Ken-ichi Shimokawa

Simultaneous determination of six adenyl purines in human plasma by high-performance liquid chromatography with fluorescence derivatization

A sensitive method was developed for the simultaneous determination of six adenyl purines in human plasma by high-performance liquid chromatography. The adenyl purines (adenine, adenosine, AMP, ADP, ATP and cyclic AMP) were derivatized using 2-chloroacetaldehyde for fluorescence detection, and the reaction and separation conditions were reinvestigated to improve sensitivity for small volume sample analysis. Each derivatized purine was separated on a Capcell Pack SG120A column with mobile phase consisting of 0.05 M citric acid-0.1 M dipotassium hydrogen phosphate (pH 4.0)-methanol (97+3). The detection limits were 100-1000 fmol/ml by fluorescence detection, some 500 times better than previous reports. The proposed method was applied to determine adenyl purines in human plasma. The purine levels were as follows: ATP (9.2-22.2 pmol/ml), ADP (5.5-22.2 pmol/ml), AMP (0.8-3.2 pmol/ml). Other purines, adenine, adenosine, cAMP were lower than 0.1 pmol/ml.

Physicochemical properties and controlled drug release of microcapsules prepared by simple coacervation.

Ternary phase diagrams of gelatin-water-methanol, gelatin-water-ethanol, and gelatin-water-propanol systems were prepared to evaluate optimal coacervation. The results of their evaluation suggested that the optimal coacervation region expands with the hydrophobicity of the added poor solvent (methanol, ethanol, 1-propanol, and 2-propanol). Microcapsules prepared based on the optimal coacervation region differed in controlled drug release among poor solvents used even when the concentration of gelatin, a membrane component, is the same. Compared with microcapsules prepared using the gelatin-water-ethanol system, those prepared using the gelatin-water-propanol system showed a 34% decrease in the drug release rate 24h after the initiation of the drug release test. These results suggested that microcapsules prepared using gelatin-water-various lower alcohol systems can readily control drug release and can be a useful drug delivery system (DDS).

Determination of β-blockers by high performance liquid chromatography coupled with solid phase microextraction from urine and plasma samples

The solid phase microextraction (SPME) method was applied to drug monitoring of β-blockers in human urine and plasma. Six β-blockers (alprenolol, atenolol, oxprenolol, pindolol, β-propranolol, and timolol) were extracted from urine and plasma samples by SPME fiber coated with 85-μm polyacrylate for 2 h at 30°C. The β-blockers extracted were separated by Capcell PackTM SG120 (4.6 × 150 mm, 5 μm) with 0.05 M phospholic acid-methanol (55 + 45, v/v) and detected by photometric detection (280 nm) and fluorometric detection (excitation wavelength 220 nm, emission wavelength at 360 nm). The recoveries of β-blockers (500 μg/ml) from urine were 84.3 to 88.4%, and from plasma they were 86.4 to 90.2%. The detection limits were 2 to 25 μg/ml by the photometric detection, 0.4 μg/ml of β-propranolol by fluorescence detection. The relative standard deviations were 10.1 to 15.3% for 500 μg/ml β-blockers in urine, 9.9 to 13.2% for 5 μg/ml of β-blockers in plasma, respectively.

Adsorption of various antimicrobial agents to endotoxin removal polymyxin-B immobilized fiber (Toraymyxin®).

The presence/absence of adsorption of 9 representative types of antimicrobial agent used in combination with a polymyxin-B immobilized fiber (PMX-F) were determined and the degree of adsorption to PMX-F was quantitatively evaluated. Various antimicrobial agents were dissolved at appropriate concentrations, and PMX-F was added to each solution and incubated at 37°C. Antimicrobial solutions without PMX-F were also similarly incubated as controls. After 2 and 4h, the concentration of each antimicrobial agent was determined using HPLC. To produce an environment closer to the in vivo state, albumin or serum was added, and similar evaluation was performed. In the presence of albumin, the rate of adsorption to PMX-F was relatively high for Cefmetazon(®), Pentcillin(®), Ciproxan(®) and Zyvox(®). In the presence of serum, the adsorption rate was 4.02±2.83% for Pentcillin(®), 5.59±1.00% for Ciproxan(®), and 22.12±3.23% for Zyvox(®). The results of this study suggest that adequate caution is necessary on the clinical use of Zyvox(®), which was adsorbed in the presence of serum as an environment close to the in vivo environment, but the use of other antimicrobial agents in combination with PMX-F may have only slight influences on adsorption to PMX-F.

