Gas-liquid chromatographic analysis of volatile short chain fatty acids in fecal samples as pentafluorobenzyl esters.

A protocol was developed for the analysis of volatile short chain fatty acids in microsamples of feces, short chain fatty acid (SCFA) extraction was from fecal samples using ethanol incorporating n-hexanoic acid as an internal standard. The SCFAs were converted to pentafluorobenzyl esters with alpha-2,3,4,5,6-pentafluorotoluene and analyzed on a gas-liquid chromatograph equipped with an electron capture detector. One hundred milligrams of sample was routinely used but analysis could be carried out on 20 mg of sample.     

High-performance liquid chromatographic chiral separation of beta2-homoamino acids

Reversed-phase high-performance liquid chromatographic methods were developed for the separation of enantiomers of eleven unnatural beta(2)-homoamino acids on chiral stationary phases containing macrocyclic glycopeptides (teicoplanin-containing Chirobiotic T and T2) or the macrocyclic peptide teicoplanin aglycone (Chirobiotic TAG) as chiral selectors. The effects of the organic modifier, the mobile phase composition, temperature, and the structures of the analytes on the separations were investigated. Separations were carried out at constant mobile phase compositions in temperature range 7-45 degrees C and the changes in enthalpy, Delta(DeltaH(o)), entropy, Delta(DeltaS(o)), and free energy, Delta(DeltaG(o)), were calculated. The -Delta(DeltaG(o)) values obtained on the three columns indicated that Chirobiotic TAG, without sugar units, may promote the interactions of the enantiomers of beta(2)-homoamino acids with branched alkyl or aryl side-chains, whereas for beta(2)-homoamino acids with alkyl side-chains Chirobiotic T and T2 seem to be more favorable. The elution sequence was determined in some cases and was observed to be R < S.     

High-performance liquid chromatographic analysis of acylated lipids containing pyrene fatty acids

A high-performance liquid chromatographic method for the separation and quantification of acylated lipids containing pyrene fatty acids is described. The method is adapted from a procedure originally developed for the analysis of tissue lipids (Christie, W. W. (1985) J. Lipid Res. 26, 507-512). Pyrenyl lipid analogs ranging in polarity from cholesteryl ester to lysophosphatidylcholine are completely resolved on a silica column in 50 min by gradient elution with a ternary solvent system. Furthermore, pyrene-labeled triglycerides are resolved according to the number of pyrene fatty acid residues incorporated. Pyrenyl lipids are detected at levels of 10(-13) mol by high-sensitivity fluorescence detection. Accurate quantification of pyrenyl lipids is obtained by correcting peak areas for mobile-phase quenching effects. The close correspondence between chromatograms obtained for the separation of labeled lipids extracted from Hep-G2 cells incubated with either 12-(1-pyrenyl)dodecanoic acid (fluorescence detection) or [1-14C]oleic acid (radioactivity detection) indicates that this HPLC method is equally suitable for analysis of native lipids.     

High-performance liquid chromatographic separation of bile acid pyrenacyl esters with cyclodextrin-containing mobile phase

The high-performance liquid chromatographic separation of bile acid pyrenacyl esters with cyclodextrin-containing mobile phase is presented. Compared with conventional methods, inclusion chromatography gives much more satisfactory separation of derivatized bile acids in a short time. The application of this method to the separation of glycine-conjugated bile acids in human bile is also described.     

High-performance liquid chromatographic separation of heparin-derived oligosaccharides

Heparin has been enzymatically depolymerized with heparinase (heparin lyase (EC 4.2.2.7)) and then separated into di-, tetra-, hexa-, octa-, and decasaccharide mixtures by low-pressure gel-permeation chromatography (GPC). These sized mixtures were resolved by strong anion-exchange (SAX) HPLC into multiple components. The fractions from the SAX-HPLC were collected and characterized for size by GPC-HPLC and sulfate content by ion chromatography. This study provides detailed methodology for the separation of larger and more highly sulfated oligosaccharides than previously reported. It describes the first use of ion chromatography for the accurate determination of the sulfate content of heparin oligosaccharides, a method which can also be applied to heparin and other glycosaminoglycans.     

