Analysis of the amino acid 3-methylhistidine by gas-liquid chromatography

A method for the quantitative separation of 3-methylhistidine from other amino acids, by gas-liquid chromatography, has been developed. This method gives complete resolution of the N-heptafluorobutyryl isobutyl esters of 20 amino acids with the use of a single column packed with 3% SE-30 on $\text{100}\text{120}$ mesh Gas Chrom Q. Using this method the 3-methylhistidine content of urine and meat has been determined.     

Superior gas-liquid chromatography of methyl cholanoate acetates on cyanopropylphenylsiloxane liquid phases

The resolution and recovery of mixtures of the methyl ester acetates of synthetic and natural bile acids are excellent on gas-liquid chromatographic columns prepared with 1–3% OV-225 on 100–200 mesh Gas Chrom Q. The columns are operated isothermally at 250–260°C with helium as carrier gas and conventional gas chromatographic equipment. Under these conditions, complete separation of the major bile acids of rat and human bile is obtained in 30–60 min. This method of analysis is superior to that based on the trifluoroacetyl or trimethylsilyl derivatives, using OV-225 or other liquid phases.     

Identification of fatty acids by GC-MS using polar siloxane liquid phases

A practical method is described for the unambiguous characterization of all fatty acids in natural mixtures from a single injection into a GC-MS instrument. The fatty acids are separated as the methyl esters on a polar siloxane column which yields resolutions similar to those commonly obtained on polyester columns, but has higher thermal stability. Any leakage of the polar siloxane into the mass spectrometer is further reduced by placing a small amount of a thermally stable methyl siloxane packing at the outlet end of the column which serves as a trap. The glc peaks emerging from such columns yield characteristic mass spectra, including molecular ions, which along with the retention data are adequate for the accurate identification of both saturated and unsaturated fatty acids and dimethyl acetals.     

Purification of fatty acid ethyl esters by solid-phase extraction and high-performance liquid chromatography

We have developed a two-step method to purify fatty acid ethyl esters (FAEE) using solid-phase extraction (SPE), with a recovery of 70±3% (mean±S.E.M.) as assessed using ethyl oleate as a recovery marker from a standard lipid mixture in hexane. The first step of the SPE procedure involves application of a lipid mixture to an aminopropyl-silica column with simultaneous elution of FAEE and cholesteryl esters from the column with hexane. Gas chromatographic analysis of FAEE without interference from cholesteryl esters may be performed using the eluate from the aminopropyl-silica column, thus eliminating the need for an octadecylsily (ODS) column in this case. The FAEE can then be separated from the cholesteryl esters, if necessary, by chromatography on an ODS column and elution with isopropanol-water (5:1, v/v). Both the aminopropyl-silica and ODS columns were found to be effective for up to four uses. To permit isolation of specific FAEE species following isolation of total FAEE by the two-step SPE method, we have also developed a purification scheme for individaal FAEE by high-performance liquid chromatography (HPLC). Thus, this simple method allows for reproducible isolation of total FAEE by SPE and isolation of individual FAEE species by HPLC.     

Gas chromatographic determination of the optical purities of amino acids using N-trifluoroacetyl menthyl esters

Nineteen α-amino acids and one β-amino acid were resolved as their N-TFA menthyl esters by gas-liquid chromatography. Neutral and basic amino acids were well resolved on some conventional packed columns. For the complete resolution of acidic amino acids, a capillary column was used. The results of the applications to some biochemical substances showed that this method is of value for practical usage. The mechanism of the resolution was also investigated and showed that the difference between the derivatives of the enantiomeric pairs in the ability to form intermolecular hydrogen bond contributes essentially to the resolution.     

Preparative liquid chromatography of carbohydrates: mono- and di-saccharides, uronic acids, and related derivatives

The general principles and practical aspects of preparative high-performance liquid chromatography (l.c.) of mono- and di-saccharides, sugars acids, lactones, and N-acetylated amino sugar derivatives are described. Milligram to gram quantities of these carbohydrates were isolated on semi-preparative (0.78 X 30 cm) or preparative (approximately 2.0 X 30 cm) columns packed with aminopropyl silica gel provided better resolution of individual mono- and di-saccharides, but columns of cation-exchange resin had higher capacity and were more durable and economical to use. Preparative, cation-exchange columns were operated at flow rates of less than 5 mL/min and pressures of approximately 1-2 MPa, allowing them to be used on unmodified analytical l.c. systems. Details are given for the efficient packing, use, and care of these columns, and on the effects of column selectivity, packing technique, and sample size on chromatographic resolution. Isolation of naturally occurring sugars from biological sources on a laboratory-packed column is described.     

