determination of lipid classes by a gas-chromatographic procedure

5
940 Analyst, November, 1970, Vol. 95, $9. 940-944 Determination of Lipid Classes by a Gas-chromatographic Procedure BY W. W. CHRISTIE, R. C. NOBLE AND J. H. MOORE (The Hannah Dairy Research Institute, A yr, Scotland) Gas chromatography of the component fatty acids with added internal standard can be used to determine a wide variety of neutral and polar lipids, after separation by thin-layer chromatography, if precautions are taken to minimise losses, Results can be expressed in terms of the relative amounts of fatty acids contained by the lipids or the actual weights and molar amounts of the lipid classes can be calculated by applying readily derived factors. The procedure is at least as accurate as others in current use and has the additional merit of allowing simultaneous determination of lipid and fatty acid com- position. THE determination of lipid classes that have been separated by thin-layer chromatography is usually accomplished by destructive methods, which involve charring of the lipids with a strong oxidising agent such as 50 per cent. sulphuric acid1 or chromic acid2 followed by photodensitometry of the carbon produced. The method has the disadvantages that the sample is completely destroyed, the yield of carbon is variable3s4p6 and appropriate reference standards are not always available. Individual phospholipid classes can readily be assayed quantitatively by phosphorus determinati~n,~ 9 ' although this method, also, is destructive. Gravimetric methods are subject to many errors8 Gas - liquid chromatography of the methyl esters of the component acids, with added internal standard, has been used successfully for the determination of single classes of lipids such as triglyceride species separated by silver nitrate chromatography9 or plasma tri- glycerides and cholesteryl esters.ll Animal and plant tissues contain a wide variety of lipid classes differing greatly in polarity and solubility in organic solvents. The feasibility of using gas - liquid chromato- graphy with added internal standard for simultaneously determining the fatty acid composi- tions and amounts of such lipid classes, after being separated by thin-layer chromatography, has been examined with a view to obtaining the maximum amount of infomation from a single analysis of a lipid sample. EXPERIMENTAL Whenever possible reactions were carried out under nitrogen, and 2,6-di-t-butyl-+-cresol was added to thin-layer chromatographic sprays and solvents to minimise autoxidation. Solvents were dried appropriately and distilled before use. A standard solution of methyl heptadecanoate (Sigma Chemical Co. Ltd., St. Louis, Mo.) in anhydrous methanol (0.285 g 1-1) was prepared. LIPID SAMPLES- Fresh pig liver was obtained from the local abattoir, and freeze-dried pig's blood was supplied by Dr. K. G. Mitchell, National Institute for Research in Dairying, Reading. Lipids were extracted with chloroform - methanol (2 + 1 v/v). THIN-LAYER CHROMATOGRAPHY- Preparative thin-layer chromatography was carried out in unlined tanks on 20 x 20-cm glass plates coated with either Kieselgel G (E. Merck) or Kieselgel G without binder (Camag) in layers 0.5 mm thick. 0 SAC and the authors. Published on 01 January 1970. Downloaded by State University of New York at Stony Brook on 24/10/2014 21:02:15. View Article Online / Journal Homepage / Table of Contents for this issue

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Page 1: Determination of lipid classes by a gas-chromatographic procedure

940 Analyst, November, 1970, Vol. 95, $9. 940-944

Determination of Lipid Classes by a Gas-chromatographic Procedure

BY W. W. CHRISTIE, R. C. NOBLE AND J. H. MOORE (The Hannah Dairy Research Institute, A yr, Scotland)

Gas chromatography of the component fatty acids with added internal standard can be used to determine a wide variety of neutral and polar lipids, after separation by thin-layer chromatography, if precautions are taken to minimise losses, Results can be expressed in terms of the relative amounts of fatty acids contained by the lipids or the actual weights and molar amounts of the lipid classes can be calculated by applying readily derived factors. The procedure is at least as accurate as others in current use and has the additional merit of allowing simultaneous determination of lipid and fatty acid com- position.

