Separation of Oxidized and Unoxidized Molecular Species of Phosphatidylcholine by High Pressure Liquid Chromatography 1 C.G. CRAWFORD, R.D. PLATTNER, O.J. SESSA, and J.J. RACKIS, Northern Regional Research Center, Agricultural Research, Science and Education Administration, U.S. Department of Agriculture, Peoria, Illinois 61604 ABSTRACT

Soy phosphatidylcholine (PC) has been separated into its major molecular species by reversephase high pressure liquid chromatography (HPLC). An aqueous methanol gradient was used that allowed detection of the various species by their absorbance at 206 nm. Oxidized species were detected by their absorbance at 234 nm and were resolved from the unoxidized species. This technique has been used to separate and purify unoxidized dilinoleyl phosphatidylcholine (di 18:2 PC) from other species of soy PC and to monitor the autoxidation of an aqueous suspension of the purified di 18:2 PC. Two oxidized products were formed from di 18:2 PC. Further analysis showed that they were PC, but one of the products contained an oxidized and an unoxidized fatty acid; in the other product, both fatty acids were oxidized. INTRODUCTION

Although high pressure liquid chromatography (HPLC) has found widespread use for separating many organic compounds, there is only a limited number of reports on the HPLC separation of phospholipids. A major deterrent to the use of HPLC as an analytical technique for lipid analysis has been the problem of rapidly detecting small amounts of phospholipid in the eluant. The 200 nm range of phospholipid absorbance limits the choice of solvents to those which do not absorb in that region, although several solvent systems have been found which allow ultraviolet (UV) detection and provide resolution of phospholipid classes by HPLC on silicic acid (1,2) or /~Bondapak-NH 2 columns (3). We have been studying oxidized PC from soy (4,5) and have found that it contributes to the bitter flavor of soy meal. In order to minimize autoxidation during isolation and to facilitate studies on the oxidiation of molecular species, we wanted a rapid method that could separate oxidized from unoxidized phosphatidylcholine (PC). The resolution of compounds by reversephase chromatography is due, in part, to the nonionic interactions of molecules with the stationary support; therefore, this chromatographic technique could provide a method to separate oxidized from unoxidized species of PC. Porter et al. (6) have recently reported the reverse-phase separation of several PC molecular species, but the inclusion of chloroform in the solvent only allows detection by refractive index, which precludes gradient elution and 206 nm detection. 1present in part at FASEB 63rd Annual Meeting, Dallas, Texas, April 1979. 91

Arvidson (7) has reported the reverse-phase separation of egg PC molecular species on an alkylated derivative of Sephadex in a methanolwater solvent system. We report here the separation of soy PC molecular species and the separation of oxidized from unoxidized species by reverse-phase HPLC in aqueous methanol while detecting the lipid species by their UV (206 nm) absorbance. MATERIALS AND METHODS

Soy PC was purified from commercial lecithin (Central Soya, Chicago, IL) by chromatography on Florisil and DEAE-cellulose columns (Supelco Inc., Bellefonte, PA) as described by Sessa et al. (4). Thin layer chromatography (TLC) was carried out on Silica Gel 60 plates (EM Laboratory, Inc., Elmsford, NY) with CHC13/CH3OH/H20 (65:35:4), and the lipids were visualized with 380 nm light after spraying with 0.1% 8-anilino-l-napthalene sulfonate in H 2 0 (8). Reverse-phase chromatography was carried out on a/2Bondapak C 18 column (3.9 mm x 30 cm) in aqueous methanol, either isocratically (95%) or with a linear gradient (91 to 95%) at 2 ml/min. Equipment for HPLC (Waters Assoc., Inc., Milford, MA) included model 6000-A pumps, UK6 injector, column, model 450 detector, and model 660 solvent programmer. Lipids were detected by their absorbance at 206 or 234 nm. Fractions were collected from the HPLC eluant, chloroform was added to make them 2:1 in CHC13/CH3OH, then sufficient 1.0% NaC1 was added to give 0.2 vol of aqueous phase (9). The organic phase was separated, washed with water, then stored at -10 C until analyzed. Samples of the isolated fractions were