Separation of neutral lipids and free fatty acids by high-performance liquid chromatography using low wavelength ultraviolet detection

Normal phase, isocratic high-performance liquid chromatography methods are described for the separation of neutral lipid and fatty acid classes using low wavelength detection. Prior to high-performance liquid chromatography, methods were developed and are described for the separation of phospholipids from neutral lipids and fatty acids using small (600 mg) silica Sep-PaksTM. Recoveries of cholesteryl esters, triglycerides, fatty acids, and phospholipids from the silica columns were greater than 95%. Two mobile phases are described for lipid class separation by high-performance liquid chromatography. The first mobile phase, hexane-2-propanol-acetic acid 100:0.5:01, resulted in incomplete separation of cholesteryl ester and triglyceride but excellent separations of fatty acids and cholesterol. The second mobile phase, hexane-n-butyl chloride-acetonitrile-acetic acid 90:10:1.5:0.01, resulted in complete separation of the four lipid classes. This mobile phase also separated individual triglycerides and fatty acids based on the number of double bonds. Recoveries of radiolabeled lipids for the four lipid classes from high-performance liquid chromatography was greater than 95% with both mobile phases.     

Improved method for simultaneous determination of alcohols, volatile fatty acids, lactic acid or 2,3-butanediol in biological samples

A rapid and sample procedure was developed to determine, by gas-liquid chromatography, the concentrations of C₂---C₄ alcohols, C₂---C₆ volatile fatty acids (VFA) and lactic acid or 2,3-butanediol in fermentation liquids. both lactic acid and 2,3-butanediol are oxidized to acetaldehyde by periodic acid and acetaldehyde was eluted before ethanol. A complete separation of the alcohols and acids was performed in <15 min on a column packed with 80/100 Chromosorb WAW, having GP 10% SP-1200/1% H₃PO₄ as the liquid phase. The method was suitable for the analysis of rumen fluid and fermentation products from microbial cultures. The detection limits for all compounds were <0.13 nmol · injection⁻¹.     

Determination of monoenoic fatty acid double bond position by permanganate-periodate oxidation followed by high-performance liquid chromatography of carboxylic acid phenacyl esters

This investigation was carried out to develop methods for a reverse-phase, high-performance liquid chromatography analysis of the monocarboxylic and dicarboxylic acids produced by permanganate-periodate oxidation of monoenoic fatty acids. Oxidation reactions were performed using [U-14C]oleic acid and [U-14C]oleic acid methyl ester in order to measure reaction yields and product distributions. The 14C-labeled oxidation products consisted of nearly equal amounts of monocarboxylic and dicarboxylic acid (or dicarboxylic acid monomethyl ester), with few side products (yield greater than 98%). Conversion of the carboxylic acids to phenacyl esters proceeded to completion. HPLC of carboxylic acid phenacyl esters was performed using a C18 column with a linear solvent gradient beginning with acetonitrile/water (1/1) and ending with 100% acetonitrile. Excellent resolution was achieved for all components of a mixture of C5 through C12 monocarboxylic acid phenacyl esters and C6 through C11 dicarboxylic acid phenacyl esters. Resolution was also achieved for all components of a mixture of C5 through C12 monocarboxylic acid phenacyl esters and C6 through C11 dicarboxylic acid monomethyl, monophenacyl esters. The resolution obtained by HPLC demonstrates that, for a wide range of monoenoic fatty acids, both products of a permanganate-periodate oxidation can be identified on a single chromatogram. Free fatty acids and fatty acid methyl esters were analyzed with equal success. Neither the oxidation nor the esterification reaction caused detectable hydrolysis of methyl ester. The method is illustrated for free acids and methyl esters of 14:1 (cis-9), 16:1 (cis-9), 18:1 (cis-6), 18:1 (cis-9), and 18:1 (cis-11).     

