Separation of brain monosialoganglioside molecular species by high-performance liquid chromatography

A method for the separation of molecular species of brain monosialogangliosides by high-performance liquid chromatography is described. GM4, GM3, GM2, and GM1 were purified from human brain and their individual molecular species were separated on a C18 reversed-phase column. Peaks were identified by mass spectrometry of the intact ganglioside, by gas-liquid chromatography of the fatty acids, and by high-performance liquid chromatography of the long chain bases. A characteristic elution sequence of molecular species permitted their identification based upon their retention times on the reversed-phase column.     

Separation of molecular species of sphingomyelin by reversed-phase high-performance liquid chromatography.

A convenient method for the separation of molecular species of sphingomyelin by reversed-phase high-performance liquid chromatography (HPLC) is described. Sphingomyelin species from bovine brain and sheep and pig erythrocytes were resolved into 10-12 separate peaks on a micro -BondaPak C(18) or Nucleosil-5-C(18) reversedphase column with methanol-5 mM potassium phosphate buffer, pH 7.4, 9:1 (v/v) as the solvent. Detection was at 203-205 nm. The sphingomyelin species were primarily resolved due to specific hydrophobic interaction of their fatty acid and sphingoid chains with the alkyl ligand of the stationary phase. The retention time of the sphingomyelin species increased progressively as the number of carbon atoms in the hydrophobic chains increased in the homologous series. The presence of one double bond in the molecule reduced the retention time significantly. Introduction of a second double bond in the fatty acid side chain did not reduce the retention time to the same extent as the first double bond. The presence of a trans double bond in the sphingoid moiety increased the retention time of sphingomyelin more than did a cis double bond in the fatty acid side chain. The differential hydrophobic interaction observed between the ligand of the stationary phase and different alkyl chains of the sphingomyelin species illustrates that reversed-phase HPLC technique can be conveniently used to study the extent of relative hydrophobicity of different types of alkyl chains.-Jungalwala, F. B., V. Hayssen, J. M. Pasquini, and R. H. McCluer. Separation of molecular species of sphingomyelin by reversed-phase high-performance liquid chromatography.     

Separation of chloroplast polar lipids and measurement of galactolipid metabolism by high-performance liquid chromatography

Procedures are described for the separation of polar lipids from plant chloroplasts by high-performance liquid chromatography, using a polar-modified silica column. Glycolipids and phospholipids were eluted with a gradient of 2-propanol/n-hexane (80:55, v/v) and 2-propanol/n-hexane/water/methanol (80:55:15:10, v/v). The lipids were detected by uv absorbance at 202 nm. Diacylglycerol and mono-, di-, and trigalactosyldiacylglycerol and phosphatidylcholine were separated on a LiChrosorb NH2 column (7-microns particles, Merck, FRG), but acidic lipids were retained. These lipids could be quantified from their 202-nm absorbance recording. The absorption coefficients obtained depended on the mean number of double bonds in the different lipid classes. The separation was applied for a rapid monitoring of the lipid composition in thylakoids and in fractionated inner and outer envelopes. The activities of galactosyltransferases involved in galactolipid metabolism, UDPGal:diacylglycerol galactosyltransferase and galactolipid:galactolipid galactosyltransferase, could be measured quantitatively in specific assays for both enzymes.     

Separation and quantitation of molecular species from plant lipids by high-performance liquid chromatography

Monogalactosyl-, digalactosyl-, and sulfoquinovosyl diacylglycerol as well as phosphatidyl glycerol were isolated by conventional TLC and then separately subjected to HPLC for resolution of molecular species. Molecular species emerge in groups from reversed-phase columns during gradient elution. The groups are separated according to the sum of carbon and double bond numbers in fatty acyl pairs in linear relation to elution times. Therefore, it is possible to identify a species group with respect to carbon and double bond numbers by its retention time. The separation is monitored by recording the absorbance at 200 nm which depends on double bond combinations in acyl pairs. Diacylglycerols released from glyco- and phospholipids were separated as rho-anisoyl derivatives according to similar criteria. In this case separation was monitored at 250 nm, at which wavelength the absorbance is directly related to molar proportions. By calculating corrected 200-nm/250-nm absorbance ratios for different molecular species of rho-anisoyl diacylglycerols, relative response factors for different double bond combinations were obtained. The 200-nm absorbances of intact lipid species can be converted to molar proportions by division with these factors.     

