High-performance liquid chromatographic determination of tauro- and glyco-conjugated bile acids in human serum

A method for the identification and individual determination of the ten tauro- and glyco-conjugated bile acids is described. It consists in a specific three-step extraction from small serum samples (500 microliters), high-performance liquid chromatographic separation and direct spectrophotometric detection at 119 nm. Extraction can be checked by the use of an internal standard. The reproducibility, recovery and separation of fractions were satisfactory.     

Determination of diagnostically important free and conjugated bile acids in blood plasma by high-performance liquid chromatography

A procedure was proposed for the determination of free bile acids and their conjugates in blood plasma by the reversed-phase HPLC using the column Lichrospher 100 RP-18 (250 + 4.6 mm) with gradient elution and UV-detection at 206 nm. The procedure allowed the simultaneous determination of diagnostically important cholic acids, tauro-and glyco-cholates in blood plasma of patients with no preliminary separation of the analytes into subtypes. The bile acids and their conjugates were isolated from the sample matrix by solid phase extraction in a Sep-Pack C18 cartridge. The limits of detection were 0.11–0.15 mM for free acids and 0.015–0.025 mM for conjugates.     

Pre-column derivatization of free bile acids for high-performance liquid chromatographic and gas chromatographic-mass spectrometric analysis

Pentachlorophenyl (PCP) esters of five free bile acids (FBA) were obtained by reacting the FBA and Kovacs' complex (KC) in a 1:8 molar ratio in acetone at 65 degrees C, and were purified by column chromatography on silica gel. The esters were crystallized from benzene-hexane, derivatized as trimethylsilyl ethers for gas chromatography on a DB-1 capillary column and for gas chromatography-mass spectrometry with a DB-5 column, and mass spectrometry (MS) in the electron-impact (EI) positive-ion mode at 70 eV. The reaction is specific for FBA even in the presence of glycine and taurine conjugates of bile acids. The PCP esters were treated with benzylamine in chloroform or methanol to produce N-benzyl derivatives of FBA. The N-benzylamides were separated by high-performance liquid chromatography (HPLC) on a 4-microns Nova-Pak C18 column, studied by thermospray-LC-MS, and in the direct insertion probe-EI positive-ion mode.     

High-performance liquid chromatographic determination of alpha-keto acids in human serum and urine using 1,2-diamino-4,5-methylenedioxybenzene as a precolumn fluorescence derivatization reagent

A simple and highly sensitive high-performance liquid chromatographic (HPLC) method for the determination of alpha-keto acids in human serum and urine is described. In an acidic solution, twelve species of alpha-keto acids examined were converted by reaction with 1,2-diamino-4,5-methylenedioxy-benzene into highly fluorescent derivatives. The derivatives were separated isocratically by reversed-phase HPLC on a TSK gel ODS-80TM column and detected fluorimetrically. Eight alpha-keto acids in human serum and eleven alpha-keto acids in human urine can be determined simultaneously. The detection limits (signal-to-noise ratio = 5) are 6-44 fmol in an injection volume of 5 microliters. The intra-assay relative standard deviations for both serum and urine sample analyses are usually ca. 5%.     

Rapid and sensitive on-line precolumn purification and high-performance liquid chromatographic assay for bile acids in serum

A simple and rapid technique for the simultaneous isolation and analysis of fifteen kinds of bile acid was developed using reversed-phase high-performance liquid chromatography with an automatic dual-precolumn switching system. The serum samples were directly injected onto a first precolumn (hydroxyapatite), which was flushed with 1 mM phosphate buffer. Serum proteins were strongly retained on the hydroxyapatite column, but bile acids were unretained. The bile acids were absorbed on a second precolumn (Serumout-25) and eluted onto the analytical column with solvent B (acetonitrile-methanol-30 mM ammonium acetate, 20:20:60, v/v/v). For the separation of each bile acid, the gradient elution technique was used (solvent A was acetonitrile-methanol-30 mM ammonium acetate, 30:30:40). After separation of the bile acids, NADH was produced by use of immobilized 3 alpha-hydroxysteroid dehydrogenase column and then determined fluorimetrically (gamma em = 460 nm, gamma ex = 350 nm). The recoveries of bile acids in serum generally approached 100%.     

