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Separation of tocopherol and tocotrienol isomers using normal- and reverse-phase liquid chromatography
ANALYTICALBIOCHEMISTRY 1 8 0 , 3 6 8 - 3 7 3 (1989)
Separation of Tocopherol and Tocotrienol Isomers Using Normal- and Reverse-Phase Liquid Chromatography Barrie Tan i and Linda Brzuskiewicz Department of Chemistry, University of Massachusetts, Amherst, Massachusetts 01003
Received N o v e m b e r 21, 1988
This optimization study for tocopherols and tocotrienols involved both normal- and reverse-phase liquid chromatography using various columns and mobile phases. Normal-phase systems showed elution of the h o m o l o g s in o r d e r o f i n c r e a s i n g p o l a r i t y w i t h s e p a r a tion based on methyl substituents on the chromanol moiety. Reverse-phase systems showed class separation based on the saturation of the phytyl side chain; the more saturated tocopherols were retained on the column longer. When the Zorbax ODS was used with an isocratic ternary acetonitrile:methanohmethylene chloride (60:35:5) mixture, the optimized resolution w a s > 2 . 0 a n d s e p a r a t i o n w a s a c h i e v e d in < 1 3 m i n , b u t t h e r e w a s n o s e p a r a t i o n o f ~- a n d 7 - t o c o p h e r o l s . T h e normal-phase silica and amino columns provided separ a t i o n o f all a v a i l a b l e i s o m e r s w i t h r e s o l u t i o n > 1.1 a n d s e p a r a t i o n t i m e s o f < 5 . 5 a n d < 1 0 m i n , r e s p e c t i v e l y . Optimized isocratic binary solvent mixtures of hexane:2propanol were used for silica (99:1) and amino (98:2) c o l u m n s . D e r i v a t i v e s p e c t r a s h o w e d d i f f e r e n c e s dep e n d i n g o n s u b s t i t u e n t s in t h e c h r o m a n o l m o i e t y b u t not the phytyl side chain. Second- and fourth-derivative spectra gave the best differentiation of the vitamin E isomers.
© 1989 Academic Press, Inc.
T h e naturally occurring tocopherols and tocotrienols constitute the majority of the vitamin E group of compounds. Other members of the vitamin E group consist of tocopherol esters and derivatives found in multivitamin supplements and as food preservatives. T he tocopherols and tocotrienols are light yellow, fat-soluble, viscous oils produced primarily in plants. T h e chloroplasts of young plants contain significant amounts of a-tocopherol, which is the most biologically active isomer. As the plant matures and fruits, various other tocopherols appear (1). Some food sources containing vitamin E ini T o w h o m correspondence should be addressed. 368
clude plant and seed oils, nuts, whole grains, green leafy vegetables, eggs, liver, and milk (2). T he basic structure of tocopherol is shown in Fig. 1. Tocopherols are methyl-substituted hydroxychromans with a phytyl side chain. Natural vitamin E is composed of two homologous series: (i) the tocopherols with a saturated side chain, and (ii) the tocotrienols with an unsaturated side chain. T h e three asymmetric carbons allow a total of eight possible diastereoisomers. T h e naturally occurring stereoisomer of a-tocopherol has all three carbons in the R-configuration and is known as D-a-tocopherol. T he structural name for this compound is 2,5,7,8tetramethyl-2-(4',8',12'-trimethyltridecyl)-6-chromanol. Even though cis-trans isomers of tocotrienols occur, it appears t h a t all of the natural tocotrienols possess the all-trans configuration (1). T he chemical name for a-tocotrienol is 2,5,7,8-tetramethyl-2-(4',8',12'-trimethyltrideca-3',7',ll'-trienyl)-6-chromanol. In addition to the difficulty of separating the eight isomers on the basis of their similar structural and physical properties, these compounds also have similar uv spectra. Their spectra are fairly nondescript and have a ~max ranging from 295 nm for a-tocopherol (literature value of 292 nm, Ref. (3)) to 298 nm for 5-tocopherol as illustrated in Fig. 2. A recent paper by Bukovits and Lezerovich (4) indicated t h a t use of second-derivative spectra for the analysis of tocopherols aided in the identification of components in a mixture due to the stepwise elimination of background absorption as derivative order was increased. T he major reason for studying vitamin E is its natural antioxidant capabilities. Vitamin E functions as an antioxidant to protect fat in membranes around cells (such as nerves, heart, muscles, and red blood cells) from damage by oxygen (2). While a-tocopherol is the most biologically active form of natural vitamin E, biological activity is not a basis for determining antioxidant activity (1). Less is known about the antioxidant capabilities of individual isomers. 0003-2697/89 $3.00 Copyright © 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.
OPTIMIZATION STUDY OF TOCOPHEROLS AND TOCOTRIENOLS R e c e n t reviews b y P a r r i s h (1) a n d Nelis et al. (5) h a v e e x t e n s i v e l y discussed various m e t h o d s for a n a l y z i n g vit a m i n E isomers r a n g i n g f r o m t h e colorimetric m e t h o d s of E m m e r i e a n d E n g e l (6), to T L C , GC, a n d finally H P L C . While H P L C is p r e s e n t l y t h e b e s t m e t h o d for a n a l y z i n g these isomers, m o s t l i t e r a t u r e refers only to t h e analysis of the four tocopherols. T h i s is n o t too surprising as m o s t vegetable oils a n d biological fluids cont a i n v a r y i n g a m o u n t s of t h e d o m i n a n t t o c o p h e r o l s a n d less of the t o c o t r i e n o l s (5). T o c o t r i e n o l s h a v e b e e n f o u n d in p a l m oil, c o c o n u t oil, a n d cereal grains such as wheat, rye, oats, a n d b a r l e y (7). C u r r e n t i n t e r e s t in the tocotrienols to aid in decreasing c a r d i o v a s c u l a r disease (8,9) a n d as a possible a n t i c a r c i n o g e n i c a g e n t (8) creates a n e e d to f u r t h e r optimize H P L C s e p a r a t i o n s y s t e m s to i n c o r p o r a t e t h e tocotrienols. T h u s , the p u r p o s e of this r e p o r t was to s i m u l t a n e o u s l y a n a l y z e t o c o p h e r o l a n d t o c o t r i e n o l i s o m e r s using various n o r m a l - a n d reversep h a s e liquid c h r o m a t o g r a p h i c systems. Use of seconda n d f o u r t h - d e r i v a t i v e s p e c t r a was also included to enh a n c e identification.
