Separation of methylated free bile acids from their taurine and methyl glycine conjugates by thin-layer chromatography

Class separation of methylated free bile acids from bile acids conjugated with taurine and methylglycine was accomplished using a solvent system of 2,2,4-trimethylpentane-absolute ethanol 10:1 (v/v). By developing a silica thin-layer plate two times with solvent in a Brinkmann sandwich tank, the difficult resolution between methyl cholate and methyl glycolithocholate was achieved. Evidence is presented that this separation system may be useful as a preparative step in the analysis of bile acids by gas-liquid chromatography or high pressure liquid chromatography.--Bolt, M. J. G. Separation of methylated free bile acids from their taurine and methyl glycine conjugates by thin-layer chromatography.     

Reverse-phase purification and silica gel thin-layer chromatography of porphyrin carboxylic acids

Thin-layer chromatography on silica gel 60 plates in the solvent N,N-dimethylformamide/methanol/ethylene glycol/glacial acetic acid/1-chlorobutane/chloroform (4/35/6/0.4/18/20 by volume) separates porphyrin carboxylic acids by the number of free carboxyl groups. Coproporphyrins I and III and isocoproporphyrin are separated in 30 min, other porphyrins in 15 min. The N,N-dimethylformamide and acetic acid in the solvent strongly increase porphyrin fluorescence on the plates. Fading and diffusion of the fluorescent patterns is prevented by storage of the plates in the cold and dark without oxygen and with desiccant. In a preliminary step, porphyrins are purified in high yields, concentrated, and deacidified rapidly (2 min) by reversephase chromatography on cartridges containing a C₁₈ spacer or on Amberlite XAD-2 columns. The methods are applied to urines of porphyria patients and for following porphyrin ester hydrolysis.     

Bile acids: XLVIII. Separation of conjugated bile acids by high-pressure liquid chromatography

Major conjugated bile acids of human bile have been resolved by high-pressure liquid chromatography. The elutions are carried out in two stages on Corasil II or μPorasil columns; first, an alkaline solvent system (2-propanol/ethyl acetate/water/7n ammonium hydroxide, 260:600:50:3) was used for separation into groups: tauro-dihydroxy derivatives, taurocholate, glyco-dihydroxy derivatives, and glycocholate. The fraction containing glyco-dihydroxy conjugates was separated by rechromatography in acetonitrile/acetic acid, 400:10, and the fraction containing tauro-dihydroxy conjugates could be partially resolved by rechromatography in acetonitrile/acetic acid/formic acid (97%)/water, 500:10:5:10. Three samples of prepared human bile have been similarly treated.     

Extraction of bile acids from rat feces containing cholestyramine

The fecal extraction procedure described by Evrard and Janssen (1) was inadequate for the complete extraction of conjugated bile acids from feces containing the bile acid sequestrant, cholestyramine. As judged by gas-liquid chromatographic analysis, substitution of 0.5 n HCl in absolute ethanol for glacial acetic acid allowed for complete recovery (98-104%) of three different conjugated bile salts in the presence of the resin.     

Aqueous and ethanolic extracts of Galinsoga parviflora and Galinsoga ciliata. Investigations of caffeic acid derivatives and flavonoids by HPTLC and HPLC-DAD-MS methods

Galinsoga ciliata Raf. Blake like Galinsoga parviflora Cav., comes from the Andes region. The chemical composition, activity and use are similar for both species. Galinsoga species are used in folk medicine as anti-inflammatory agents and accelerators for wound healing. Extracts are applied topically onto the skin to treat dermatological diseases, eczemas, lichens and hard-healing-wounds, and also to treat snakebites. Orally they used to cure flu and colds.In the studies using HPTLC method, different stationary phases, including unmodified silica gel, silica gels modified with CN, NH₂, DIOL and RP18 groups were tried. The best separation of the tested compounds was achieved on silica gel plates, when as mobile phases mixtures – ethyl acetate–acetic acid–formic acid–water (100:11:11:26, v/v/v/v), ethyl acetate–methanol–formic acid–water (50:3:4:6, v/v/v/v) and ethyl acetate–methyl ethyl ketone–formic acid–water (30:9:3:3, v/v/v/v) – were used. Using reference substances, in the examined extracts the presence of flavonoids: patulitrin, quercimeritrin, quercitagetrin, and phenolic acids – caffeic and chlorogenic acids was found.HPLC analyses of extracts were carried out on a reversed-phase Zorbax SB column (150 mm × 2.1 mm, 1.9 μm). The mobile phase (A) was water/acetonitrile/formic acid (95:5:0.1, v/v/v) and the mobile phase (B) was acetonitrile/formic acid (100:0.1, v/v). A linear gradient system was used: 0–30 min, 1–30% B. Application of HPLC-DAD-MS method confirmed the results obtained by HPTLC method. Moreover, in the tested extracts the presence of caffeoylglucaric acids as dominating compounds was detected.     