C.G. CRAWFORD, R.D. PLATTNER, D.J. SESSA AND J.J. RACKIS

92

transmethylated with 0.5 M KOH in anhydrous methanol, neutralized with glacial acetic acid, and after addition of H 2 0 , the methyl esters were extracted with petroleum ether/diethyl ether (1 : 1). Part of each sample that contained oxidized fatty acids was transmethylated in the presence of NaBH 4. The methyl esters were analyzed on a Packard 428 gas chromatograph using a 6 ft x 2 mm glass column packed with 10% DEGS or a 3 ft x 2 mm glass column packed with 5% Apiezon L (Supelco Inc., Bellefonte, PA). Oxidized fatty acid methyl esters were analyzed by gas chromatography-mass spectroscopy (GC-MS) after silylation as described elsewhere (10). Dilinoleyl phosphatidylcholine (di 18:2 PC) was prepared in the following manner. The eluant from HPLC containing di 18:2 PC was collected and combined from a series of runs of soy PC and then extracted as described above. This material was rechromatographed, reextracted into chloroform, and used for the oxidation studies. The di 18:2 PC was oxidized as an aqueous suspension. The lipid was taken to dryness under nitrogen; then water was added to make the suspension 1 pmole/ml. This suspension was mixed vigorously for 5 rain and then stirred gently in air at room temperature. Aliquots (25 /al) were analyzed by HPLC at various time intervals to monitor the reaction; after oxidizing for 60 hr, the lipids were extracted with CHC13/CH3OH (2:1). The extract was then chromatographed by reverse-phase HPLC in 90% CH3OH , and the separated oxidized products and unoxidized material were collected. Collected fractions were taken to dryness in vacuo with addition of CH3OH and then stored at -10 C in CHC13 until analyzed.

RESULTS

AND

DISCUSSION

When soy PC is chromatographed by reversephase in a gradient of methanol/water, it is resolved into a number of species as shown in Figure 1. Fatty acid analysis of the material in the major peaks indicated in Figure 1-A is given in Table I, along with the major molecular species found in each peak. Peak V contains a number of fatty acids other than 18:3, which may be derived from contaminating oxidized species (see below). Peak VI appears to be pure 18:2-18:3 PC, but if the sample is contaminated by equal amounts of di 18:3 from the preceding peak and di 18:2 from the following peak, the contamination would be impossible to detect by fatty acid analysis. The di 18:2 LIPIDS, VOL. 15, NO. 2

0xpd~zedSpec=es 05t

~] d118:2

B

A E

0025 ell 18V3

10 Min

1 20

3t0 Mm

1 40

50

FIG. 1. Elution profile of UV absorbing species of soy PC. Conditions: 2.5 mg PC, pBondapack C18 eluted at 2 ml/min with 91% aqueous CH3OH for 15 min, then with a linear gradient to 95% methanol for 20 min. A, absorbance at 206 nm, B, absorbance at 234 nm. fraction (Peak VII) appears to contain a small amount of 16:0-18:3 species even after rechromatography on reverse-phase HPLC. No attempt was made to resolve the 16:0-18:2 species (Peak VIII) from the 18:1-18:2 species (Peak IX), but they may be separated on the Fatty Acid Analysis C o l u m n as described by Porter et al. (6) who report the separation of the analogous molecular species 16:0-18:1 from di I8:1. The component of Peak X was a mixture of all the fatty acids listed in Table I; due to the small amount found in soy PC, it was not studied further. Analysis of the fatty acids in the leading edge of Peak XI showed it to contain mostly 18:1, indicating that under these conditions di 18:1 PC may not be completely resolved from 18:0-18:2 PC. Reverse-phase HPLC of fatty acid methyl esters (11) and triglycerides (12) has shown that species differing by two carbons can be separated and that a double bond decreases the retention time equivalent to a saturated species with two less carbons. This separation is also seen in our reverse-phase chromatography of phosphatidylcholine and, because of the comparatively simple fatty acid composition of soy PC, the major molecular species of soy PC can be resolved. Figure 1-B shows the chromatogram of the same sample measuring the absorbance at 234 nm. Absorbance at 234 nm was taken as an indication of oxidized fatty acid moieties and by this criterion, the oxidized species of soy PC have been resolved from the unoxidized except in the area of the di 18:3. Analysis by GC-MS of the fatty acid methyl esters in the first

