Youn-Bok Chung

Comparative study of naphthalene-2,3-dicarboxaldehyde and o-phthalaldehyde fluorogenic reagents for chromatographic detection of sphingoid bases.

Naphthalene-2,3-dialdehyde (NDA) was developed as a precolumn labeling reagent for the fluorescent determination in a HPLC system of bioactive sphingoid bases, including sphingosine, sphinganine, and C20-sphinganine. Cellular sphingoid bases generally exist in the range of 10 to approximately 100 pmol/10(6) cells in a wide variety of cell types and tissues. This study aimed to obtain stable fluorescent derivatives of sphingoid bases and to increase their detectability at low concentrations. Sphingoid bases were reacted with NDA in the presence of cyanide ion to readily make an intensely fluorescent structure, 1-cyano-2-alkyl-benz[f]isoindole (CBI) and were then eluted separately on a reversed-phase C18 column with a simple mobile phase of 90% acetonitrile. For evaluating the NDA method, we compared the fluorescent intensity, elution profile, stability, and detectability of NDA derivatives with those of corresponding o-phthalaldehyde (OPA) derivatives. By monitoring the fluorescent intensity at the excitation wavelength of 252 nm and emission wavelength of 483 nm, NDA derivatives were sensitively determined at concentrations below 1.0 pmol of sphingoid bases in 1 x 10(5) U937 cells and were more stable than OPA derivatives. Linear calibration plots were obtained in the range studied (0.5 to approximately 500 nM). The limit of detection for NDA derivatives of sphingoid bases was approximately 0.1 pmol (S/N=3). The method successfully measured the accumulation of sphingosine in U937 cells following N,N-dimethylsphingosine treatment, and of sphinganine following fumonisin B1 treatment.

Pharmacokinetics of propentofylline and the quantitation of its metabolite hydroxypropentofylline in human volunteers

Propentofylline (PPF, 3-methyl-1-(5-oxohexyl)-7-propylxanthine) has been reported to be effective for the treatment of both vascular dementia and dementia of the Alzheimer type. The pharmacological effects of PPF may be exerted via the stimulation of nerve growth factor, increased cerebral blood flow, and inhibition of adenosine uptake. The objectives of this experiment are to determine the kinetic behavior of PPF, to identify, and to quantify its metabolite in human. Blood samples were obtained from human volunteers following oral administration of 200 mg of PPF tablets. For the identification and quantification of the metabolite, 3-methyl-1-(5-hydroxyhexyl)-7-propylxanthine (PPFOH), PPFOH was synthesized and identified by gas chromatography/mass spectroscopy (GC/MS) and1H-nuclear magnetic resonance spectroscopy. The molecular weight of synthesized metabolite is 308 dalton. The PPF and PPFOH in plasma were extracted with diethyl ether and identified by electron impact GC/MS. The plasma concentrations of PPF and PPFOH were determined by gas chromatography/nitrogen phosphorus detector in plasma and their pharmacokinetic parameters were determined. The mean half-life of PPF was 0.74 hr. The areas under the curve (AUCs) of PPF and PPFOH were 508 and 460 ng.hr/ml, respectively. Cmax of PPF was about 828.4 ng/ml and the peak concentration was achieved at about 2.2 hr (Tmax). These results indicate that PPF is rapidly disappeared from blood due to extensive metabolism into PPFOH.

An HPLC Determination of Trimetazidine in Human Plasma Using Liquid‐Liquid Extraction for Sample Clean‐Up

Abstract Trimetazidine dihydrochloride has been used as an antianginal drug that possess protective properties against ischemia‐induced damage to heart. A simple and sensitive analytical method of trimetazidine dihydrochloride in human plasma by using high performance liquid chromatography (HPLC) was developed. The method employs a liquid‐liquid extraction for isolation and sample concentration, followed by reversed‐phase liquid chromatography (RPLC) analysis using ultraviolet (UV) detection at 207 nm. Analytes were extracted from plasma samples that previously were mixed with 300 µL saturated K2CO3 solution into an ethyl acetate phase. HPLC separation was accomplished at 40°C on a reversed‐phase column using a mobile phase, 15% acetonitrile in 50 mM potassium dihydrogen phosphate and phosphoric acid (pH=4.0), at a flow‐rate of 1.0 mL/min. The linear range of the method was between 10‐ and 150 ng/mL of tirmetazidine dihydrochloride in human plasma and the quantification limit was 10 ng/mL. The intra‐ and inter‐day relative standard deviation (RSD) were less than 7.6% and the accuracy was in the range of 98–107%. Extraction recoveries ranged from 71.5 to 84.6% and the C.V. values showed between 1.7 and 10.4% in the same concentration range. This method has been sought for carrying out pharmacokinetic studies and for assessing bioavailabiltiy. Such a method would be ideally suitable for pharmacokinetic studies in human volunteers after oral administration of different types of dosages of the drug.

