Separation of Monosulfated Bile Acids by High-Performance Liquid Chromatography

Abstract

Separation of 3-, 7- and 12-monosulfates of cholate, chenodeoxycholate, deoxycholate, lithocholate and ursodeoxycholate and their glyco- and tauro-conjugates by high-performance liquid chromatography on a reversed phase column has been carried out. Effects of pH and salt concentration of a mobile phase on the k' value of sulfated bile acids were investigated with the μBondapak C₁₈ and ODS SC-02 columns. The 3-sulfated bile acids were efficiently separated on ODS SC-02 using three aqueous ammonium carbonate/acetonitrile systems. The chromatographic behaviors of 7-and 12-sulfated bile acids are also briefly discussed.     

Enzymatic Determination of Urinary Total 7α-Hydroxy Bile Acids

Abstract

An enzymatic determination of urinary 7^α-hydroxy bile acids is described. The principle of the method is as follows: after hydrolysis with β-glucuronidase and solvolysis, the ethyl acetate extract is washed with alkalin solution and water, then the alkali and water washes of the ethyl acetate extract are combined and the solution is acidified to pH 1 and sodium chloride added. Shake solution with ethyl acetate to re-extract the acidic fraction and the ethyl acetate layer is evaporated. Add enzyme color solution of 7_α-hydroxysteroid dehydrogenase (7_α-HSD, from E. coli) to a tube of extract residue and then after incubation, measure the absorbance of the solution.

Excretion values of urinary total 7_α-hydroxy bile acids was measured with patients of acute hepatitis and normal subjects and excretion pattern of 7_α-hydroxy bile acids by the use of chromatographic fractionation was also shown.     

Specificity and Cross Reactivity in Bile Acid Radioimmunoassays Serum Bile Acids,Radioimmunoassay

Abstract

In man there are four main bile acid fractions and within each fraction there is the possibility of at least three bile acid moities - two conjugated and one unconjugated. Bile acids thus present a considerable challenge to radioimmunoassay techniques. Few of the antisera described to date are satisfactory in that they do not show equal reactivity to each of the moities to be assayed, and many have unacceptable cross reactivity properties. Clearly there is need for caution and development in this field. The first application of radioimmunoassay (RIA) techniques to the measurement of serum bile acids was made by Simmonds, Korman, Go and Hofmann in 1973¹. Because of its unique sensitivity and ease of application, RIA has been used not only to determine the increased serum bile acid levels found in liver disease, but also to monitor the clearance of bile acids from the peripheral circulation of normal subjects, to provide 24 hour serum bile acid profiles in normal subjects and even to assay the low serum bile acid levels found in patients in whom the bile acid enterohepatic circulation has been interrupted. Some 20 preparations of bile acid antisera have been described to date and commercial kits are now available, for RIA of each of the 4 major bile acid fractions found in human sera. Considerable differences however, in the specificity of these antisera are indicated by their reported cross reactivities and the analytical validity of their use is often questionable.     

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.     

Studies on Steroids Clxix. High-Performance Liquid Chromatographic Behavior of Sulfated Bile Acids

Abstract

The elution behavior of sulfated bile acids in high-performance liquid chromatography on the octadecylsilyl bonded column with acetonitrile/0.5% phosphate buffer has been investigated. A significant influence of pH of the mobile phase on the capacity ratio (κ′) was observed in the higher pH region for bile acid 12-sulfates. Blockage of the 12α-hydroxyl group in sulfated bile acids by acetylation produced a marked decrease in the κ′ values relative to their parent compounds in the pH range above 6.0. The κ′ values of dehydrocholate monosulfates and their acetates were measured and the increments exerted by transformation into the acetates were estimated. Remarkable increments were observed for dehydrocholate monosulfates with the 3α-hydroxyl group but not for those with the 7α- or 12α-hydroxyl group. The effect of pH of the mobile phase on chromatographic behavior has been discussed from the stereochemical point of view.     

