Separation of neutral lipid,free fatty acid and phospholipid classes by normal phase HPLC

Normal phase high performance liquid chromatography methods are described for the separation of neutral lipid, fatty acid and five phospholipid classes using spectrophotometric detection at 206 nm. Separations were accomplished in less than 10 min for each lipid class. A mobile phase consisting of hexane/methyltertiarybutylether/acetic acid (100∶5∶0.02) proved effective in separating cholesteryl ester and triglyceride with recoveries of 100% for radiolabeled cholesteryl oleate and 98% for radiolabeled triolein. Free fatty acid and cholesterol were separated by two different mobile phases. The first, hexane/methyltertiarybutylether/acetic acid (70∶30∶0.02) effectively separated free fatty acids and cholesterol, but did not separate cholesterol from 1,2-diglyceride. A mobile phase consisting of hexane/isopropanol/acetic acid (100∶2∶0.02) effectively separated free fatty acid, cholesterol, 1,2-diglyceride and 1,3-diglyceride. Recoveries of oleic acid and cholesterol were 100% and 97%, respectively. Five phospholipid classes were separated using methylteriarybutylether/methanol/aqueous ammonium acetate (pH 8.6) (5∶8∶2) as the mobile phase. The recoveries of phosphatidylinositol, phosphatidylethanolamine, phosphatidylcholine, sphingomyelin and lysophosphatidylcholine were each greater than 96%.     

High-performance liquid chromatography of human milk triacylglycerols and gas chromatography of component fatty acids

Human milk triacylglycerols were separated by high-performance liquid chromatography. A 5-μ Supelcosil LC-18 column (Supelco, Inc., Bellefonte, PA) was used with acetone/acetonitrile (64∶36, vol/vol) as mobile phase. Triacylglycerols were tentatively identified based on theoretical carbon number and relative retention time. Despite changes resulting from dietary fat variation, the major component triacylglycerols were those composed of palmitic, oleic and linoleic acids. Triacylglycerols with palmitic, stearic and oleic acids were present as minor components. Fatty acids were quantified by gas chromatography relative to an internal standard. Ratios of n−6/n−3 fatty acids were found to be high than previously reported. Based on a paper presented at the Symposium on Milk Lipids held at the AOCS Annual Meeting, Baltimore, MD, April 1990.     

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 sulfated bile acids by high-performance liquid chromatography

In order to establish a new method for simultaneous determination of sulfated bile acids without deconjugation, separation of 3-sulfates of unconjugated, glyco- and tauroconjugated bile acids by high-performance liquid chromatography has been undertaken. The preliminary experiment indicated that reversed phase chromatography on an ODS SC-02 column using ammonium carbonate aqueous solution/acetonitrile as a mobile phase would be promising. The ratio of peak width to retention time was varied depending upon the salt concentration. The content of water in mobile phase exerted an influence on the capacity ratio to a certain extent, but not on the resolution. The use of 0.5% ammonium carbonate/acetonitrile (26∶8 and 20∶8, v/v) was found to be suitable for complete resolution of sulfated cholate, chenodeoxycholate, deoxycholate, lithocholate and their glyco- and tauro-conjugates.     

Rapid separation of the major phospholipid classes on a single aminopropyl cartridge

A rapid method for the separation of the individual phospholipid classes phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylinositol (PI) by a single solid-phase extraction was developed. PC, PE, PS and PI were sequentially eluted from aminopropyl bonded silica with acetonitrile/n-propanol (2∶1, vol/vol), methanol, isopropanol/methanolic HCl (4∶1, vol/vol) and methanol/methanolic HCl (9∶1, vol/vol). Standard recoveries were over 95% for PC and PE and over 85% for PS and PI with undistorted fatty acid composition. The separation of complex lipid mixtures on aminopropyl minicolumns can be refined to the level of individual phospholipid classes.     