Reagents and materials. All solvents were H P L C grade a n d were p u r c h a s e d f r o m Fisher Scientific Co. (Fair Lawn, N J ) . T h e four t o c o p h e r o l s t a n d a r d s were o b t a i n e d f r o m the H e n k e l Corp. (La Grange, IL), a n d t h e t h r e e t o c o t r i e n o l s t a n d a r d s were o b t a i n e d f r o m P O R I M ( K u a l a L u m p u r , M a l a y s i a ) . / 3 - T o e o t r i e n o l was t h e only c o m p o u n d which could n o t be obtained. T h e
TOCOPHEROL 5
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TOCOTRIENOL
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Position of Methyl Group Tocopherols Tocotrienols 5,7,8-Trimethyl a-T a-T3 5,8-Dimethyl I~-T 13-T3 7,8-Dimethyl ¥-T ¥-T3 8-Monomethyl 8- T 8- T3 FIG. 1.
28~
290
Navelen~th
308
31~
(nm)
320
33{3
FIG. 2. Ultraviolet-visible spectra for tocopherols and tocotrienols, where 1 is a-T, 2 is a-T3, 3 is fl-T, 4 is ?-T, 5 is ~/-T3, 6 is 5-T, and 7 is ~-T3.
t o c o p h e r o l a n d t o c o t r i e n o l s t a n d a r d s were k e p t in a m b e r vials, flushed with nitrogen, a n d stored at - 2 0 ° C .
MATERIALS AND METHODS
FO~
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369
Structures oftocopherols and tocotrienols.
Sample preparation. T h e seven s t a n d a r d s were dissolved in m e t h a n o l for r e v e r s e - p h a s e c h r o m a t o g r a p h y with c o n c e n t r a t i o n s r a n g i n g f r o m 0.131 to 0.197 m g / m l . T h e s e s a m e s t a n d a r d s were dissolved in h e x a n e for norm a l - p h a s e w o r k with a c o n c e n t r a t i o n range of 0.142 to 0.167 m g / m l . F r o m these stock solutions, two m i x t u r e s were made: one for each p h a s e type. E a c h m i x t u r e consisted of all s e v e n s t a n d a r d s w i t h a final c o n c e n t r a t i o n which was o n e - t e n t h of its original stock c o n c e n t r a t i o n (approxim a t e l y 0.015 m g / m l ) . All solutions were stored in a m b e r vials w r a p p e d with a l u m i n u m foil a n d k e p t at - 2 0 ° C . Chromatography. A n a l y t i c a l i n s t r u m e n t a t i o n consisted of an H P 1090M ( H e w l e t t - P a c k a r d ) liquid chrom a t o g r a p h w i t h a n H P 9000 c o m p u t e r w o r k s t a t i o n a n d a u v - v i s d i o d e a r r a y detector. T h i s included a R h e o d y n e 7010 m a n u a l injection switching valve (20-#1 s a m p l e loop), a PV-5 t e r n a r y solvent delivery system, a n d a 4.5ttl flow cell, a n d the s y s t e m was recorded on a T h i n k j e t p r i n t e r or a C o l o r P r o 8 - p e n plotter. T h e u v - v i s diodearr a y d e t e c t o r m o n i t o r e d a w a v e l e n g t h of 295 nm, with a b a n d w i d t h of 4 nm. T h e analytical c o l u m n s used were all of the s a m e d i m e n s i o n , 25 × 0.46 cm: Vydac C-18 (Hesperia, CA); Z o r b a x ODS, CN, NH2, a n d S I L (DuP o n t Co., W i l m i n g t o n , DE). E a c h c o l u m n was used with its respective guard c o l u m n (1.5 × 0.32 cm, 7 #m) f r o m B r o w n l e e L a b s ( R a i n i n I n s t r u m e n t Co., W o b u r n , MA). T h e isocratic mobile p h a s e s for n o n a q u e o u s reversep h a s e ( N A R P ) 2 c h r o m a t o g r a p h y consisted of various bi2 Abbreviations used: NARP, nonaqueous reverse-phase; LRT, longest retention time; ACN, acetonitrile.
370
TAN AND BRZUSKIEWICZ a
mal-phase system (Fig. 3a). The lower polarity isomers (a-T and a-T3) eluted first and the higher polarity isomers (5-T and 5-T3) eluted last. Thus, the normal-phase column provided separation based on the number as well as the position of methyl substituents on the chromanol moiety. Silica columns are generally more capable of separating positional isomers such as #- and ~-tocopherol. Also, the separation indicated t h a t the polarity of the tocopherols and tocotrienols decreased with increased number of methyl groups. Use of the reverse-phase column (Fig. 3b) showed the class separation of the saturated and unsaturated side chains. The less polar but more saturated tocopherols were retained in the stationary phase longer. The order of elution within each class of compounds (i.e., tocotrienols) was from higher polarity (6-T3) to lower polarity (a-T3), which was expected for reverse-phase systems. It appeared that the side chain interacted with the stationary phase in such a way that caused the more saturated tocopherols to remain on the column longer than the unsaturated tocotrienols. Of the two reverse-phase columns used, neither one could separate the two positional isomers of #-T and ~/T. However, the Zorbax SIL and NH2 columns could separate all seven vitamin E standards. Therefore, normal-phase columns were preferred for isomeric separation. These conclusions are described later in more detail.
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F I G . 3. T y p i c a l c h r o m a t o g r a m s for t o c o p h e r o l s a n d tocotrienols. (a) N o r m a l - p h a s e , u s i n g a Zorbax S I L w i t h a 99:1 ( h e x a n e : 2 - p r o p a n o l ) mobile p h a s e , a n d (b) R e v e r s e - p h a s e , u s i n g a Z o r b a x O D S w i t h 60:35: 5 (ACN:CH~OH:CH2C12), w h e r e 1 is a - T , 2 is a - T 3 , 3 is S-T, 4 is -y-T, 5 is ~ - T 3 , 6 is 6-T, a n d 7 is 5-T3.
nary acetonitrile:methanol (ACN:CH3OH) combinations, and a ternary system of ACN:CH3OH:CH2C12 (60: 35:5). The isocratic mobile phase systems for normalphase chromatography consisted of 99% hexane and 1% polar modifier. These modifiers included methylene chloride (CH2C12), tetrahydrofuran (THF), n-butanol, and 2-propanol. All chromatographic separations were performed at ambient temperatures with a flow rate of 2.0 ml/min. Nitrogen was used as the H P L C solvent degasser. RESULTS AND DISCUSSION
Normal-phase vs reverse-phase.
Some general comments can be made regarding NARP and normal-phase chromatography. Two typical chromatograms are illustrated in Fig. 3. The first chromatogram depicted the elution order for toeopherols and tocotrienols in a nor-
Reverse-phase chromatography was used primarily for the analysis of a-tocopherol in the presence of its esters, other fat-soluble vitamins, carotenes, and other reducing substances (1,5). Although this article pertains to the analysis of tocopherol and tocotrienol isomers, it was necessary to optimize a NARP system to obviate these interferents in actual samples. This would eliminate some preparative procedures which could lead to sample loss or degradation.
