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Quantitative determination of α-tocopherol on thin layers of silica gel
ANALYTICAL
BIOCHEMISTRY
83, @l-407
(1977)
quantitative determination of a-Tocopherol on Thin Layers of Silica Gel1 JOHN L. HESS,~ MARK A. PALLANSCH, AND G. E. BUNCE department
KIM
HARICH,
of Biochemjst~ and nutrition, ~irg~~i~ F~~techn~c Institute and State University, Blacksb~rg, Virginia 24061
Received December 2, 1976; accepted August 12, 1977 cY-Tocopherol in the range of l-10 pg may be directly quantified on thin layers of silica gel containing fluorescent indicator. Following chromatography, the plate is heated at 110°C for 18 hr so that ty-tocopherol is qu~titatively converted to its oxidized products. The chromatogram is scanned at 270 nm. These products absorb this excitation radiation so that reduced fluorescence correlates with a-tocopherol content. The technique takes advantage of the facile oxidation ofcu-tocopherol and eliminates the steps of sample transfer, chromophore development, and quantification normally employed. This direct, sensitive method is suitable for biological samples and yields 99% recovery.
In the laboratory dealing with a-tocopherol as a variable in dietary experiments or other applications, it becomes necessary to quantify small amounts of the material. Techniques described by Bieri (1) provide quantitative extraction and definitive separation of a-tocopherol from serum and tissue samples. We have used the technique described in this report to evaluate successfully the levels of serum and liver a-tocopherol levels in rats maintained on diets deficient in this tocopherol as reported by Bunce and Hess (2). Although techniques,of liquid chromatography and gas chromatography (gc)-mass spectrometry have been used to quantify this compound, extensive initial cleanup or derivatization of the sample is required for such work (3,4). The technique we describe provides a sensitive, accurate, direct measure of the a-tocopherol. MATERIALS
AND METHODS
The NBC division of ICN served as a source for a-tocopherol and a-tocopherol acetate. Solvents were redistilled, and commercially prepared layers of silica gel containing fluorescent binder, Baker-flex lB2-F, were obtained from A. H. Thomas. ’ Supported by PWS Grant No. EY 01060. Z To whom correspondence should be addressed. 401 Copyright D 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
ISSN 0003.2697
402
HESS ETAL.
RELATIVE
DISTANCE
1. Densitometer scan of a thin-layer chromatogram of a saponified extract from serum. The sample was transferred to an activated thin layer of silica gel with fluorescent indicator, Baker-flex 1B2-F, and developed with benzene:ethanol @Xl, v/v) in the dark. Following heating for 18 hr at llO”C, the chromatogram was scanned with broad-band uv radiation at 270 nm. Reduction in fluorescence was measured as an absorbance ratio in which the denominator was determined by the background fluorescence from an adjacent area of the thin layer which contained no sample. R, = 0.57 or-0.02 for BHT. Rf = 0.31 2 0.02 for cu-tocopherol. FIG.
The method of Bieri (1) was employed throughout using ethanol during initial fragmentation of the sample (serum or liver), followed by saponification and extraction with hexane. Two milliliters of a freshly prepared solution, 2% pyrogallol in ethanol, were added to 0.5 g of tissue (wet weight). Fo~owing homogenization the temperature was increased to 7O”C, and OS ml of 60% NaOH was added under a stream of purified N?. The stoppered tube was occasionally shaken throughout the 20- to 25min reaction at 70°C. Note that during these steps, losses occur related to N2 quality and high incident light so that we scrubbed N, with Fieser’s solution (5) in order to remove traces of 0, and carried out the extraction and saponification under low light intensity. Following saponification, the tube was cooled and an equal volume of HZ0 was added. Then 3 ml of 0.00125% BHT3 in hexane was shaken vigorously with the sample for 2 min. This extraction was repeated and the hexane extracts were transferred to a conical centrifuge and dried at 50°C under N,. The extracted material was quantitatively transferred as a benzene solution to activated thin-layer plates; spot size should be unifo~ly small (<5 mm) and should not contain more than 35 mg of material. Plates were then developed in the dark using benzene:ethanol(99: l), az-tocopherol R, = 0.31 zt 0.021. Two-dimensional separation using hexane:ethanol s Abbreviation
used: BHT butylated hydroxytoluene.
