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Separation of complex lipids of cereals by high-performance liquid chromatography with mass detection
Journal of Chromatography, 436 (1988)5 10-5 13 Elsevier Science Publishers B.V., Amsterdam - Printed CHROM.
in The Netherlands
20 200
Note
Separation of complex lipids of cereals chromatography with mass detection
by high-performance
liquid
W. W. CHRISTIE* The Hannah Research Institute,
Ayr KA6 SHL
(U.K.)
and W. R. MORRISON Food Science Division (Received
October
University
of Strathclyde,131
A&ion St., Glasgow Gl 1SD (U.K.)
5th, 1987)
The analysis of the complex lipids of cereals presents particular problems, because of the presence of appreciable amounts of glycolipids in addition to the phospholipids normally encountered in plant and animal tissues. Only a few applications of high-performance liquid chromatography (HPLC) to the analysis of plant lipids have been described. Gradients of hexane-isopropanol-water1,2 and isocratic elution with acetonitrile-methanol-sulphuric acid3 have been used as mobile phases with columns of silica gel and UV spectrophotometric detection at 205 nm to separate a range of complex lipids of plants, and conditions similar to the former have been employed with an amine-bonded phase 4. These and related methods have been revieweds. With UV detection methods, direct quantification is rarely possible, and traces of oxidised lipids can give an intense signal which swamps that of interest; a limited range of solvents can be used in the mobile phase and this can impose a restriction on the quality of the separation. Recently, a rapid HPLC method for the analysis of lipid classes from animal tissues was described, in which a column of silica gel was used with a ternary gradient elution scheme 6,7. Lipids ranging in polarity from cholesterol esters to lysophosphatidylcholine were eluted separately in only 20 min. The by Applied Chromatodetector employed was the “mass detector” manufactured graphy Systems (Macclesfield, U.K.), also known as an “evaporative analyser” or “light-scattering detector”. With this system, almost any organic solvent can be employed in the mobile phase and good quantification is possible with careful calibration. The procedure has now been adapted for the sequential separation of the individual glycolipids and phospholipids in cereal extracts. EXPERIMENTAL
Lipids were extracted from unbleached wheat flour with water-saturated butanols. The individual classes of glycolipids and phospholipids were analysed by a thin-layer chromatography (TLC) procedureQ, and then were isolated by preparative TLC to provide reference lipids for identification of components emerging from the 0021-9673/88/$03.50
0
1988 Elsevier Science Publishers
B.V.
511
NOTES
TABLE
I
TERNARY GRADIENT ELUTION SYSTEM REQUIRED CLASSES AND REACTIVATION OF THE PROBLEM
FOR
THE
SEPARATION
OF LIPID
For A, B and C, see Experimental. Step
Time (min)*
Mobile phases A
B
C
1 2
0 8
100 -
loo
_ _
3 4 5 6
23 28 28.1 33
_ _ 100 100
_ _ _ _
100 100 _ _
between
the compositions
* A linear gradient
was produced
specified at each time interval.
HPLC system. The lipid extract used as the test mixture in developing the elution system consisted of so-called “free lipids”, extracted from wheat flour with light petroleum (b.p. 40-60°C) at 20°C f O.l”C using 16 ml solvent per g of flourlo. The HPLC equipment, including the column of silica gel, was as described previously6,‘. No guard column was used, but an Upchurch filter with a stainlesssteel frit (2-pm pores; Scotlab Instrument Sales, Bellshill, U.K.) was incorporated between the injector and the column to trap microparticulate impurities. All natural samples were filtered through Acrodisc TM-CR disposable filters (0.45~pm pores, Gelman Sciences, MI, U.S.A.) prior to analysis. In the gradient elution scheme finally adopted, the three reservoirs in the ternary solvent mixer contained the following: (A) hexane-butan-2-oneeacetic acid (35:65:0.4, v/v); (B) hexanechloroformisopropanol-aqueous buffer (42:5:45:3, v/v); (C) as B but 32:5:50:8 (v/v). The aqueous buffer consisted of 0.5 mM serine adjusted to pH 7.5 with ethylamine. The flow-rate was 2 ml/min, and the solvent elution programme is shown in Table I. RESULTS
AND DISCUSSION
When cereal lipids were examined by the ternary gradient scheme developed
TABLE
II
COMPOSITION
OF THE POLAR
LIPIDS
IN THE TEST MIXTURE
Lipid class
Abbreviation
wt.%
Monogalactosyldiacylglycerol Monogalactosylmonoacylglycerol Digalactosyldiacylglycerol Digalactosylmonoacylglycerol N-Acylphosphatidylethanolamine N-Acyllysophosphatidylethznolamine Others