TABLE 1
Comparison of Zorbax ODS and Vydac C-18 Columns for Reverse-Phase Chromatographya Zorbax O D S b
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a V a l u e s e x p r e s s e d in t e r m s of m e a n _+ SD are b a s e d o n v a r i o u s isocratic A C N : C H 3 O H mobile p h a s e s , w h e r e n for t h e Z o r b a x a n d Vydac c o l u m n s were 8 a n d 11, respectively. b P e a k n u m b e r s c o r r e s p o n d to t h o s e in t h e legend to Fig. 3b: 7, 5-T3; 5, ~/-T3; 3, #- a n d ~ - T w h i c h coeluted; a n d 1, a - T . c a, selectivity; a n d Rs, resolution.
OPTIMIZATION
STUDY OF TOCOPHEROLS
TABLE 2 Chromatographic Optimization on Various Normal-Phase Columns and Polar Modifiers" 2 -
Propanol Column c Cyano d (1,2) (6,7) Amino (1,2) (3,4) {6,7) Silica (1,2) (3,4) (6,7)
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1.19 1.14 1.20
1.7 2.0 2.8
1.17 1.12 1.17
1.4 1.7 2.2
1.18 1.21 1.45
1.17 1.11 1.16
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371
AND TOCOTRIENOLS
essary to find an optimal mobile phase which could reduce this gap and still maintain the resolution within a class of compounds. Isocratic ternary solvent compositions which utilized the organic modifier CH2C12 were tried. This solvent increased the mobile phase solubility of the tocopherols and caused t hem to elute faster. Small amounts of modifier were added until a system of 60:35: 5 (ACN:CH3OH:CH2C12) was reached. This system decreased the gap between the tocopherols and the tocotrienols, yet maintained resolution values within each class similar to t hat of the 60:40 (ACN:CH3OH) mobile phase. Hence, the use of CH2Cle decreased the longest retention time (LRT) from 13 to about 10 min and the retention gap from 3.5 to 2.5 min without the sacrifice of peak resolutions.
Normal-phase chromatography. Two reviews indicated t hat normal-phase chromatography was preferred for the separation oftocopherol isomers (1,5). There has
Values are b a s e d on t h e average of duplicate r u n s , a n d all mobile p h a s e s c o n s i s t e d of 99% h e x a n e a n d 1% modifier. b ,,,, m e a n s t h a t t h e s o l v e n t gave no p e a k s after 15 m i n or t h a t r e s u l t s were n o t reproducible. c P e a k n u m b e r s in p a r e n t h e s e s refer to t h o s e in t h e legend to Fig. 3a: 1, a - T ; 2, a - T 3 ; 3, ~-T; 4, ~ - T ; 6, 5-T; a n d 7, &-T3. P e a k p a i r s 3 a n d 4 were n o t i n c l u d e d because/3- a n d ? - T coeluted.
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Th e columns utilized for N A R P chromatography were the Vydac C-18 and the Zorbax ODS. Various mobile phases ranging from 100% acetonitrile to 100% methanol were used. Th er e were no major trends in selectivity or resolution, with an increase in methanol concentration, for either column. A summary of these two factors is presented in Table 1. T he selectivity and resolution between the first two eluting compounds and the last two compounds were studied. Figure 3b shows the elution pairs tabulated in Table 1. For the Zorbax ODS, the selectivity between the first two components was slightly higher th an th at between the last two. T h e variations of the eluting pair selectivities were less when the Zorbax column was used t h a n when the Vydac column was used. W h en the Vydac C-18 was used, lower resolutions between both elution pairs were observed. Therefore, the Zorbax ODS was chosen as the more suitable reversephase column for the analysis of tocopherol and tocotrienol isomers. Mobile phase optimization was t hen performed with the Zorbax column. Utilization of this column with 100% methanol caused all seven components to elute in about 7 min. While resolution between peaks was fairly good using this mobile phase, better resolution could be obtained using a 60:40 (ACN:CH3OH) solvent mixture. T h e retention time for the last component was reached in less th an 13 min. As methanol concentration increased, the retention gap between the two classes of compounds narrowed, but the resolution between peaks within each class became poorer. Therefore, it was nec-
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F I G . 4. Derivative s p e c t r a of t o c o p h e r o l s a n d tocotrienols. (a) Seco n d derivative a n d (b) f o u r t h derivative, w h e r e 1 is a - T , 2 is a - T 3 , 3 is /3-T, 4 is ~ - T , 5 is -y-T3, 6 is 5-T, a n d 7 is 5-T3.
372
TAN AND BRZUSKIEWICZ
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F I G . 5. Chromatographic separation of vitamin E isomers in crude palm oil: (a) using the optimal normal-phase of hexane:2-propanol (99:1) with the Zorbax SIL, and (b) using the optimal reverse-phase of ACN:CH8OH:CH2C12 (60:35:5) with the Zorbax ODS, where l is aT, 2 is a-T3, 5 is ~,-T3, and 7 is 5-T3.
been little to no H P L C separation of tocotrienols alone or tocotrienols with tocopherols reported. T h e optimization study for normal-phase systems was more complex. T he Zorbax cyano, amino, and silica columns were employed. T h e mobile phases consisted of 99% hexane and 1% of a polar modifier. These modifiers included 2-propanol, n-butanol, T H F , and CH2C12. A summary of results for selectivity and resolution between the first two, last two, and f~- and ~/-T is given in Table 2. T h e cyano bonded-phase LC column could be used in both normal- and reverse-phase systems, depending on the compound of interest. In this particular study, the elution order of the isomers followed t h a t of normalphase (i.e., least polar ones eluted first). However, the cyano column was similar to reverse-phase due to its inability to separate fl- and -~-tocopherol. No separation efficiency was gained by using one modifier over another
as far as selectivity and resolution were concerned (Table 2) except t h a t 2-propanol caused all seven isomers to elute with a L R T of about 6 min. T e t r a h y d r o f u r a n and n-butanol modifiers gave similar L R T s of about 6.5 to 7 min, with CH2C12 at 10 min. Also, the components have better peak shapes when 2-propanol was used. T h e use of n-butanol offered slightly better column efficiency, and the use of CH2C12 showed higher capacity factors (k') t han the use of other solvents. T he cyano column was not useful for reverse-phase work as all isomers eluted as one peak in less t han 2 min for both mobile phases of 100% methanol and 100% acetonitrile. Vitamin E isomeric separations using the bondedphase amino column gave the same elution order as th a t of Fig. 3a. Table 2 indicates t hat 2-propanol as a modifier offered better selectivity and resolution between the three sets of peaks under investigation. No values were obtained for CH2C12 because no peaks were detected within the first 15 min of the chromatographic run. This was probably due to its low solvent strength. T h e retention time for the last component in 2-propanol and nbutanol was about 13 to 15 min, whereas the retention time for T H F was 23 min. When the amount of modifier was increased to 2% 2-propanol, the total retention time decreased to less t han 10 min and good resolution between peaks was maintained. T h e silica column was most commonly used for normal-phase chromatography. Table 2 shows that while selectivities and resolutions for systems using 2-propanol and n-butanol were similar, the use of 2-propanol offered better resolution between ~-T and ~-T3. T h e lower polarity modifiers of T H F and CH2C12 were not useful for the silica column. T he T H F was very unstable and chromatograms were not reproducible, and CH2C12 was not strong enough to elute the isomers. T h e total retention time for all seven isomers was about 5.5 min for 2-propanol and about 6 min for n-butanol. T h e k' for each component was generally higher for the system using n-butanol. However, the column efficiencies using 2-propanol with the silica column were superior to those of any of the systems tested for the other two columns. T he resolution between f~- and ~-T was lower for the silica column t han for the amino, but still at an acceptable value of 1.0. Resolution could be improved using 2-propanol modifier below 1%. However, it was difficult to accurately reproduce this system with small modifier composition, and a new solution was needed each day. For the optimal normal-phase system of Zorbax SIL with 99:1 (hexane:2-propanol), derivative spectra were examined. T h e zeroth-order spectra are found in Fig. 2. Figure 4 shows second- and fourth-derivative spectra, respectively. Differences occurred only in substituents in the chromanol moiety but not in the phytyl side chain (e.g., ~-T and ~-T3 were indistinguishable in the zeroth, second, and fourth derivatives). T here was a bathochromic shift of about 4 nm from the ~ to/3 to ~ to ~ isomers.