DETERMINATION
40
O---a M
0
4
403
OF (Y-TOCOPHEROL
6
12
OXYGEN AIR
lb
20
TIME, itOURS FIG. 2. Conversion of a-tocopherol to its oxidized products. Samples of 10, 20, and 40 pg of standard cY-tocopherol were spotted on an activated thin layer of silica gel, Baker-flex lB2-F, and the chromatogram was developed with benzene:ethanol (99:1, v/v) in the dark. The chromatograms were scanned with broad-band uv radiation at 270 nm at different times after removal of the chromatogram from the solvent tank. During this time the plate was incubated at 110°C in air (II 0) or oxygen (0 - - - 0).
(9:1), a-tocopherol R, = 0.37 & 0.063, in the second dimension was essential for samples containing other quinols (1). Routinely the plate was heated in an oven at 1 IO-120°C for 18-20 hr, cooled, and scanned with the densitometer. Scans were performed on a Schoeffel SD 3000 densitometer (Schoeffel Instrument Corporation, Westwood, N.J.) in the transmission mode using a 1 x 5-mm slit with the monochromator slit at 1.5 nm and the wavelength at 270 nm; fullscale absorbance was calibrated to 0.4 for samples containing less than TABLE REPLICATE
DETERMINATIONS
1
OF CX-TOCOPHEROL
Sample
n
Rat serum H,O* Rat liver, 100-g male rat Rat liver, 600-g male rat
6 3 3 4
ON INDIVIDUAL
SAMPLE@
a-Tocopherol (mean 2 SD) 6.6 10.1 23.6 14.0
+ ? 2 ?
0.7 &ml 0.6 pg/ml 1.5 FLgig fresh weight 1.4 fig/g fresh weight
u The samples were divided into equal portions; a-tocopherol was determined as peak area using standards of a-tocopherol run on the chromatograms at the same time as the reference. a A IO-pg sample of a-tocopherol in ethanol was added to a l-ml aliquot of H,O.
404
HESS
ET
AL.
10 pg of a-tocopherol. A portion of the plate containing no compounds was used in the reference beam of the instrument during the scan; 12 samples were run on a 20 x 20-cm plate. Peak heights and areas correlated well with the amount of a-tocopherol standard placed on the chromatogram; peak areas were calculated from the recorder integrator information from the base of the peak rise to the return to baseline. Mass spectra of the tocopherol and its oxidized products were determined with the Varian MAT 112 double-focusing mass spectrometer. Samples were eluted from thin-layer plates, redissolved in hexane, and evaporated to dryness on the sample boat with NB. Electron-impact spectra were obtained with a direct probe at 72 eV and a temperature of 55°C. RESULTS
Observing the plate with uv illumination immediately following development revealed a spot in the position of the cY-tocopherol standard which had minimal uv-absorbing properties. Heating the plate at 110°C for at least 18 hr provided a quantitative conversion of a-tocopherol to the quinone, and possibly other oxidized forms, having greater uv absorbance. A typical scan of a heated chromatogram of a saponified serum sample extract is given in Fig. 1. In other experiments tocopheryl acetate, which migrates just ahead of a-tocopherol with a
log a-TOCOPHEROL.pg
FIG. 3. Standard curve. Log of peak area as determined from densitometer thin-layer chromatograms plotted as the ordinate vs log of the concentration of a-tocopherol in the spot. Values for this curve were obtained consistently over period. Intercept is 1.055, slope is 0.62, and the correlation coefficient for the the data points with a Wang programmable calculator is 0.952.