MGDG MGMG DGDG DGMG NAPE NALPE
16.0 4.4 41.6 14.5 10.4 5.1 1.4
512
NOTES
previously for the separation of the lipids of animal tissues6,7, the glycolipids and the less polar phospholipids emerged together from the column with the introduction of the aqueous component of the mobile phase and individual components could not be distinguished. Glycolipids are commonly separated from phospholipids by means of adsorption chromatography with mobile phases containing solvents such as acetone’ l. Various elution schemes were, therefore, tried in which acetone and solvents in the same selectivity groupi2, such as ethyl acetate or butan-2-one, were employed as constituents of the mobile phase in the early stages of the procedure. Rather than using a complete lipid extract from a cereal, a glycolipid-rich fraction was used as the test mixture, since it contained mostly those lipid classes that had not been resolved by HPLC in previous studies. Its composition was determined by a TLC procedure9 and is shown in Table II. In addition to glycolipids, it contained Nacylphosphatidylethanolamine and its lyso derivative, but trace amounts only of more polar phospholipids. To control the overall polarity of the initial mobile phase, hexane in various concentrations was added. Later, it was found advantageous to add a small amount of acetic acid as this gave sharper peaks. In general, it was a relatively easy matter to separate the glycolipid classes from each other, but it proved more difficult to resolve DGMG from NAPE. The best results were obtained with a gradient of hexane-butan-2-oneeacetic acid (35:65:0.4, v/v), changed by means of a linear gradient to a solvent mixture corresponding to that devised earlier6,7 for the start of the separation of individual phospholipids (step 1 to 2 in Table I). The nature of the
, 5
10 time
15 (mid
Fig. 1, Separation of the glycolipid-rich fraction from wheat flour, detailed in Table II, by HPLC on a column of silica gel by the ternary gradient elution system described in the Experimental section. Details of the abbreviations are given in Table II.
NOTES
513
separation is shown in Fig. 1. Unfortunately, the individual simple lipids were not resolved, as they were not retained by the adsorbent, and they emerged together at the start of the analysis. It would no doubt be possible to correct this if a quaternary solvent delivery system were available. The next step in the elution sequence (step 2 to 3 in Table I) corresponds to the stage in the elution system devised previously in which the individual phospholipid classes are separated6s7. Step 3 to 4 is only required if lysophosphatidylcholine is present in appreciable concentrations, while the final steps are necessary to restore the activity of the column for the analysis of the next sample. Preliminary work on the calibration of the system for the analysis of cereal lipids appears to indicate that the response of the detector to glycolipids resembles that for phospholipids, i.e. that a sigmoidal relationship holds and tails off for components present at amounts below 10 pg. A different calibration line is obtained for each component of the standard mixture. This problem and other difficulties associated with sample extraction are now being addressed. REFERENCES 1 C. Demandre, A. Tremolieres, A.-M. Justin and P. Mazliak, Phytochemistry, 24 (1985) 481. 2 N. Sotirhos, C.-T. Ho and S. S. Chang, Fette Seifen Anstrichm., 88 (1986) 6. 3 D. Marion, G. Gandemer and R. Douillard, in P.-A. Siegenthaler and W. Eichenberger (Editors), Structure, Function and Metabolism of Plant Lipids, Elsevier, Amsterdam, 1984, p. 139. 4 J. W. M. Heemskerk, G. Bogemann, M. A. M. Scheijen and J. F. G. M. Wintermans, Anal. Biochem., 154 (1986) 85. 5 W. W. Christie, High-Performance Liquid Chromatography and Lipids, Pergamon, Oxford, 1987. 6 W. W. Christie, J. Lipid Res., 26 (1985) 507. 7 W. W. Christie, J. Chromatogr., 361 (1986) 396. 8 W. R. Morrison, D. L. Mann, S. Wong and A. M. Coventry, J. Sci. Food Agric., 26 (1975) 507. 9 W. R. Morrison, S. L. Tan and K. D. Hargin, J. Sci. Food Agric., 31 (1980) 329. 10 W. R. Morrison, L. J. Wylie and C. N. Law, J. Cereal. Sci., 2 (1984) 145. 11 W. W. Christie, Lipid Analysis, Pergamon, Oxford, 2nd ed., 1982. 12 L. R. Snyder, J. Chromatogr. Sci., 16 (1978) 223.
in The Netherlands
20 200
Note
Separation of complex lipids of cereals chromatography with mass detection
by high-performance
liquid
W. W. CHRISTIE* The Hannah Research Institute,
Ayr KA6 SHL
(U.K.)
and W. R. MORRISON Food Science Division (Received
October
University
of Strathclyde,131
A&ion St., Glasgow Gl 1SD (U.K.)