Separation of dimethylphosphatidates of alkylacyl glycerophosphocholine and their molecular species by high-performance liquid chromatography

A high-performance liquid chromatography (HPLC) method for separation of the alkylacyl and diacyl analogs of choline glycerophospholipids (CGP) of guinea pig polymorphonuclear leukocytes and their molecular species is described. CGP were hydrolyzed with phospholipase D and then methylated with diazomethane to convert them to dimethylphosphatidates. The dimethylphosphatidates were then separated into the alkylacyl and diacyl subclasses by HPLC on a silica gel column within 15 min. The alkylacyl and diacyl analogs were then separated into individual molecular species by reverse-phase HPLC. Dimethylphosphatidates were resolved into 15 separate peaks, and 11-16 different molecular species of alkylacyl and diacyl glycerophosphocholines were identified on gas-liquid chromatography. The present results indicate that the CGP of polymorphonuclear leukocytes are composed of 27 major molecular species. In the alkylacyl subclass, the most predominant species was the 16:0-18:2 species (32%), followed by the 18:1-18:2 (18%), 16:0-16:0 (16%), and 16:0-18:1 (15%) species. The diacyl type consisted mainly of species with 18:2 at the 2-position, such as the 16:0-18:2, 18:0-18:2, and 18:1-18:2 species, the total percentage of which was 57%.     

Separation of the major species of interstitial collagen by reverse-phase high-performance liquid chromatography

Results presented here demonstrate a further application of reverse-phase high-performance liquid chromatography to the separation of large proteins. At a pH near 4.5 with a high pyridine concentration, we have completely separated three major types of human collagen (Types I, II, and III) from mixtures. We illustrate the application of this technique to the preparation of Types I and II collagen from lathyritic chick cartilage extracts.     

Separation of neutral oligosaccharides by high-performance liquid chromatography

We have developed a method for separating reduced, neutral oligosaccharides by high-performance liquid chromatography on columns of MicroPak AX-5 (Varian Associates) with a mobile phase consisting of acetonitrile:H₂O. Individual glucose oligomers containing from 1 to 20 glucose moieties can be separated in a single 1-h analysis with a solvent program of decreasing acetonitrile concentration. We have applied this method to both the analysis and preparative isolation of glycoprotein-derived oligosaccharides obtained by enzymatic release with endoglycosidases or chemical release by hydrazinolysis. Introduction of ³H by reduction with NaB³H₄ permits the detection of subnanomole quantities of oligosaccharides. This method offers previously unattainable rapidity and resolution for the analysis of oligosaccharides.     

Separation of phosphohydroxyamino acids by high-performance liquid chromatography

A high-performance liquid chromatography system has been developed which resolves O-phosphoserine, O-phosphothreonine, and O⁴-phosphotyrosine. Separation is performed on an anion-exchange resin eluted with phosphate buffer of low pH. Detection of the amino acid derivatives is accomplished using O-phthalaldehyde in an in-stream fluorometric system. This highly sensitive method has been applied to the detection of phosphohydroxyamino acids in bovine myelin basic protein phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase and in whole bovine myelin phosphorylated by endogenous kinases.     