BLOOD COAGULATION FACTOR X (FX) IN HUMAN SEMINAL PLASMA

The active form of human blood coagulation factor X (FXa, EC 3.4.21.6) showing N - f -Benzoyl-L-isoleucyl-L-glutamyl-L-glycyl-L-arginine- p -nitroanilide (S-2222) hydrolyzing activity was first detected in human semen (seminal plasma) by affinity chromatography using anti-human coagulation factor X, and this enzyme activity was inhibited by anti-human FX. This enzyme has been associated with the human coagulation factor X (FX) in human semen (seminal plasma) by Western blot analysis, and the molecular mass of mature FX was also estimated to be 59 KDa by SDS-PAGE and Western blot analysis.

Preliminary Monitoring of Bisphenol A and Nonylphenol in Human Semen by Sensitive High Performance Liquid Chromatography and Capillary Electrophoresis After Proteinase K Digestion

Abstract To investigate human exposure by bisphenol A (BPA) and nonylphenol (NP), BPA and NP in human semen were preliminary monitored by sensitive high performance liquid chromatography (HPLC) by fluorescence pre-labeling with 2-(4-carboxyphenyl)-5,6-dimethylbenzimidazole (CDB) and capillary electrophoresis (CE) based on miceller electrokinetic chromatography after proteinase K digestion. The semen samples were collected from 57 patients who attended in vitro embryo fertilization program (IVF-ET). They were classified into nine groups according to their properties by semen character (e.g., normozoospermia) and statistical analysis (e.g., sperm count and sperm mobility). The proteinase K digestion was effective in obtaining high recovery value on the extraction process. In this time, BPA and NP were not detected in nine groups (57 samples, limits of detections = 1 and 7 pg/mL). It was suspected that BPA and NP levels in these semen were less than 1 pg/mL and 7 pg/mL, respectively. There were no significant exposures to BPA and NP on semen samples in this time experiments. Further large scale monitoring was needed for diagnosis effect of endocrine disruptor to human sperm production.

Adsorption of various antimicrobial agents to endotoxin removal polymyxin-B immobilized fiber (Toraymyxin®). Part 2: Adsorption of two drugs to Toraymyxin PMX-20R cartridges.

In our previous study, the degree of adsorption of 9 representative antimicrobial agents to Toraymyxin(®) PMX-F sheets was quantitatively evaluated. As a result, the adsorption rate was 22.1% for Linezolid in the presence of serum. Therefore, we investigated whether two types of antimicrobial agents (Ciprofroxacin and Linezolid) can be better adsorbed on PMX-F sheets. When the number of PMX-F sheets was increased in a step wise manner, specifically 2, 4, 6, 8 and 12, the adsorption rate increased linearly. In addition, the adsorption to polymyxin-B immobilized fiber (Toraymyxin(®) PMX-20R) cartridges, widely used to remove endotoxins from circulating blood in the treatment of sepsis, was quantitatively evaluated. As a result, in the presence of serum, Linezolid showed adsorption to PMX-20R, and the adsorption rate after 2h was 54.5%, and that after 4h was 65.8%. The results of this study suggest the necessity of monitoring blood antimicrobial concentration during treatment for sepsis with Linezolid, which showed adsorption to PMX-20R in an environment close to a clinical environment.