Separation of [1-3H]cellooligosaccharides by thin-layer chromatography: assay for cellulolytic enzymes

A thin-layer chromatographic method for the separation with high resolution of [1-3H]cellooligosaccharides on silica gel plates has been developed. Reducing end-labeled glucose through cellohexaose were separated on silica gel plates with three ascents of ethyl acetate:water: methanol (40:15:20; v:v) and each was extracted with an efficiency of 88 +/- 3%. Separations of cellooligosaccharides using other adsorbents, solvents, and impregnants are also described. This thin-layer chromatographic method facilitated analysis of the activity of cellulolytic enzymes.     

Separation of glycoprotein-derived oligosaccharides by thin-layer chromatography.

A thin-layer chromatography (tlc) system has been developed for the separation of glycoprotein-derivedoligosaccharides. The method involves chromatography on silica gel using n-propanol/acetic acid/water (3:3:2 v/v) as the solvent. This tlc method was used to separate pathological oligosaccharides isolated from individuals with G_M1 gangliosidosis and with neuraminidase deficiency. The results indicate the potential usefulness of the system in the analysis of complex carbohydrates.     

Chromatography of permethylated oligosaccharide alditols

A new chromatographic method which enables the separation of permethylated oligosaccharide alditols has been developed. The method entails chromatography on precoated silica gel plates using benzene-methanol (16:1, v/v or 10:1 v/v) as developing solvent. Separations of disaccharides were obtained on the basis of glycosidic linkage and anomeric configuration; the method can accomodate oligosaccharides containing up to 15 glycose units. The combined use of thin-layer chromatography and gas-liquid chromatography provides a powerful approach for the characterization of oligosaccharides. Retention indices are given of permethylated oligosaccharide alditols on a fused-silica capillary column bonded with DB-1.     

Separation of [1-H]cellooligosaccharides by thin-layer chromatography: Assay for cellulolytic enzymes

A thin-layer chromatographic method for the separation with high resolution of [1-³H]-celloligosaccharides on silica gel plates has been developed. Reducing end-labeled glucose through cellohexaose were separated on silica gel plates with three ascents of ethyl acetate:water:methanol (40:15:20; v:v) and each was extracted with an efficiency of 88 ± 3%. Separations of cellooligosaccharides using other adsorbents, solvents, and impregnants are also described. This thin-layer chromatographic method facilitated analysis of the activity of cellulolytic enzymes.     

Liquid chromatographic direct resolution of beta-amino acids on a doubly tethered chiral stationary phase containing N--H amide linkage based on (+)-(18-crown-6)- 2,3,11,12-tetracarboxylic acid

A doubly tethered chiral stationary phase (CSP) prepared by bonding (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid to doubly tethered primary aminoalkyl silica gel was used for the resolution of various beta-amino acids. All the beta-amino acids tested were resolved quite well, the separation (alpha) and the resolution factors (RS) being in the ranges 1.34-2.09 and 2.52-7.45, respectively, with a mobile phase of methanol-water (50:50, v/v) containing 10 mM acetic acid. The chiral recognition efficiency of the doubly-tethered CSP was found to be generally superior to that of the corresponding singly-tethered CSP in the resolution of beta-amino acids. The chiral recognition behaviors for the resolution of beta-amino acids on the doubly tethered CSP were examined by varying the type and content of organic and acidic modifiers in the aqueous mobile phase and the column temperature.     

Lactic acid enantiomers: separation by thin-layer chromatography on silica gel plates impregnated with Cu2+.

