Optimized determination of thiochrome derivatives of thiamine and thiamine phosphates in whole blood by reversed-phase liquid chromatography with precolumn derivatization

A liquid chromatographic method involving precolumn derivatization for determining thiamine and its phosphate esters in human blood has been optimized. Blood sample stored at - 20 degrees C were haemolysed and deproteinized by perchloric acid. The supernatants of the samples were oxidized by addition of potassium ferricyanide-sodium hydroxide, and phosphoric acid was added to obtain a neutral pH in order to extend the column life. The samples were stable after derivatization for at least 24 h, if protected from light and kept at room temperature. Gradient separation with 140 mmol phosphate buffer (pH 7.0), and methanol, tert-butylammonium hydroxide and dimethylformamide as modifiers, on a 3-microns Chromsphere octadecylsilica column gave an analysis time of 15 min. The method was found to be very suitable for the determination of thiamine components in whole blood. The minimal detectable amount is 0.5 nmol/l and the method is linear to at least 1000 nmol/l. The recovery (98 +/- 3%) and precision are very good.     

The pregnane derivatives of the roots from Asclepias lilacina Weimarck. II. Structure determination

Partial formulae have been derived for the nine lilacinosides which were isolated, partly in pure form, and for two related substances which were only obtained as a mixture. These substances are derived basically from two pregnane derivatives (sarcostin and deacetyl-metaplexigenin) present in partially benzoylated form. Of these were isolated genin B = 12,20-di-O-benzoyl-sarcostin ( 1 ), genin C = 12-O-benzoyl-20-O-acetylsarcostin ( 2 ), and genin D, which could be identified with 12-mono-O-benzoyl-desacetyl-metaplexigenin ( 4 ). These three genins are bound to different sugars, the latter being present as mono-, di-, tri- and probably tetra-saccharides. The structures of these were partially derived: after mild hydrolysis three new disaccharides were obtained, which are named asclepobiose (U1), lilacinobiose (U2) and digalilobiose (U5). U1 and U2 were isolated in crystalline form. On hydrolysis U1 yielded 3-O-methyl-6-deoxy-D-allose (U3), U2 yielded 3-O-methyl-6-deoxyglucose (thevetose), and digitalose was obtained from U5.     

Pharmacokinetics of protein-unbound linezolid in the blood and the mechanism of hepatobiliary excretion in the rat

Linezolid (Zyvox), an oxazolidinones antibiotic, was developed for the treatment of infectious diseases caused by gram-positive pathogens. To investigate the mechanism of hepatobiliary excretion of linezolid, a parallel study design used two groups; in the control group, rats received linezolid alone (3 or 10 mg/kg, i.v.). In the drug-treated groups, 10 min prior to linezolid administration, cyclosporin A (CsA; 10 mg/kg, i.v.), a P-glycoprotein (P-gp) inhibitor, was given in the rats. The microdialysis probes were implanted into the jugular vein toward right atrium and the bile duct of Sprague-Dawley rats for multiple biological fluid sampling. Separation was performed using a reversed phase C₁₈ (4.6 mm × 150 mm i.d., 5 μm) with mobile phase of acetonitrile-methanol-1% 1-octanesulfonic acid in water of 30:10:60 (v/v/v) at flow rate of 1 ml/min. The UV detection for linezolid was set at a wavelength of 260 nm. Following linezolid (10 mg/kg, i.v.) administration, the concentration of linezolid in the brain was less than the limit of quantification and the area-under the concentration curve versus time curve (AUC) of blood and bile were 1780 ± 50 and 2850 ± 276 (min μg/ml), respectively. The bile-to-blood distribution ratio was 1.6 ± 0.2 (n = 6), which was defined as AUC_bile/AUC_blood. The results demonstrated that the transportation of linezolid into bile might be mediated by active transport. However, after treatment with CsA, the linezolid AUC in bile was 3060 ± 411 (min μg/ml) which did not indicate a significant difference with linezolid alone. These results suggest that the hepatobiliary excretion of linezolid might not be regulated by P-gp transportation.     

Determination of thiamine and thiamine phosphates as thiochrome derivatives by reversed-phase chromatography on polystyrene packing materials

Summary The chromatography of thiochrome and related phosphate esters was studied on two different polystyrene packing materials.The adsorbents were compared with respect to capacity factors, plate heights and resolution factors measured with different mixtures of phosphate buffer (pH 8.5) and methanol.In contrast with derivatized silica, these phases were found to be stable using an alkaline mobile phase and sample. Fast separations were possible with good peak symmetry at all organic modifier concentrations.