A highly effective one-minute thin-layer chromatographic separation of unconjugated tri- and dihydroxy bile acids

A rapid, miniature thin-layer chromatographic procedure has been developed for the separation of unconjugated tri- and dihydroxy bile acids. The new solvent system consisted of isooctane:diisopropyl ether:methanol:acetic acid:formamide (2:1:0.13:0.07:0.02,$\text{v}\text{v}$). Complete separation of tri- and dihydroxy bile acid fractions was easily achieved within 1 min on ITLC type SG chromatography sheets. A sensitive fluorescent visualization technique was employed. The reflected fluorescence intensity of the bile acids fluorophore spots was measured with a Farrand Mark I spectrofluorometer equipped with a thin-layer scanning attachment. The excitation and emission wavelengths were 375 and 436 nm, respectively. The above procedure has been adapted for the separation of unconjugated bile acids from serum samples. The bile acids were adsorbed onto Amberlite XAD-2, eluted with ethanol, and concentrated. Further purification was performed by a simple 3-min thin-layer chromatographic technique. This new procedure for the separation of tri- and dihydroxy bile acids is rapid, sensitive, and less complex than conventional procedures presently employed in clinical laboratories.     

Optimization of the visualization of thin-layer chromatograms by charring

Summary A factorial experiment was used to optimize the effects of temperature and time on the charring of thin layer chromatograms prepared for quantification. The method is demonstrated by the charring of sodium oleate. It turns out that this technique makes it possible to select reliable charring conditions. The determined optimum temperatures and times are very similar for all the tested levels of sodium oleate.Principle author.     

Determination of bile acids in serum and bile by a modified gas chromatographic glass capillary technique

Summary A modified gas chromatographic glass capillary technique for determination of five major bile acids (Cholic acid: CA, Chenodeoxycholic acid: CDCA, Deoxycholic acid: DCA, Lithocholic acid: LCA and Ursodeoxycholic acid: UDCA) has been developed after preliminary extraction with XAD-2 resin. Enzymatic hydrolysis prevents the formation of interferring degradation products. Ether extraction with centrifugation eliminates water soluble interferring substances. Derivatization to hexafluorpropyl- instead of methyl esters results in better separation, shortens analysis time and prolongs the life span of the column.     

High-performance liquid chromatographic analysis of bile acids in hamster bile

A high-performance liquid chromatographic procedure has been developed for the determination of pentamidine concentrations in serum samples. A microbore, reversed-phase column was used with a mobile phase consisting of methanol and water with sodium heptanesulfonate and triethylamine as modifiers. Pentamidine could be extracted from serum only by the addition of an ion-pairing agent, di(2-ethylhexyl) phosphoric acid, to the chloroform used for extraction. The method can be used to reliably detect levels as low as 5 ng/ml. The pentamidine concentration in the serum of eleven patients 24 h after their tenth daily dose of pentamidine averaged 60 +/- 34 ng/ml.     

Retention and separation studies of cholesterol and bile acids using thermostated thin-layer chromatography.

The influence of temperature on retention and separation of cholesterol and bile acids, using reversed-phase thin-layer chromatography, was studied. As mobile phases methanol-water mixtures of various compositions were used. Chromatographic experiments were performed using vapor-saturated chambers at temperatures ranging from 5 to 60 degrees C. A linear relationship between R(M) values and temperature (1/T) as well as mobile phase composition was observed. The elution order of steroids under the conditions investigated was discussed. Each chromatogram was evaluated using simple optimization parameters and the best chromatographic conditions for the separation of multicomponent samples were chosen.     

Improved thin-layer chromatographic method for sugar separations

A method is described for the utilization of pre-coated, non-impregnated silical gel thin layers in one- and two-dimensional separations of carbodhyrates and related compounds. Boric and phenylboronic acids were added to the organic elution systems in different concentrations and their interactions with the sugar molecules during the chromatographic process were studied. A comparison was made between solvent systems containing boric or phenylboronic acid and systems devoid of both acids as eluents. With boric acid-containing solvents the migration of some sugars was considerably inhibited, whereas phenylboronic acid produced an increase in the RF values of certain sugars. The combination of these two types of solvent in two dimensional development resulted in the clear separation of a group of mono- and disaccharides of bioclinical interest.     

Development of a reversed-phase thin-layer chromatographic method for artemisinin and its derivatives

In this study a clear separation between seven analogues of artemisinin on thin-layer chromatography (TLC) is presented. The developed TLC method is carried out on a RP-C18 thin-layer plate using acetonitrile-water (50:25 v/v) as the mobile phase. Spots are visualized by derivatization with an acidified 4-methoxybenzaldehyde reagent in methanol-water. This method allows the separation of a diverse group of compounds that have versatile hydrophilic/lipophilic characteristics; namely artemisinin, artesunate (AS), artelinic acid (AL), arteether (AE), both isomers of artemether (AM) (alpha and beta), dihydroartemisinin, and desoxyartemisinin. Separation of some degradation products and impurities, down to 2%, allows quality control and stability investigation of all actives in raw material and pharmaceutical formulations. The method is further developed via densitometric measurement for quantitative determination purposes for AL and AS. The derivatization technique is evaluated, showing good stability and reproducibility of the coloring process. Percent relative standard deviation values are less than 5% for replicates, and linearity is obtained in the range of 0.5 to 8 microg. A comparative study with high-performance liquid chromatography (HPLC) on a C18 column, applying the same mobile phase, proves the suitability of the TLC method, in which almost all presented analytes are separated from each other. In contrast, HPLC requires at least a 20-min analysis to chromatograph all of the compounds and only betaAM and AE are clearly separated from each other and from the other compounds.     

Simultaneous quantification of withanolides in Withania somnifera by a validated high-performance thin-layer chromatographic method

This paper describes a sensitive, selective, specific, robust, and validated densitometric high-performance thin-layer chromatographic (HPTLC) method for the simultaneous determination of 3 key withanolides, namely, withaferin-A, 12-deoxywithastramonolide, and withanolide-A, in Ashwagandha (Withania somnifera) plant samples. The separation was performed on aluminum-backed silica gel 60F254 HPTLC plates using dichloromethane-methanol-acetone-diethyl ether (15 + 1 + 1 + 1, v/v/v/v) as the mobile phase. The withanolides were quantified by densitometry in the reflection/absorption mode at 230 nm. Precise and accurate quantification could be performed in the linear working concentration range of 66-330 ng/band with good correlation (r2 = 0.997, 0.999, and 0.996, respectively). The method was validated for recovery, precision, accuracy, robustness, limit of detection, limit of quantitation, and specificity according to International Conference on Harmonization guidelines. Specificity of quantification was confirmed using retention factor (Rf) values, UV-Vis spectral correlation, and electrospray ionization mass spectra of marker compounds in sample tracks.