OPTIMIZATION STUDY OF TOCOPHEROLS AND TOCOTRIENOLS T h i s was seen m o r e readily in the s e c o n d - d e r i v a t i v e t h a n in the z e r o t h - o r d e r spectra. T h e a- a n d fl-isomer groups were distinguished f r o m all o t h e r groups in the second derivative, b u t t h e 7- a n d 5-isomer groups were n o t (Fig. 4a). M i n o r d i f f e r e n t i a t i o n was observed b e t w e e n t h e 3~ a n d 5 i s o m e r s w i t h t h e f o u r t h derivative (Fig. 4b). A specially p a c k e d a l u m i n a c o l u m n ( W a t e r s - M i l l i pore, Milford, MA) was also tested. H o w e v e r , all isocratic solvent s y s t e m s a t t e m p t e d on this c o l u m n were u n s t a b l e a n d results were n o t reproducible. Of t h e four c o l u m n s t e s t e d for n o r m a l - p h a s e , t h e silica a n d a m i n o were m o s t suitable for the s e p a r a t i o n of tocopherols a n d tocotrienols. Applications. T h e o p t i m a l s y s t e m s for e a c h p h a s e t y p e were applied to vegetable oils such as a l m o n d , safflower, corn, w h e a t g e r m , apricot, a n d p a l m oil. Vegetable oils, for the m o s t p a r t , required little s a m p l e cleanup; therefore, the only s a m p l e p r e t r e a t m e n t was to dissolve t h e oil in a suitable solvent (ethyl a c e t a t e for r e v e r s e - p h a s e , a n d h e x a n e for n o r m a l - p h a s e ) a n d chill o v e r n i g h t at - 2 0 ° C. T h i s c a u s e d s o m e of the sterols a n d triglycerides to precipitate. S a m p l e s were t h e n centrifuged at 4°C to r e m o v e t h e solids, a n d t h e lipid-soluble s u p e r n a t a n t was c h r o m a t o g r a p h e d . Crude p a l m oil, which c o n t a i n s significant a m o u n t s of tocotrienols, b e s t exemplified the s i m u l t a n e o u s s e p a r a t i o n of t o c o p h e r o l s a n d tocotrienols. T h e s e results are s h o w n in Fig. 5. Q u a n t i t a t i o n was a c c o m p l i s h e d u s i n g the calibration of standards. CONCLUSIONS On the basis of the c h r o m a t o g r a p h i c factors discussed, the s y s t e m of 99:1 ( h e x a n e : 2 - p r o p a n o l ) in c o n j u n c t i o n
373
with the Z o r b a x S I L c o l u m n was the o p t i m a l n o r m a l p h a s e s y s t e m for the analysis of t o c o p h e r o l a n d tocotrienol isomers. T h e s y s t e m of 60:35:5 (ACN:CH3OH: CH2C12) w i t h a Z o r b a x O D S could be utilized p r o v i d e d t h a t either/3- or ~/-tocopherol (but n o t both) was p r e s e n t along w i t h o t h e r t o c o p h e r o l s or tocotrienols. I t is h o p e d t h a t the results of this s t u d y will decrease the a m o u n t of t i m e s p e n t by r e s e a r c h e r s on o p t i m i z i n g a s y s t e m so t h a t a p p l i c a t i o n s can be pursued, a n d provide the o p p o r t u n i t y to identify the t o c o p h e r o l s a n d tocotrienols concomitantly.
REFERENCES
1. Parrish, D. B. (1980) CRC Crit. Rev. Food Sei. Nutr. 13,161-187. 2. Hegarty, V. (1988) Decisions in Nutrition, pp. 181-182, Times Mirror/Mosby College Pub., St. Louis, MO. 3. Morton, R. A. (1975) Biochemical Spectroscopy, pp. 410-417, Wiley, New York. 4. Bukovits, G. J., and Lezerovich, A. (1987) d. Amer. Oil Chem. Soc. 64(4), 517-520. 5. Nelis, H. J., DeBevere, V. O. R. C., and DeLeenheer, A. P. (1985) in Modern Chromatographic Analysis of the Vitamins (DeLeenheer, A. P., Lambert, W. E., and DeRuyter, M. G. M., Eds.), pp. 129-200, Marcel Dekker, New York. 6. Emmerie, A., and Engel, C. (1938) Rec. Trav. Chim. Pays-Bas. 57, 1351. 7. Qureshi, A. A., Burger, W. C., Peterson, D. M., and Elson, C. E. (1986) J. Biol. Chem. 261, 10,544-10,550. 8. Qureshi, A. A., Ahmad, Y., and Elson, C. E. (1988) in International Oil Palm/Palm Oil Conference: Tech. Progress and Prospects (Ong, A. S. H., Ed.), PORIM Press, Kuala Lumpur, Malaysia. 9. Editorial (1987) Nutr. Rev. 45,205-207.