Identification of natural diacylglycerols as the 3,5-dinitrophenylurethanes by chiral phase liquid chromatography with mass spectrometry

This study reports a facile identification of the molecular species of enantiomeric diacylglycerols by combining chiral phase high-performance liquid chromatography with positive chemical ionization mass spectrometry. For this purpose the 3,5-dinitrophenylurethane (DNPU) derivatives ofsn-1,2(2,3)-diacylglycerols are separated on an (R+)-naphthylethylamine polymer column (25 cm × 0.46 cm ID) using an isocratic solvent system made up of hexane/dichloroethane/acetonitrile (85∶10∶5, by vol) or isooctane/tert-butyl methyl ether/acetonitrile/isopropanol (80∶10∶5∶5, by vol). About 1% of the column effluent (1 mL/min) was admitted to a quadrupole mass spectrometer (Hewlett-Packard, Palo Alto, CA)via direct liquid inlet interface, and positive chemical ionization spectra were recorded over the range of 200–900 mass units. The DNPU derivatives of diacylglycerols yield characteristic [M-DNPU]⁺ and [RCO+74]⁺ ions for each diacylglycerol species from which the molecular weight and exact pairing of fatty acids can be unequivocally obtained. The characteristic ions appear to be generated in nearly correct mass proportions as indicated by preliminary quantitative comparisons. The abbreviations 14∶0, 16∶1, 18∶2, etc. represent normal chain fatty acids of 14, 16, 18, etc. acyl carbons and 0, 1, 2, etc. double bonds, respectively; 16∶0–18∶1, etc. represent diacylglycerols containing 16∶0 and 18∶1 fatty acids of unspecified positional distribution;sn indicates stereospecific numbering of glycerol carbons;sn-1,2-diacylglycerols andsn-2,3-diacylglycerols are enantiomeric diacylglycerols of unspecified fatty acid composition;rac-1,2-diacylglycerols are racemic diacylglycerols representing equal amounts ofsn-1,2-andsn-2,3-enantiomers;sn-1,2(2,3)-diacylglycerols are a mixture ofsn-1,2-andsn-2,3-diacylglycerols of unspecified proportion of enantiomers and unspecified fatty acid compisition and positional distribution; X-1,3-diacylglycerols are diacylglycerols of unspecified fatty acid composition and reverse isomer content.     

Chromatographic separation of glucopyranosyl sinapate from canola meal

The compound 1-O-β-D glucopyranosyl sinapate (GPS), a phenolic glycoside, was separated from ethanolic extracts of defatted canola meal by a two-step chromatographic method. The first step involved Sephadex LH-20 chromatography with methanol as the eluting solvent. The solvent from the fraction containing GPS was evaporated, and glucopyranosyl sinapate was subsequently separated by a semi-preparative high-performance liquid chromatography method with an RP-18 column and a mobile phase consisting of water/acetonitrile/acetic acid (88∶10∶2, vol/vol/vol).     

Analysis of north atlantic and baltic fish oil triacylglycerols by high-performance liquid chromatography with a silver ion column

Triacylglycerols from Atlantic herring (Clupea harengus), sandeel (Ammodytes sp.) and Baltic herring (Clupea harengus membras) have been fractionated by silver ion high-performance liquid chromatography. An ion exchange column loaded with silver ions was the stationary phase, and a gradient in the mobile phase from 1,2-dichloroethane/dichloromethane (1∶1, v/v) to acetone and then to acetone/acetonitrile (2∶1, v/v) was used to effect the separation with light-scattering (i.e., mass) detection. Fractions were collected via a streamsplitter, and fatty acid methyl esters were prepared by transesterification in the presence of an internal standard for identification and quantification by gas liquid chromatography. Triacylglycerols were separated according to the number of double bonds in the fatty acyl residues. Resolution was excellent at first, when the least unsaturated molecules eluted (trisaturated to dimonoene-monodiene fractions). Base-line resolution could no longer be achieved when molecules containing trienoic or more highly-unsaturated fatty acids began to elute because of overlapping components. Nonetheless, some valuable separations of species containing two saturated and/or monoenoic fatty acids and one polyenoic fatty acid were achieved. Double bond indices (average number of double bonds in each triacylglycerol molecule) were calculated to estimate the separations possible. Fractions containing at least 11–14 double bonds per molecule were obtained.     

