Ken-ichi Hara

Determination of montelukast sodium in human plasma by column-switching high-performance liquid chromatography with fluorescence detection

MK-0476 (montelukast sodium) is a potent and selective cysteinyl leukotriene receptor antagonist that is being investigated in the treatment of asthma. A simple and sensitive method for the determination of MK-0476 in human plasma was developed using column-switching high-performance liquid chromatography (HPLC) with fluorescence detection. A plasma sample was injected directly onto the HPLC system consisting of a pre-column (Capcell pak MF) and an analytical column (Capcell pak C18) which were connected with a six-port switching valve. The column eluate was monitored with a fluorescence detector (excitation at 350 nm; emission at 400 nm). The calibration curve was linear in a concentration range of 1-500 ng ml(-1) for MK-0476 in human plasma. The intra-day coefficients of variation of all concentrations within the range was less than 9.2%, and the intra-day accuracy values were between 97.2 and 114.6%. This method was used to measure the plasma concentration of MK-0476 following oral administration of the drug in humans.

Method for the simultaneous determination of losartan and its major metabolite, EXP-3174, in human plasma by liquid chromatography–electrospray ionization tandem mass spectrometry

A liquid chromatography-electrospray ionization tandem mass spectrometric method was developed for the simultaneous determination of losartan and its major active metabolite, EXP-3174, in human plasma. The two analytes and the internal standard (DuP-167) were extracted from plasma under acidic conditions by using solid-phase extraction cartridges containing a sorbent of copolymer, poly(divinylbenzene-co-N-vinylpyrrolidone). The analytes were separated by LC equipped with a reversed-phase C18 column, and introduced into the mass spectrometer via the electrospray ion source with pneumatically-assisted nebulization. For LC-MS-MS samples, an isocratic mobile phase consisting of [0.1% triethylamine-0.1% acetic acid (pH 7.1)]-acetonitorile (65:35, v/v) was used, and the assay was monitored for the negative fragment ions of the analytes. The method demonstrated linearity from 1 to 1000 ng/ml for both losartan and EXP-3174. The limit of quantification for both compounds in plasma was 1 ng/ml. This assay method may be useful for the measurement of levels of the two compounds in clinical studies of losartan.

Hepatobiliary transport of a nonpeptidic endothelin antagonist, (+)-(5S,6R,7R)-2-butyl-7-[2((2S)-2-carboxypropyl)-4-methoxyphenyl]-5-(3,4-methylenedioxyphenyl) cyclopentenol[1,2-b]pyridine-6-carboxylic acid: uptake by isolated rat hepatocytes and canalicular membrane vesicles.

AbstractPurpose. Hepatobiliary excretions of drugs from the blood to the bile include two essential transmembrane processes: uptake into hepatocytes and secretion from hepatocytes. The purpose of this study was to clarify the transport mechanisms underlying these processes for a new non-peptide endothelin antagonist, (+)-(5S,6R,7R)-2-butyl-7-[2((2S)-2-carboxypropyl)-4-methoxyphenyl]-5-(3,4-methylenedioxy- phenyl)cyclopentenol[1,2-b]pyridine-6-carboxylic acid (J-104132). Methods. Biliary excretion of J-104132 was assessed in rats after intravenous injection. To evaluate the hepatic uptake process, J-104132 was incubated with freshly isolated rat hepatocytes and the uptake of J-104132 was calculated. To evaluate the biliary secretion process, the uptake of J-104132 into rat canalicular membrane vesicles that were isolated from normal Sprague-Dawley rats or Eisai hyperbilirubinemic rats was measured. Results. After intravenous injection, J-104132 was recovered from the bile quantitatively (99.7 ± 1.3%) as its intact form. J-104132 was taken up by isolated rat hepatocytes in a time- and temperature-dependent manner. The uptake was saturable with Km and Vmax of 5.7 μM and 564 pmol/min/106 cells, respectively. The uptake was Na+ independent and was reduced in the presence of ATP depleters (rotenone and carbonyl cyanide-p-(trifluoromethoxy)-phenylhydra- zone), organic anions (dibromosulfophthalein, indocyanine green, BQ-123, and pravastatin), and bile acids (taurecholate and cholate). In Sprague-Dawley rats, J-104132 was taken up by canalicular membrane vesicle ATP-dependently with Km and Vmax values of 6.1 μM and 552 pmol/min/mg protein, respectively. However, ATP-dependent uptake disappeared in Eisai hyperbilirubinemic rats. Conclusions. These data suggest that energy-dependent and carrier-mediated transport systems play important roles in hepatobiliary excretion of J-104132 (both uptake and secretion processes), which is the main excretion route in rats. As for the secretion process of J-104132, an involvement of mrp2 was demonstrated.

Simultaneous Determination of Omeprazole and its Metabolites (5′‐Hydroxyomeprazole and Omeprazole Sulfone) in Human Plasma by Liquid Chromatography‐Tandem Mass Spectrometry

Abstract A method for the simultaneous determination of omeprazole (OME) and its two metabolites (5′‐hydroxyomeprazole (H‐OME) and omeprazole sulfone (OME‐S)) in human plasma is described. OME and its metabolites were extracted from plasma with phosphate buffer (pH 7.4) by using solid phase extraction system, Oasis HLB μ Elution plates, and the eluate was directly injected into LC‐MS/MS system. The analytes were chromatographed with a reversed‐phase column, XTerra MS C18 column (150×4.6 mm) and an HPLC mobile phase which consisted of a mixture of acetonitrile/water (45/55, v/v) containing 10 mM ammonium hydroxide (pH 8.0). A Sciex API 4000 tandem mass spectrometer equipped with a heated nebulizer atmospheric pressure chemical ionization interface was used as a detector and was operated in the positive ion mode. Multiple reaction monitorings using the precursor product ion combinations of m/z 346.2→198.2, 362.0→214.0, 362.0→150.0, and 349.2→201.0 were used to detect OME, H‐OME, OME‐S, and internal standard (OME‐d3), respectively. The method was validated in the concentration range of 1–1000 ng/mL plasma with adequate assay precision, accuracy, and reproducibility. This sensitive and selective method with a rapid and simple sample preparation procedure was applied to a clinical pharmacokinetic study of OME.