HPLC OF SOY PC MOLECULAR SPECIES

93

TABLE I Mole Percent Fatty Acid Composition of the Soy PC Species Separated by Reverse-Phase HPLC Peak a V VI VII VIII 1X XI

16:0

18:0

5.0

1.5

2.2 39.2 22.5 0.8

0.9

43.8

18 : 1

18:2

18 : 3

2.7

3.2 49.5 93.2 51.7 51.4 45.4

87.0 49.6 3.6

8.9 26.0 7.3

Major species di-18:3 18:2-18:3 di-18:2 16:0-18:2 18:1-18:2 18:0-18:2

2.6

aSee Figure 1 for peaks analyzed. f o u r p e a k s s h o w e d t h a t in a d d i t i o n to uno x i d i z e d f a t t y acids, t h e r e was a c o m p l e x m i x t u r e o f oxo, e p o x y , a n d h y d r o x y f a t t y acids as previously described (5). T h e di 18:2 PC f r a c t i o n o f soy PC was collected, t h e n purified b y r e c h r o m a t o g r a p h y a n d allowed to u n d e r g o a u t o x i d a t i o n in water. Figure 2-A a n d B s h o w respectively t h e H P L C spectra b e f o r e a n d a f t e r o x i d a t i o n , i n d i c a t i n g a decrease in u n o x i d i z e d di 18:2 PC w i t h t h e g e n e r a t i o n o f t w o n e w species t h a t have abs o r b a n c e at 234 n m (Figure 2-C). TLC analysis o f t h e c o m p o u n d s c o m p r i s i n g t h e t h r e e p e a k s is s h o w n in Figure 3, and a l t h o u g h t h e y c a n n o t b e s e p a r a t e d b y TLC u n d e r t h e c o n d i t i o n s used, t h e r e is a slight d i f f e r e n c e in p o l a r i t y in t h e o r d e r e x p e c t e d f r o m t h e reverse-phase HPLC separation. T h e t o t a l i o n i z a t i o n c h r o m a t o g r a m of t h e silylated m e t h y l esters f r o m t h e PC in Peak 2 (Figure 2) is p r e s e n t e d in Figure 4. Mass spectra 93, 104, a n d 108 ( i n d i c a t e d b y arrows) are all similar w i t h m a j o r i o n peaks at 382, 311, 225, 130, a n d 73, i n d i c a t i n g t h a t t h e o x i d i z e d f a t t y acids are 9 or 13 h y d r o x y dienes ( 1 0 , 1 3 ) . T h e i r chromatograptiic b e h a v i o r allows t e n t a t i v e i d e n t i f i c a t i o n as 9 or 13 cis-transand 9 or 13 trans-trans h y d r o x y d i e n e (13). GC-MS o f t h e silylated m e t h y l esters f r o m t h e PC in Peak 1 (Figure 2) s h o w e d t h e same f r a g m e n t a t i o n p a t t e r n , e x c e p t t h a t t h e a m o u n t of u n o x i d i z e d 18:2 was m a r k e d l y r e d u c e d . By q u a n t i t a t i o n of t h e m e t h y l esters o n A p e i z o n L., t h e 18:2 to h y d r o x y d i e n e r a t i o was f o u n d t o b e 1:1 in Peak 2 a n d 1 5 : 8 5 in Peak 1, w h i c h i n d i c a t e s t h a t a single f a t t y acid is o x i d i z e d in t h e PC in Peak 2, b u t b o t h f a t t y acids are o x i d i z e d in t h e PC in Peak 1. T h e o x i d i z e d PC m a y b e h y d r o p e r o x i d e as isolated, b u t lack o f m a t e r i a l has p r e v e n t e d a c h e m i c a l d e t e r m i n a t i o n . T h e r e was n o qualitative d i f f e r e n c e b e t w e e n t h e o x y g e n a t e d f a t t y acids e x t r a c t e d f r o m u n t r e a t e d o r N A B H 4 t r e a t e d s a m p l e s ; b u t m o r e h y d r o x y d i e n e was

IJ

'i--

4

8 12 Min.