THE determination of lipid classes that have been separated by thin-layer chromatography is usually accomplished by destructive methods, which involve charring of the lipids with a strong oxidising agent such as 50 per cent. sulphuric acid1 or chromic acid2 followed by photodensitometry of the carbon produced. The method has the disadvantages that the sample is completely destroyed, the yield of carbon is variable3s4p6 and appropriate reference standards are not always available. Individual phospholipid classes can readily be assayed quantitatively by phosphorus determinati~n,~ 9' although this method, also, is destructive. Gravimetric methods are subject to many errors8

Gas - liquid chromatography of the methyl esters of the component acids, with added internal standard, has been used successfully for the determination of single classes of lipids such as triglyceride species separated by silver nitrate chromatography9 or plasma tri- glycerides and cholesteryl esters.ll

Animal and plant tissues contain a wide variety of lipid classes differing greatly in polarity and solubility in organic solvents. The feasibility of using gas - liquid chromato- graphy with added internal standard for simultaneously determining the fatty acid composi- tions and amounts of such lipid classes, after being separated by thin-layer chromatography, has been examined with a view to obtaining the maximum amount of infomation from a single analysis of a lipid sample.

EXPERIMENTAL Whenever possible reactions were carried out under nitrogen, and 2,6-di-t-butyl-+-cresol

was added to thin-layer chromatographic sprays and solvents to minimise autoxidation. Solvents were dried appropriately and distilled before use. A standard solution of methyl heptadecanoate (Sigma Chemical Co. Ltd., St. Louis, Mo.) in anhydrous methanol (0.285 g 1-1) was prepared.

LIPID SAMPLES-

Fresh pig liver was obtained from the local abattoir, and freeze-dried pig's blood was supplied by Dr. K. G. Mitchell, National Institute for Research in Dairying, Reading. Lipids were extracted with chloroform - methanol (2 + 1 v/v).

THIN-LAYER CHROMATOGRAPHY-

Preparative thin-layer chromatography was carried out in unlined tanks on 20 x 20-cm glass plates coated with either Kieselgel G (E. Merck) or Kieselgel G without binder (Camag) in layers 0.5 mm thick.

0 SAC and the authors.

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Page 2: Determination of lipid classes by a gas-chromatographic procedure

CHRISTIE, NOBLE AND MOORE 941

GAS - LIQUID CHROMATOGRAPHY-

Gas - liquid chromatographic analyses were carried out on columns of 15 per cent. w/v poly(ethyleneglyco1 adipate) on Chromosorb W (100 to 120-mesh, acid washed and silanised ; Phase Separations Ltd., Rock Ferry, Cheshire) in a Pye 104 chromatograph. The amount of each ester present (per cent. w/w) was calculated from the product of the peak height and retention time, and results obtained were converted into mole per cent. by multiplying these values by the appropriate factors. Fatty acids were identified by comparison of reten- tion times with those of authentic standards or by using the “separation factors” of Ackmann and Burgher.12 DETERMINATION OF MAJOR LIPID CLASSES BY THIN-LAYER AND GAS - LIQUID CHROMATO-

GRAPHY-

Total lipid extracts (5 to 10 mg) were chromatographed on Kieselgel G plates with the solvent system hexane - diethyl ether - formic acid (80 + 20 + 2 v/v). The component lipid classes were detected by spraying with a 0.1 per cent. w/v solution of 2,4-dichloro- fluorescein in methanol and identified by comparison with standard mixtures. Bands were scraped off on to small chromatographic columns from which the cholesteryl esters, tri- glycerides and free fatty acids were eluted with 100 ml of diethyl ether and the phospholipids with 25 ml of methanol containing 5 per cent. of anhydrous hydrogen chloride; 1 ml of the standard solution of methyl heptadecanoate was added to each fraction. The cholesteryl esters and triglycerides were dissolved in a small amount of benzene and transesterified with 0 . 5 ~ sodium methoxide in dry methanol, free fatty acids were methylated with a 5 per cent. w/v solution of boron trifluoride in methanol, and the phospholipids transesterified by refluxing for 2 hours in the methanolic hydrogen chloride used to elute them from the Kieselgel G. Methyl esters were dissolved in hexane for gas - liquid chromatographic analysis.

GRAVIMETRIC DETERMINATION OF MAJOR LIPID CLASSES-

The lipid extracts (50 mg) were each chromatographed on a single Kieselgel G plate as before. The cholesteryl ester, triglyceride, free fatty acid and free cholesterol bands were scraped on to small chromatographic columns from which they were eluted with 100ml of diethyl ether. The solvent was removed and the samples were thoroughly driedunder reduced pressure before being weighed. The phospholipids were recovered from a column by elution with 100 ml of methanol. The methanol was evaporated off and the phospholipids were re-dissolved in chloroform and filtered to remove any silica gel or calcium sulphate eluted from the plate before the solvent was removed to enable the phospholipids to be weighed. Recoveries ranged from 98 to 103 per cent.