Separation of Tocopherol and Tocotrienol Isomers Using Normal- and Reverse-Phase Liquid Chromatography Barrie Tan i and Linda Brzuskiewicz Department of Chemistry, University of Massachusetts, Amherst, Massachusetts 01003
Received N o v e m b e r 21, 1988
This optimization study for tocopherols and tocotrienols involved both normal- and reverse-phase liquid chromatography using various columns and mobile phases. Normal-phase systems showed elution of the h o m o l o g s in o r d e r o f i n c r e a s i n g p o l a r i t y w i t h s e p a r a tion based on methyl substituents on the chromanol moiety. Reverse-phase systems showed class separation based on the saturation of the phytyl side chain; the more saturated tocopherols were retained on the column longer. When the Zorbax ODS was used with an isocratic ternary acetonitrile:methanohmethylene chloride (60:35:5) mixture, the optimized resolution w a s > 2 . 0 a n d s e p a r a t i o n w a s a c h i e v e d in < 1 3 m i n , b u t t h e r e w a s n o s e p a r a t i o n o f ~- a n d 7 - t o c o p h e r o l s . T h e normal-phase silica and amino columns provided separ a t i o n o f all a v a i l a b l e i s o m e r s w i t h r e s o l u t i o n > 1.1 a n d s e p a r a t i o n t i m e s o f < 5 . 5 a n d < 1 0 m i n , r e s p e c t i v e l y . Optimized isocratic binary solvent mixtures of hexane:2propanol were used for silica (99:1) and amino (98:2) c o l u m n s . D e r i v a t i v e s p e c t r a s h o w e d d i f f e r e n c e s dep e n d i n g o n s u b s t i t u e n t s in t h e c h r o m a n o l m o i e t y b u t not the phytyl side chain. Second- and fourth-derivative spectra gave the best differentiation of the vitamin E isomers.
© 1989 Academic Press, Inc.
T h e naturally occurring tocopherols and tocotrienols constitute the majority of the vitamin E group of compounds. Other members of the vitamin E group consist of tocopherol esters and derivatives found in multivitamin supplements and as food preservatives. T he tocopherols and tocotrienols are light yellow, fat-soluble, viscous oils produced primarily in plants. T h e chloroplasts of young plants contain significant amounts of a-tocopherol, which is the most biologically active isomer. As the plant matures and fruits, various other tocopherols appear (1). Some food sources containing vitamin E ini T o w h o m correspondence should be addressed. 368
clude plant and seed oils, nuts, whole grains, green leafy vegetables, eggs, liver, and milk (2). T he basic structure of tocopherol is shown in Fig. 1. Tocopherols are methyl-substituted hydroxychromans with a phytyl side chain. Natural vitamin E is composed of two homologous series: (i) the tocopherols with a saturated side chain, and (ii) the tocotrienols with an unsaturated side chain. T h e three asymmetric carbons allow a total of eight possible diastereoisomers. T h e naturally occurring stereoisomer of a-tocopherol has all three carbons in the R-configuration and is known as D-a-tocopherol. T he structural name for this compound is 2,5,7,8tetramethyl-2-(4',8',12'-trimethyltridecyl)-6-chromanol. Even though cis-trans isomers of tocotrienols occur, it appears t h a t all of the natural tocotrienols possess the all-trans configuration (1). T he chemical name for a-tocotrienol is 2,5,7,8-tetramethyl-2-(4',8',12'-trimethyltrideca-3',7',ll'-trienyl)-6-chromanol. In addition to the difficulty of separating the eight isomers on the basis of their similar structural and physical properties, these compounds also have similar uv spectra. Their spectra are fairly nondescript and have a ~max ranging from 295 nm for a-tocopherol (literature value of 292 nm, Ref. (3)) to 298 nm for 5-tocopherol as illustrated in Fig. 2. A recent paper by Bukovits and Lezerovich (4) indicated t h a t use of second-derivative spectra for the analysis of tocopherols aided in the identification of components in a mixture due to the stepwise elimination of background absorption as derivative order was increased. T he major reason for studying vitamin E is its natural antioxidant capabilities. Vitamin E functions as an antioxidant to protect fat in membranes around cells (such as nerves, heart, muscles, and red blood cells) from damage by oxygen (2). While a-tocopherol is the most biologically active form of natural vitamin E, biological activity is not a basis for determining antioxidant activity (1). Less is known about the antioxidant capabilities of individual isomers. 0003-2697/89 $3.00 Copyright © 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.
OPTIMIZATION STUDY OF TOCOPHEROLS AND TOCOTRIENOLS R e c e n t reviews b y P a r r i s h (1) a n d Nelis et al. (5) h a v e e x t e n s i v e l y discussed various m e t h o d s for a n a l y z i n g vit a m i n E isomers r a n g i n g f r o m t h e colorimetric m e t h o d s of E m m e r i e a n d E n g e l (6), to T L C , GC, a n d finally H P L C . While H P L C is p r e s e n t l y t h e b e s t m e t h o d for a n a l y z i n g these isomers, m o s t l i t e r a t u r e refers only to t h e analysis of the four tocopherols. T h i s is n o t too surprising as m o s t vegetable oils a n d biological fluids cont a i n v a r y i n g a m o u n t s of t h e d o m i n a n t t o c o p h e r o l s a n d less of the t o c o t r i e n o l s (5). T o c o t r i e n o l s h a v e b e e n f o u n d in p a l m oil, c o c o n u t oil, a n d cereal grains such as wheat, rye, oats, a n d b a r l e y (7). C u r r e n t i n t e r e s t in the tocotrienols to aid in decreasing c a r d i o v a s c u l a r disease (8,9) a n d as a possible a n t i c a r c i n o g e n i c a g e n t (8) creates a n e e d to f u r t h e r optimize H P L C s e p a r a t i o n s y s t e m s to i n c o r p o r a t e t h e tocotrienols. T h u s , the p u r p o s e of this r e p o r t was to s i m u l t a n e o u s l y a n a l y z e t o c o p h e r o l a n d t o c o t r i e n o l i s o m e r s using various n o r m a l - a n d reversep h a s e liquid c h r o m a t o g r a p h i c systems. Use of seconda n d f o u r t h - d e r i v a t i v e s p e c t r a was also included to enh a n c e identification.
Reagents and materials. All solvents were H P L C grade a n d were p u r c h a s e d f r o m Fisher Scientific Co. (Fair Lawn, N J ) . T h e four t o c o p h e r o l s t a n d a r d s were o b t a i n e d f r o m the H e n k e l Corp. (La Grange, IL), a n d t h e t h r e e t o c o t r i e n o l s t a n d a r d s were o b t a i n e d f r o m P O R I M ( K u a l a L u m p u r , M a l a y s i a ) . / 3 - T o e o t r i e n o l was t h e only c o m p o u n d which could n o t be obtained. T h e
TOCOPHEROL 5
7
~
~
]
I~
5
CH3
-" "~
H
CH 3
4'
H
8'
TOCOTRIENOL
3'
7'
11'
Position of Methyl Group Tocopherols Tocotrienols 5,7,8-Trimethyl a-T a-T3 5,8-Dimethyl I~-T 13-T3 7,8-Dimethyl ¥-T ¥-T3 8-Monomethyl 8- T 8- T3 FIG. 1.
