Triglyceride composition of ewe,cow, and goat milk fat

The separation and identification of the components in milk fat, which are mainly triglycerides, is a challenge due to its complex composition. A reverse-phase high-performance liquid chromatography (HPLC) method with gradient elution and light-scattering detection is described in this paper for the triglyceride analysis in ewes’ milk fat. Triglyceride identification was carried out by combining HPLC, gas-liquid chromatography (GLC), and the calculated equivalent carbon numbers of several triglyceride standards. Quantitation of partially resolved peaks in the HPLC chromatogram was accomplished by applying a peak deconvolution program. Forty-four fatty acids were identified by GLC analysis, but only 19 were used for the following prediction of triglyceride molecular species; 181 triglycerides were identified, some of which were grouped at the same peak and needed application of the deconvolution program. Consequently, coefficients of variation were close to or lower than 5%. Moreover, the triglyceride composition of ewe, cow, and goat milk fat were compared by using these methods. These results show that ewe milk fat is richer in short- and medium-chain triglycerides, and cow milk fat is richer in long-chain and unsaturated triglycerides.     

Detection of trace fatty acids in fats and oils by urea fractionation and gas-liquid chromatography

The detection of trace fatty acids (<0.1%) in a fat or oil by gas-liquid chromatography is possible when the methyl esters are fractionated with urea to provide a number of less complex fractions. Identification and estimation of trace fatty acids is simplified by quantitative removal of other fatty acids having similar gas chromatographic retention times. A detailed knowledge of the order in which inclusion compounds are formed was obtained by fractionating a complex mixture of marine and vegetable fatty acids. In addition, lanolin was fractionated to determine the preferential order in which saturated, branched chain (iso-, anteiso-) and hydroxy acids form inclusion compounds. Using urea fractionation and gas chromatography, 52 trace fatty acids were tentatively identified in butter, 30 in lard, and 26 in walnut oil. Presented at the AOCS Meeting in New Orleans, 1964.     

Determination of milk fat in chocolates by gas-liquid chromatography of triglycerides and fatty acids

The combination of two routine methods is proposed to determine the content of milk fat (MF) in chocolates, which is applicable even in the presence of lauric fats or others. The content of MF is obtained from the sum of C₄₀, C₄₂, and C₄₄ medium-chain triglycerides, determined by capillary gas-liquid chromatography (GLC). A new method, based on methyl esters of lauric acid and on minor acids situated between myristic and palmitic, is proposed. It enables detection and estimation of potential lauric fats, as well as the determination of the actual content of MF. The influence of other vegetable and animal fats is discussed. We analyzed 45 MF samples extracted from industrial milk powders and from pure or fractionated MF for chocolate manufacturing or pastry by GLC of triglycerides. We also analyzed by capillary GLC the methyl esters from 22 of those fats. Mixtures of these 22 MF samples with a cocoa butter also were used for chromatographic analyses of methyl esters and triglyceride. Results from the various analytical methods have been presented.     

Determination of trace amounts of fatty acids in edible oils by capillary gas-liquid chromatography

The effect of using different gas-liquid chromatography (GLC) hardware to quantify low concentrations of fatty acids was studies. A fused-silica capillary column was operated in two different chromatographs (A and B) that were interfaced to three different chromatographic data systems to process the flame-ionization detector signals (systems A, B1, and B2). A test routine was developed that allowed the proper selection of peak processing parameters for the automatic recognition and integration of fatty acids occurring at trace levels. However, agreement of analytical results between the three analytical systems was not satisfactory; components at concentrations <0.10 g/100 g could not be quantified with high reliability, although the same capillary column and identical sample solutions were used (quasi-repeatability conditions). Even for major fatty acids, deviations up to 1.0 g/100 g were noted, which could only be attributed to the use of different GLC hardware. Attention should be paid to these technical restrictions when formulating product specifications based on fatty acid profile parameters.     