Polymeric Sorbents for Bile Acids: II. Oligopeptide-Containing Resins with Quaternary Amine Groups

Abstract

Polymeric sorbents for bile acids have been prepared by attaching lysine-containing peptide sequences onto a water-swellable polyamide resin, by solid phase peptide synthesis, and then attaching a terminal N,N-dimethyl glycine residue that was subsequently quaternized. The resins with relatively longer peptide sequences demonstrated a higher binding capacity, on a per active site basis, for bile acids in pH 7.4 aqueous buffer solutions at 20°C than cholestyramine and colestipol when tested under the same in vitro conditions. In solutions of low ionic strength, they also have a degree of specificity for bile acid anions. The resins have a higher binding affinity for cholic acid than for glycocholic acid, which indicates the importance of the hydrophobic interactions in the binding.     

Functionally Substituted N- and P-Containing Salts of Resinous and Bile Acids

New functionally substituted N- and P-containing salts of resinous and bile acids 1b, c, 2b-f, 3b-f, 4b-f, 5b, c, and 6b, c were prepared by reaction of the corresponding acids with amines or triphenylphosphine in anhydrous acetone.     

Structural and Functional Implications of the Interaction between Macrolide Antibiotics and Bile Acids

Macrolide antibiotics, such as azithromycin and erythromycin, are in widespread use for the treatment of bacterial infections. Macrolides are taken up and excreted mainly by bile. Additionally, they have been implicated in biliary system diseases and to modify the excretion of other drugs through bile. Despite mounting evidence for the interplay between macrolide antibiotics and bile acids, the molecular details of this interaction remain unknown. Herein, we show by NMR measurements that macrolides directly bind to bile acid micelles. The topology of this interaction has been determined by solvent paramagnetic relaxation enhancements (solvent PREs). The macrolides were found to be bound close to the surface of the micelle. Increasing hydrophobicity of both the macrolide and the bile acid strengthen this interaction. Both bile acid and macrolide molecules show similar solvent PREs across their whole structures, indicating that there are no preferred orientations of them in the bile micelle aggregates. The binding to bile aggregates does not impede macrolide antibiotics from targeting bacteria. In fact, the toxicity of azithromycin towards enterotoxic E. coli (ETEC) is even slightly increased in the presence of bile, as was shown by effective concentration (EC₅₀) values.     

Microdetermination of Bile Acids by High-Performance Liquid Chromatography Equipped with a High Sensitive Conductance Detector

Abstract

Conductimetric detection of bile acids in reversed phase high-performance liquid chromatography is described. The solvent system of the mixture of water and organic solvent containing small amount of basic salts such as ammonium carbonate is found useable by removing the cation with the cation exchange column inserted between the ODS column and the conductance detector. Thus, a few ng of tauro-and glyco-conjugated bile acids can be detected without tedious derivatization and hydrolysis.     

Dominant Factors Affecting the Chromatographic Behaviour of Bile Acids

With the aim of optimizing the chromatographic process by avoiding any preliminary derivatizing step, we examined the chromatographic behaviour of a selected set of unconjugated bile acids looking at the dominant factors that affect the performances of three different stationary phases: RP-8, RP-18 and RP-18 Base Deactivated (RP-18-BD). Accordingly to its structural peculiarity, the RP-18-BD column combined with a specific mobile phase has proved to be the most suitable one, in enhancing both separation factor α and resolution R _S within the selected set of analytes. Pronounced changes in the chromatographic profiles by only slightly changing the mobile phase composition (pH, buffer concentration, percentage and kind of organic modifier) prompted us to achieve satisfactory results in the separation and resolution of the selected set of bile acids.Presented at: CE in the Biotechnology & Pharmaceutical Industries: 7th symposium on the practical applications for the analysis of proteins, nucleotides and small molecules, Montreal, Canada, August 12–16, 2005.An erratum to this article can be found at     

Determination of Free Bile Acids in Raw Materials and Bulk Products by HPLC and GC

Abstract

A procedure based on two chromatographic methods with different selectivities (HPLC and GC) was developed for the quality control assay of free bile acids in raw materials from animals and bulk products utilized in the pharmaceutical industry. HPLC was carried out without preliminary derivatization using an Ultrasphere ODS column with UV detection at 210 nm and methanol-acetonitrile-acetate buffer as the mobile phase. For GC, bile acids were converted into their trifluoroacetyl-hexafluoroisopropyl derivatives and analysed on a SE-52 capillary column with flame-ionization detection. Bile acid levels in hydrolysed ox bile, in bulk cholic and deoxycholic acid determined by HPLC correlated with results obtained by GC, with the exception of the analytes present in low concentrations (less than 3% w/w) detectable only by GC. HPLC-UV is the more suitable technique for routine analyses of free bile acids in pharmaceutical matrices owing to its simplicity and rapidity. However, because of the low sensitivity and specificity of the UV detection, the accuracy of the HPLC assay should be verified by comparison with GC.     