28~
290
Navelen~th
308
31~
(nm)
320
33{3
FIG. 2. Ultraviolet-visible spectra for tocopherols and tocotrienols, where 1 is a-T, 2 is a-T3, 3 is fl-T, 4 is ?-T, 5 is ~/-T3, 6 is 5-T, and 7 is ~-T3.
t o c o p h e r o l a n d t o c o t r i e n o l s t a n d a r d s were k e p t in a m b e r vials, flushed with nitrogen, a n d stored at - 2 0 ° C .
MATERIALS AND METHODS
FO~
27~
369
Structures oftocopherols and tocotrienols.
Sample preparation. T h e seven s t a n d a r d s were dissolved in m e t h a n o l for r e v e r s e - p h a s e c h r o m a t o g r a p h y with c o n c e n t r a t i o n s r a n g i n g f r o m 0.131 to 0.197 m g / m l . T h e s e s a m e s t a n d a r d s were dissolved in h e x a n e for norm a l - p h a s e w o r k with a c o n c e n t r a t i o n range of 0.142 to 0.167 m g / m l . F r o m these stock solutions, two m i x t u r e s were made: one for each p h a s e type. E a c h m i x t u r e consisted of all s e v e n s t a n d a r d s w i t h a final c o n c e n t r a t i o n which was o n e - t e n t h of its original stock c o n c e n t r a t i o n (approxim a t e l y 0.015 m g / m l ) . All solutions were stored in a m b e r vials w r a p p e d with a l u m i n u m foil a n d k e p t at - 2 0 ° C . Chromatography. A n a l y t i c a l i n s t r u m e n t a t i o n consisted of an H P 1090M ( H e w l e t t - P a c k a r d ) liquid chrom a t o g r a p h w i t h a n H P 9000 c o m p u t e r w o r k s t a t i o n a n d a u v - v i s d i o d e a r r a y detector. T h i s included a R h e o d y n e 7010 m a n u a l injection switching valve (20-#1 s a m p l e loop), a PV-5 t e r n a r y solvent delivery system, a n d a 4.5ttl flow cell, a n d the s y s t e m was recorded on a T h i n k j e t p r i n t e r or a C o l o r P r o 8 - p e n plotter. T h e u v - v i s diodearr a y d e t e c t o r m o n i t o r e d a w a v e l e n g t h of 295 nm, with a b a n d w i d t h of 4 nm. T h e analytical c o l u m n s used were all of the s a m e d i m e n s i o n , 25 × 0.46 cm: Vydac C-18 (Hesperia, CA); Z o r b a x ODS, CN, NH2, a n d S I L (DuP o n t Co., W i l m i n g t o n , DE). E a c h c o l u m n was used with its respective guard c o l u m n (1.5 × 0.32 cm, 7 #m) f r o m B r o w n l e e L a b s ( R a i n i n I n s t r u m e n t Co., W o b u r n , MA). T h e isocratic mobile p h a s e s for n o n a q u e o u s reversep h a s e ( N A R P ) 2 c h r o m a t o g r a p h y consisted of various bi2 Abbreviations used: NARP, nonaqueous reverse-phase; LRT, longest retention time; ACN, acetonitrile.
370
TAN AND BRZUSKIEWICZ a
mal-phase system (Fig. 3a). The lower polarity isomers (a-T and a-T3) eluted first and the higher polarity isomers (5-T and 5-T3) eluted last. Thus, the normal-phase column provided separation based on the number as well as the position of methyl substituents on the chromanol moiety. Silica columns are generally more capable of separating positional isomers such as #- and ~-tocopherol. Also, the separation indicated t h a t the polarity of the tocopherols and tocotrienols decreased with increased number of methyl groups. Use of the reverse-phase column (Fig. 3b) showed the class separation of the saturated and unsaturated side chains. The less polar but more saturated tocopherols were retained in the stationary phase longer. The order of elution within each class of compounds (i.e., tocotrienols) was from higher polarity (6-T3) to lower polarity (a-T3), which was expected for reverse-phase systems. It appeared that the side chain interacted with the stationary phase in such a way that caused the more saturated tocopherols to remain on the column longer than the unsaturated tocotrienols. Of the two reverse-phase columns used, neither one could separate the two positional isomers of #-T and ~/T. However, the Zorbax SIL and NH2 columns could separate all seven vitamin E standards. Therefore, normal-phase columns were preferred for isomeric separation. These conclusions are described later in more detail.
6
2~"
15"
3
i
4 5 E
2
L
5-
3 Tlrn~
4
5
(mln.)
3
4
-j
G 31
2
Nonaqueous reverse-phase chromatography (NARP). ,
.
,
.
4 Time
,
•
8 (mln.)
.
.
,
S
F I G . 3. T y p i c a l c h r o m a t o g r a m s for t o c o p h e r o l s a n d tocotrienols. (a) N o r m a l - p h a s e , u s i n g a Zorbax S I L w i t h a 99:1 ( h e x a n e : 2 - p r o p a n o l ) mobile p h a s e , a n d (b) R e v e r s e - p h a s e , u s i n g a Z o r b a x O D S w i t h 60:35: 5 (ACN:CH~OH:CH2C12), w h e r e 1 is a - T , 2 is a - T 3 , 3 is S-T, 4 is -y-T, 5 is ~ - T 3 , 6 is 6-T, a n d 7 is 5-T3.
nary acetonitrile:methanol (ACN:CH3OH) combinations, and a ternary system of ACN:CH3OH:CH2C12 (60: 35:5). The isocratic mobile phase systems for normalphase chromatography consisted of 99% hexane and 1% polar modifier. These modifiers included methylene chloride (CH2C12), tetrahydrofuran (THF), n-butanol, and 2-propanol. All chromatographic separations were performed at ambient temperatures with a flow rate of 2.0 ml/min. Nitrogen was used as the H P L C solvent degasser. RESULTS AND DISCUSSION
Normal-phase vs reverse-phase.
Some general comments can be made regarding NARP and normal-phase chromatography. Two typical chromatograms are illustrated in Fig. 3. The first chromatogram depicted the elution order for toeopherols and tocotrienols in a nor-
Reverse-phase chromatography was used primarily for the analysis of a-tocopherol in the presence of its esters, other fat-soluble vitamins, carotenes, and other reducing substances (1,5). Although this article pertains to the analysis of tocopherol and tocotrienol isomers, it was necessary to optimize a NARP system to obviate these interferents in actual samples. This would eliminate some preparative procedures which could lead to sample loss or degradation.