scans of standard a 3-year line fit to
DETERMINATION
405
OF (r-TOCOPHEROL
slightly higher&, did not respond to the heating step by showing increased uv absorbance. The ester, of course, is less sensitive to oxidation than is the free tocopherol so that observed differences in uv-absorbing properties of the chromatogram before and after heating are attributed to oxidation reactions. The time dependence of this change in c+tocopherol uv-absorption properties is presented in Fig. 2. Heating the plate in a Nz atmosphere prevented conversion of the tocopherol, indicating a requirement for 0,. Using 0, atmospheres while heating the chromatograms, however, slightly accelerated the rate of tocopherol conversion but did not alter the end point as determined by densitometer measurements (Fig. 2). In attempts to identify products following heating, the chromatogram samples from the a-tocopherol spot were eluted and rechromatographed. The quantity of tocopherol decreased as a function of time so that less than 1% remained after 20 hr of incubation at 110°C. The eluted material contained compounds with much reduced migration rates in the same solvent system, one with an R,expected for the quinone (1); however, much of this oxidized material also remained at or near the origin on rechromatography. The putative a-tocopherol after chromatography, but before heating, was eluted and its mass spectrum was compared to that for authentic a-tocopherol. We observed an exactly comparable spectrum with characteristic ions of masses 430, 205, and 165 (6). Material extracted from the plate after oxidation yielded a mass spectrum devoid of the parent ion, mass 430, of cr-tocopherol. Rather, ions of masses 446 and 462 indicated that extensive oxidation had occurred. Also, the predominant tetramethylcatechol fragment, mass 165, characteristic of reduced cy-tocopherol was barely detectable in the oxidized product reflecting the expected change in the oxidized ring fragmentation. All spectra were evaluated following subtraction of masses due to background as measured on an extract from a blank area of the same plate used to TABLE RECOVERY
2
OFLY-TOCOPHEROL"
Recovery Sample
H-A Rat serum Liver
n 3 7 3
(%"
SD)
100.0 k 6.2 95.2 -t 14.7 98.0 + 3.5
(LTen micrograms of ru-tocopherol in ethanol were added to samples. Extraction, saponification, and separation were performed and areas were quantified. Standards were chromatographed on these thin layers so that the standard curve was established with identical solvent and oxidation conditions as used for the samples. Recovery is reported as percentage of total added to the original sample.
406
HESS
ET AL.
separate the tocopherol. The area on the oxidized chromatogram, presumed to be cr-tocopherol in the two-dimensional system used with the liver samples, yielded mass spectra similar to those of the oxidized authentic cr-tocopherol. In Fig. 3 we report a linear correlation between the log values for peak area vs the log of a-tocopherol content in the spot. Such linearity was consistently observed and validates quantitative aspects of the technique. Approximation of 10% accuracy may be achieved if the slope of this standard curve, 0.62, is applied to data for which an intercept value is calculated using the standard quantity of tocopherol added to a particular chromatogram as a reference point. Areas for a given spot were integrated with a precision greater than 3% when the plate was reoriented in the densitometer between repetitive scans. In Table 1 we report the reproducibility of the technique as determined on liquid and tissue samples. The experimental standard deviation for samples with a-tocopherol content less than 10 pg is approximately 0.67 pg. Tissue samples reported in the table were run at 0.5-g quantities so that measured quantity on the plate was actually between 6 and 12.5 pg. In Table 2 we report recoveries of a-tocopherol from various samples of serum and liver. Excellent recoveries were consistently observed when attention was given to the quality of nitrogen used in evaporation steps and to minimal ambient light intensity, particularly during extraction, spotting, and chromatography steps of the procedure. Natural daylight most effectively reduced yields so that window shades were drawn and fluorescent lamps were not used during these procedures, although fluorescent illumination was not particularly deleterious. DISCUSSION
