore columns with a diameter of 4.6 mm. The method offers high sensitivity and selectivity with short-wavelength ultraviolet detection at 230 nm. The detection limits of both I and II are 0.1 ng/ml using 1 ml of serum. The method is suitable for the pharmacokinetic study of I.2HCl after oral administration to man.

Complete two-dimensional separation for analysis of acidic compounds in plasma using column-switching reversed-phase liquid chromatography

A complete two-dimensional separation technique for the determination of acidic compounds in plasma was developed by using column-switching reversed-phase liquid chromatography. This technique was based on solute peak enrichment at the top of the second column during heart-cutting and an ion-pair chromatographic separation in the second column using tetrabutylammonium ion, where different separation modes in the first and second columns and solute peak enrichment at the top of the second column during heart-cutting were achieved coincidentally. Retention behaviors of two solutes, zidovudine-beta-D-glucuronide (AZT-beta-D-Gluc) and probenecid, in the first and second column and solute peak enrichment at the top of the second column were investigated for establishment of the system. Different retention behaviors of the solutes in the first and second column, which were evaluated by changes in capacity factor versus acetonitrile concentration in the mobile phases, and peak enrichment could be accomplished by using ion-pair chromatography in the second column. System suitability was confirmed by assessing the number of theoretical plates (N) of the second column for the solutes after heart-cutting. The N values in the second column after column switching were almost same as those in the case that the solutes were directly injected onto the second column. These results indicate that complete two-dimensional separation should be achieved by using this system. Furthermore, this technique was applied to method development for the determination of AZT-beta-D-Gluc and probenecid in rat plasma. The peaks of each analyte in the plasma extract obtained by deproteinization were well separated from those of endogenous substances, and easy determination of the analytes could be accomplished at the ng/ml level only by changing the acetonitrile concentration in the mobile phases.

Sensitive determination of methotrexate in monkey plasma by high-performance liquid chromatography using on-line solid-phase extraction

A high-performance liquid chromatographic method was developed for the sensitive determination of methotrexate (MTX) in monkey plasma using direct injection and on-line solid-phase extraction. After application of a 100-microliters aliquot of plasma to a pre-treatment column, the column was washed with 0.02 M phosphate buffer (pH 7) to eliminate plasma proteins and endogenous substances, and subsequently the adsorbed MTX was eluted. The MTX fraction was transferred to an analytical column by a column-switching (heart-cutting) technique, and MTX was analyzed using ion-pair chromatography with tetrabutylammonium bromide. More than 50 injections could be performed onto one pretreatment column. The accuracy, precision, reproducibility and linearity were satisfactory over a wide range of MTX concentrations (5-1000 ng/ml). The quantitation limit was 5 ng/ml with a signal-to-noise ratio of 5. The method was suitable for the pharmacokinetic study of MTX in monkey.

Sensitive high-performance liquid chromatographic determination of EM523, a new erythromycin derivative, in human plasma and urine with ultraviolet detection using column switching

A sensitive high-performance liquid chromatographic (HPLC) method using column switching is described for the determination of EM523 (I), a new erythromycin derivative, in human plasma and urine. The analyte was extracted from alkalinized plasma or urine with a mixture of n-hexane and acetone. After the evaporation of the organic layer, the reconstituted residue was injected into the HPLC system and separated on the first column. After column switching, the heart-cut fraction contamination the analyte was further separated on the second column and monitored by ultraviolet absorbance at 210 nm. The detection limits were 1 ng/ml in plasma and 10 ng/ml in urine. The method was applied to the clinical trials of I.