6-oxy-(acetyl piperazine) fluorescein as a new fluorescent labeling reagent for free fatty acids in serum using high-performance liquid chromatography

A new fluorescein-based fluorescent derivatizating reagent, 6-oxy-(acetyl piperazine) fluorescein (APF), has been designed, synthesized and developed for carboxylic acid labeling. It was used as a pre-column derivatizing reagent for the determination of seven free fatty acids (lauric acid, myristic acid, arachidonic acid, linoleic acid, palmitic acid, oleic acid, and stearic acid) with high-performance liquid chromatography (HPLC). The derivatization reaction of APF with seven fatty acids was completed at 60 degrees C for 1 h using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) as the condensing reagent. On a C18 column, the derivatives of APF with seven free fatty acids could be separated completely in 22 min using a mobile phase of methanol-water (88:12, v/v) containing 7 mmol L(-1) pH 6.5 Na2HPO4-H3Cit3 buffer with fluorescence detection at lambdaex/lambdaem=467/512 nm. The detection limits could reach 0.1-6.4 nmol L(-1) (signal-to-noise=3). This reagent was applied to the determination of the free fatty acids in human serum samples with satisfying recovery efficiencies varying from 93 to 105%.     

A highly sensitive determination of individual serum bile acids using high-performance liquid chromatography with immobilized enzyme

We have developed a highly sensitive method for the simultaneous determination of individual bile acids in serum using high-performance liquid chromatography (HPLC) combined with immobilized 3 alpha-hydroxysteroid dehydrogenase. Both the HPLC column and the immobilized-enzyme column are suitable for use with alkaline solutions necessary in working with this enzyme system. With this method we were able to determine simultaneously fifteen different serum bile acids.     

Rapid and accurate reversed-phase high-performance liquid chromatographic determination of conjugated bile acids in human bile for routine clinical applications. Therapeutic control during gallstone dissolution therapy

This paper introduces a new method to detect the taurine and glycine conjugates of five different bile acids (cholic acid, deoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid and lithocholic acid) in human bile. Advantages of this method are sufficient separation of compounds within a short period of time and a high rate of reproducibility. Using a mobile phase gradient of acetonitrile and water, modified with tetrabutylammonium hydrogen sulphate (0.0075 mol/l), we were able to maximize the differentiation between ursodeoxycholic acid and lithocholic acid, which is of primary interest during conservative gallstone dissolution therapy. Use of this gradient reduced analysis time to less than 0.5 h. Recovery rates for this modified method ranged from 94% to 100%, and reproducibility was 98%, sufficient for routine clinical applications.     

Quantitative high-performance liquid chromatographic determination of sulfur amino acids in protein hydrolysates

A rapid, quantitative high-performance liquid chromatographic procedure for the determination of methionine and cystine after oxidation to methionine sulfone and cysteic acid is described. The Dns derivatives of the amino acids are separated by reversed-phase chromatography with a phosphate buffer-acetonitrile gradient and detected by UV absorption at 254 nm. The procedure is validated by confirming the methionine and cystine content of ribonuclease A. The average yields of cysteic acid and methionine sulfone from triplicate analyses of ribonuclease A were 98.1% (±3.3) and 106.1% (±2.4) of the theoretical values, respectively.     