Data presented in Figs. 1 and 2 indicate the value of observing a-tocopherol directly on thin layers of silica gel following separation from other hydrophobic substances. The well-established separation techniques of Bieri (1) are sufficiently definitive so that a double-beam scanning densitometer may be used with no interference from other uv-absorbing materials. The technique is direct and less tedious than the commonly used calorimetric method for determination of a-tocopherol. Since one cannot use the ferric chloride-bathophenanthroline technique directly on serum or tissue extracts, i.e., without first extraction and chromatography, we see no advantages to this calorimetric technique. The quantitative conversion of a-tocopherol on the plate enables use of the technique over extended experimental periods with minimal variation. The standard curve in Fig. 2 was determined from data extending over a 3-year period. However, if accuracy greater than 10% is required, we recommend that a set of standards which bracket the expected concentration range be run at the same time in order to accomodate experimental
DETERMINATION
OF LU-TOCOPHEROL
407
variation due to specific parameters related to the thin-layer chromatography and oxidation conditions. The theoretical basis for the log-log relationship between the recorded “absorbance” and the amount of the compound in a spot remains unclear. The technique indirectly measures uv-absorbing properties of the oxidized tocopherol by monitoring decreased fluorescence at that location on the thin layer. The plate is illuminated from above by broad-band-pass illumination at 270 nm. The resulting fluorescence of the plate is monitored as transmitted light through the polymer support of the thin layer. This support also functions as a filter for uv irradiation, so that only photons from the fluorescing plate reach the photomultipliers. Thus, we are measuring a fluorescence intensity which decreases as a function of the thickness of the plate, which for most commercial plates is constant, as well as uvabsorbing properties of the oxidized tocopherol which competes for the radiation used for fluorescence excitation. Dark areas on the plate occur not because tocopherol quenches the fluorescent radiation but because it prevents excitation of the fluor. The log-log relationship is an excellent empirical description of these observations. As described, this technique permits rapid, accurate detection of l-pg quantities of cr-tocopherol. Instrumentation permits additional sensitivity; signal to noise ratio, however, decreases so that accurate detection of samples of less than 500 ng becomes di~cult. Quality of such data may be improved through use of a signal averaging device. In agreement with Nishiyama et al. (7), we found that recoveries of tocopherol were lower if the amount of lipid in a sample were large, e.g., when low cY-tocopherol content required processing of larger samples as in the case of diets deficient in a-tocopherol. The large quantity of fatty acids extracted with the nonsaponi~able lipids prevented effective chromatographic separation. ACKNOWLEDGMENTS The authors thank Ms. Teena Cochran for excellent technical assistance in obtaining the mass spectra of our samples and Dr. J. R. Vercellotti for consultation regarding the mass spectra.
REFERENCES 1. Bieri, J. B. (1969) in Lipid Chromatographic Analysis (Marinetti, G. V., ed.), Vol. 2, pp. 459-478, Dekker, New York. 2. Bunce, G. E., and Hess, J. L. (1976) f. N~rr. 106, 222-228. 3. Lovelady, H. G. (1973) J. Cfiromatogr. 85, 81-92. 4. Rao, M. K. G., and Perkins, E. G. (1972) J. Agr. Food Chem. 20, 240-245. 5. Fieser, L. F., and Fieser, M. (eds.) (1967) in Reagents for Organic Synthesis, Vol. 1, p. 393, Wiley, New York. 6. Elliot, W. H., and Wager, G. R. (1972) in Biochemical Applications of Mass Spectrometry(Wailer, G. R., ed.), pp. 510-513, Wiley Interscience, New York. 7. Nishiyama, J., E&son, E. C., Mizuno, G. R., and Chipault, J. R. (1976) J. Nurr. Sci. Vi~~~~~~~.
21, 355-361.