A simple and fast method for the separation of D- and L-lactic acid enantiomers by thin-layer chromatographic silica gel precoated plates impregnated with Cu(II) is proposed. The two lactic acid enantiomers, dissolved in acetone, are deposed on these plates. D- and L-lactic acids form complexes with Cu(II), with different Rf values, suitable for radiochemical measurements. The real separation was confirmed by enzymatic tests carried out on spots of D and L forms scraped from the chromatographic plates. Total radioactivity of 14C-labeled D- and L-lactic acids was recovered. A Cu/lactic acid complex is evidenced.     

Assay for polyunsaturated fatty acids by argentation-thin-layer chromatography using commercial thin-layer plates

A simple, rapid, and convenient procedure for silver nitrate impregnation of commercial precoated silica gel plates is described. Silica-gel plates (Silica gel 60, E. Merck) were sprayed with 40% silver nitrate in water, dried in air, and activated at 100°C for 30 min. Samples containing fatty acid methyl esters were applied as 0.5- to 1.0-cm streaks and developed with a solvent system of benzene:ethyl acetate (9:1, v/v). The plates were sprayed with 70% sulfuric acid saturated with potassium dichromate, and the spots were detected by careful heating at 120°C for 90 min. This procedure is useful for separation and isolation of various species of fatty acid methyl esters and for simple, rapid, and reproducible estimation of microgram quantities of materials by spectrodensitometry of the chromatogram.     

Determination of individual conjugated bile acids in human bile

A method has been developed and validated for the determination of the six major conjugated bile acids, cholesterol, and total phospholipids in bile of human subjects previously injected with 4-(14)C-cholesterol. The procedure is designed for use with 5-10 ml of duodenal or T-tube bile and eliminates difficulties associated with existing methods for bile acid determination, in particular the requirement for preliminary saponification under pressure or the use of paper chromatography. Saponification under pressure is employed only in steps where partial destruction of the steroid moiety of conjugated bile acids is not a crucial matter. A preliminary Folch extraction and washing step separated free cholesterol and phospholipids (bottom layer) from the six major conjugated bile acids (top layer). The conjugated bile acids were then fractionated cleanly by thin-layer chromatography to give four groups, the (14)C content of each of which was determined. A second aliquot of the top layer was used to determine (after deconjugation) the radioactivity ratio of deoxycholic acid to chenodeoxycholic acid for the two unresolved groups (dihydroxycholanoic acid conjugates with glycine and taurine, respectively). A third aliquot was used for determination of specific activities of the methyl esters of cholic, chenodeoxycholic, and deoxycholic acids derived from the total bile salts. Appropriate calculations yielded the concentration in bile of all six major bile acid conjugates.     

Gaschromatographische analyse von sauren urinmetaboliten nach trennung auf kieselgelfertigsäulen

Gas chromatographic estimation of acidic urinary metabolites after separation on prepacked silica gel columnsThe acidic ethylacetate extracts of 24-h urine specimens are evaporated and redissolved in chloroform—methanol—acetic acid. The resulting solution is transferred to a prepacked silica gel column. Elution takes 160 min using a specially designed chloroform—methanol—acetic acid gradient. The eluate is divided into fractions (16 min each) which are evaporated to dryness. The residues are silylated and determined quantitatively by gas chromatography. The capacity of the silica gel column allows analysis of 30% of a 24-h urine specimen. In consequence, metabolites can be quantitated at concentrations less than 1 mg per 24 h. The method is suitable to obtain more detailed metabolic profiles of the carboxylic acids in urine.     

Analysis of bile acids in conventional and germfree rats.

The well-known bile acid analysis technique used by us and others (Grundy, Ahrens, and Miettinen. 1965. J. Lipid Res. 6:397-410) does not allow for the detection of hyodeoxycholic acid, a product of quantitative importance in rodent feces. Using updated methodology, it was established that hyodeoxycholic acid and omega-muricholic acid, both apparent conversion products of beta-muricholic acid, occur in apppreciable amounts in intestinal contents and feces of conventional Wistar type Lobund rats. In conventional rats, these bile acids comprise about 50% of fecal bile acids; they are not found in intestinal contents or feces of germfree rats. Others have demonstrated that hyodeoxycholic acid if formed by combined action of gut flora and liver. A new method for the separation of conjugated and free bile acids in biological samples was developed. Results with this method confirmed the total conjugation of bile acids in the germfree rat, and the almost total deconjugation that takes place in the cecum of the conventional rat.     

