Improved high-performance liquid chromatographic determination of thiamine and its phosphate esters in animal tissues

An improved method for the determination of thiamine and its phosphate esters in animal tissues using reversed-phase high-performance liquid chromatography with precolumn derivatization is described. Thiamine and its phosphate esters were converted into fluorophores by alkaline cyanogen bromide, and the derivatives were applied to an ODS packed column. Then the effluent obtained by an acidic mobile phase was mixed with an alkaline methanol solution to increase the fluorescence intensity of the derivatives which was determined spectrofluorometrically. A complete, rapid and quantitative separation of thiamin and its phosphate esters was achieved and the use of the acidic buffer as a mobile phase improved the column stability. The fluorophores of thiochrome ester peaks on the chromatogram were sensitive to pretreatment with thiamine triphosphatase or acid phosphatase. The applicability of the method to the determination of the form of thiamin in various tissues of rat is demonstrated.     

Spectrofluorometric Determination of Thiamin and Riboflavin in Vegetable Foods

Abstract

Modifications of the AOAC thiamin and riboflavin methods allow effective and simple determination of both vitamins from aliquots of the digested sample of vegetable foods. Study of the minimum amount required for a complete hydrolysis of the vitamers corresponding to each vitamin have been carried out. A critical study of thiocrome formation which was later used in the spectrofluorimetric determination of thiamin was done. In order to separate riboflavin from the digested sample, a new liquid chromatographic method based on adsorption in florisil is proposed. The aforementionated methods were applied to different samples of several vegetable foods.     

Thiamin ylide: isolation and identification

The action of two equivalents of NaOEt on thiamin hydrochloride in EtOH gives a neutral compound. Spectroscopic data and its chemical behavior indicated that the compound is thiamin ylide $\text{1}$. The chemical behaviors of $\text{1}$ are entirely consistent with those of in situ generated thiamin ylide.     

High Performance Liquid Chromatography of Cholesteryl Esters

Abstract

The separation of cholesteryl esters by high performance liquid chromatography was facilitated by cooling of the column, followed by spontaneous return to ambient conditions. the resolution of the esters was increased. the method was applied to detection of the cholesteryl esters in plasma.     

Separation of a Homologous Series of Long-Chain Fatty Alcohols (C49-C58) From Mycobacterium Tuberculosis H37Ra by High Performance Liquid Chromatography

Abstract

Fatty alcohols were obtained from the saponified chloro-form-methanol extract of Mycobacterium tuberculosis H37Ra by extraction with diethyl ether and partially purified by silicic acid column chromatography. The long-chain fatty alcohols (C₄₉-C₅₈) were separated from the shorter-chain alcohols by Sephadex LH-20 column chromatography and further fractionated into saturated and unsaturated alcohols by argentation thin-layer chromatography. These two fractions were analyzed by proton nuclear magnetic resonance spectro-scopy, derivatized to the 3,5-dinitrobenzoyl esters, and fractionated on a C₁₈-bonded silica column by high performance liquid chromatography (HPLC). Complete separation of esters differing by two carbon units and partial separation     

Concomitant determination of thiamin and its phosphate esters in human blood and serum by high-performance liquid chromatography

A high-performance liquid chromatographic method for the simultaneous determination of thiamin and thiamin phosphate esters in human blood or serum has been developed. The eluent consists of acetonitrile and phosphate buffer, in the ratios 90:10 (v/v) for the elution of thiamine and 60:40 (v/v) for the phosphate esters. The four compounds are eluted within 15 min. The detection limit is 13-16 fmol. Between-assay variation is 5-11%. Samples of whole blood and serum from 30 healthy adults were analysed. The following reference values were obtained for 15 females 15 males (nM, mean +/- S.D.). In serum: thiamin, 10.9 +/- 2.9/16.9 +/- 3.3; thiamin monophosphate, 8.3 +/- 1.5/3.7 +/- 1.5. In whole blood: thiamin, 29.6 +/- 10.0/33.4 +/- 10.4; thiamin monophosphate, 9.7 +/- 2.3/10.9 +/- 5.1; thiamin diphosphate, 121 +/- 29.6/165 +/- 40.4.     

Free Radical Formation of p-Nitrophenacyl Esters of Carboxylic Acids and Their Detection by High-Performance Liquid Chromatography with Electron Spin Resonance

Abstract

p-Nitrophenacyl esters derived from p-nitrophenacyl bromide and carboxylic acids were determined by high-performance liquid chromatography with electron spin resonance. p-Nitrophenacyl bromide was used as the pre-column reagent to determine the carboxylic acids. The p-nitrophenacyl esters gave free radicals by hydrolysis with alkaline solution, and then the free radicals were detected with electron spin resonance. The separation of these esters was achieved within 20 min.     

Separation of Prostaglandins on a Hydrophobic Sephadex Derivative

Abstract

Liquid-gel chromatography on a hydrophobic Sephadex derivative has been used for the separation of prostaglandins. Solvent systems are given for separation of PGE and PGF compounds either as free acids or as methyl esters, using a Sephadex derivative containing 12% hydroxyalkyl groups. The columns are simple to prepare and can be used repeatedly over long periods of time. The recovery is high and the technique should be a useful complement to other methods used for the isolation of prostaglandins.     