Separation and determination of saturated very-long-chain free fatty acids in plasma of patients with adrenoleukodystrophy using solid-phase extraction and high-performance liquid chromatographic analysis

An improved method was developed for the isolation of very-long-chain free fatty acids (VLCFFAs) in plasma and their separation and determination by high-performance liquid chromatography (HPLC). The method includes sample clean-up using solid-phase extraction, fluorophoric labelling of the FFAs and reversed-phase HPLC separation. The solid-phase extraction was carried out with aminopropyl-bonded phase columns. The FFAs were then derivatized with 9-anthryldiazomethane (fluorescent) reagent and separated by HPLC on an RP-18 column with methanol as the mobile phase. Using this method, the concentrations of C20:0, C22:0, C24:0 and C26:0 were determined in the plasma of five adrenoleukodystrophy (ALD) patients, one obligatory heterozygote, four healthy male volunteers and one child with cerebral leukodystrophy but without any other ALD symptoms. Statistically significant differences were found in the levels of C24 and C26 and in the ratios C24/C22 and C26/C22 in ALD patients and in normal controls. The values were higher in patients with X-ALD. This method therefore provides a rapid and accurate procedure for the laboratory confirmation of X-ALD.     

High-performance liquid chromatographic determination of free and total polyamines in human serum as fluorescamine derivatives

A highly sensitive and simple fluorimetric method for the determination of free and total polyamines, spermidine, spermine, putrescine and cadaverine, in human serum by high-performance liquid chromatography is described. The polyamines, obtained after clean-up of deproteinized serum by Cellex P column chromatography, are converted to their fluorescamine derivatives in the presence of nickel ion which inhibits the reaction of interfering amines with fluorescamine, and the derivatives are separated simultaneously by reversed-phase chromatography (LiChrosorb RP-18) with a linear gradient elution. The lower limits of detection are 10 and 15 pmole for spermine and the others in 0.5 ml of serum, respectively.     

Improved high-performance liquid chromatographic assay for the determination of ethionamide in serum

A solid-phase extraction (SPE) method was developed to simplify the preparation of human serum prior to high-performance liquid chromatography of ethionamide (ETA). Octadecyl SPE columns were used. Serum constituents were removed from the column with water, and ETA was eluted with methanol. Samples were evaporated to dryness, reconstituted in mobile phase, and assayed. The method is reproducible, with a recovery of ETA of 64%, comparable to the more tedious liquid-liquid extraction method for ETA.     

Gas chromatographic determination of serum bile acids — Application of a novel extraction procedure

Summary The profile of serum bile acids is a result of their liver metabolism and enterohepatic circulation.In the present work size exclusion chromatography is used for extraction of serum bile acids to optimize the methodology for analyzing serum bile acids by high resolution gas chromatography.Compared to other extraction methods like adsorption-[1–3] or reversed phase chromatography [4,5], this novel technique yielded a satisfactory recovery (75–104%) with high reproducibility. Therefore a reliable determination of serum bile acids is possible.     

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.     

Highly sensitive determination of free fatty acids in human serum by high-performance liquid chromatography with fluorescence detection

Endotoxins from four bacterial species extracted by three different procedures were acid-methanolyzed and the methyl esters of the fatty acids were analyzed by packed-column gas chromatography. There were qualitative and quantitative differences in the fatty acid profiles of the lipopolysaccharides isolated from four Gram-negative bacteria. Our data show considerable lot-to-lot variations in amounts of four methyl esters from the same bacterial serotype extracted by the same procedure and in the same bacterial serotype extracted by different procedures. These results indicate that extraction and perhaps culture conditions, as well as bacterial species, affect the fatty acid composition of endotoxins, hydrolyzed and derivatized by these procedures.     

Normal-phase high-performance liquid chromatographic determination of phenols

Normal-phase high-performance liquid chromatography has been used for separation of phenol and its monoderivatives. Multi-component mixtures of hexane (non-polar component) with butan-1-ol, chloroform, butyl bromide, butyl chloride or diethyl ether (polar additives) were used as selective eluents. Silica gel "Silasorb 600" with specific surface area of about 600 m(2)/g and average particle size of $ 10 mum was used as the sorbent. Phenol and the o-, m-, p-isomers of cresol were concentrated by extraction with n-butyl acetate from aqueous solutions. A method for determination of microamounts of phenols in aqueous solutions in the presence of 160-fold amounts of aromatic hydrocarbons has been developed.