Generic Selection Criteria for Safety and Patient Benefit [IV] − Physicochemical and pharmaceutical properties of brand-name and generic ketoprofen tapes

The physicochemical and pharmaceutical properties (pH, peel force, water-vapor permeability, and stretchability) of brand-name and generic ketoprofen products were evaluated and compared. The pHs of Mohrus as a brand-name product and Teikoku as a generic product were low (about 4). Among the other generic products, Patell and Nichi-Iko had a pH of about 4.3 while Frestol, Raynanon, BMD, and Touchron showed a pH of 4.6-5.2, which was in the pH range of normal healthy skin (4.5-6.5). The adhesive force was high (≥ 1.38) for Mohrus as a brand-name product as well as for Teikoku and Patell as generic products, but it was low (≤ 0.57) for the other 5 generic products. The water-vapor permeabilities of Mohrus as a brand-name product and Teikoku and Patell as generic products were low, being less than 1/6 of those for the other 5 generic products. Among the 5 generic products, BMD showed the highest water-vapor permeability (1,330 g/m²), and the other products also showed a value ≥ 1,100 g/m². The elongatedness of Mohrus was the lowest (15.5 cm), and that of Raynanon was the highest (24.5 cm); the difference was 9 cm. In this study, the physiochemical and pharmaceutical properties of ketoprofen tapes were clarified, which will allow pharmacists to provide products according to the needs of each patient when a brand-name product is changed to a generic one.

Physicochemical properties of magnesium aluminum silicate (smectone) gels prepared using electrolytic-reduction ion water (2): Effects of various salts on the phase diagram.

We produced gels using electrolytic-reduction ion water and magnesium aluminum silicates (smectone), and evaluated in detail gel properties in the presence of various types of salt (NaCl, KCl, CaCl(2), MgCl(2), and AlCl(3)). Each salt was added to deionized-distilled water or electrolytic-reduction ion water, and phase diagrams for the smectone concentration (2.0-4.0%) were produced. The areas of the three phases of smectone (gel, sol, and separation) at each salt concentration were expressed as percentages of the total area. As a result, uni- and polyvalent cations (excluding Ca(2+) ions) affected the stability of gels produced using electrolytic-reduction ion water, and, particularly, univalent cations (Na(+), K(+)) markedly improved gel stability. Using electrolytic-reduction ion water as a dispersal medium, drug delivery systems (DDS) that can maintain the gelling state can be prepared. Thus, gel preparations with maintained functions or controlled-release transdermal drugs can be obtained.

Rheological properties of reversible thermo-setting in situ gelling solutions with the methylcellulose–polyethylene glycol–citric acid ternary system (2): Effects of various water-soluble polymers and salts on the gelling temperature

The influences of various salts and water-soluble polymers on the phase transition temperature of thermo-setting gels prepared by combining methylcellulose (MC)-sodium citrate (SC)-polyethylene glycol (PEG) at appropriate ratios (the MC-SC-PEG system) were investigated. Concerning cations, comparison of the phase transition temperature between SC and tripotassium citrate (PC) showed a rapid increase in the viscosity of SC between 20 degrees C and 25 degrees C and an increase in the viscosity of PC between 30 degrees C and 35 degrees C. Concerning the valency of anions, comparisons among SC, disodium tartrate dihydrate (ST), disodium maleate hemihydrates (SM), and sodium sulfate (SS) showed a rapid increase in the viscosity of trivalent SC between 20 degrees C and 25 degrees C and changes in the viscosity of the three bivalent sodium salts (ST, SM, and SS) at > or =30 degrees C. Thus the phase transition temperature decreased with an increase in the valency of anions. Subsequently, the influences of various water-soluble polymers on the gelling temperature were compared. Using polyvinylpyrrolidone (PVP) instead of PEG, the gelling temperature decreased with an increase in the PVP concentration even without the addition of SC. Unlike PVP, the addition of xanthan gum as a viscosity-increasing polysaccharide did not reduce the gelling temperature irrespective of its concentration. Temperature-associated changes in viscosity were observed at a fixed SC concentration with changes in the concentration of PVP or PEG. The gel phase transition temperature increased from 46 degrees C to 50 degrees C in gels not containing PVP or PEG. The viscosity did not differ between the addition of PVP or PEG at a low concentration and its absence. However, the viscosity clearly changed after the addition of each agent at a high concentration.