Separation of bile acid methyl esters by high-performance liquid chromatography

Conjugated bile acids, namely glyco- and tauro-3α,6α-dihydroxy-5β-cholanoic acid (hyodeoxycholic acid), 3α,7α-dihydroxy-5β-cholanoic acid (chenodeoxycholic acid), 3α,6α,7α-trihydroxy-5β-cholanoic acid (hyocholic acid) and 3α-hydroxy-6-oxo-5β-cholanoic acid (6-keto-litocholic acid) were isolated from pig bile, and subsequently transformed into the corresponding methyl esters. Separation of the methyl esters of the isolated bile acids by high-performance liquid chromatography (HPLC) was accomplished on a ZORBAX-CN column (Dupont, Boston, MA) withn-hexane/2-propanol/methylene chloride (89∶6∶5, by vol) as the mobile phase containing traces (≈1%) of amyl alcohol and water as moderators. HPLC analysis of the methyl esters also showed the presence of methyl 3α-hydroxy-6-oxo-5α-cholanoate, which was probably produced in the course of alkaline hydrolysis of the conjugated bile acids.     

Separation of monoacylglycerols by high-performance liquid chromatography on nitrile-bonded phase

Monoacylglycerol molecular species, as their di-3,5-dinitrophenylurethane derivatives, were well separated by normalphase high-performance liquid chromatography on nitrilebonded phase. The peaks emerged in the order 20∶0, 18∶0, 16∶0, 18∶1, 16∶1, 18∶2, and 18∶3. The peaks of 1- and 2-monoacylglycerols with the same acyl group showed complete overlapping. This method could be applied to get acyl compositions in the three positions of triacyl-sn-glycerols in their stereospecific analysis. Presented partially at the 83rd AOCS Annual Meeting held in Toronto, Canada, May 10–14, 1992.     

Separation and identification of isomeric conjugated fatty acids by high-performance liquid chromatography with photodiode array detection

A simple, high-performance liquid chromatographic method is described for the separation of tetraenoic, trienoic and dienoic conjugated fatty acids on a Zorbax ODS reversed-phase column using acetonitrile/tetrahydrofuran (95∶5, vol/vol) at a flow rate of 1.2 mL/min as mobile phase. Also described is the separation of the isomeric conjugated fatty acids with acetonitrile/water/tetrahydrofuran (90∶90∶1, by vol) as mobile phase. The simultaneous detection and identification of the separated geometrical isomers in the eluant was accomplished using photodiode array detection.     

Separation of conjugated bile acids by reverse phase thin layer chromatography

Seven conjugated bile acids were separated by 1-dimensional reverse phase thin layer chromatography using ethanol/0.3 M calcium chloride/dimethylsulfoxide (25∶25∶2) as mobile phase.     

Separation of wax esters from olive oils by high-performance liquid chromatography

To detect adulteration of olive oil with solvent-extracted oils, the determination of the wax ester content has become more important during recent years. Hence, a greater number of wax ester analyses need to be performed by quality control laboratories. The most common method in use requires a liquid chromatographic (LC) separation of the less polar fraction, which contains the wax esters, from the glyceride matter on a hand-filled silica gel column. The aim of this project was to verify the possibility of replacing LC with high-performance liquid chromatography by taking advantage of the greater reliability and repeatability of this technique, as well as of the possibility of making the separation automatic. The paper describes how to perform the analysis and the statistical test that was carried out; furthermore, a comparison has been made with the usual method and results are in good agreement.     

Ion-pair high-performance liquid chromatography of bile salt conjugates: Application to pig bile