A Fully Automatic Method for Analysis of Individual Bile Acids in the Serum Using High-Performance Liquid Chromatography for Clinical Use

Abstract

We have reported highly sensitive methods for the analysis of individual bile acids in the serum using high-performance liquid chromatography coupled with an enzymatic fluorometric system. In this report, a new system equipped with a sample treatment mechanism for chromatographic analysis of serum bile acids is detailed. Most of the protein and other hydrophilic components of the injected serum sample are removed in the pretreatment system, so that only the remaining bile acids are introduced into the chromatographic system and eluted with irrigants containing a coenzyme (NAD) for fluorometric detection. With this method, we are able to simultaneously determine 15 different serum bile acids in an hour without the tedious manual sample processing steps. This system opens up an approach to fully automated analysis of bile acids in the blood.     

High-Performance Liquid Chromatographic Behavior of 2-,4- and 6-Hydroxylated Bile Acid Stereoisomers

Abstract

High-performance liquid chromatographic behavior has been investigated for 40 stereoisomeric 2-, 4- and 6-hydroxylated bile acids, most of which possess a vicinal glycol structure, as their 4-nitrophthalimidomethyl (NPM) ester derivatives on a reversed-phase column using methanol-water systems as a mobile phase. The bile acid NPM esters were further derivatized to the so-called “mixed” acetonide-NPM esters, whereby the stereochemical relationships (α, α-and β,β-cis, diaxial trans or diequatorial trans) of 2,3-, 3,4-and 6,7-diol groups in the molecules were reflected to changes in the capacity factors of bile acids.     

Selective Binding Thermodynamics of Bile Acids by Oligo (ethylenediamino)-β-Cyclodextrins and Their Copper (II) Complexes

Complex stability constants (K _S), standard molar enthalpy changes (ΔH°) and entropy changes (TΔS°) for the inclusion complexation of native β-cyclodextrin (β-CD) (1) and some modified β-CDs, i.e., mono(6-ethylenediamino-6-deoxy)-β-CD (3), mono[6-diethylenetriamino-6-deoxy]-β-CD (4) and their corresponding copper complexes 5 and 6, with four representative bile acid guests, i.e., cholate (CA), deoxycholate (DCA), glycocholate (GCA) and taurocholate (TCA), were determined at 25 °C in aqueous phosphate buffer solution (pH 7.20) by means of isothermal titration microcalorimetry (ITC). The stoichiometry of resulting inclusion complexes between CDs and bile acids was demonstrated by UV and conductivity as well as ITC experiments, showing 1:1 binding model upon all inclusion complexation except for metal-mediated dimer 5. The complex stability constants for modified β-CD 2–4 are dramatically magnified with the extended length of amino tether. As compared with 3 and 4, copper(II) complexes 5 and 6 significantly enhance not only binding ability but also molecular selectivity toward bile guest molecule CA through multipoint recognition, but decreased complexes stability toward TCA could be attributed to the decreased hydrophobic microenvironment of CDs cavity due to the introduction of copper(II) coordination center. Thermodynamically, the resulting complexes between hosts and bile guests are driven absolutely by enthalpy, accompanied by entropy gain or loss. Using the present data and those previously reported for mono(6-amino-6-deoxy)-β-CD (2), thermodynamic behavior and enhanced molecular selectivity could be discussed from the viewpoint of hydrophobic interactions, electrostatic cooperation and van der Waals between the hosts and guests.     