Content and distribution oftrans-18:1 acids in ruminant milk and meat fats. Their importance in european diets and their effect on human milk

Thetrans-18:1 acid content and distribution in fats from ewe and goat milk, beef meat and tallow were determined by a combination of capillary gas-liquid chromatography and argentation thin-layer chromatography of fatty acid isopropyl esters. Thetrans isomers account for 4.5 ± 1.1% of total fatty acids in ewe milk fat (seven samples) and 2.7±0.9% in goat milk fat (eight samples). In both species, as in cow, the main isomer is vaccenic (trans-11 18:1) acid. The distribution profile oftrans-18:1 acids is similar among the three species. The contribution of ewe and goat milk fat to the daily intake oftrans-18:1 acids was estimated for people from southern countries of the European Economic Community (EEC): France, Italy, Greece, Spain, and Portugal. It is practically negligible for most of these countries, but in Greece, ewe and goat milk fat contributeca. 45% of the daily consumption oftrans-18:1 acids from all dairy products (0.63 g/person/day for a total of 1.34 g/person/day). Thetrans-18:1 acid contents of beef meat fat (ten retail cuts, lean part) and tallow (two samples) are 2.0 ± 0.9% and 4.6%, respectively, of total fatty acids (animals slaughtered in winter). Here too, the main isomer is vaccenic acid. Othertrans isomers have a distribution pattern similar to that of milk fat. Beef meat fat contributes less than one-tenth of milk fat to thetrans-18:1 acid consumed. The daily per capita intake oftrans-18:1 acids from ruminant fats is 1.3–1.8 g for people from most countries of the EEC, Spain and Portugal being exceptions (ca. 0.8 g/person/day). In France, the respective contributions of ruminant fats and margarines to the daily consumption oftrans-18:1 acids are 1.7 and 1.1 g/person/day (60 and 40% of total, respectively). These proportions, based on consumption data, were confirmed by the analysis of fat from milk of French women (ten subjects). The mean content oftrans-18:1 acids in human milk is 2.0 ± 0.6%, with vaccenic acid being the major isomer. Based on the relative levels of thetrans-16 18:1 isomer, we could confirm that milk fat is responsible for the major part of the daily intake oftrans-18:1 acids by French people. The daily individual intake oftrans-18:1 isomers from both ruminant fats and margarines for the twelve EEC countries varies from 1.5 g in Spain to 5.8 g in Denmark, showing a well-marked gradient from the southwest to the northeast of the EEC.     

Separation of petroselinic (cis-6 18:1) and oleic (cis-9 18:1) acids by gas-liquid chromatography of their isopropyl esters

Petroselinic (cis-6 18:1) and oleic (cis-9 18:1) acids that occur together in Umbelliferae seeds can be resolved by gasliquid chromatography (GLC) of their methyl or isopropyl esters on a 50 m × 0.25 mm fused-silica capillary column coated with a 100% cyanopropyl polysiloxane stationary phase (CP Sil 88). The use of isopropyl esters instead of methyl esters increases the difference between equivalent chainlengths from 0.06 carbon unit up to 0.08. This is sufficient to obtain an almost base-line resolution between the two components.cis-Vaccenic acid is completely separated from oleic acid in both derivative forms. GLC of fatty acid isopropyl esters on an appropriate capillary column thus appears to be the simplest means to simultaneously and accurately quantitate petroselinic, oleic andcis-vaccenic acids.     

Improvement in the resolution of individualtrans-18:1 isomers by capillary gas-liquid chromatography: Use of a 100-m CP-Sil 88 column

Doubling the length of a CP-Sil 88 capillary column (Chrompack, Middelburg, The Netherlands) from 50 to 100 m remarkably improves the resolution of individualtrans-18:1 isomers from either ruminant fats or partially hydrogenated oils. Although the use of a 50-m column gives interesting results, it does not allow sufficient resolution of thetrans-10 andtrans-11 18:1 isomers. Moreover, thetrans-6 totrans-9 18:1 isomers emerge as a single group of peaks, whereas thetrans-12 isomer is only partly resolved from the adjacenttrans-11 andtrans-13 plustrans-14 isomers. With the 100-m column, thetrans-9,trans-10, andtrans-12 18:1 isomers are almost base-line resolved from other isomers. However, with both columns, it is not possible to separate the critical pair oftrans-13 andtrans-14 18:1 acids which co-elute under a single peak. Despite this minor drawback, the 100-m CP-Sil 88 column appears to be of great interest for the separation and the quantitation of most individualtrans-18:1 acids. Except for the use of argentation thin-layer chromatography, there is no need for complementary techniques, such as ozonolysis. This simple and powerful tool may be applied to ruminant fats, partially hydrogenated oils, and human tissue lipids.     