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.     

A multivariate optimization of triacylglycerol analysis by high-performance liquid chromatography

In the present study we have used statistical experimental design and multivariate optimization to formally optimize a reversed-phase high-performance liquid chromatography method for the analysis of triacylglycerol molecular species of natural oils. The optimal conditions found were, on an octadecylsilan-column, from acetonitrile/isooctane (90∶10, vol/vol) to acetonitrile/ethanol/isooctane (40∶35∶25, by vol), at a column temperature of 50°C and a flowrate of 1.5mL/min using a negative exponential gradient profile. Several examples of separations of natural seed and animal oils,i.e., soybean oil, rapeseed oil, palm oil, linseed oil, tallow and fish oil, are given. A version of the equivalent carbon number concept, utilizing the Snyder polarity index, was used to identity the molecular species.     

Optimization and validation of high-performance liquid chromatographic method for the determination of dowtherm ATM in edible oils and oleochemicals

A method using high-performance liquid chromatography and fluorescence detection is optimized and validated for the determination of Dowtherm A^TM in spiked oleochemicals and edible oils. The samples are directly injected into a reversed-phase C18 column, and Dowtherm A is detected using a fluorescence detector set at 247 nm excitation and 310 nm emission wavelengths. The simple isocratic mobile phase used is a mixture of methanol and water (90∶10, vol/vol) at a flow rate of 1 mL/min. The limits of quantitation are from 0.1 to 0.2 μg/g. Mean recoveries ranged from 93.0 to 116% with reproducibilities of 1.29–3.84%. The procedure provides a simple, reliable and sensitive method for determining Dowtherm A residue in oleochemicals and edible oils without prior sample cleanup or extraction.     

The fatty acids ofEntomophthora coronata

The total lipids and fatty acid composition ofEntomophthora coronata were determined. The fungus was grown on a chemically defined medium and a chemically nondefined medium (Sabouraud dextrose yeast extract) for a period of 26 days. The organism contained from 16.2% to 44.6% total lipids depending upon the days of growth. The major fatty acids were 12∶0 (5.5–9.0%), 13∶0 (1.2–8.2%), 14∶0 (33.5–43.5%), 16∶0 (9.7–13.9%), 18∶1₉ (20.4–22.4%), and 18∶2_9,12 (3.5–10.5%). Lesser amounts of 15∶0, 16∶1, 16∶2, 17∶0, 18∶0, two other 18∶2 (both having conjugated double bonds), 18∶3_6,9,12, another 18∶3 (conjugated double bonds), 20∶3_8,11,14, 20∶4_5,8,11,14, another 20∶4 (conjugated double bonds), and 24∶1 acids were found. Trace amounts of 20∶0, 20∶1, 20∶2, 22∶0 and 24∶0 were also present. The relative percentage of most of the fatty acids did not vary appreciably with growth. However, 18∶2_9,12 and 20∶4_5,8,11,14 increased with age of the chemically defined culture. Peak E (18∶2, conjugated double bonds) increased and 13∶0 and 18∶3_6,9,12 decreased with age of the chemically nondefined culture. The fatty acids were predominately saturated (56.9–69.1%) and contained a high percentage of shorter chain fatty acids (C 12 to C 15). The fatty acids of the chemically defined culture were more unsaturated than the Sabouraud culture and the unsaturation increased with age of the culture.     