The high-performance liquid chromatographic separation and quantitation of conjugated bile salts from pig bile is reported. Synthetic standards and bile samples were chromatographed on a C₁₈ reversed phase column using acetonitrile/water/tetrabutyl ammonium phosphate as an isocratic mobile phase at a flow rate of 1 mL/min. Detection of the ion-pairs was at 214 nm. The method permits efficient separation of all conjugated pig biliary bile salts without prior modification or treatment of the samples. Analysis of 12 pig biles showed that 85% of the bile salts are conjugated to glycine. The three main conjugated bile salts were glyco-3α,6α,7α-trihydroxy-5β-cholanoic acid (GHC), glyco-3α,7α-dihydroxy-5β-cholanoic acid (GCDC), and glyco-3α,6α-dihydroxy-5β-cholanoic acid (GHDC). Glyco-3α-hydroxy-6-oxo-5β-cholanoic acid (G3α6oxo), tauro-3α,7α-dihydroxy-5β-cholanoic acid (TCDC), tauro-3α,6α,7α-trihydroxy-5β-cholanoic acid (THC), and tauro-3α,6α-dihydroxy-5β-cholanoic acid (THDC) were found to contribute each for 4 to 5% ot the total. An excellent correlation was found between the sum of conjugated bile salts quantitated by high-performance liquid chromatography (HPLC) and values obtained by conventional enzymatic assay. Simplicity, efficiency and relative rapidity of the method render it suitable for routine analyses.     

Separation of tripalmitin from its hydrolysis products by simple isocratic reversed-phase high-performance liquid chromatography

An isocratic reversed-phase high-performance liquid-chromatographic method was developed for monitoring the lipase-catalyzed hydrolysis of solid triglycerides in supercritical carbon dioxide. The reaction products obtained consist of free fatty acids, monoglycerides, diglycerides, and unreacted triglycerides. For the method, developed with tripalmitin, a mobile phase of acetonitrile/chloroform/acetic acid (50:50:1, vol/vol/vol), a C₁₈ column, and refractive index detection were used. Analysis time is 7 min. Response factors were determined for each neutral lipid class, which permitted quantitation of the hydrolysis product mixture.     

Analysis of conjugated bile acids by high performance liquid chromatography and mass spectrometry

Because of the known advantages of coupling high performance liquid chromatography with mass spectrometry (HPLC-MS) in biological fluids, studies on the reversed-phase HPLC-MS system for direct analysis of conjugated bile acids in human bile samples are described. Ten samples of gallbladder bile of apparently healthy subjects were examined. The amounts of each tauro- and glycoconjugated bile acid as trifluoracetate were determined by mass fragmentography. Quantitation of at least 1 ng of each bile acid was possible.     

Separation of molecular species ofcis- andtrans-triacylglycerols intrans-hardened confectionery fats by silver-ion high-performance liquid chromatography

Silver-phase high-performance liquid chromatography (HPLC) on silver nitrate-loaded silica achieves incomplete separation of major triacylglycerol (TAG) classes present intrans-hardened fats. The “ChromSpher Lipids” silverloaded cation exchange HPLC column has been found to yield good separations oftrans-hardened TAG, with molecular species well resolved. Separations comparable to those previously possible for nonhardened fats are now possible fortrans-hardened fats. The separation is on the basis of number and type (i.e.cis/trans) of double bonds only; the position of the double bond along the acyl group appears not to influence the separation significantly. The analysis of a palm fraction, hardened to a slip melting point of 37°C and chemically randomized, is presented as an example. This technique offers a new approach to understanding and controlling the hydrogenation and processing oftrans-hardened fats.     

Bile acids LVII. Analysis of bile acids by high pressure liquid chromatography and mass spectrometry

Several high pressure liquid chromatographic methods for the separation of conjugated and free bile acids are presented. A mixture of synthetic conjugated bile acids has been separated by reverse-phase systems consisting of either a Waters Associates’ “fatty-acid analysis” or a μBondapak/C₁₈ column eluted with a mixture of 2-propanol/potassium phosphate buffer (pH 2.5 or 7.0). The major conjugated bile acids present in the gallbladder bile of obese subjects have been analyzed each in less than 30 min and quantitated with a U.V. detector set at 193 nm. Some of the 5α- and 5β-isomers of conjugated bile salts could be resolved in straight-phase systems on Corasil II or μPorasil columns. Mass spectra of the conjugated bile acids obtained by electron impact were characteristic of the type of amino acid attached to the side chain, and the number of hydroxyl substituents on the nucleus. Most of the isomers could readily be differentiated by the relative intensities of the fragment ions.     