Determination of Eperisone in Human Plasma by Liquid Chromatography-ESI-Tandem Mass Spectrometry

A sensitive and selective method for the determination of 4′-ethyl-3-methyl-3-piperidinopro-piophenone hydrochloride (eperisone hydrochloride) in human plasma was developed and validated. The procedure employed an internal standard and a solvent extraction step followed by chromatography on a Xterra C18 minibore column. Detection was by electrospray ionization tandem mass spectrometry with multiple reaction monitoring. The mass transitions of eperisone and tolperisone (IS) were m/z 260 → 98 and m/z 246 → 98, respectively. The method has a limit of detection of 0.1 pg/mL for eperisone based on the three times signal-to-noise value with a linear range from 0.01 to 10.0 ng/mL for the analyte. Extraction recovery was on average 98.6±7.2% (SD) for eperisone. The Intra- and inter-day assay accuracy ranged from 93 to 114% and precision (RSD) was better than 8.5%. The method was successfully employed to analyze plasma samples and evaluate pharmacokinetics of eperisone in healthy male volunteers.

Effect of excipients on the stability and transport of recombinant human epidermal growth factor (rhEGF) across Caco-2 cell monolayers.

The effect of sixteen excipients on the transport of recombinant human epidermal growth factor (rhEGF) across Caco-2 cell monolayers was examined at 37°C. The apparent apical to basolateral (A-B) permeability (P{onapp}) of 30 μM rhEGF was 8.15x10-7 cm/sec, indicative of a poor level of absorption in the Gl tract. The Papp was 1.7- and 6.3-fold greater than the Papp in the basolateral to apical (B-A) direction and the A-B permeability of mannitol, respectively, and decreased dramatically to a negligible level at 4°C, consistent with a receptor mediated transcytosis of rhEGF. The stability of rhEGF was very poor, undergoing more than 85% degradation in 2 h in the transport medium at 37°C. A significant increase in the Papp could be achieved by the addition of certain excipients, as exemplified by 23, 21, 20 and 16-fold increases, in the presence of sodium taurochenodeoxycholate (NaTCDC), sodium taurodeox-ycholate (NaTDC), sodium glycodeoxycholate (NaGDC) and sodium laurylsulfate (SLS) (all at a concentration of 1% w/v), respectively. A significant increase in stability could also be achieved by the addition of some of the excipients, as represented by 1% SLS, which nearly completely stabilized the rhEGF. Unfortunately, however, an increase in the Papp of rhEGF could not be achieved without a simultaneous and extensive decrease in the integrity of the cell membranes. Thus, more efficient excipients, that specifically enhance the permeation of rhEGF and do not alter the membrane integrity, should be pursued in order to safely enhance the permeation of rhEGF.

Endogenous Sphingolipid Metabolites Related to the Growth in Sphingomonas chungbukensis

Sphingolipids are present in animals, plants, fungi, yeasts and some bacteria. In mammalian cells sphingolipids act as lipid mediators for cell growth, differentiation, apoptosis and angio-genesis. In contrast, in bacteria the biological significance of sphingolipids has not been fully elucidated and sphingolipid metabolism has not been investigated. The aim of this study was to compare the pattern of sphingolipid metabolites in HIT-T15 ß cells originating from hamster pancreas to that in the bacterial strainSphingomonas chungbukensis DJ77, under various culture conditions. It was found that the concentration of cellular sphinganine (Sa) in S.chungbukensis was higher than that of sphingosine (So), while the level of cellular So in HIT-T15 cells was higher than that of Sa. Aeration and shaking during culture increased bacterial growth in S.chungbukensis, and the contents of So and Sa were also elevated. These results indicate that a denovo sphingolipid pathway appeared to be active in bacteria and that bacterial growth may be closely related to Sa levels.