DETERMINATION OF PHOSPHOLIPID CLASSES BY THIN-LAYER AND GAS - LIQUID CHROMATO-

The phospholipids were separated from the lipid extract by chromatography on silicic acid (Mallinckrodt; 100 mesh) by using the method of Hanahan, Dittmer and Warashinala; 1 to 5 mg of phospholipids were chromatographed on plates coated with Kieselgel G, without binder, with the solvent system chloroform - methanol - acetic acid - water (25 + 15 + 4 + 2) .14

The phospholipid classes were detected by spraying with 2,4-dichlorofluorescein and eluted from the plates with methanol. Identification was by comparison with known standards. One millilitre of the standard solution of methyl heptadecanoate was added to each fraction before trans-esterification with a 5 per cent. w/v solution of hydrogen chloride in methanol.

DETERMINATION OF PHOSPHORUS-

Phospholipid classes obtained as above were analysed for phosphorus by the procedure of Chen, Toribara and Warner.6

GRAPHY-

RESULTS Pig liver and blood were chosen for this study as neither appears to have been analysed

in detail before and as they were expected to have differing fatty acid and lipid compositions. Preliminary analysis of the fatty acids of the total lipid extracts from these tissues confirmed that they did not contain significant amounts of heptadecanoic acid, so that this acid was suitable for use as an internal standard.

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Page 3: Determination of lipid classes by a gas-chromatographic procedure

942 CHRISTIE, NOBLE AND MOORE DETERMINATION OF LIPID [Anh!&St, vol. 95

The response of the flame-ionisation detectors in the gas chromatographs used was proportional to the amount of each component present. The total amount of fatty acids in each lipid class could then be related directly to the amount of internal standard added to obtain a measure of the weight of fatty acids present in that class. The molar proportions of fatty acids were readily calculated by multiplying the detector responses by the appro- priate arithmetical factors derived from the molecular weights of the components. The relative amounts of each lipid class could then be expressed most simply in terms of their fatty acids as “weight per cent. of the total fatty acids” or “mole per cent. of the total fatty acids.” (In practice, it has been found that the relative amounts obtained by both methods are numerically almost identical.) A close approximation to the actual weights of the lipid classes themselves can then be obtained by multiplying the weight of fatty acids in each lipid class by a factor calculated by dividing the molecular weight of the heptadecanoic acid derivative of the lipid class by the molecular weight of methyl heptadecanoate. For this purpose, the phospholipids were considered to be diheptadecanoyl-lecithin. Factors for the most common lipid classes are listed in Table I.

TABLE I FACTORS FOR QUANTITATIVE ASSESSMENT OF LIPIDS

Lipid class Factor Cholesteryl esters . . . . 2.246 Triglycerides . . .. . . 0.995 Diglycerides . . .. . . 1.049

Free fatty acids .. . . 0.951 Phospholipids . . .. . . 1-371

Monoglycerides . . .. . . 1.211

The weight per cent. of each lipid class obtained by using these factors agreed well with the value obtained gravimetrically (Table 11). Standard deviations for three determinations by the gas-chromatographic procedure were low and were much smaller than those for the gravimetric method.

Individual phospholipid classes separated by thin-layer chromatography were determined in a similar way and the results for the major phospholipids of pig liver and blood are listed in Table 111.

In this instance, the results agreed well with those of phosphorus determinations on similar samples, and standard deviations for three determinations were of comparable magnitude. The gas-chromatographic procedure was probably more accurate than the phosphorus method, especially with minor components, because of the proportionately higher blank readings obtained with the latter method. For example, in the determination of sphingomyelin in these samples the optical density of the blank was equal to 80 per cent. of the value obtained for the sphingomyelin.

DISCUSSION The major difficulties experienced in using gas chromatography of component fatty acids

for the quantitative determination of lipid classes of widely differing polarities lie in obtaining quantitative recovery from the thin-layer plates and in ensuring that no opportunity occurs for selective loss of the lipid or the internal standard in any of the subsequent procedures. Diethyl ether or chloroform can be used to elute most neutral lipid classes quantitatively from silica-gel plates, and we have found that with methanol or methanol containing 5 per cent. w/v of hydrogen chloride or 2 per cent. v/v of sulphuric acid more than 95 per cent. of the phospho- lipids are recovered. Other losses can be minimised by adding the internal standard methyl ester solution directly to the eluate, which is collected in a vessel suitable for carrying out methylation, so that no further transfer is necessary until all of the fatty acids are present as methyl esters. The above results then confirm that the method can be used for the quantitative determination of a wide variety of lipid classes as well as individual species of single neutral lipid classes to which the method has hitherto been confined.