16

4

,

8 12 Min.

16

cI

8 12 Min.

16

FIG. 2. Reverse-phase HPLC elution pattern of 20 pg di 18:2 PC resolved in 95% CH3OH at 2 ml/min. A, 206 nm absorbance before oxidation; B, 206 nm absorbance after 50 hr oxidation; C, 234 nm absorbance after 50 hr oxidation.

FIG. 3. Silica gel TLC of di 18:2 oxidation products resolved in CHC13/CH3OH/H20 (65:35:4). Lane 1, lyso PC standard- lane 2, di 18 2 PC before oxidation ; lane 3, total extract of di 18:2 PC oxidized 50 hr; lane 4, PC in Peak 1 (Fig. 2);lane 5, PC in Peak 2 (Fig. 2); lane 6, PC in Peak 3 (Fig. 2). LIPIDS, VOL. 15, NO. 2

94

C.G. CRAWFORD, R.D. PLATTNER, D.J. SESSA AND J.J. RACKIS

recovered from samples that had been reduced with NaBH4, indicating that some of the fatty acids were hydroperoxides. Although oxidized soy PC contains many forms of oxygenated fatty acid(s), hydroxy diene was the only product found in the aqueous autoxidation of di 18:2 PC. This implies that interaction between different molecular species can modify the oxidative products; results of studies on these interactions will be the subject of future reports.

100 18:2

75

i2= 50

ACKNOWLEDGMENTS We thank H.W. Gardner, G.E. Spencer, and R. Kleiman for helpful discussions during the course of this work. REFERENCES 1. Jungalwala, F.B., J.E. Evans, and R.H. McCluer, Biochem. J. 155:55 (1976). 2. Guerts van Kessel, W.S.M., W.M.A. Hax, R.A. Demel, and J. de Gier, Biochim. Biophys. A c t a 486:524 (1977). 3. Kiuchi, K., T. Ohta, and H. Ebins, J. Chromatogr. 133:226 (1977). 4. Sessa, D.J., K. Warner, and J.J. Rackis, J. Agric. Food Chem. 24:16 (1976). 5. Sessa, D.J., H.W. Gardner, R. Kleiman, and D. Weisleder, Lipids 12:613 ( 1977). 6. Porter, N.A., R.A. Wolf, and J.R. Nixon, Lipids 14:20 (1979). 7. Arvidson, G.A.E., J. Chromatogr. 103:201 (1975). 8. Gitler, C., Anal. Biochem. 50:324 (1972). 9. Folch, J., M. Lees, and G.H. Sloane-Stanley, J. Biol. Chem. 226:497 (1957). 10. Kleiman, R., and G.F. Spencer, J. Am. Oil Chem. Soc. 50:31 (1973). 11. Scholfield, C.R., J. Am. Oil Chem. Soc. 52:36

LIPIDS, VOL. 15, NO. 2

18:0

20

40

60 80 Spectrum Number

100

120

FIG. 4. GC-MS total ion chromatogram of the silylated methyl esters from the PC in Peak 2 (Fig. 2).

12. 13.

(1975). Plattner, R.D., G.F. Spencer, and R. Kleiman, J. Am. Oil Chem. Soc. 54:511 (1977). Frankel, E.N., W.E. Neff, W.K. Rohwedder, B.P.S. Khambay, R.F. Garwood, and B.C.L. Weedon, Lipids 12:908 (1977).

[Received August 20, 1979]