Determination of squalene in olive oil using fractional crystallization for sample preparation

A simple, rapid method for the determination of squalene in virgin olive oil was developed using RP-HPLC with detection at 208 nm. Fractional crystallization from methanol/acetone (7∶3, vol/vol) was applied to obtain squalene in the liquid fraction of the oil prior to HPLC. Elution of squalene was then carried out isocratically with acetone/acetonitrile (40∶60 vol/vol) within 11 min. The detection limit was 23 mg/kg, and the limit of quantification 79 mg/kg. The precision of the crystallization procedure (CV%=3.76, n=7) and the mean recovery (92.5 and 81.5% for the 7,000 and 700 mg/kg levels of addition, respectively) were satisfactory. The method is easily applicable to fulfill future needs for nutrition labeling.     

Quantitative determination of diesel oil in contaminated edible oils using high-performance liquid chromatography

A simple and reliable high-performance liquid chromatography method for the analysis of diesel oil in contaminated edible oils is described. Analysis performed using a diol column with a mobile phase of heptane and isopropanol (94∶6, vol/vol). Although baseline separation between diesel and other background fluorescent components was not achieved, quantitation was still possible using baseline integration. The method is linear over the range of 5–1000 μg/g with a correlation coefficient (r ²) of 0.9984. Average recoveries from spiked edible oils were 94.4–101.3%, with a limit of quantitation (LOQ) of 5 μg/g for sunflower oil, palm olein, and groundnut oil. Corn oil has a higher content of ester components, thus, LOQ was slightly worse (40 μg/g). The applicability of the method was confirmed by gas chromatography-mass spectroscopic detection to show the presence of diesel hydrocarbons in the suspected contaminated crude palm oil. This procedure provides a simple and sensitive method for determining diesel oil concentration in contaminated edible oils without prior sample cleanup or extraction.     

High-performance liquid chromatography of selected phenolic compounds in olive oils

A reverse-phase high-performance liquid chromatographic technique with isocratic elution has been developed to separate and quantitate the major phenolic compounds of the hydroalcoholic extracts of olive oils. Hydroxytyrosol, tyrosol, caffeic acid,p-hydroxyphenylacetic acid and homovanillic acid were analyzed on a μBonapak C18 column with an acetonitrile/water/acetic acid (20:90:0.18, vol/vol/vol) mixture as a mobile phase. Electrochemical detection provided selectivity as well as sensitivity. The method was applied to the analysis of the most important phenolic compounds in olive oils.     

A simple HPLC method for the simultaneous analysis of phosphatidylcholine and its partial hydrolysis products 1- and 2-acyl lysophosphatidylcholine

A simple HPLC method for the simultaneous analysis of phosphatidylcholine (PC), 1-acyl lysophosphatidylcholine (1-acyl LPC), and 2-acyl lysophosphatidylcholine (2-acyl LPC) with refractive index detection is described. The separation of these three compounds was achieved on a Waters (Milford, MA) Spherisorb amino phase column using a mixture of ethanol and an aqueous oxalic acid solution as eluent. The optimal mixture of ethanol to oxalic acid solution was 92∶8 (vol/vol). PC and the two regioisomers of LPC eluted within 15 min. The calibration curves were linear in a concentration range from 0.05 to 2.5 mM. Natural PC or LPC eluted in a single peak, despite the diversity in the fatty acid composition. There was no rearrangement between 1-acyl LPC and 2-acyl LPC during analysis or storage in ethanol within 23 h. This method is thus especially suitable for studying reactions on PC and acyl migration in LPC.     

Determination of the amounts of free arachidonic acid in resident and activated rabbit alveolar macrophages by fluorometric high performance liquid chromatography

An improved method of high performance liquid chromatography (HPLC) has been developed for the separation and quantitation of low levels of free fatty acids as they occur in mammalian tissues. The fatty acid analysis is based on the esterification of the carboxylic group with 9-anthryldiazomethane (ADAM). HPLC separation and fluorescence measurement of fatty acid ADAM esters allow the determination of pmole amounts of fatty acids. The amounts of free fatty acids of resident and activated alveolar macrophages were determined by the fluorometric HPLC method. There were approximately 2 μg/10⁶ cells of free fatty acids. In resident macrophages, free 20∶4 was a minor component (0.8% of total free fatty acids), while significant amounts of 20∶4 were found in the total glycerophospholipids, representing 16.6% of the total fatty acids. A marked increase in amounts of 20∶4 (8 times) occurred in activated macrophages stimulated for 1 hr with opsonized zymosan. Small but significant increases (1.5 times) also occurred in other fatty acids. These results show that the release reaction of fatty acids was not selective for 20∶4 in alveolar macrophages after the challenge with opsonized zymosan.     