Differentiated quantification of human bile acids in serum by high-performance liquid chromatography-tandem mass spectrometry

Determination of quantitative changes in the pattern of serum bile acids is important for the monitoring of diseases affecting bile acid metabolism. A sensitive and specific high-performance liquid chromatography (HPLC)-MS/MS method was developed for the differentiated quantification of unconjugated as well as glycine- and taurine-conjugated cholic, chenodeoxycholic (CDCA), deoxycholic (DCA), ursodeoxycholic (UDCA) and lithocholic acid (LCA) in serum samples. After solid-phase extraction and reversed-phase HPLC separation, detection of the conjugated bile acids was performed using electrospray ionization (ESI)-MS/MS and selected reaction monitoring mode, whereas unconjugated bile acids were determined by ESI-MS and selected ion monitoring mode. The within-day and between-day coefficients of variation were below 7% for all bile acids and the recovery rates of the extraction procedure were between 84.9 and 105%. The developed method was applied to a group of 21 healthy volunteers and preliminary reference intervals in serum were established. In patients with drug-induced cholestasis, an elevation of primary bile acids has been shown.     

Analysis of conjugated bile acids by packed-column supercritical fluid chromatography

A rapid method has been developed for the simultaneous separation of the polar glycine- and taurine-conjugated bile acids by packed-column supercritical fluid chromatography. Samples were analysed on a cyanopropyl-bonded silica column with ultraviolet detection at 210 nm and carbon dioxide modified with methanol as the mobile phase. The influence of the stationary phase, modifier concentration, temperature, column pressure and modifier identity on retention was also studied. This new chromatographic method is applicable to the assay of conjugated bile acids in duodenal bile samples from patients with hepatobiliary diseases.     

Estimation of boswellic acids from market formulations of Boswellia serrata extract and 11-keto beta-boswellic acid in human plasma by high-performance thin-layer chromatography

A rapid and sensitive high-performance thin-layer chromatographic (HPTLC) method was developed and validated for the quantitative estimation of boswellic acids in formulation containing Boswellia serrata extract (BSE) and 11-keto beta-boswellic acid in human plasma. Simple extraction method was used for isolation of boswellic acid from formulation sample and acidified plasma sample. The isolated samples were chromatographed on silica gel 60F(254)-TLC plates, developed using ternary-solvent system (hexane-chloroform-methanol, 5:5:0.5, v/v) and scanned at 260 nm. The linearity range for 11-KBA spiked in 1 ml of plasma was 29.15-145.75 ng with average recovery of 91.66%. The limit of detection and limit of quantification for 11-KBA in human plasma were found to be 8.75 ng/ml and 29.15 ng/ml. The developed method was successfully applied for the assay of market formulations containing BSE and to determine plasma level of 11-keto beta-boswellic acid in a clinical pilot study.     

Separation of individual sulfated bile acid conjugates as calcium complexes using reversed-phase partition thin-layer chromatography.

A method for separating individual monosulfated primary bile acid conjugates by reversed-phase partition thin-layer chromatography on octadecyl-bonded silica gel is described. The solvent system is acetonitrile containing calcium, probably as calcium carbamate. Excellent resolution of the 3- and 7-monosulfated glycine conjugates, as well as 3- and 7-monosulfated taurine conjugates of cholic and chenodeoxycholic acids is reported. A convenient class separation of sulfated from nonsulfated primary bile acid conjugates by adsorption thin-layer chromatography on low-polarity silica gel is also described.     

A simple method for the separation of bile acid groups by ion-exchange chromatography

A simple ion-exchange chromatographic method for the separation of bile acid mixtures is described. The method employs Dowex 1 as anion exchanger and mixtures of aqueous ethanol and hydrochloric acid as eluants. The use of mixtures in which both the ethanol and acid concentrations are varied has permitted a clear separation of the major bile acid groups (nonconjugated, glycine conjugated, and taurine conjugated bile acids).