High-performance liquid chromatographic separation of bilirubin conjugates

A fast sensitive method for the isolation and quantitation of biliary bile pigments by reverse-phase high-performance liquid chromatography has been developed. Nine conjugates of bilirubin as well as unconjugated bilirubin and an internal standard, unconjugated mesobilirubin IX alpha, were all separated to baseline by gradient elution. The following sequence of eluted compounds was chemically identified by separating their ethyl anthranilate derivatives by thin-layer chromatography and by their enzymatic formation with UDP-bilirubin transferase and cosubstrate: bilirubin diglucuronide, bilirubin monoglucuronide monoglucoside, bilirubin monoglucuronide monoxyloside, bilirubin monoglucuronide (C-8, C-12), bilirubin diglucoside, bilirubin monoglucoside monoxyloside, bilirubin dixyloside, bilirubin monoglucoside (C-8, C-12), and bilirubin monoxyloside. The use of the commercially available mesobilirubin IX alpha as an internal standard was found to facilitate quantitation of the bilirubin conjugates.     

High-performance liquid chromatographic separation of fluorescent esters of hepoxilin enantiomers on a chiral stationary phase

Fluorescent anthryl (ADAM) derivatives of hepoxilins have been shown to possess good chromatographic properties affording good sensitivity for the high-performance liquid chromatographic analysis and detection of these compounds and related eicosanoids (12-hydroxyeicosatetraenoic acid) in biological samples. We report herein the separation of all possible stereoisomers of hepoxilins A₃ and B₃ as their methyl esters as well as their ADAM ester and acetate derivatives on a cellulose trisdimethylphenylcarbamate chiral stationary phase (Chiracel OD) in the normal-phase mode. This methodology is important to address the mechanistic route of biosynthesis of these products.     

High-performance liquid chromatographic determination of α-keto acids

A high-performance liquid chromatographic method has been developed for the determination of eight kinds of α-keto acids. These acids were derivatized with o-phenylenediamine into 2-quinoxalinol derivatives, which were extracted into chloroform. The quinoxalinol derivatives were separated by reversed-phase high-performance liquid chromatography using a 250 mm × 2.1 mm I.D. column packed with LiChrosorb RP-8 (5 μm). This method could be satisfactorily applied to urine samples without any prepurification.     

High-performance liquid chromatographic analysis and separation of N-feruloylserotonin isomers

The N-feruloylserotonin containing fraction was isolated from seeds of Leuzea carthamoides (Willd.) DC by solvent extraction followed by column chromatography on silica gel or on Sephadex LH-20. Nuclear magnetic resonance spectroscopic analysis of the isolated fraction showed the presence of four structurally related compounds. These compounds were identified as four isomers of N-feruloylserotonin: N-(Z)-feruloylserotonin, N-(Z)-isoferuloylserotonin, N-(E)-feruloylserotonin and N-(E)-isoferuloylserotonin. They were analyzed by HPLC on Separon SGX C18, Separon SGX and Separon SGX phenyl, using various mobile phases. Separon SGX phenyl phase was found the most efficient for a rapid analysis and for the final separation of the N-feruloylserotonin isomers.     

High-performance liquid chromatographic separation and electrochemical detection of cephalosporins

Pulsed amperometric detection (PAD) is useful for detection of cephalosporins following separation on a C₁₈ column using an acetate buffer solvent with a small percentage of organic modifier. Under these conditions, the indirect PAD mode worked better than direct PAD, with IPAD outperforming both. A gradient program was demonstrated that allowed separation and sensitive electrochemical detection of eleven different cephalosporins with widely differing side chain structures. The cephalosporins could be detected to sub-micromolar levels with this separation. Applications of the method for quantitation of pharmaceutical formulations and for monitoring cephalexin in porcine serum were demonstrated. To improve the detectability of cephalexin, an on-column concentration scheme using separate concentration and elution solvents was applied to porcine serum.     