Separation of bilirubin species in serum and bile by high-performance reversed-phase liquid chromatography

A high-performance, reversed-phase liquid chromatographic (HPLC) procedure has been developed for the separation of at least three major bilirubin fractions in bile and four fractions in human serum. This procedure was unlike most others, in that serum was not totally deproteinized prior to injection onto the HPLC column; instead, serum was treated with an excess of sodium sulfate solution to precipitate primarily proteins larger than albumin. Injection of the filtered and diluted supernatant onto a reversed-phase column then resulted in the separation of the bilirubin species in a 24-min gradient elution run. Both the initial aqueous acidic mobile phase and the final isopropyl alcohol-based mobile phase contained 5% methoxyethanol (v/v) to facilitate elution of albumin still present in the treated sample. Bilirubin species eluting from the column were detected by absorbance at 450 nm.Results of a number of chromatographic separations of pathological sera indicated a wide variation in the relative proportions of the four bilirubin fractions observed. A correlation of the sum of the areas of the bilirubin peaks observed by HPLC was found with the total bilirubin value obtained by a standard reference procedure.     

Separation of porphyrin isomers by high-performance liquid chromatography.

A reversed-phase gradient elution system is described for the simultaneous separation of the type I and type III isomers of 8-, 7-, 6-, 5- and 4-carboxylated porphyrins and isocoproporphyrins. The method, adaptable for isocratic and stepwise separation of individual groups of isomers, is also suitable for preparative isolation of pure porphyrins. The analyses of porphyrin isomers in the urine and faeces of porphyric patients are examples of applications.     

Separation of hemoglobin types by cation-exchange high-performance liquid chromatography

The use of a recently developed cation-exchange HPLC packing material for the separation of hemoglobin types in human blood has been investigated. Adult and newborn hemolysates from normal individuals and from subjects with hemoglobin disorders were analyzed using a weak cation carboxymethyl-bonded phase on 5-micron-particle-size silica. Elution was accomplished using a Bistris (2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-1, 3-propanediol) gradient. Seven well-resolved HbA1 fractions eluted before the major HbA peak. Hbs A1a, A1b, A1c and an HbA1 fraction that increased with aging of the hemolysates were separately eluted. HbF when present or when added to the hemolysates eluted as a distinct peak. HbA was followed by Hbs A2, S, and C when present. An early-eluting peak corresponding to Hb Bart's was identified in newborn hemolysates. It is concluded that cation-exchange HPLC provides a new tool for the reliable separation of minor hemoglobin components.     

Separation of derivatized glycosphingolipids into individual molecular species by high performance liquid chromatography

The high performance liquid chromatography separation of the perbenzoyl derivatives of the neutral glycosphingolipids (GlcCer, LacCer, GbOse3Cer, GbOse4Cer, and GgOse3Cer) and the p-bromophenacyl and 2,4-dinitrophenyl hydrazide derivatives of the gangliosides (GM4, GM3, GM2, GM1, GD1a) into individual molecular species on a C18 reversed-phase column is described. Peaks were identified by comparing their relative retention times to the relative retention time of the corresponding glycosphingolipid of known molecular species composition. As little as 5 to 10 pmol of each molecular species of neutral glycosphingolipids and 3 to 5 pmol of the gangliosides can be detected. The effects of changes in the proportion of acetonitrile, methanol, and water in the mobile phase and of column temperature on the molecular species separation are described. A procedure for the tentative identification of glycosphingolipid molecular species based on their relative retention times is presented.     

Analysis of molecular species of plant polar lipids by high-performance and gas liquid chromatography

Total lipid extracts from potato tubers and tobacco leaves are separated into lipid classes by two step HPLC using a silicic column. Elution is first performed for 20 min with a programmed linear gradient of two mixed solvents running from 100% of solution A (isopropanol-hexane, 4:3) to 100% of solution B (isopropanol-hexane-water, 8:6:1.5); the column is then eluted with pure solution B in an isocratic mode for 20 min more. The main polar lipids (MGDG, DGDG, PC, PE, PG) from both plant tissues can be collected and further separated into component molecular species on a simplified HPLC system with a C₁₈ column eluted in an isocratic mode with a polar solvent. Molecular species separations are achieved within 35 min; quantifications are made through GLC analysis of attached fatty acids. Three to five main molecular species are thus clearly identified in each lipid class. In potato tuber, phospholipids (PC, PE) 18:2/18:2 species are predominant. In tobacco leaf, six double bond species (18:3/18:3 and 16:3/18:3) are predominant in galactolipids, whereas PC contains a greater number of molecular species varying by their degree of unsaturation (from 18:3/18:3 to 16:0/18:2). Only certain molecular species of PG contain Δ³-trans-hexadecenoic acid.     