The results can be expressed in several ways. For simplicity they can be related solely to the amounts of component fatty acids found in each lipid class and expressed as “weight per cent. of fatty acids” or “mole per cent. of fatty acids,” which are quantities that we have

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Page 4: Determination of lipid classes by a gas-chromatographic procedure

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Page 5: Determination of lipid classes by a gas-chromatographic procedure

944 CHRISTIE, NOBLE AND MOORE

found to be numerically almost identical. A good approximation to the weight of each lipid class can then be obtained from this relationship by multiplying them by readily derived factors. Weights of lipid classes thus obtained are similar to those found gravimetrically and are more reproducible. It seems probable that they are at least as accurate as results obtained by photodensitometry.

Individual phospholipids can also be quantitatively determined by the gas-chromato- graphic method described and the results expressed in a similar way. The molar amount of all phospholipids that can be positively identified can be simply derived from the “mole per cent. fatty acids” found.

Although methyl heptadecanoate proved to be a suitable internal standard for the analysis of pig lipids, there may be instances when it is less useful, for example, in the analysis of ruminant lipids in which significant amounts of this acid occur naturally. However, if a sufficient excess of standard over the endogenous acid is added, this need not introduce a large error. The choice of standard will depend on the nature of the sample and on the liquid phase used in the gas-chromatographic columns. Errors may also be introduced into the determination of phospholipids if significant amounts of glyceryl ethers or plasmalogens are present. However, these are minor components in most tissues. The method can only be used, of course, to determine lipids that contain fatty acids. Free cholesterol, for example, must be determined independently.

Most methods of lipid analysis require separate determinations of lipid and fatty acid composition. This study demonstrates that gas chromatography of the component fatty acids of lipid classes with added internal standard permits both of these determinations to be performed simultaneously with accuracy. The method should be particularly suited to computer analysis of the results. It enables the maximum amount of information to be obtained from a single analysis, which may be important when only very small amounts of material are available.

gave valuable technical assistance. Miss M. Hunter, Miss J. Laidlaw, Miss E. Skinner, Miss A. Wallace and Mrs. I. Knowles

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.

10. 11.

12. 13. 14.

Privett, 0. S., Blank, M. L., and Lundberg, W. O., J . Amer. Oil Chem. Soc., 1961, 38, 312. Rouser, G., Galli, C., Lieber, E., Blank, M. L., and Privett, 0. S., Ibid., 1964, 41, 836. Privett, 0. S., and Blank, M. L., Ibid., 1963, 40, 70. Blank, M. L., Schmit, J. A., and Privett, 0. S., Ibid., 1964, 41, 371. Rouser, G., Kritchevsky, G., Galli, C., and Heller, D., Ibid., 1965, 42, 215. Chen, P. S., Toribara, T. Y. , and Warner, H., Analyt. Chem., 1956, 28, 1756. Rouser, G., Siakotos, A. N., and Fleischer, S., Lip ids , 1966, 1, 85. Komarek, R. J., Jensen, R. G., and Pickett, B. W., J . L i p i d Res., 1964, 5, 268. Gunstone, F. D., Padley, F. B., and Qureshi, M. I., Chew. & Ind . , 1964, 483. Blank, M. D., Verdino, B., and Privett, 0. S., J . Amer. Oil Chem. SOC., 1965, 42, 87. Boyers, C. Y., Hamilton, J. G., Muldrey, J. E., Miyamasu, W. T., Reynolds, G. A., and Schally,

Ackman, R. G., and Burgher, R. D., J . Chromat., 1963, 11, 185. Hanahan, D. J., Dittmer, J. C., and Warashina, E., J . BioZ. Chem., 1957, 228, 685. Skipski, V. P., Peterson, R. F., and Barclay, M., Biochern. J.. 1964, 90, 374.

A. V., Ibid. , 1966, 43, 2.

Received February 16th, 1970 Accepted July Sth, 1970

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