Synthesis of lipids from acetate by human preputial and abdominal skin in vitro

Lipogenesis in vitro from acetate-1-(14)C was studied in human preputial skin and abdominal skin. Radioactive lipids were separated by column chromatography on Florisil and by thin-layer chromatography on silica gel. Radioactivity was incorporated chiefly into the triglyceride, sterol, and polar lipid fractions, while lesser amounts of (14)C were found in the hydrocarbon, wax, diglyceride, monoglyceride, and fatty acid fractions; labeling of steryl esters was minimal. On thin-layer chromatography, the radioactive polar lipids had mobilities similar to lysolecithin, phosphatidyl choline, phosphatidyl ethanolamine, and phosphatidic acid. The radioactive fatty acids of the different lipid fractions were separated by gas-liquid chromatography. The major (14)C-labeled acids were 16:0 and 18:0. Radioactivity was also detected in acids 14:0, 15:0, 16:1, 18:1, 18:2, 20:0, 20:1, 22:0, 24:0, 24:1, and 26:0. No radioactivity could be detected in arachidonic acid, although this fatty acid comprises 9% of the chromatographed fatty acids. The pattern of incorporated (14)C was different from the percentage mass composition of the fatty acids. Skin is therefore active in the biosynthesis of a wider variety of lipids than previously demonstrated.     

A technique for the determination of enantiomeric amino acids in biological samples.

Racemic amino acids can be separated into their enantiomers by means of gas-liquid chromatography. The most applied technique, today, is the conversion of chiral compunds into diastereoisomers with optically active reagents and subsequent chromatography on conventional optically inactive stationary phases. In previous studies it has been realized that this technique is associated with various problems. We studied the use of optically active stationary phases for separating enantiomers directly via a diastereoisomeric association complex. The optically acitve stationary phases employed are N- and C-terminal substituted dipeptides of the type N-trifluoroacetyl-dipeptide-cyclohexyl esters and have been synthesised by the I-hydroxibenztriazole dicyclohexylcarbodiimide method. The quality of these phases with respect to separation factors, resolution factors, and thermodynamical properties have been evaluated. All synthetic phases show excellent properties; however, when attempting separation of mixtures of naturally occurring amino acids extensive overlap in the elution diagram was detected. Only one phase - N-TFA-L-chi-amino-n-butyryl-L-chi-amino butyric acid cyclohexyl ester - gave complete resolution of the naturally occurring amino acids alanine, valine, glycine, threonine, leucine, isoleucine, serine and proline on a 400 ft x 0.02 in capillary column. Less volatile amino acids such as aspartic acid, phenylalanine, methionine, glutamic acid, tyrosine, arginine, and tryptophan can be resolved at a 100 ft x 0.02 in column.     

Chromatographic analysis of carotenol fatty acid esters in Physalis alkekengi and Hippophae rhamnoides

The carotenol fatty acid esters of two potentially valuable sources of plant carotenoids, sepals of Physalis alkekengi (Chinese lantern) and fruits of Hippophae rhamnoides (sea buckthorn), were separated by column chromatography and identified by HPLC-DAD and HPLC-MS. A chemical and an enzymatic hydrolysis were employed to identify the parent carotenoids and to remove the lipid components. Zeaxanthin and beta-cryptoxanthin esters represented the main fraction in P. alkekengi sepals and an important one in H. rhamnoides fruits. Beta-Cryptoxanthin palmitate and zeaxanthin dipalmitate were identified as major compounds in both plants. In P. alkekengi, the carotenoids were mainly (> 90%) esterified with palmitic acid, and a high proportion (> 80%) of saturated medium chain fatty acids was found (by GC-MS) in the total lipid extract. Although the total lipid extract of H. rhamnoides contained significant amounts of unsaturated fatty acids, especially oleic and palmitoleic acids, the xanthophylls were mainly esterified with saturated fatty acids. The oleoresins of both species represent potential sources of carotenoid esters and can be used as food additives, cosmetic ingredients or nutraceuticals.     

Methodology in the study of organic acids in children

A method is described for the extraction of urinary organic acids in children, their conversion to TMS and oxime TMS derivatives and their separation by gas liquid chromatography on a packed column of 10% OV 101. The compounds are identified by mass spectrometry. Profiles of urinary organic acids in normal neonates and in several metabolic disorders are presented.     