TABLE 1
Comparison of Zorbax ODS and Vydac C-18 Columns for Reverse-Phase Chromatographya Zorbax O D S b
Vydac C-18 b
Item ~
Nos. 7, 5
Nos. 3, 1
Nos. 7, 5
Nos. 3, 1
R~
1.225 _+ 0.005 2.08 +_ 0.20
1.200 - 0.007 3.39 -+ 0.31
1.20 -- 0.02 0.91 _+ 0.09
1.20 ± 0.01 1.87 -+ 0.21
a V a l u e s e x p r e s s e d in t e r m s of m e a n _+ SD are b a s e d o n v a r i o u s isocratic A C N : C H 3 O H mobile p h a s e s , w h e r e n for t h e Z o r b a x a n d Vydac c o l u m n s were 8 a n d 11, respectively. b P e a k n u m b e r s c o r r e s p o n d to t h o s e in t h e legend to Fig. 3b: 7, 5-T3; 5, ~/-T3; 3, #- a n d ~ - T w h i c h coeluted; a n d 1, a - T . c a, selectivity; a n d Rs, resolution.
OPTIMIZATION
STUDY OF TOCOPHEROLS
TABLE 2 Chromatographic Optimization on Various Normal-Phase Columns and Polar Modifiers" 2 -
Propanol Column c Cyano d (1,2) (6,7) Amino (1,2) (3,4) {6,7) Silica (1,2) (3,4) (6,7)
n-Butanol
THF b
CH2CI2 b
a
R~
a
R~
a
R~
a
R~
1.29 1.23
1.4 1.4
1.28 1.22
1.8 1.8
1.29 1.23
1.5 1.5
1.28 1.23
1.8 1.7
1.19 1.14 1.20
1.7 2.0 2.8
1.17 1.12 1.17
1.4 1.7 2.2
1.18 1.21 1.45
1.17 1.11 1.16
1.9 1.2 2.0
1.16 1.09 1.14
1.3 1.1 2.1
----
1 . 2
m
1.6 2.2
--m
_ _
m
m
E
371
AND TOCOTRIENOLS
essary to find an optimal mobile phase which could reduce this gap and still maintain the resolution within a class of compounds. Isocratic ternary solvent compositions which utilized the organic modifier CH2C12 were tried. This solvent increased the mobile phase solubility of the tocopherols and caused t hem to elute faster. Small amounts of modifier were added until a system of 60:35: 5 (ACN:CH3OH:CH2C12) was reached. This system decreased the gap between the tocopherols and the tocotrienols, yet maintained resolution values within each class similar to t hat of the 60:40 (ACN:CH3OH) mobile phase. Hence, the use of CH2Cle decreased the longest retention time (LRT) from 13 to about 10 min and the retention gap from 3.5 to 2.5 min without the sacrifice of peak resolutions.
Normal-phase chromatography. Two reviews indicated t hat normal-phase chromatography was preferred for the separation oftocopherol isomers (1,5). There has
Values are b a s e d on t h e average of duplicate r u n s , a n d all mobile p h a s e s c o n s i s t e d of 99% h e x a n e a n d 1% modifier. b ,,,, m e a n s t h a t t h e s o l v e n t gave no p e a k s after 15 m i n or t h a t r e s u l t s were n o t reproducible. c P e a k n u m b e r s in p a r e n t h e s e s refer to t h o s e in t h e legend to Fig. 3a: 1, a - T ; 2, a - T 3 ; 3, ~-T; 4, ~ - T ; 6, 5-T; a n d 7, &-T3. P e a k p a i r s 3 a n d 4 were n o t i n c l u d e d because/3- a n d ? - T coeluted.
%
2-
I-
Th e columns utilized for N A R P chromatography were the Vydac C-18 and the Zorbax ODS. Various mobile phases ranging from 100% acetonitrile to 100% methanol were used. Th er e were no major trends in selectivity or resolution, with an increase in methanol concentration, for either column. A summary of these two factors is presented in Table 1. T he selectivity and resolution between the first two eluting compounds and the last two compounds were studied. Figure 3b shows the elution pairs tabulated in Table 1. For the Zorbax ODS, the selectivity between the first two components was slightly higher th an th at between the last two. T h e variations of the eluting pair selectivities were less when the Zorbax column was used t h a n when the Vydac column was used. W h en the Vydac C-18 was used, lower resolutions between both elution pairs were observed. Therefore, the Zorbax ODS was chosen as the more suitable reversephase column for the analysis of tocopherol and tocotrienol isomers. Mobile phase optimization was t hen performed with the Zorbax column. Utilization of this column with 100% methanol caused all seven components to elute in about 7 min. While resolution between peaks was fairly good using this mobile phase, better resolution could be obtained using a 60:40 (ACN:CH3OH) solvent mixture. T h e retention time for the last component was reached in less th an 13 min. As methanol concentration increased, the retention gap between the two classes of compounds narrowed, but the resolution between peaks within each class became poorer. Therefore, it was nec-
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372
TAN AND BRZUSKIEWICZ
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F I G . 5. Chromatographic separation of vitamin E isomers in crude palm oil: (a) using the optimal normal-phase of hexane:2-propanol (99:1) with the Zorbax SIL, and (b) using the optimal reverse-phase of ACN:CH8OH:CH2C12 (60:35:5) with the Zorbax ODS, where l is aT, 2 is a-T3, 5 is ~,-T3, and 7 is 5-T3.