High-performance liquid chromatographic separation and on-line mass spectrometric detection of saturated and unsaturated oligogalacturonic acids

A method for the simultaneous determination of saturated and unsaturated oligogalacturonic acids up to degree of polymerization (dp) of 7 by high-performance liquid chromatography (HPLC) is presented. For this purpose, a Cyclobond I 2000 column and a volatile mobile phase consisting of ammonium formate and methanol were used, allowing direct coupling of HPLC to a mass spectrometer via an electrospray interface (ESI-MS) without additional desalting. The analytical system was used for the characterization of digests obtained by incubation of polygalacturonic acid with commercial enzyme preparations.     

High-performance liquid chromatographic separation of the biotransformation products of oxaliplatin

A novel single reversed-phase HPLC system was developed for separating oxaliplatin and its biotransformation products formed in rat plasma. The major stable biotransformation products of oxaliplatin formed in rat plasma were identified as Pt(dach)(Cys)₂, Pt(dach)(Met) and free dach. The minor biotransformation products Pt(dach)Cl₂, Pt(dach)(GSH) and Pt(dach)(GSH)₂ could also be resolved from other Pt-dach complexes. Among these biotransformation products, the identification of Pt(dach)(Met) was further confirmed by LC–ESI-MS, and the identification of Pt(dach)(Cys)₂, Pt(dach)(GSH), Pt(dach)(GSH)₂ and free dach was confirmed by atomic absorption and double isotope labeling. This HPLC technique should prove useful for separating and identifying the biotransformation products of Pt-dach drugs such as oxaliplatin, ormaplatin and Pt(dach)(mal) in biological fluids. This will allow a more complete characterization of the pharmacokinetics and biotransformations of these Pt-dach drugs, which should in turn lead to a better understanding of the mechanisms leading to their toxicity and efficacy.     

High-performance liquid chromatographic separation of leghemoglobins from soybean root nodules

A crude fraction of soybean nodule ferri-leghemoglobin was absorbed onto a commercial DEAE HPLC column at pH 8.0, and resolved into eight isoprotein fractions. The identity of the leghemoglobins were determined by their order of elution from the DEAE column and by isoelectric focusing, using isoprotein standards isolated by conventional procedures. The three isoproteins of the c complex, c1, c2, c3, were not resolved. Unexpected heme containing proteins eluted just after leghemoglobin a and the c complex. These components possessed proteins similar to leghemoglobin a and the c complex, respectively, as judged by isoelectric focusing and absorbance spectra of the ferri and ferrous forms. The components designated leghemoglobin a' and leghemoglobin c' were also differentiated from leghemoglobin a and c by reverse-phase HPLC in a C18 column. Amounts of protein for the DEAE HPLC column ranged from 10 micrograms to 20 mg and sample volumes ranged from 2 to 250 microliters. The time required for chromatography varied depending on the gradient used, but never exceeded 40 min for samples up to 5 mg protein or 120 min for samples containing 5 to 20 mg protein. Due to the sensitivity of detection at 403 nm and leghemoglobins being the predominant chromophore at that wavelength, it was possible to quantitate levels of individual leghemoglobins in samples extracted from single nodules (ca. 15 to 65 mg fresh weight tissue). Quantitation was performed by interfacing the spectrophotometer output (10 mV) to a microcomputer and using commercially available software.     

High-performance liquid chromatographic separation of fluoroquinolone enantiomers: a review

Fluoroquinolones are antibacterial agents widely used clinically. In recent years, there has been an important development of new derivatives, and more than 7000 analogues have been described today. Different fluoroquinolones (FQ) have one or two chiral centers in their chemical structure and are available as racemates, diastereoisomers, or pure enantiomers. The clinical and pharmaceutical uses of these compounds need effective analytical procedures for quality control and pharmacodynamic and pharmacokinetic studies. This review article focuses on the high-performance liquid chromatographic separation of fluoroquinolone stereoisomers by the use of derivatization methods and ligand exchange (LE) or chiral liquid chromatography.     