BIOCHEMISTRY
83, @l-407
(1977)
quantitative determination of a-Tocopherol on Thin Layers of Silica Gel1 JOHN L. HESS,~ MARK A. PALLANSCH, AND G. E. BUNCE department
KIM
HARICH,
of Biochemjst~ and nutrition, ~irg~~i~ F~~techn~c Institute and State University, Blacksb~rg, Virginia 24061
Received December 2, 1976; accepted August 12, 1977 cY-Tocopherol in the range of l-10 pg may be directly quantified on thin layers of silica gel containing fluorescent indicator. Following chromatography, the plate is heated at 110°C for 18 hr so that ty-tocopherol is qu~titatively converted to its oxidized products. The chromatogram is scanned at 270 nm. These products absorb this excitation radiation so that reduced fluorescence correlates with a-tocopherol content. The technique takes advantage of the facile oxidation ofcu-tocopherol and eliminates the steps of sample transfer, chromophore development, and quantification normally employed. This direct, sensitive method is suitable for biological samples and yields 99% recovery.
In the laboratory dealing with a-tocopherol as a variable in dietary experiments or other applications, it becomes necessary to quantify small amounts of the material. Techniques described by Bieri (1) provide quantitative extraction and definitive separation of a-tocopherol from serum and tissue samples. We have used the technique described in this report to evaluate successfully the levels of serum and liver a-tocopherol levels in rats maintained on diets deficient in this tocopherol as reported by Bunce and Hess (2). Although techniques,of liquid chromatography and gas chromatography (gc)-mass spectrometry have been used to quantify this compound, extensive initial cleanup or derivatization of the sample is required for such work (3,4). The technique we describe provides a sensitive, accurate, direct measure of the a-tocopherol. MATERIALS
AND METHODS
The NBC division of ICN served as a source for a-tocopherol and a-tocopherol acetate. Solvents were redistilled, and commercially prepared layers of silica gel containing fluorescent binder, Baker-flex lB2-F, were obtained from A. H. Thomas. ’ Supported by PWS Grant No. EY 01060. Z To whom correspondence should be addressed. 401 Copyright D 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
ISSN 0003.2697
402
HESS ETAL.
RELATIVE
DISTANCE
1. Densitometer scan of a thin-layer chromatogram of a saponified extract from serum. The sample was transferred to an activated thin layer of silica gel with fluorescent indicator, Baker-flex 1B2-F, and developed with benzene:ethanol @Xl, v/v) in the dark. Following heating for 18 hr at llO”C, the chromatogram was scanned with broad-band uv radiation at 270 nm. Reduction in fluorescence was measured as an absorbance ratio in which the denominator was determined by the background fluorescence from an adjacent area of the thin layer which contained no sample. R, = 0.57 or-0.02 for BHT. Rf = 0.31 2 0.02 for cu-tocopherol. FIG.
The method of Bieri (1) was employed throughout using ethanol during initial fragmentation of the sample (serum or liver), followed by saponification and extraction with hexane. Two milliliters of a freshly prepared solution, 2% pyrogallol in ethanol, were added to 0.5 g of tissue (wet weight). Fo~owing homogenization the temperature was increased to 7O”C, and OS ml of 60% NaOH was added under a stream of purified N?. The stoppered tube was occasionally shaken throughout the 20- to 25min reaction at 70°C. Note that during these steps, losses occur related to N2 quality and high incident light so that we scrubbed N, with Fieser’s solution (5) in order to remove traces of 0, and carried out the extraction and saponification under low light intensity. Following saponification, the tube was cooled and an equal volume of HZ0 was added. Then 3 ml of 0.00125% BHT3 in hexane was shaken vigorously with the sample for 2 min. This extraction was repeated and the hexane extracts were transferred to a conical centrifuge and dried at 50°C under N,. The extracted material was quantitatively transferred as a benzene solution to activated thin-layer plates; spot size should be unifo~ly small (<5 mm) and should not contain more than 35 mg of material. Plates were then developed in the dark using benzene:ethanol(99: l), az-tocopherol R, = 0.31 zt 0.021. Two-dimensional separation using hexane:ethanol s Abbreviation
used: BHT butylated hydroxytoluene.