A method for the simultaneous analysis of unconjugated and glycine-conjugated bile acids by capillary gas-liquid chromatography

A method for the simultaneous analysis of unconjugated and glycine-conjugated bile acids by means of capillary gas-liquid chromatography without need for prior deconjugation is described. The method involves: i) the use of an aluminum-clad fused-silica capillary column coated with a very thin film (0.1 μm) of a highly thermostable bonded and crosslinked methyl polysiloxane, and ii) the analysis of the bile acids as their methyl ester-dimethylethylsilyl ether derivatives. This method, used to separate the major free and glycine-conjugated bile acids from human gall bladder bile, should be applicable for the analysis of other biological fluids.     

Quantitative determination of trans-fatty acids in oils and fats by capillary gas chromatography: results of a JOCS collaborative study

Excessive intake of trans-fatty acids increases the risk of cardiovascular disease. Much attention is drawn to the consumption of trans-fatty acids worldwide, and regulations for trans-fatty acids are instituted in several countries. Precise and convenient methods for determination of trans-fatty acid level are required, but there is no standard method using capillary Gas Chromatography in Japan. Therefore, for the new standard method, collaborative studies were carried out. The results were as follows: 1) Heptadecanoate (C17:0 free fatty acid) was chosen for internal standard substance. 2) Two Gas Chromatography columns, SP2560 (100 m) column (100% cyanopropyl polysiloxane liquid phase) and TC-70 (60 m) column (70% cyanopropyl polysilphenylene-siloxane liquid phase), were examined in the collaborative studies. We measured the edible oil samples containing 2-45 g/100 g of trans-fatty acids, and trans-fatty acid contents were quantitatively the same with both columns. The range of reproducibility coefficient of variation were below 10%. 3) Fats and oils sampled were soybean, rapeseed, palm, palm kernel, beef tallow, pork fat and their hydrogenated forms, for which good peak resolution was achieved. From the above results, the technique evaluated in the present study was considered to be suitable for determination of the content of trans-fatty acids in fats and oils exclusive of fish oil and milk fat.     

Analysis of isomeric monoethylenic fatty acids in a partially hydrogenated herring oil by glass capillary gas liquid chromatography

The use of a glass capillary column coated with Silar 10 CP for gas liquid chromatographic analysis of geometric and positional isomers of monoethylenic fatty acids of a partially hydrogenated herring oil is reported. The results are in agreement with previous studies performed by different methods and demonstrate the usefulness of this technique in detecting and determining many types of isomeric fatty acids.     

Determination of the glyceride structure of fats: Distribution of individual saturated and unsaturated acids1

A method has been devised which gives the distribution of saturated and unsaturated fatty acids. It involves fractionation of the triglycerides into groups on the basis of total unsaturation by employing chromatography on a silicic acid-silver nitrate column. The glyceride composition of each fraction is then determined by gas-liquid chromatography (GLC) of the oxidized glycerides. Using this method, the glyceride composition of lard and cocoa butter was determined to give quantitative amt of 24 and 18 glycerides, respectively. Duplicate analyses agreed to within ±0.5%. The fatty acid composition calculated from the glyceride composition agreed to within ±1.5% with that of the original fat. This approach provides a new basis for the evaluation of the glyceride tyes in natural fats and for the first time permits the quantitative determination of all the chemically different glycerides of myristic, palmitic, stearic, oleic, linoleic and linolenic acids in a fat. Presented at AOCS Meeting in Minneapolis, 1963. Issued at NRC 7947. National Research Council Postdoctorate Fellow. Prairie Regional Laboratory, Saskatoon, Sask.     

Quantitative determination of traces of free gossypol in fats,oils, and fatty acids by paper chromatography

Summary A quantitative method for the determination of traces of free gossypol in oils and fatty acids was developed. The method is based on the concentration of gossypol by extraction and quantitative paper chromatography of the extract. The method is specific for free gossypol and is not subject to interferences. The new method is both accurate and reproducible. The lower limit of detection is 10 p.p.m. The method is intended primarily for p.p.m. levels but is suitable for all concentrations. With slight modifications the method is applicable to meals. Presented at the fall meeting of the American Oil Chemists’ Society, Cincinnati, O., September 30 to October 2, 1957.     