Determination of Rebamipide in Human Plasma by HPLC

Abstract A simple determination method of rebamipide in human plasma by using high performance liquid chromatography (HPLC) was developed. The method involves a single liquid–liquid extraction and reversed‐phase chromatography with fluorometric detection (excitation, 320 nm; emission, 380 nm). Analytes were extracted from plasma samples that contain an internal standard (ofloxacin) into ethylacetate with a high yield after adjustment to pH 2–3. Separation was accomplished at 60°C on a reversed‐phase column using a mobile phase of acetonitrile–water–acetic acid (30:70:5, v/v, pH 2.4), at a flow‐rate of 1.0 mL/min. The linear range of the assay was 2–500 ng/mL of the drug in plasma and the limit of quantitation was 2.0 ng/mL. The intra‐ and inter‐day relative standard deviation (RSD) were less than 10% and the accuracy of the assay was in the range of 97–104%. Analysis of the drug in human plasma indicates that the procedure can be carried out conveniently and quickly and, therefore, is suitable for obtaining pharmacokinetic profiles in human subjects after oral administration of different types of the drug.

Determination of recombinant human epidermal growth factor (rhEGF) in a pharmaceutical preparation by capillary electrophoresis

A simple assay method of recombinant human epidermal growth factor (rhEGF) in a pharmaceutical preparation was studied and validated by capillary electrophoresis (CE) using micellar electrokinetic chromatography (MEKC) techniques. Factors affecting the migration behavior and separation performances of the peptide; type of buffer, pH, buffer concentration, and concentration of sodium dodecyl sulfates (SDS) were investigated to optimize the analytical performance. CE was performed using running buffer, 50.0 mM borate (pH 8.5) containing 12.5 mM SDS at 20 kV of the applied voltage. Calibration curves for the rhEGF showed good linearity (r>0.999) over the wide dynamic range from 1.25 to 100 μg/ml. Sample analysis was performed by using standard addition method to eliminate the matrix effects of dosage vehicle. This method is assumed to be useful for quality control (QC) of various forms of pharmaceutical products of the peptide.

Solubilization of IH-901, a Novel Intestinal Metabolite of Ginseng Saponin, in Aqueous Solution

The purpose of the present study was to formulate the aqueous solution of , an intestinal bacterial metabolic derivative from Ginseng protopanaxadiol saponin. For this purpose, the effects of various solubilization agents such as cosolvents [ethanol, propylene glycol (PG), polyethylene glycol 300 (PEG 300), polyethylene glycol 400 (PEG 400), glycerin], surfactants and a complexation agent , on the solubility of IH-90l in aqueous solution were evaluated. The solubility of IH-901 in water was under . Cosolvents such as ethanol, PG, PEG 300, PEG 400 and glycerin did not enhance the solubility of IH-901 at the 0 - 40% concentration range. The solubility of IH-901 was significantly elevated by the addition of cosolvents over the 80% concentration range. On the other hand, tween 80, and HPBCD showed enhanced effects on the solubility of IH-901. The enhanced effects of Poloxamer 407 or Poloxamer 188 on the IH-901 solubility were less pronounced compared with . As a results, aqueous solution was selected as an optimum solvent system. The aqueous solutions containing 10% and 7% were formulated as dosing solutions containing 5.0 mg/ml of IH-901 for its intravenous and oral administration, respectively. The formular showed physical stability after stored for 7 days at .

Determination of Recombinant human epidermal growth factor (rhEGF) in a pharmaceutical formulation by high performance liquid chromatography with electrochemical detection

A novel HPLC method with electrochemical detection has been developed for the determination of recombinant human epidermal growth factor (rhEGF) in pharmaceutical products. rhEGF was separated from other components in formulation on a reversed-phase C18 column with 24% acetonitrile in 0.1 M phosphate buffer (pH 4.75). The optimum electrochemical oxidation of EGF was obtained at 0.85 V vs. Ag/AgCl in a glassy carbon working electrode due to electroactive tyrosine, tryptophan, methionine, and arginine residues. The quantitation range was from 1.0 to 200 ng of rhEGF with the linear correlation coefficient greater than 0.999. The method was successfully applied for the quantitation of rhEGF in a pharmaceutical preparation.