The use of SEP-PAKTM C18 Cartridges in the Preparation of Bile Acid Methyl Ester Acetates

Abstract

A method is described for the rapid and Quantitative extraction of bile acid derivates by Sep-Pak^TM C₁₈ cartridge. The method is used for the preparation of bile acid methyl ester acetates. The method was validated by determining the efficiency and the recovery of radiolabelled taurine-conjugated and free bile acids and of bile acid containing biological samples, by thin-layer chromatography with zonal scanning after each step and by internal standardization withing the gas-chromatographic analysis. The recovery of bile acids after hydrolysis amounted to 93.7% ± 2.7%, 97.7% ± 3.6% and 100% ± 2.3% for gallbladder bile, serum bile and mixtures of pure bile acids resp. The recovery of cholic acid methyl ester acetate and chenodeoxycholic acid methyl ester acetate after the entire procedure, including hydrolysis Sep-Pak^TM -extraction, methylation, acetylation and again Sep-Pak^TM -extraction, amounted to 85.6% ± 4.6%, 88.4% ± 5.3% and 89.3% ± 3.5% for gallbladder bile acids, serum bile acids and bile acid mixtures resp. It is concluded that Sep-Pak^TM can efficiently be used in the preparation of bile acid methyl ester acetates, thereby avoiding time-consuming and inconsistent extractions.     

CaII‐Catalyzed Alkenylation of Alcohols with Vinylboronic Acids

Direct alkenylation of a variety of alcohols with vinylboronic acids has been accomplished using the air‐stable calcium(II) complex Ca(NTf₂)₂ under mild conditions with short reaction times. For reluctant transformations, an ammonium salt was used as an additive to circumvent the reactivity issue.     

Evaluation of Mobile and Stationary Phases in Reversed-Phase High-Performance Liquid Chromatography of Conjugated Bile Acids

Abstract

The reversed-phase high-performance liquid chromatographic separation of the ten major conjugated bile acids of man using isocratic conditions is described. Each component of the mobile and stationary phases was examined for its ability to influence the separation selectivity. Manipulation of pH, buffer species, organic modifier and different types of packings showed that optimal resolution was obtained with a mobile phase of methanol-0.02M sodium acetate (60:30) adjusted to pH 4.2 with phosphoric acid, on a Supelcosil LC-18-DB column. Advantages of the optimized phase system are the complete baseline separation of compounds within a short period of time, improved peak symmetry and a high rate of reproducibility. This new chromatographic method, coupled with UV detection at 205 nm, is suitable for the simultaneous determination of bile acid conjugates in routine clinical analysis.     

Unnatural Amino Acids. 2. Simple Method of Obtaining Esters of Aziridine-2-carboxylic Acids by a Transesterification Reaction

A series of N-unsubstituted esters of aziridine-2-carboxylic acid has been obtained by transesterification in basic medium using primary, secondary, and tertiary alcohols. Methods of transesterification using various bases (K₂CO₃, ROLi, t-BuOK) have been compared. Transesterification with lithium alcoholates also affords the possibility of obtaining esters of N-substituted aziridine-2-carboxylic acids. Transesterification of chiral esters proceeds with retention of the configuration of the chiral center.     

Glycidyl Esters of Aromatic Acids

Abstract

The reaction of substituted benzoic acids, dicarboxylic acids such as phthalic, terephthalic, and isophthalic acids, and the sodium or potassium salts of these acids with equimolar or excess epichlorohydrin in the presence of benzyltrimethylammonium chloride has been studied using various solvents such as toluene, dioxane, monochlorobenzene, and tetrachloroethylene. Use of the free carboxylic acids gave only fair to low yields of glycidyl esters, while sodium or potassium salts of the carboxylic acids gave excellent yields of materials of high oxirane content. The epoxidation of chlorohydrin esters of these acids by the dehydrochlorination was also studied using various dehydrochlorinating reagents such as NaOH, KOH, Na₂CO₃, and NaAlO₂ in such solvents as water, dichloromethane, dioxane, and monochlorobenzene at various temperatures. Reaction time, reaction temperature, and water content were found to influence the yield of glycidyl esters. It is suggested that the reaction path involves nucleophilic attack upon the terminal position of the epoxide or epichlorohydrin. The resulting alkoxide then reacts further to give either a glycidyl ester or a chlorine-containing by-product, the predominant course depending upon reaction conditions.     

HPLC-Analysis of PTH-Amino Acids

Abstract

Some of the problems that affect the usefulness of HPLC-analysis of PTH-amino acids have been examined. Four different C₁₈-reversed phase packings have been tested, together with different mobile phases. The separation problems of the Met/Val-group, the Phe/Leu/Ile/Lys-group and of the basic amino acids His and Arg on the reversed phase packings have been examined and solutions to some of the problems have been demonstrated. The sensitivity of the quantitative analysis of amino acids from a hydrolysate was determined.