DETERMINATION
40
O---a M
0
4
403
OF (Y-TOCOPHEROL
6
12
OXYGEN AIR
lb
20
TIME, itOURS FIG. 2. Conversion of a-tocopherol to its oxidized products. Samples of 10, 20, and 40 pg of standard cY-tocopherol were spotted on an activated thin layer of silica gel, Baker-flex lB2-F, and the chromatogram was developed with benzene:ethanol (99:1, v/v) in the dark. The chromatograms were scanned with broad-band uv radiation at 270 nm at different times after removal of the chromatogram from the solvent tank. During this time the plate was incubated at 110°C in air (II 0) or oxygen (0 - - - 0).
(9:1), a-tocopherol R, = 0.37 & 0.063, in the second dimension was essential for samples containing other quinols (1). Routinely the plate was heated in an oven at 1 IO-120°C for 18-20 hr, cooled, and scanned with the densitometer. Scans were performed on a Schoeffel SD 3000 densitometer (Schoeffel Instrument Corporation, Westwood, N.J.) in the transmission mode using a 1 x 5-mm slit with the monochromator slit at 1.5 nm and the wavelength at 270 nm; fullscale absorbance was calibrated to 0.4 for samples containing less than TABLE REPLICATE
DETERMINATIONS
1
OF CX-TOCOPHEROL
Sample
n
Rat serum H,O* Rat liver, 100-g male rat Rat liver, 600-g male rat
6 3 3 4
ON INDIVIDUAL
SAMPLE@
a-Tocopherol (mean 2 SD) 6.6 10.1 23.6 14.0
+ ? 2 ?
0.7 &ml 0.6 pg/ml 1.5 FLgig fresh weight 1.4 fig/g fresh weight
u The samples were divided into equal portions; a-tocopherol was determined as peak area using standards of a-tocopherol run on the chromatograms at the same time as the reference. a A IO-pg sample of a-tocopherol in ethanol was added to a l-ml aliquot of H,O.
404
HESS
ET
AL.
10 pg of a-tocopherol. A portion of the plate containing no compounds was used in the reference beam of the instrument during the scan; 12 samples were run on a 20 x 20-cm plate. Peak heights and areas correlated well with the amount of a-tocopherol standard placed on the chromatogram; peak areas were calculated from the recorder integrator information from the base of the peak rise to the return to baseline. Mass spectra of the tocopherol and its oxidized products were determined with the Varian MAT 112 double-focusing mass spectrometer. Samples were eluted from thin-layer plates, redissolved in hexane, and evaporated to dryness on the sample boat with NB. Electron-impact spectra were obtained with a direct probe at 72 eV and a temperature of 55°C. RESULTS
Observing the plate with uv illumination immediately following development revealed a spot in the position of the cY-tocopherol standard which had minimal uv-absorbing properties. Heating the plate at 110°C for at least 18 hr provided a quantitative conversion of a-tocopherol to the quinone, and possibly other oxidized forms, having greater uv absorbance. A typical scan of a heated chromatogram of a saponified serum sample extract is given in Fig. 1. In other experiments tocopheryl acetate, which migrates just ahead of a-tocopherol with a
log a-TOCOPHEROL.pg
FIG. 3. Standard curve. Log of peak area as determined from densitometer thin-layer chromatograms plotted as the ordinate vs log of the concentration of a-tocopherol in the spot. Values for this curve were obtained consistently over period. Intercept is 1.055, slope is 0.62, and the correlation coefficient for the the data points with a Wang programmable calculator is 0.952.