Separation and identification of eugenol in ethanol extract of cloves by reversed-phase high-performance liquid chromatography

A method has been developed for the separation and identification of eugenol in the alcohol extract of cloves directly without having to previously separate other components. Extraction of eugenol with alcohol is followed by analysis with high-performance liquid chromatography. Separation by reversed-phase chromatography on low-polarity μBondapak C₁₈ was achieved with isocratic elution. Tentative identification is based on chromatographic appearance of a peak with retention time 5.54 min for pure standard reference eugenol. The identification of eugenol was confirmed by gas chromatography/mass spectrometry analysis.     

Gas-liquid chromatography of bile acids

Methods are reviewed for the preparation, gasliquid chromatographic (GLC) separation, identification, and quantitative estimation of the trifluoroacetyl derivatives of bile acid methyl esters. Of the stationary phases tried (SE-30, QF-1 and XE-60) methylfluoroalkyl silicone (QF-1) was best suited for analysis of the trifluoroacetates. This phase (1–2% QF-1 on 100–120 mesh Gas Chrom P) allowed an orderly resolution of conformational isomers and consistently gave GLC columns (stainless steel tubes, 1/8 in. O.D. × 3 ft) from which the bile acid derivatives could be recovered in high yield. Applications to biological materials are illustrated with bile acid samples from animal biles and from human duodenal drainage and feces. Identifications of the major bile acids made by the GLC of the trifluoroacetates were confirmed by results obtained with bile acid methyl esters and bile acid methyl ester acetates on QF-1 and the other liquid phases investigated. For most mixtures of bile acids, however, it appears that GLC of methyl esters and methyl ester trifluoroacetates on QF-1 is sufficient for a reliable recognition of common bile acids. Overall accuracy of the estimates was of the order of ± 5%, but it varied with the nature and concn of the component.     

Direct analysis of trivernolin by high-performance liquid chromatography

A rapid liquid chromatographic method for the quantitation of trivernolin in epoxy seed oils has been developed. A weighed sample of the oil is dissolved in chloroform (～100–300 mg/ml), and a 1–20µl portion of the oil is chromatographed on a 2 ft×1/4 in. OD C₁₈ µ-Bondapak column with acetonitrile-acetone (2:1, v/v). The area of the trivernolin peak from the differential refractometer recorder tracing is converted toµg trivernolin by use of a calibration curve of peak area vs. trivernolin weight.     

Determination of free fatty acids in marine phytoplankton causing red tides by fluorometric high-performance liquid chromatography

The proportion and content of free fatty acids (FFA) in four species of causative phytoplankton of red tides, cultured in axenic conditions, were obtained by fluorometric high-performance liquid chromatography. Twelve FFA in phytoplankton were identified. Raphidophyte flagellates,Chattonella antiqua andHeterosigma akashiwo, contained highly unsaturated fatty acids and 16:0 acid as the predominant FFA. Major FFA in diatoms,Skeletonema costatum andChaetoceros didymum, were 14:0, 16:0, 16:1, and 20:5 acids.     

Scale-up purification for rutin hyrdrolysates by high-performance counter-current chromatography coupled with semi-preparative high-performance liquid chromatography

The study made the isolation of three constituents from rutin and exemplified how to achieve quercetin separation and quantification in rapid and scale-up processes by high-performance counter-current chromatography (HPCCC). Meanwhile, we also isolated the other two constituents, kaempferol and isorhamnetin, by semi-preparative high-performance liquid chromatography. After systematic optimization of solvent systems, sample concentration and flow rate on analytical Mini-DE centrifuge, the same conditions were scale-up by 50-fold in preparative Midi-DE centrifuge to purify quercetin, and the maximum sample loading achieved 1.65 g. Eventually, we successfully isolated quercetin, kaempferol and isorhamnetin with the purity of 99.80%, 99.84% and 99.95%, respectively. The process, therefore, provided an efficient method of obtaining sufficient quantities of highly purified quercetin, as well as kaempferol and isorhamnetin.     