5th, 1987)
The analysis of the complex lipids of cereals presents particular problems, because of the presence of appreciable amounts of glycolipids in addition to the phospholipids normally encountered in plant and animal tissues. Only a few applications of high-performance liquid chromatography (HPLC) to the analysis of plant lipids have been described. Gradients of hexane-isopropanol-water1,2 and isocratic elution with acetonitrile-methanol-sulphuric acid3 have been used as mobile phases with columns of silica gel and UV spectrophotometric detection at 205 nm to separate a range of complex lipids of plants, and conditions similar to the former have been employed with an amine-bonded phase 4. These and related methods have been revieweds. With UV detection methods, direct quantification is rarely possible, and traces of oxidised lipids can give an intense signal which swamps that of interest; a limited range of solvents can be used in the mobile phase and this can impose a restriction on the quality of the separation. Recently, a rapid HPLC method for the analysis of lipid classes from animal tissues was described, in which a column of silica gel was used with a ternary gradient elution scheme 6,7. Lipids ranging in polarity from cholesterol esters to lysophosphatidylcholine were eluted separately in only 20 min. The by Applied Chromatodetector employed was the “mass detector” manufactured graphy Systems (Macclesfield, U.K.), also known as an “evaporative analyser” or “light-scattering detector”. With this system, almost any organic solvent can be employed in the mobile phase and good quantification is possible with careful calibration. The procedure has now been adapted for the sequential separation of the individual glycolipids and phospholipids in cereal extracts. EXPERIMENTAL
Lipids were extracted from unbleached wheat flour with water-saturated butanols. The individual classes of glycolipids and phospholipids were analysed by a thin-layer chromatography (TLC) procedureQ, and then were isolated by preparative TLC to provide reference lipids for identification of components emerging from the 0021-9673/88/$03.50
0
1988 Elsevier Science Publishers
B.V.
511
NOTES
TABLE
I
TERNARY GRADIENT ELUTION SYSTEM REQUIRED CLASSES AND REACTIVATION OF THE PROBLEM
FOR
THE
SEPARATION
OF LIPID
For A, B and C, see Experimental. Step
Time (min)*
Mobile phases A
B
C
1 2
0 8
100 -
loo
_ _
3 4 5 6
23 28 28.1 33
_ _ 100 100
_ _ _ _
100 100 _ _
between
the compositions
* A linear gradient
was produced
specified at each time interval.
HPLC system. The lipid extract used as the test mixture in developing the elution system consisted of so-called “free lipids”, extracted from wheat flour with light petroleum (b.p. 40-60°C) at 20°C f O.l”C using 16 ml solvent per g of flourlo. The HPLC equipment, including the column of silica gel, was as described previously6,‘. No guard column was used, but an Upchurch filter with a stainlesssteel frit (2-pm pores; Scotlab Instrument Sales, Bellshill, U.K.) was incorporated between the injector and the column to trap microparticulate impurities. All natural samples were filtered through Acrodisc TM-CR disposable filters (0.45~pm pores, Gelman Sciences, MI, U.S.A.) prior to analysis. In the gradient elution scheme finally adopted, the three reservoirs in the ternary solvent mixer contained the following: (A) hexane-butan-2-oneeacetic acid (35:65:0.4, v/v); (B) hexanechloroformisopropanol-aqueous buffer (42:5:45:3, v/v); (C) as B but 32:5:50:8 (v/v). The aqueous buffer consisted of 0.5 mM serine adjusted to pH 7.5 with ethylamine. The flow-rate was 2 ml/min, and the solvent elution programme is shown in Table I. RESULTS
AND DISCUSSION
When cereal lipids were examined by the ternary gradient scheme developed
TABLE
II
COMPOSITION
OF THE POLAR
LIPIDS
IN THE TEST MIXTURE
Lipid class
Abbreviation
wt.%
Monogalactosyldiacylglycerol Monogalactosylmonoacylglycerol Digalactosyldiacylglycerol Digalactosylmonoacylglycerol N-Acylphosphatidylethanolamine N-Acyllysophosphatidylethznolamine Others
MGDG MGMG DGDG DGMG NAPE NALPE
16.0 4.4 41.6 14.5 10.4 5.1 1.4
512
NOTES