High-performance liquid chromatographic separation of glycopeptides from Nereis cuticle collagen

To facilitate the structural studies of invertebrate collagens, a sensitive and effective method was developed, using reverse-phase high-performance liquid chromatography for preparative isolation of the collagen subunits and their clostridial collagenase-derived peptides; the methods have been applied to Nereis cuticle collagen. The two subunits of denatured Nereis cuticle collagen, termed A and B, were initially separated by high-performance liquid chromatography. These polypeptides, with Mr of about 0.5 million, were each exhaustively digested with clostridial collagenase. The digest of the A subunit, which contains all of the uronic acid, was enriched for the uronic acid-containing glycopeptides by means of gel filtration. These glycopeptides were resolved into 23 major peaks, using reverse-phase HPLC, over a 5-h elution time, with an acetonitrile gradient (0-20%) containing 0.1% TFA. The amino acid composition data suggests that the peptides are of variable length, from 5 to 17 residues, while beta-elimination studies show that the uronic acid-containing moieties are all O-glycosidically linked to threonine residues, in the peptides examined. The amino acid sequence of one of the major glycopeptides was determined and found to be Gly-Hyp-Ala-Gly-Gly-Ile-Gly-Glu-Thr-Gly-Ala-Val-Gly-Leu-Hyp. The amino acid compositions of glycosylated and nonglycosylated peptides which had eluted, numbering about 100, showed a correspondence between hydrophobicity or hydrophilicity and emergence time from the column. We also found that the peptides most enriched in 4-hydroxyproline emerged earliest. These studies provide a foundation for elucidating the detailed structures of the large, unusual subunits of a well-characterized cuticle collagen.     

High-performance liquid chromatographic analyses of hydroxymonocarboxylic acids and dicarboxylic acids in urine as their 2-nitrophenylhydrazides

Both hydroxymonocarboxylic acids and dicarboxylic acids in urine were converted into their 2-nitrophenylhydrazides without lengthy and cumbersome sample workup and were separated from each other by two-step extraction with diethyl ether at different pH values. HPLC analysis of each acid group was achieved isocratically within 30 min. By the use of a visible-range detector (400 nm) the detection limits ranged from 1 to 2 pmol and from 2 to 5 pmol per injection for the hydroxymonocarboxylic acids and dicarboxylic acids, respectively. The analytical results showed good recovery and reproducibility. Analysis profiles of the two acid groups in normal and diabetic subjects could be performed with 200 microliters of urine. The present method is superior over previously published methods because of its great simplicity and its time-, cost-, and labor-saving nature.     

High-performance liquid chromatographic separation and detection methods for anabolic compounds

The role of high-performance liquid chromatography (HPLC) in methods of analysis for anabolic compounds in biological samples is reviewed. Special attention is given to both the separation and detection of anabolic compounds. A distinction is made between on-line detection systems, such as ultraviolet detection and diode-array detection, and off-line detection methods with special emphasis on immunochemical detection methods using non-isotopic labels. A number of applications are given to elucidate the possibilities of HPLC in the analysis of anabolic compounds.     

High-performance liquid chromatographic separation and quantitation of tetrapyrroles from biological materials

We describe a rapid, reverse-phase HPLC procedure for separating and quantifying tetrapyrroles of biological interest. This procedure uses a 5-micron C18 column and the mobile phase is ammonium phosphate (pH 3.5) with a methanol gradient that is increased from 61 to 100%. Detection is by absorbance at 405 nm or by fluorescence. Porphyrins, heme, and the heme breakdown products, biliverdin and bilirubin, can be separated from a single injection in 25 min. Injections can be made every 40 min. Limits of detection are about 0.1 pmol for porphyrins, 5 pmol for heme, and 10 pmol for biliverdin and bilirubin. We present examples of the use of the system for separating tetrapyrroles formed by primary cultures of chick embryo hepatocytes and homogenates of rat liver.     

High-performance liquid chromatographic separation of lipoxygenase isozymes in crude soybean extracts

A mixture of the soybean lipoxygenase isozymes purified by conventional methods was readily resolved by high-performance liquid chromatography using a SynChropak AX-300 anion-exchange column. Analysis of crude soybean extract by this procedure showed the presence of four different lipoxygenase activities. Mutant soybeans lacking in the isozymes lipoxygenase-1 and -3 were used to test the application of this procedure.