Analysis ofcis- andtrans-fatty acid isomers in hydrogenated and refined vegetable oils by capillary gas-liquid chromatography

For quantitation ofcis- andtrans-fatty acid isomers, infrared (IR) spectroscopy, gas-liquid chromatography (GLC) on highly polar stationary phases or the combination (GLC-IR) may be used. IR offers the advantage of simplicity and speed, but the lower determination limit of 5% and the lack of detailed information limit its use. Detailed fatty acid information, required for, e.g., food-labeling purposes, can only be obtained with GLC methods. Most of the GLC methods are optimized for partially hydrogenated samples. AOCS Official Method Ce 1c-89 prescribes a single, highly polar stationary phase, SP2340, but underestimates the amount oftrans isomers due to 18∶1 positional isomer overlap. The combined GLC-IR method may circumvent this problem but at the cost of time, effort, and precision.Trans isomers in refined (deodorized or stripped) oils are different in type and levels from isomers in partially hydrogenated oils; theirtrans isomers are mono-trans trienoic and dienoic isomers, occurring at levels up to about 1–3%. GLC conditions for hydrogenated samples are often not suitable for refined oils because of overlap problems, but this time in the 18∶3 region. Through careful selection of stationary phase and temperature program optimization (Drylab^®GC), we have developed a single method that is suitable for hydrogenated, as well as refined, processed oils. The accuracy was checked withcis andtrans fatty acid fractions isolated by silverion exchange high-performance liquid chromatography. Thetrans values obtained with the optimized method are in good agreement with the results obtained for the isolated fractions. We propose that recommended methods describe GLC conditions in terms of separation criteria rather than recommending only a fixed combination of stationary phase and temperature program.     

Hepatoma,host liver,and normal rat liver lipids: Distribution of isomeric monoene fatty acids in individual lipid classes

Monoenoic acid fractions were isolated from phosphatidycholine, phosphatidylethanolamine, triglycerides, and cholesteryl esters of hepatoma 7288CTC, host liver, and normal liver from animals maintained on chow and fat free diets. Hexadecanoate (16:1), octadecenoate (18:1), and eisosenoate (20:1) fractions were analyzed quantitatively for their isomeric composition. The fat free diet had little or no effect relative to the chow diet on the isomeric composition of 16:1, 18:1, and 20:1 from any lipid class in either heptoma, host liver, or normal liver. Host livers were reduced in palmitoleic acid, and oleic and eicos-11-enoic acids were increased relative to normal liver. The 16:1 fraction from triglyceride of normal liver, host liver, and hepatoma contained 90, 80, and 75% palmitoleic acid, respectively. The 20:1 fraction from triglycerides of normal liver, host liver, and hepatoma contained ca. 55, 70, and 60% eicos-11-enoic acid, respectively, with the remainder consisting of eicos-13-enoic acid. The proportion of vaccenic acid in the 18:1 fraction was 60, 50, 20, and 25% for phosphatidylethanolamine, phosphatidylcholine, triglycerides, and cholesteryl esters, respectively, with oleic acid making up the balance. In contrast, all hepatoma lipid classes exhibited the same proportion of oleic (70%) and vaccenic (30%) acids. These data appear to be the first to demonstrate lipid class specificity for isomeric octadecenoic acids in normal liver and the loss of this specificity in a neoplasm.     

Short method for the detection of chick edema factor in fats,oils and fatty acids by electron capture gas chromatography

The electron capture gas liquid chromatography test method for chick edema factor has been modified by replacing the alumina column step with an additional sulfuric acid cleanup and a caustic wash which permits a reduction in sample cleanup time. Samples are subjected to double preliminary sulfuric acid cleanup and caustic wash and extracted each time with trimethyl pentane. The final extract is washed with sulfuric acid and examined by electron capture gas chromatography. Gas chromatographic peaks with retention times vs. Aldrin (Ra values) between 8 and 45 are indicative of the presence of chick edema factor.     