been little to no H P L C separation of tocotrienols alone or tocotrienols with tocopherols reported. T h e optimization study for normal-phase systems was more complex. T he Zorbax cyano, amino, and silica columns were employed. T h e mobile phases consisted of 99% hexane and 1% of a polar modifier. These modifiers included 2-propanol, n-butanol, T H F , and CH2C12. A summary of results for selectivity and resolution between the first two, last two, and f~- and ~/-T is given in Table 2. T h e cyano bonded-phase LC column could be used in both normal- and reverse-phase systems, depending on the compound of interest. In this particular study, the elution order of the isomers followed t h a t of normalphase (i.e., least polar ones eluted first). However, the cyano column was similar to reverse-phase due to its inability to separate fl- and -~-tocopherol. No separation efficiency was gained by using one modifier over another
as far as selectivity and resolution were concerned (Table 2) except t h a t 2-propanol caused all seven isomers to elute with a L R T of about 6 min. T e t r a h y d r o f u r a n and n-butanol modifiers gave similar L R T s of about 6.5 to 7 min, with CH2C12 at 10 min. Also, the components have better peak shapes when 2-propanol was used. T h e use of n-butanol offered slightly better column efficiency, and the use of CH2C12 showed higher capacity factors (k') t han the use of other solvents. T he cyano column was not useful for reverse-phase work as all isomers eluted as one peak in less t han 2 min for both mobile phases of 100% methanol and 100% acetonitrile. Vitamin E isomeric separations using the bondedphase amino column gave the same elution order as th a t of Fig. 3a. Table 2 indicates t hat 2-propanol as a modifier offered better selectivity and resolution between the three sets of peaks under investigation. No values were obtained for CH2C12 because no peaks were detected within the first 15 min of the chromatographic run. This was probably due to its low solvent strength. T h e retention time for the last component in 2-propanol and nbutanol was about 13 to 15 min, whereas the retention time for T H F was 23 min. When the amount of modifier was increased to 2% 2-propanol, the total retention time decreased to less t han 10 min and good resolution between peaks was maintained. T h e silica column was most commonly used for normal-phase chromatography. Table 2 shows that while selectivities and resolutions for systems using 2-propanol and n-butanol were similar, the use of 2-propanol offered better resolution between ~-T and ~-T3. T h e lower polarity modifiers of T H F and CH2C12 were not useful for the silica column. T he T H F was very unstable and chromatograms were not reproducible, and CH2C12 was not strong enough to elute the isomers. T h e total retention time for all seven isomers was about 5.5 min for 2-propanol and about 6 min for n-butanol. T h e k' for each component was generally higher for the system using n-butanol. However, the column efficiencies using 2-propanol with the silica column were superior to those of any of the systems tested for the other two columns. T he resolution between f~- and ~-T was lower for the silica column t han for the amino, but still at an acceptable value of 1.0. Resolution could be improved using 2-propanol modifier below 1%. However, it was difficult to accurately reproduce this system with small modifier composition, and a new solution was needed each day. For the optimal normal-phase system of Zorbax SIL with 99:1 (hexane:2-propanol), derivative spectra were examined. T h e zeroth-order spectra are found in Fig. 2. Figure 4 shows second- and fourth-derivative spectra, respectively. Differences occurred only in substituents in the chromanol moiety but not in the phytyl side chain (e.g., ~-T and ~-T3 were indistinguishable in the zeroth, second, and fourth derivatives). T here was a bathochromic shift of about 4 nm from the ~ to/3 to ~ to ~ isomers.
OPTIMIZATION STUDY OF TOCOPHEROLS AND TOCOTRIENOLS T h i s was seen m o r e readily in the s e c o n d - d e r i v a t i v e t h a n in the z e r o t h - o r d e r spectra. T h e a- a n d fl-isomer groups were distinguished f r o m all o t h e r groups in the second derivative, b u t t h e 7- a n d 5-isomer groups were n o t (Fig. 4a). M i n o r d i f f e r e n t i a t i o n was observed b e t w e e n t h e 3~ a n d 5 i s o m e r s w i t h t h e f o u r t h derivative (Fig. 4b). A specially p a c k e d a l u m i n a c o l u m n ( W a t e r s - M i l l i pore, Milford, MA) was also tested. H o w e v e r , all isocratic solvent s y s t e m s a t t e m p t e d on this c o l u m n were u n s t a b l e a n d results were n o t reproducible. Of t h e four c o l u m n s t e s t e d for n o r m a l - p h a s e , t h e silica a n d a m i n o were m o s t suitable for the s e p a r a t i o n of tocopherols a n d tocotrienols. Applications. T h e o p t i m a l s y s t e m s for e a c h p h a s e t y p e were applied to vegetable oils such as a l m o n d , safflower, corn, w h e a t g e r m , apricot, a n d p a l m oil. Vegetable oils, for the m o s t p a r t , required little s a m p l e cleanup; therefore, the only s a m p l e p r e t r e a t m e n t was to dissolve t h e oil in a suitable solvent (ethyl a c e t a t e for r e v e r s e - p h a s e , a n d h e x a n e for n o r m a l - p h a s e ) a n d chill o v e r n i g h t at - 2 0 ° C. T h i s c a u s e d s o m e of the sterols a n d triglycerides to precipitate. S a m p l e s were t h e n centrifuged at 4°C to r e m o v e t h e solids, a n d t h e lipid-soluble s u p e r n a t a n t was c h r o m a t o g r a p h e d . Crude p a l m oil, which c o n t a i n s significant a m o u n t s of tocotrienols, b e s t exemplified the s i m u l t a n e o u s s e p a r a t i o n of t o c o p h e r o l s a n d tocotrienols. T h e s e results are s h o w n in Fig. 5. Q u a n t i t a t i o n was a c c o m p l i s h e d u s i n g the calibration of standards. CONCLUSIONS On the basis of the c h r o m a t o g r a p h i c factors discussed, the s y s t e m of 99:1 ( h e x a n e : 2 - p r o p a n o l ) in c o n j u n c t i o n
373
with the Z o r b a x S I L c o l u m n was the o p t i m a l n o r m a l p h a s e s y s t e m for the analysis of t o c o p h e r o l a n d tocotrienol isomers. T h e s y s t e m of 60:35:5 (ACN:CH3OH: CH2C12) w i t h a Z o r b a x O D S could be utilized p r o v i d e d t h a t either/3- or ~/-tocopherol (but n o t both) was p r e s e n t along w i t h o t h e r t o c o p h e r o l s or tocotrienols. I t is h o p e d t h a t the results of this s t u d y will decrease the a m o u n t of t i m e s p e n t by r e s e a r c h e r s on o p t i m i z i n g a s y s t e m so t h a t a p p l i c a t i o n s can be pursued, a n d provide the o p p o r t u n i t y to identify the t o c o p h e r o l s a n d tocotrienols concomitantly.
REFERENCES
1. Parrish, D. B. (1980) CRC Crit. Rev. Food Sei. Nutr. 13,161-187. 2. Hegarty, V. (1988) Decisions in Nutrition, pp. 181-182, Times Mirror/Mosby College Pub., St. Louis, MO. 3. Morton, R. A. (1975) Biochemical Spectroscopy, pp. 410-417, Wiley, New York. 4. Bukovits, G. J., and Lezerovich, A. (1987) d. Amer. Oil Chem. Soc. 64(4), 517-520. 5. Nelis, H. J., DeBevere, V. O. R. C., and DeLeenheer, A. P. (1985) in Modern Chromatographic Analysis of the Vitamins (DeLeenheer, A. P., Lambert, W. E., and DeRuyter, M. G. M., Eds.), pp. 129-200, Marcel Dekker, New York. 6. Emmerie, A., and Engel, C. (1938) Rec. Trav. Chim. Pays-Bas. 57, 1351. 7. Qureshi, A. A., Burger, W. C., Peterson, D. M., and Elson, C. E. (1986) J. Biol. Chem. 261, 10,544-10,550. 8. Qureshi, A. A., Ahmad, Y., and Elson, C. E. (1988) in International Oil Palm/Palm Oil Conference: Tech. Progress and Prospects (Ong, A. S. H., Ed.), PORIM Press, Kuala Lumpur, Malaysia. 9. Editorial (1987) Nutr. Rev. 45,205-207.