Hee-Yong Lee

Narrowbore high-performance liquid chromatography for the simultaneous determination of sildenafil and its metabolite UK-103,320 in human plasma using column switching

A fully automated narrowbore high-performance liquid chromatography method with column switching was developed for the simultaneous determination of sildenafil and its active metabolite UK-103,320 in human plasma samples without pre-purification. Diluted plasma sample (100 microl) was directly introduced onto a Capcell Pak MF Ph-1 column (20x4 mm I.D.) where primary separation occurred to remove proteins and concentrate target substances using 15% acetonitrile in 20 mM phosphate solution (pH 7). The drug molecules eluted from the MF Ph-1 column were focused in an intermediate column (35x2 mm I.D.) by a valve switching step. The substances enriched in the intermediate column were eluted and separated on a phenyl-hexyl column (100x2 mm I.D.) using 36% acetonitrile in 10 mM phosphate solution (pH 4.5) when the valve status was switched back. The method showed excellent sensitivity (detection limit of 10 ng/ml), good precision (RSD < or = 2.3%) and accuracy (bias: +/-2.0%) and speed (total analysis time 17 min). The response was linear (r2 > or = 0.999) over the concentration range 10-1000 ng/ml.

Column-switching high-performance liquid chromatographic determination of clarithromycin in human plasma with electrochemical detection.

A column-switching HPLC method was described for the direct analysis of clarithromycin in human plasma using electrochemical detector without sample pre-purification step. Plasma samples were diluted with washing solvent, i.e. acetonirile-methanol-0.05 M potassium phosphate buffer (pH 7.0) (5:2:93, v/v) and then, injected to the precolumn. After plasma proteins had flowed out from the precolumn, clarithromycin and internal standard (roxithromycin) were eluted to a Luna 2 C(18) column and separated with acetonitrile-methanol-0.05 M potassium phosphate buffer (pH 7.0) (41:6:53, v/v). Electrochemical oxidation of clarithromycin occurred at 0.87 V vs. Ag/AgCl reference electrode with glassy carbon electrode. The calibration curve was linear in the concentration range 0.1-4 mug ml(-1) with correlation coefficient of 0.998. This method showed excellent precision (RSD 3.8% at 0.1 mug ml(-1)) and accuracy (+/-2%) with the total analysis time per sample of 30 min. The present method was successfully applied to the pharmacokinetic study of clarithromycin in volunteers receiving a single oral administration of clarithromycin.

Simultaneous determination of ampicillin and metampicillin in biological fluids using high-performance liquid chromatography with column switching

A new high-performance liquid chromatographic method with column switching has been developed for the simultaneous determination of metampicillin and its metabolite ampicillin in biological fluids. The plasma, urine and bile samples were injected onto a precolumn packed with LiChrosorb RP-8 (25-40 microns) after simple dilution with an internal standard solution in 0.05 M phosphate buffer (pH 7.0). The polar plasma components were washed out using 0.05 M phosphate buffer (pH 7.0). After valve switching, the concentrated drugs were eluted in the back-flush mode and separated by an Ultracarb 5 ODS-30 column with a gradient system of acetonitrile-0.02 M phosphate buffer (pH 7.0) as the mobile phase. The method showed excellent precision, accuracy and speed with a detection limit of 0.1 microgram/ml. The total analysis time per sample was less than 40 min and the coefficients of variation for intra- and inter-assay were less than 5.1%. This method has been successfully applied to plasma, urine and bile samples from rats after intravenous injection of metampicillin.

Roles of human liver cytochrome P450 3A4 and 1A2 enzymes in the oxidation of myristicin

The aim of this work was to identify the form(s) of human liver cytochrome P450 (CYP) involved in the hepatic transformation of myristicin to its major metabolite, 5-allyl-1-methoxy-2,3-dihydroxybenzene. When microsomes prepared from different human liver samples were compared, the activity of 5-allyl-1-methoxy-2,3-dihydroxybenzene formation was well correlated (r(2)=0.87) with nifedipine oxidation (a marker of CYP3A4). With a microsomal sample having high CYP3A4 activity, microsomal oxidation of myristicin to the major metabolite (5-allyl-1-methoxy-2,3-dihydroxybenzene) was markedly inhibited by gestodene and ketoconazole, selective inhibitors of CYP3A enzymes, but not by any of several other P450 inhibitors. Antibodies raised against CYPs 3A4 and 1A2 could also inhibit the oxidation of myristicin, but antibodies recognizing other CYPs had no effect. The oxidation of myristicin to 5-allyl-1-methoxy-2,3-dihydroxybenzene was catalyzed by purified bacterial recombinant CYPs 3A4 and 1A2. These results provide evidence that CYP3A4 (and possibly other CYP3A enzymes) and CYP1A2 play roles in the formation of the major metabolite, 5-allyl-1-methoxy-2,3-dihydroxybenzene.

Determination of salmon calcitonin in formulations by high-performance liquid chromatography with electrochemical detection

SummaryA novel HPLC method with electrochemical detection has been developed for the determination of salmon calcitonin (sCT). sCT was separated from other components in its formulations on a narrow-bore reversed-phase C18 column with 33% acetonitrile in 0.05m phosphate buffer (pH 6.5) at a flow rate of 0.2 mL min−1. Electrochemical oxidation of sCT at a glassy carbon electrode occurred at 0.87 V relative to the Ag/AgCl reference electrode. The limit of quantitation was 0.1 ng and the calibration curve was linear in the concentration range 0.5–20 ng; the correlation coefficient was 0.998. The method was successfully applied for the quantitation of sCT in commercial injection and nasal spray formulations.