Analysis of C18:1 cis and trans fatty acid isomers by the combination of gas-liquid chromatography of 4,4-dimethyloxazoline derivatives and methyl esters

Trans fatty acids in foods are usually analyzed by gas-liquid chromatography (GLC) of fatty acid methyl esters (FAME). However, this method may produce erroneously low values because of insufficient separation between cis and trans isomers. Separation can be optimized by preceding silver-ion thin-layer chromatography (Ag-TLC), but this is laborious. We have developed an efficient method for the separation of 18-carbon trans fatty acid isomers by combining GLC of FAME with GLC of fatty acid 4,4-dimethyloxazoline (DMOX) derivatives. We validated this method against conventional GLC of FAME, with and without preceding Ag-TLC. Fatty acid isomers were identified by comparison with standards, based on retention times and mass spectrometry. Analysis of DMOX derivatives allowed the 13t, 14t, and 15t isomers to be separated from the cis isomers. The combination of the GLC analyses of FAME and DMOX derivatives gave results comparable with those obtained by GLC of FAME after preceding Ag-TLC, while saving about 100 h of manpower per 25 samples. It allowed the identification and quantitation of 11 trans and 8 cis isomers and resulted in 25% higher values for total C_18:1 trans, compared with the analysis of FAME alone. The combination of DMOX and FAME analyses, as applied to the analysis of 14 foods that contained ruminant fat and partially hydrogenated vegetable and fish oils, indicated that the most common isomers were 11t in ruminant fats, 9t in partially hydrogenated fish fats, and either 9t or 10t in partially hydrogenated vegetable fats. The combination of GLC analyses of FAME and DMOX derivatives of fatty acids improves the quantitation of 18-carbon fatty acid isomers and may replace the laborious and time-consuming Ag-TLC.     

Analysis of alpha-linolenic acid geometrical isomers in deodorized oils by capillary gas-liquid chromatography on cyanoalkyl polysiloxane stationary phases: A note of caution

Analysis of alpha-linolenic acid geometrical isomers in deodorized or heated oils by capillary gas-liquid chromatography (GLC) on polar cyanoalkyl polysiloxane stationary phases requires some care to avoid interferences with other fatty acids. Depending on the temperature of the column, thecis-11 20∶1 acid may elute before, with or after thecis-9,cis-12,cis-15 18∶3 acid during GLC. In some instances [temperature higher than 180°C with a CP Sil 88 column (Chrompack, Middelburg, The Netherlands)], the 20∶1 acid coelutes with thetrans-9,cis-12,cis-15 18∶3 acid, leading to abnormally high levels of this last isomer. Consequently, the degree of isomerization of alpha-linolenic acid will be over-estimated under such conditions. It is recommended that the behavior ofcis-11 20∶1 acid relative to temperature be checked carefully prior to the determination of alpha-linolenic acid geometrical isomers by GLC. Temperatures lower than 160°C seem appropriate to separate all of these components from each other and fromcis-11 20∶1 acid in a 50 m×0.25 mm i.d. CP Sil 88 capillary column.     

The regiospecific position of 18∶1 cis and trans monoenoic fatty acids in milk fat triacylglycerols

TAG of butterfat were fractionated according to the type and degree of unsaturation into six fractions by silver-ion HPLC. The fractions containing TAG with either cis-or trans-monoenoic FA were collected and fractionated further by reversed-phase HPLC to obtain fractions containing cis TAG of ACN:DB (acyl carbon number:double bonds) 48∶1, 50∶1, and 52∶1 as well as trans 48∶1, 50∶1, and 52∶1. The FA compositions of these fractions were elucidated by GC. The MW distribution of each fraction was determined by ammonia negative-ion CI-MS. Each of the [M-H]⁻ parent ions was fractionated further by collision-induced dissociation with argon, which gave information on the location of cis-and trans-FA between the primary and secondary positions of TAG. The results suggest that the sn-positions of the monoenoic cis-and trans-FA depend on the two other FA present in the molecule. With 14∶0 FA in the TAG molecule, the 18∶1 FA in the sn-2 position are mostly present as cis-isomers. When there is no 14∶0 in the TAG molecule, the trans-18∶1 isomers seem to be more common in the sn-2 position. Also when other long-chain FA are present, the trans-isomers are more likely to be located in the secondary (sn-2) position.