Ion Chromatographic Determination of Sodium Isethionate

Abstract

Single column anion chromatography with conductivity detection has been used for the determination of sodium isethionate. A mobile phase of aqueous phthalic acid - methanol (pH 2.5) allows for the separation of isethionate from chloride and alkyl isethionate ester surfactants. Commercially available samples of sodium isethionate and alkyl esters were analyzed by the described procedure.     

Recycling HPLC of p-Bromophenacyl Esters of Saturated C35–56 Fatty Acids from Mycobacterium Tuberculosis H37Ra on a Silica Column

Abstract

The p-bromophenacyl esters of saturated C_35–56 fatty acids from Mycobacterium tuberculosis H37Ra were separated according to structural classes on a silica column by high performance liquid chromatography (HPLC). The sample was cycled five times during HPLC. Highly purified C_35–38 esters were obtained by this method. Further HPLC fractionation on a reverse-phase column (C₁₈-bonded silica) gave complete separation of most of the remaining fractions. By combining mass spectrometry with HPLC separations, many of the fatty acid esters were tentatively identified.     

Separation of Alkaloids from Datura Metel. and Sophora Flavescens Ait by High-Speed Countercurrent Chromatography

Abstract

High-speed countercurrent chromatography was successfully applied to the separation of alkaloids from two medicinal herbs. Matrine and oxymatrine were isolated from the root of Sophora flavescens Ait with a two-phase solvent system composed of chloroform-0.07 M sodium phosphate (pH 6.4), while atropine and scopolamine were purified from the flowers of Datura mete L. with a similar solvent system by modifying the phosphate buffer pH at 6.5. Identification of each alkaloid was made by either TLC or paper partition chromatography using authentic pure compounds.     

High-Performance Liquid Chromatography of 2′-5′Oligoadenylates and Related Oligonucleotides

Abstract

HPLC has been used for the analysis and separation of the components of p_x (A2′p)_n A (x = 1 to 3, n = 1 to ≥4). Weak anion exchange columns give excellent resolution, but their instability in phosphate buffers makes them impractical for routine use. Reverse phase chromatography using C18 columns provides a satisfactory alternative method. For preliminary analysis of crude material, ammonium phosphate pH7.0 with a linear 1:1 methanol/H₂O gradient gives a good basic separation of the individual oligomers. Resolution of the 5′ mono-, di-and triphosphorylated oligomers or of the nonphosphorylated components can be obtained using ammonium phosphate pH6.0 and potassium phosphate pH6.5 buffers respectively. The C18 columns are very stable and any one column will give retention times reproducible within 0.2%.     

Separation of Deoxyribonucleotides Using Ion Suppression High Performance Liquid Chromatography with a Reversed-Phase Column

Abstract

Ion suppression-reversed phase high performance liquid chromatography, using 0.6 M ammonium dihydrogen phosphate as eluent, produces base-line separations of deoxyribonucleotides. The effects of pH and ionic strength are described. This isochratic system is simple, reproducible and fast, requiring less than 30 min for a complete separation, and is suitable for in vitro studies.     

The Reaction of Phosphoryl Chlorides with Enamines - A New Approach to β-Ketophosphonates

Abstract

Several approaches are available for the preparation of β-ketophosphonate esters. A Michaelis-Arbuzov approach using the reaction of an α-halo ketone with a trialkyl phosphite may in certain instances be controlled to favor the preparation of the target materials. However, an alternative pathway leading to the formation of vinyl phosphate esters often predominates. (This reaction system has recently been reviewed by Borowitz and Borowitz.¹) An alternative approach is that developed in recent years by Wiemer, et al.² involving the rearrangement of vinyl phosphate esters to the β-ketophosphonates.     

New Principles of Ion-Exchange Techniques Suitable to Sample Preparation and Group Separation of Natural Products Prior to Liquid Chromatography

Abstract

A Gentle method of group separation of low molecular weight hydrophilic natural products is reported. The method is based on separation of the compounds according to their net charge at different pH values using different types of ion-exchange columns connected in series. Precolumns retaining interfering compounds are used in some cases. Elution of the compounds retained on the columns is performed by use of volatile eluents. The elution principle for two of the ion-exchangers in question is removal of the charges on the column materials while for the third column the positive net charge on the compounds retained is removed. Thereby, the total amount of ions retained on the different columns is released and eluted into small volumes, which after evaporation leaves the ions as well defined salts. The method is experimentally simple and efficient to separation of natural products into groups suitable to direct use in sensitive methods of analysis as e.g. high-performance liquid chromatography and gas chromatography. Combinations of these column chromatographic methods have been adpated for micro or semimicro determinations of naturally occurring compounds, e.g., aromatic choline esters, amines, amino acids and esters of phenolic carboxylic acids. The methods seem to be general practicable for group separation of low molecular weight hydrophilic compounds.     