previously for the separation of the lipids of animal tissues6,7, the glycolipids and the less polar phospholipids emerged together from the column with the introduction of the aqueous component of the mobile phase and individual components could not be distinguished. Glycolipids are commonly separated from phospholipids by means of adsorption chromatography with mobile phases containing solvents such as acetone’ l. Various elution schemes were, therefore, tried in which acetone and solvents in the same selectivity groupi2, such as ethyl acetate or butan-2-one, were employed as constituents of the mobile phase in the early stages of the procedure. Rather than using a complete lipid extract from a cereal, a glycolipid-rich fraction was used as the test mixture, since it contained mostly those lipid classes that had not been resolved by HPLC in previous studies. Its composition was determined by a TLC procedure9 and is shown in Table II. In addition to glycolipids, it contained Nacylphosphatidylethanolamine and its lyso derivative, but trace amounts only of more polar phospholipids. To control the overall polarity of the initial mobile phase, hexane in various concentrations was added. Later, it was found advantageous to add a small amount of acetic acid as this gave sharper peaks. In general, it was a relatively easy matter to separate the glycolipid classes from each other, but it proved more difficult to resolve DGMG from NAPE. The best results were obtained with a gradient of hexane-butan-2-oneeacetic acid (35:65:0.4, v/v), changed by means of a linear gradient to a solvent mixture corresponding to that devised earlier6,7 for the start of the separation of individual phospholipids (step 1 to 2 in Table I). The nature of the
, 5
10 time
15 (mid
Fig. 1, Separation of the glycolipid-rich fraction from wheat flour, detailed in Table II, by HPLC on a column of silica gel by the ternary gradient elution system described in the Experimental section. Details of the abbreviations are given in Table II.
NOTES
513
separation is shown in Fig. 1. Unfortunately, the individual simple lipids were not resolved, as they were not retained by the adsorbent, and they emerged together at the start of the analysis. It would no doubt be possible to correct this if a quaternary solvent delivery system were available. The next step in the elution sequence (step 2 to 3 in Table I) corresponds to the stage in the elution system devised previously in which the individual phospholipid classes are separated6s7. Step 3 to 4 is only required if lysophosphatidylcholine is present in appreciable concentrations, while the final steps are necessary to restore the activity of the column for the analysis of the next sample. Preliminary work on the calibration of the system for the analysis of cereal lipids appears to indicate that the response of the detector to glycolipids resembles that for phospholipids, i.e. that a sigmoidal relationship holds and tails off for components present at amounts below 10 pg. A different calibration line is obtained for each component of the standard mixture. This problem and other difficulties associated with sample extraction are now being addressed. REFERENCES 1 C. Demandre, A. Tremolieres, A.-M. Justin and P. Mazliak, Phytochemistry, 24 (1985) 481. 2 N. Sotirhos, C.-T. Ho and S. S. Chang, Fette Seifen Anstrichm., 88 (1986) 6. 3 D. Marion, G. Gandemer and R. Douillard, in P.-A. Siegenthaler and W. Eichenberger (Editors), Structure, Function and Metabolism of Plant Lipids, Elsevier, Amsterdam, 1984, p. 139. 4 J. W. M. Heemskerk, G. Bogemann, M. A. M. Scheijen and J. F. G. M. Wintermans, Anal. Biochem., 154 (1986) 85. 5 W. W. Christie, High-Performance Liquid Chromatography and Lipids, Pergamon, Oxford, 1987. 6 W. W. Christie, J. Lipid Res., 26 (1985) 507. 7 W. W. Christie, J. Chromatogr., 361 (1986) 396. 8 W. R. Morrison, D. L. Mann, S. Wong and A. M. Coventry, J. Sci. Food Agric., 26 (1975) 507. 9 W. R. Morrison, S. L. Tan and K. D. Hargin, J. Sci. Food Agric., 31 (1980) 329. 10 W. R. Morrison, L. J. Wylie and C. N. Law, J. Cereal. Sci., 2 (1984) 145. 11 W. W. Christie, Lipid Analysis, Pergamon, Oxford, 2nd ed., 1982. 12 L. R. Snyder, J. Chromatogr. Sci., 16 (1978) 223.