A gas-liquid-chromatographic procedure for separating a wide range of metabolites occuring in urine or tissue extracts

1. A gas–liquid-chromatographic procedure is described which permits separation and identification on the same chromatogram of a wide range of substances occurring in urine or tissue extracts. The method uses hydrogen flame ionization, which detects organic compounds whether free or conjugated with no requirement for specific reactive groups. 2. For chromatography, carboxyl groups are quantitatively converted into methyl esters or trimethylsilyl esters. Phenolic, alcoholic and potential enolic groups are converted into trimethylsilyl ethers. Separations are carried out on a 6ft. column of either 10% F-60 (a polysiloxane) or 1% F-60, temperature programming at 2°/min. being used over such part of the temperature range 30°–260° as is required. Propionyl derivatives of hydroxy compounds can also be used, but only on a non-quantitative basis. Derivatives and columns have been selected for optimum range of usefulness when large numbers of samples are examined by using automated gas chromatography. 3. The method is applicable to: fatty acids above butyric acid; di- and tri-carboxylic acids; hydroxy acids and keto acids; polyhydroxy and alicyclic compounds such as glycerol, inositol, quinic acid, shikimic acid, ascorbic acid and sugar alcohols; aromatic hydroxy and acidic compounds, both benzenoid and indolic; sesquiterpenes; steroids; glycine conjugates; mercapturic acids; glucuronides. It is not satisfactory for sulphate conjugates, iminazoles or polypeptides. 4. Methylene units provide an accurate and reproducible parameter for characterizing peak position. Methylene unit values are reported for a large variety of substances occurring in, or related to those occurring in, urine and tissue extracts. 5. The nature of derivatives was confirmed by combining gas chromatography with mass spectrometry. Combined gas chromatography–mass spectrometry gives a diagnostic tool of great power in the evaluation of metabolic patterns, and various uses are discussed.     

Lipids and sterols of Pinus sylvestris L. sapwood and heartwood

Summary The lipid and sterol content and composition of three lipid fractions (free fatty acids/ sterols, triacylglycerols and sterol/triterpenoid esters) extracted from three stem discs of Pinus sylvestris were assessed to investigate metabolic changes related to heartwood formation. The wood was separated into (1) cambial zone, (2) outer sapwood, (3) inner sapwood, (4) transition zone, (5) outer heartwood and 6) inner heart-wood. The fractions were separated by thin-layer chromatography (TLC) and analysed by gas-liquid chromatography (GLC). The amount of fatty acids of sapwood triacylglycerols was about 1.5% (dry wt.) but a large reduction occurred in the transition zone. In contrast, noticeable amounts of free fatty acids were present only in the heart-wood. The most important fatty acids in the sapwood fractions were 16:0, 18:0, 18:1, 18:2 (the dominant fatty acid in all fractions), 18:3 and 20:3. Together 18:1 and 18:2 formed about 70% of the total triacylglycerol fatty acids. Of the sterol/ triterpenoid esters, 18:2 and 18:3 were predominant. The fatty acid composition of all fractions changed in the transition zone. The sterols found were sitosterol, stigmastanol, campesterol and campestanol. The amount of sterol esters increased towards the heartwood, and the amount of free sterols was lowest in the inner sapwood. Sitosterol was the dominant sterol in both free sterols and sterol esters.     

Dry-column chromatographic isolation of fatty acid esters of phorbol from croton oil

The family of mixed fatty acid esters of 4,9,12,13,20-pentahydroxy-tiglia-1,6-dien-3-one (phorbol) has been isolated from croton oil by column chromatography on silica gel and preparative dry-column chromatography on silica gel HF/254 packed in a cellophane membrane. The tumorpromoting agents are obtained rapidly and with minimum difficulty.     

Effects of freezing and drying grass products prior to fatty acid extraction on grass fatty acid and lipid class composition--a technical note

Grass and grass silage represent a rich and natural source of omega-3 polyunsaturated fatty acids, in particular linolenic acid, for ruminants. Recent research, focusing on improving the content of these beneficial fatty acids in grass, requires storage of the forage samples prior to analysis. In this study, we evaluated whether conservation of fresh grass and grass silage by freezing (1 and 4 weeks,--18 degrees C) and/or drying (24h, 50 degrees C) affected its fatty acid content and induced shifts between lipid classes. FA were extracted using chloroform/methanol (2/1, v/v) and triacylglycerols (TAG), free fatty acids (FFA) and polar lipids (PL) were separated by thin layer chromatography. Fatty methyl esters (FAME) were identified by gas chromatography. Loss of thawing liquor might provoke a dramatic decrease in extractable lipid after frozen storage of both grass and grass silage. Morever, after frozen storage, fatty acids in grass but not in grass silage seem subjected to a higher rate o f lipolysis and oxidation, as suggested by increased quantities of FFA (3.1, 7.6, 8.4 % of total FAME) and reduced proportions of poly-unsaturated fatty acids (79.5, 73.6 and 74.1 % of total FAME) when analysing fresh grass samples directly or after 1 and 4 weeks of frozen storage, respectively. Drying of fresh grass did not provoke changes in FA composition, but distribution of FA over lipid classes was significantly altered, with an increase in TAG (5.1 to 17.9 % of total FAME) and FFA (2.4 to 14.9 % of total FAME) and lower proportions of PL (90.7 to 55.7 % of total FAME).     