High performance liquid chromatographic analysis of a new proton pump inhibitor KR60436 and its active metaboliteO-demethyl-KR60436 in rat plasma samples using column-switching

A fully automated high performance liquid chromatography with column-switching was developed for the simultaneous determination of KR60436, a new reversible proton pump inhibitor, and its active metabolite O-demethyl-KR60436 from rat plasma samples. Plasma sample (50 μl) was directly introduced onto a Capcell Pak MF Ph-1 column (10 × 4 mm I.D.) where primary separation was occurred to remove proteins and concentrate target substances using acetonitrile-potassium phosphate (pH 7, 0.1 M) (2:8, v/v). The drug molecules eluted from MF Ph-1 column were focused in an intermediate column (10 × 2 mm I.D.) by the valve switching step. The substances enriched in intermediate column were eluted and separated on a Vydac 218MR53 column (250 × 3.2 mm I.D.) using acetonitrile-potassium phosphate (pH 7, 0.02 M) (47:53, v/v) at a flow rate of 0.5 ml/min when the valve status was switched back to A position. The method showed excellent sensitivity (detection limit of 2 ng/ml) with small volume of samples (50 μl), good precision and accuracy, and speed (total analysis time 24 min) without any loss in chromatographic efficiency. The response was linear (r2 ≥ 0.999) over the concentration range of 5-500 ng/ml.

Direct Analysis of Clomipramine in Human Plasma by Microbore High Performance Liquid Chromatography with Column-Switching

SummaryAn automated microbore, liquid chromatographic method with column-switching was developed for the determination of clomipramine from human plasma samples. After direct injection of samples (60 μL), plasma proteins and clomipramine were separated in size-exclusion mode using 20% acetonitrile in 20 mM phosphate buffer (pH 7.0) on Capcell Pak MF Ph-1 precolumn (10×4 mm I.D.). By valve switching, a fraction containing clomipramine was directed to an intermediate column for subsequent main separation on a microbore C18 column (250×1.5 mm I.D.) using 50% acetonitrile in 20 mM phosphate buffer (pH 2.5) at 0.1 mL min−1. The method was advantageous for rapidity (total analysis time: 15 min), reproducibility (C.V.<4.8%), and increased sensitivity (1 ng mL−1). The linearity of response was good (r2≥0.999) over the concentration range 1–250 ng mL−1.

Simultaneous determination of ursodeoxycholic acid and its glycine-conjugate in serum as phenacyl esters using multidimensional liquid chromatography

SummaryA narrowbore high-performance liquid chromatographic (HPLC) method using column switching is described for the simultaneous determination of ursodeoxycholic acid (UDCA) and glyco-UDCA (GUDCA) from serum samples as their phenacyl esters. Serum samples were subjected to a preliminary clean-up using octadecylsilane reversed-phase extraction and derivatized with phenacylbromide. The purification, fractionation and concentration of UDCA and GUDCA from the esterified serum sample were performed on-line by appropriate switching of columns. Limit of detection (LOD) of UDCA and GUDCA were 5 ng and the absolute mean recoveries averaged 84.4±8.2% and 85.2±8.4%, respectively. This method was successfully applied to the pharmacokinetic study of UDCA in rats and human.

Determination of growth hormone-releasing hexapeptide by reversed-phase high-performance liquid chromatography with electrochemical detection.

A novel HPLC method with electrochemical detection is described for the determination of a growth-hormone-releasing hexapeptide (GHRP-6). HPLC conditions, such as the column, mobile phase, and oxidation potential, were optimized for sensitivity and selectivity of analysis. GHRP-6 was separated on a reversed-phase CN column with 37% acetonitrile in 100 mM phosphate buffer (pH 7.0) as the mobile phase. The optimum electrochemical oxidation signal was obtained at 0.85 V vs. Ag/AgCl in a glassy carbon working electrode due to two electroactive tryptophans and a histidine residue. Solid-phase extraction using octadecyl cartridges was optimized for sample cleanup of GHRP-6 from serum samples and the method was successfully applied over the concentration range of 5 to 100 ng/ml of analyte. reserved.

On-line trace enrichment for the determination of insulin in biological samples using reversed-phase high performance liquid chromatography with column switching

Column-switching technique with a reversed-phase high performance liquid chromatographic method has been developed for the routine analysis of radioiodinated insulin and its degradation products in biological fluids. The diluted biological samples were loaded onto a precolumn packed with LiChrosorb RP-8 (25–40μm) using 0.1% trifluoroacetic acid (TFA) in water as a washing solvent. After valve switching, the concentrated insulins were eluted in the back-flush mode and separated by a W-Porex C18 column with a gradient of 0.1% TFA in water and 0.1% TFA in acetonitrile as the mobile phase. The method showed good precision, accuracy and speed with the detection limit of 20pg/ml. Total analysis time per sample was about 40 min and the coefficients of variation were less than 8.2%.