A sensitive method for quantitative analysis of phospholipid molecular species by high-performance liquid chromatography

A simple and sensitive method was developed for quantitative analysis of phospholipid molecular species. Diacylglycerols were prepared from phospholipids by phospholipase C treatment and converted to the corresponding dinitrobenzoyl derivatives, which could be sensitively detected at 254 nm. The derivatives of 21 molecular species were resolved by high-performance liquid chromatography with an octadecylsilyl reversed-phase column. All the derivatives had the same peak area per mol, and peak areas were proportional to the amounts of the derivatives. Quantification was carried out at the picomole level.     

Separation and purification of sphingomyelin diastereomers by high-performance liquid chromatography

All naturally occurring sphingomyelins have the d-erythro-(2S,3R) configuration of the sphingoid base. We have developed a normal-phase HPLC method for the separation of this natural stereoisomer from the l-threo-sphingomyelin, which is the other stereoisomer commonly present in semisynthetic preparations of acyl-chain defined sphingomyelins. The chromatographic method was developed by modification of a previously reported method for phospholipid separation on a normal-phase diol column. The separation was accomplished by a binary gradient of solvent mixtures (A) hexane:isopropanol:acetic acid (82:17:1.0 by vol) and (B) isopropanol:water:acetic acid (85:14:1.0 by vol) with 0.08 vol% triethylamine added to both solvent mixtures. The program of gradient elution was optimized for maximal separation of sphingomyelin diastereomers. For detection of the lipids, a light-scattering detector was used. This analytical scale HPLC method was also used for purification of the stereoisomers (up to 0.5 mg of N-oleoyl-sphingomyelin in a single injection). The purified stereoisomers were at least 99% pure according to high-performance thin-layer chromatography and analytical HPLC.     

Quantitation of individual molecular species of phosphatidylcholines by reversed-phase high-performance liquid chromatography with fluorometric detection

The multiplicity of phosphatidylcholines is caused by the presence of different pairs of fatty acids in their individual molecular species and at least 27 miscellaneous fatty acids were identified in phosphatidylcholines in the serum of healthy individuals by combined gas–liquid chromatography and mass spectrometry in our present experiments. A method is described for the separation and quantitation of molecular species of phosphatidylcholine in human serum. Total phosphatidylcholine is isolated from lipids extracted from the serum with chloroform–methanol (2:1) by reversed-phase liquid–liquid extraction and subjected to reversed-phase high-performance liquid chromatography with a discontinuous descending gradient of water. Separation is monitored by fluorometry (340/460 nm) and absorption at 205 nm, if required. Up to 25 different molecular species of phosphatidylcholine may be quantified with a satisfactory reproducibility (±5–8%). Data on the distribution of individual molecular species in phosphatidylcholine of 53 normal serums are presented. The method may be used for quantitation of these phospholipids also in other biological materials (cell lines, leukemic cells from patients), and on a micropreparative scale to isolate individual compounds. The speed of separation as well as a satisfactory reproducibility are its principal advantages.     

A highly sensitive method for quantitative analysis of phospholipid molecular species by high-performance liquid chromatography

A highly sensitive method was developed for quantitative analysis of phospholipid molecular species. Diradylglycerols prepared from phospholipids with phospholipase C were converted to the anthroyl-diradylglycerol derivatives, which could be separated into molecular species and sensitively quantified by reverse-phase HPLC using a fluorescence detector. All the molecular species of the derivatives had the same peak area per mole, and the peak areas were proportional to the amounts of the derivatives. Quantification could be carried out at the femtomole level.