Oxidative degradation of model lipids representative for main paper pulp lipophilic extractives by the laccase–mediator system

Different model lipids-alkanes, fatty alcohols, fatty acids, resin acids, free sterols, sterol esters, and triglycerides-were treated with Pycnoporus cinnabarinus laccase in the presence of 1-hydroxybenzotriazole as mediator, and the products were analyzed by gas chromatography. The laccase alone decreased the concentration of some unsaturated lipids. However, the most extensive lipid modification was obtained with the laccase-mediator system. Unsaturated lipids were largely oxidized and the dominant products detected were epoxy and hydroxy fatty acids from fatty acids and free and esterified 7-ketosterols and steroid ketones from sterols and sterol esters. The former compounds suggested unsaturated lipid attack via the corresponding hydroperoxides. The enzymatic reaction on sterol esters largely depended on the nature of the fatty acyl moiety, i.e., oxidation of saturated fatty acid esters started at the sterol moiety, whereas the initial attack of unsaturated fatty acid esters was produced on the fatty acid double bonds. In contrast, saturated lipids were not modified, although some of them decreased when the laccase-mediator reactions were carried out in the presence of unsaturated lipids suggesting participation of lipid peroxidation radicals. These results are discussed in the context of enzymatic control of pitch to explain the removal of lipid mixtures during laccase-mediator treatment of different pulp types.     

Thehalose mycolates from Nocardia asteroides,Nocardia farcinica,Gordona lentifragmenta and Gordona bronchialis

Isolation of glycolipids from Nocardia asteroides, N. farcinica, Gordona lentifragmenta and G. bronchialis, by column chromatography of lipid extracts on a 50% (w/w) mixture of silicic acid and silica gel H, is described. The isolated materials were partially characterized by infrared spectroscopy, optical rotation and refractive index measurements, and by identifying the products of alkaline hydrolysis. Analytical studies showed that the glycolipids released only trehalose in the aqueous phase while mycolic acids were the constituent fatty acids identified.The isolated lipids are trehalose esters in which the trehalose molecule is esterified with mycolic acids.     

Synergistic roles of acyl‐CoA binding protein (ACBP1) and sterol carrier protein 2 (SCP2) in Toxoplasma lipid metabolism

Toxoplasma gondii relies on apicoplast‐localised FASII pathway and endoplasmic reticulum‐associated fatty acid elongation pathway for the synthesis of fatty acids, which flow through lipid metabolism mainly in the form of long‐chain acyl‐CoA (LCACoAs) esters. Functions of Toxoplasma acyl‐CoA transporters in lipid metabolism remain unclear. Here, we investigated the roles of acyl‐CoA‐binding protein (TgACBP1) and a sterol carrier protein‐2 (TgSCP2) as cytosolic acyl‐CoA transporters in lipid metabolism. The fluormetric binding assay and yeast complementation confirmed the acyl‐CoA binding activities of TgACBP1 and TgSCP2, respectively. Disruption of either TgACBP1 or TgSCP2 caused no obviously phenotypic changes, whereas double disruption resulted in defects in intracellular growth and virulence to mice. Gas chromatography coupled with mass spectrometry (GC–MS) results showed that TgACBP1 or TgSCP2 disruption alone led to decreased abundance of C18:1, whereas double disruption resulted in reduced abundance of C18:1, C22:1, and C24:1. ¹³C labelling assay combined with GC–MS showed that double disruption of TgACBP1 and TgSCP2 led to reduced synthesis rates of C18:0, C22:1, and C24:1. Furthermore, high performance liquid chromatography coupled with high resolution mass spectrometry (HPLC‐HRMS) was used for lipidomic analysis of parasites and indicated that loss of TgACBP1 and TgSCP2 caused serious defects in production of glycerides and phospholipids. Collectively, TgACBP1 and TgSCP2 play synergistic roles in lipid metabolism in T. gondii.