Separation and Determination of Bile Acids in Human Bile by High-Performance Liquid Chromatography

Abstract

A method for simultaneous determination of major bile acids in human bile is described. The unconjugated, glycine- and taurine-conjugated bile acids are extracted with Sep-pak C¹⁸ and separated into groups by ion-exchange chromatography on a lipophilic gel. Subsequently, resolution of each group into ursodeoxycholate, cholate, chenodeoxycholate, deoxycholate and lithocholate is attained into two stages by high-performance liquid chromatography on a Radial-PAK A column. First, 0.3% ammonium phosphate (pH 7.7)/acetonitrile (19:8, v/v) is used for separation of the latter three as a mobile phase. Ursodeoxycholate and cholate are efficiently separated in 0.3% ammonium phosphate (pH 7.7)/acetonitrile (23:8, v/v). The present method is applicable to quantitation of bile acids in human bile with satisfactory accuracy and precision.     

Biosynthesis of Vitamin B1 (Thiamin): An Instance of Biochemical Diversity

The elucidation of the biosynthetic pathway to thiamin (Vitamin B₁) and its pyrophosphate ester, the important coenzyme “cocarboxylase”, has challenged researchers for many years and continues to do so. The problem of the origin of thiamin can be separated into three parts: the independent pathways to the pyrimidine moiety 4-amino-5-hy-droxymethyl-2-methylpyrimidine and to the thiazole moiety 5-(2-hydroxyethyl)-4-methylthiazole, and the route from these subunits to the vitamin. The steps in the latter process were fully established some twenty years ago, and it was shown that the route in aerobic bacteria and yeast differs to some extent from that in enteric bacteria. The pathways to the subunits, on the other hand, are still not clarified. Significant differences exist in the routes whereby each of the two subunits, the pyrimidine moiety and the thiazole moiety, originate in bacteria and yeast. One difficulty that delayed progress was that the incorporation patterns of labeled precursors, which were observed by different research groups in different microorganisms, could not be reconciled on the basis of a single pathway to each of the two subunits. It is now accepted that in each case different pathways exist in enteric bacteria and yeast, and that the biosynthesis of Vitamin B₁ represents an instance of biochemical diversity. A second factor that added to the difficulties is the minute amount of thiamin synthesized in microbiological cultures (about 15 μg per L culture). This limited the investigations until very recently either to the use of radioactive tracers or to the use of stable isotopes in conjunction with mass spectrometric analysis. It is widely recognized that both methods are associated with pitfalls in the interpretation of results. High-field ¹³C NMR, the most powerful modern method available for the determination of incorporation patterns, has only very recently been successfully employed in investigations of thiamin biosynthesis. As a result of the conceptual and experimental problems, even the primary precursors of each of the two relatively simple heterocyclic subunits of thiamin are still not completely established. A search for committed intermediates, the study of the enzymes, and identification of the genes that are involved are the matter of current research.     

Separation of Biological Pyridines by High Pressure Liquid Chromatography

Abstract

The separation of the six pyridine compounds which comprise the pyridine nucleotide cycle, nicotinamide adenine dinucleotide phosphate and para-aminobenzoic acid, a compound biologically related to these pyridines, can be achieved rapidly utilizing high pressure liquid chromatography. Optimum separation is accomplished using ion-ion pairing in reverse phase chromatography with a C₁₈ stationary phase and an aqueous mobile phase of 5mM pentanesulfonic acid and 25 mM KH₂PO₄. The effect of temperature on the separation is minimal. As little as 10 ng of these compounds is detected via absorption of ultraviolet light at a wavelength of 254 nm.     

Isocratic Separation of Fatty Acid Derivatives by Reversed Phase Liquid Chromatography. Influence of the Solvent on Selectivity and Rules for Elution Order

Abstract

The p-bromophenacyl esters of 16 fatty acids (C₁₂-C₂₂) have been separated by isocratic chromatography on a Radial Pak A cartridge (Reverse phase C₁₈ material). The separation factors α were measured using two solvent mixtures of comparable strength and the superiority of methanol-water to acetonitrile-water becomes evident.

Five precise rules are established, which indicates the retention of every fatty acid. They explain the chromatographic process i.e. elution order, resolution and selectivity.     

High Resolution Separation of Urinary Organic Acids by High Performance Liquid Chromatography

Abstract

Reverse phase, anion exchange, and two-dimensional HPLC techniques were studied in order to increase resolution of organic urinary acids for eventual quantitative measurements. Reverse phase HPLC with a phosphate buffer/acetonitrile gradient yielded a separation of over 85 components in forty minutes and a peak area reproducibility of better than 5%. Connecting two reverse phase columns together resulted in the separation of 110 components. Anion exchange chromatography was determined to be of little use in resolving urinary acids in a resonable time except as the first stage in two-dimensional chromatography where fractions from the anion exchange column were injected into a reverse phase column. Over 139 components were separated by this two-dimensional method.