Effect of extraction methods on lipid yield and fatty acid composition of lipid classes containing γ-linolenic acid extracted from fungi

The effect of extraction procedures on the lipid yield and fatty acid composition of total lipid and main lipid structures (phospholipids, diacylglycerols, triacylglycerols, free fatty acids, and sterol esters) of fungal biomass (Mucor mucedo CCF-1384) containing γ-linolenic acid (GLA) was investigated. Seventeen extraction methods, divided into three groups, were tested: six with chloroform/methanol, five with hexane/alcohols, and six with common solvents or mixtures. The chloroform/methanol procedure (2∶1) was selected as standard, where lipid yield (TL/DCW, total lipid per dry cell weight) was 17.8%, considered to be 100% of lipids present. All chloroform/methanol extractions yielded more than 83% recorvey of lipids. Use of hexane/isopropanol solvent systems led to a maximum of 75% recovery. The best lipid yield was achieved by a two-step extraction with ethanol and hexane (120%). Extraction efficiency of the other solvent systems reached a maximum of 73%. Triacylglycerols were the main structures of lipid isolated; only methanol-extracted lipid contained 58.5% phospholipids. The fatty acid content of total recovered lipid was variable and depended on both the lipid class composition and the solvent system. GLA concentrations in total lipids isolated by hexane/alcohol procedures (7.3–10.7%) are comparable with classical chloroform/methanol systems (6.5–10.0%). The maximal GLA yield was obtained with chloroform/methanol/n-butanol/water/0.1 M ethylenediaminetetraacetic acid (EDTA) (2∶1∶1∶1∶0.1, by vol) and after two-step extraction with ethanol and hexane (14.3 and 13.7 g GLA/kg DCW, respectively). The highest GLA content was analyzed in the phospholipid fraction (16.1%) after using chloroform/methanol/n-butanol/water/0.1 M EDTA (2∶1∶1∶1∶0.1, by vol). Remarkably low concentrations of polyunsaturated fatty acids were determined in the free fatty acid fraction.     

Electron impact mass spectra of the oxazoline derivatives of some conjugated diene and triene C18 fatty acids

The location of the double-bond systems of some conjugated diene and triene C₁₈ fatty acids (C18∶2[9,11], C18∶2[10,12], C18∶3[9,11,13] and C18∶3[10,12,14]) derived from alkaline isomerization has been determined by gas chromatography/mass spectroscopy analysis of their 4,4-dimethyloxazoline derivatives. The positions of the double bonds were indicated by a characteristic mass separation of 12 atomic mass units for each olefinic bond. Furthermore, the structure assignments were supported by the presence of prominent formal allylic cleavage peaks.     

Isolation and purification of perfluorodecanoic and perfluorooctanoic acids from rat tissues

A procedure for the extraction, separation, and isolation of perfluorodecanoic and perfluorooctanoic acids from biological samples is described. The use of conventional lipid extraction procedures leads to substantial loss of the perfluorinated fatty acids added to tissue. The presence of sulfuric acid in aqueous saline during phase partitioning is essential for the recovery of perfluorodecanoic and perfluorooctanoic acids in the organic phase following their extraction from tissue. The perfluorinated fatty acids are co-eluted with simple lipids from silica gel columns using diethyl ether/trifluoroacetic acid (100∶1, v/v). Simple lipids are separated by thin layer chromatography. By substituting trifluoroacetic acid for acetic acid in the developing solvents, perfluorodecanoic and perfluorooctanoic acids migrate with other free fatty acids.