高效液相色谱法测定咪·三氟水剂中咪草烟和三氟羧草醚的含量

本文报道了采用高效液相色谱法同时测定咪·三氟水剂中咪草烟和三氟羧草的方法 ,采用C18不锈钢柱 ,流动相乙腈∶水 (75∶2 5 ) ,检测波长 2 5 4nm ,咪草烟和三氟羧草醚平均回收率分别为 98 5 %～10 0 2 %和 99 4 %～ 10 0 2 % ,标准偏差分别为 0 10和 0 2 3,线性相关系数分别为 2 4 7%和 1 0 7%。该方法准确、方便。     

Stereospecific analysis of triacyl-sn-glycerols by chiral high-performance liquid chromatography

A method for the stereospecific analysis of triacyl-sn-glycerols by high-performance liquid chromatography (HPLC) on a chiral column is described. Triacyl-sn-glycerols were partially degraded with ethyl magnesium bromide, and the monoacylglycerols produced were separated as 1- and 2-monoacylglycerol fractions by thin-layer chromatography on boric acid-impregnated silica gel plates. The 1-monoacylglycerols were resolved intosn-1 andsn-3 monoacylglycerol fractions by HPLC on a chiral column (Sumichiral OA-4100) after derivatization with 3,5-dinitrophenyl isocyanate. Fatty acid methyl esters obtained from the original triacyl-sn-glycerols, 1- and 2-monoacylglycerol fractions, andsn-1 andsn-3 monoacylglycerol fractions were analyzed by gas chromatography on open-tubular columns. Stereospecific acyl distributions in triacyl-sn-glycerols were calculated from the data. The acyl distributions of several oils were obtained. The method is rapid, simple and gave reproducible results.     

Acidic hydrolysis of plasmalogens followed by high-performance liquid chromatography

A simple, quantitative method for determining the plasmalogen content of small samples is reported here. The method uses the different susceptibility to acid-catalyzed hydrolysis of the alkyl, alkenyl and acyl linkages to separate the plasmalogen subclass from the other two non-labile subclasses. Hydrolysis of plasmenylethanolamine and plasmenylcholine was complete after 4 and 1 min of acid treatment, respectively. The acid-catalyzed hydrolysis did not alter the phospholipid fatty acid composition, making this method useful for fatty acid compositional analysis of the plasmalogen subclass. High-performance liquid chromatography was used for separations, and phospholipids were quantitated by assay of lipid phosphorus or by direct quantitation of peak area. Using this method, small amounts (10 nmol) of ethanolamine glycerophospholipid and choline glycerophospholipid are subjected to acid-catalyzed hydrolysis and subsequent separation of the resulting lysocompounds obtained from plasmalogens from the more acid-stable alkylacyl and diacyl glycerophospholipid fractions. Our values for plasmalogens from commercial preparations of choline and ethanolamine glycerophospholipids agree with literature values. The usefulness of the method is demonstrated for small glycerophospholipid samples that are equivalent to samples from cultured neural cells.     

Separation and determination of glycolipids from edible plant sources by high-performance liquid chromatography and evaporative light-scattering detection

Glycolipids from edible plant sources were accurately quantified by silica-based, normal-phase high-performance liquid chromatography using an evaporative light-scattering detector. Five major glycolipid classes (acylated steryl glucoside, steryl glucoside, ceramide monohexoside, monogalactosyldiacylglycerol, and digalactosyldiacylglycerol) were separated and determined with a binary gradient system consisting of chloroform and methanol/water (95∶5, vol/vol) without any interference from other lipid classes and pigments. The described method was applied to 48 edible plants available in Japan including cereals, legumes, vegetables, and fruits. Examined plant species contained glycolipids in wide concentration ranges, such as 5–645 mg/100 g tissue.     

Separation and purification of six isoflavones from Iris tectorum Maxim by macroporous resin-based column chromatography coupled with preparative high-performance liquid chromatography

ABSTRACT

A rapid and robust preparation method of six isoflavones from ethanol aqueous extract of Iris tectorum Maxim (I. tectorum) was established by using macroporous resin column chromatography and preparative high-performance liquid chromatography (Prep-HPLC). After separation by AB-8 resins, total flavonoids content increased from 10.60% in the crude extract to 54.20% with a recovery yield of 75.12%. Subsequently, the extracts were purified by Prep-HPLC, and the purities were more than 82.2% after assessment by analytical HPLC and characterization by mass spectrometry. The established method is expected to be used for preparing available quantities of pure isoflavones from I. tectorum.