scans of standard a 3-year line fit to
DETERMINATION
405
OF (r-TOCOPHEROL
slightly higher&, did not respond to the heating step by showing increased uv absorbance. The ester, of course, is less sensitive to oxidation than is the free tocopherol so that observed differences in uv-absorbing properties of the chromatogram before and after heating are attributed to oxidation reactions. The time dependence of this change in c+tocopherol uv-absorption properties is presented in Fig. 2. Heating the plate in a Nz atmosphere prevented conversion of the tocopherol, indicating a requirement for 0,. Using 0, atmospheres while heating the chromatograms, however, slightly accelerated the rate of tocopherol conversion but did not alter the end point as determined by densitometer measurements (Fig. 2). In attempts to identify products following heating, the chromatogram samples from the a-tocopherol spot were eluted and rechromatographed. The quantity of tocopherol decreased as a function of time so that less than 1% remained after 20 hr of incubation at 110°C. The eluted material contained compounds with much reduced migration rates in the same solvent system, one with an R,expected for the quinone (1); however, much of this oxidized material also remained at or near the origin on rechromatography. The putative a-tocopherol after chromatography, but before heating, was eluted and its mass spectrum was compared to that for authentic a-tocopherol. We observed an exactly comparable spectrum with characteristic ions of masses 430, 205, and 165 (6). Material extracted from the plate after oxidation yielded a mass spectrum devoid of the parent ion, mass 430, of cr-tocopherol. Rather, ions of masses 446 and 462 indicated that extensive oxidation had occurred. Also, the predominant tetramethylcatechol fragment, mass 165, characteristic of reduced cy-tocopherol was barely detectable in the oxidized product reflecting the expected change in the oxidized ring fragmentation. All spectra were evaluated following subtraction of masses due to background as measured on an extract from a blank area of the same plate used to TABLE RECOVERY
2
OFLY-TOCOPHEROL"
Recovery Sample
H-A Rat serum Liver
n 3 7 3
(%"
SD)
100.0 k 6.2 95.2 -t 14.7 98.0 + 3.5
(LTen micrograms of ru-tocopherol in ethanol were added to samples. Extraction, saponification, and separation were performed and areas were quantified. Standards were chromatographed on these thin layers so that the standard curve was established with identical solvent and oxidation conditions as used for the samples. Recovery is reported as percentage of total added to the original sample.
406
HESS
ET AL.
separate the tocopherol. The area on the oxidized chromatogram, presumed to be cr-tocopherol in the two-dimensional system used with the liver samples, yielded mass spectra similar to those of the oxidized authentic cr-tocopherol. In Fig. 3 we report a linear correlation between the log values for peak area vs the log of a-tocopherol content in the spot. Such linearity was consistently observed and validates quantitative aspects of the technique. Approximation of 10% accuracy may be achieved if the slope of this standard curve, 0.62, is applied to data for which an intercept value is calculated using the standard quantity of tocopherol added to a particular chromatogram as a reference point. Areas for a given spot were integrated with a precision greater than 3% when the plate was reoriented in the densitometer between repetitive scans. In Table 1 we report the reproducibility of the technique as determined on liquid and tissue samples. The experimental standard deviation for samples with a-tocopherol content less than 10 pg is approximately 0.67 pg. Tissue samples reported in the table were run at 0.5-g quantities so that measured quantity on the plate was actually between 6 and 12.5 pg. In Table 2 we report recoveries of a-tocopherol from various samples of serum and liver. Excellent recoveries were consistently observed when attention was given to the quality of nitrogen used in evaporation steps and to minimal ambient light intensity, particularly during extraction, spotting, and chromatography steps of the procedure. Natural daylight most effectively reduced yields so that window shades were drawn and fluorescent lamps were not used during these procedures, although fluorescent illumination was not particularly deleterious. DISCUSSION
Data presented in Figs. 1 and 2 indicate the value of observing a-tocopherol directly on thin layers of silica gel following separation from other hydrophobic substances. The well-established separation techniques of Bieri (1) are sufficiently definitive so that a double-beam scanning densitometer may be used with no interference from other uv-absorbing materials. The technique is direct and less tedious than the commonly used calorimetric method for determination of a-tocopherol. Since one cannot use the ferric chloride-bathophenanthroline technique directly on serum or tissue extracts, i.e., without first extraction and chromatography, we see no advantages to this calorimetric technique. The quantitative conversion of a-tocopherol on the plate enables use of the technique over extended experimental periods with minimal variation. The standard curve in Fig. 2 was determined from data extending over a 3-year period. However, if accuracy greater than 10% is required, we recommend that a set of standards which bracket the expected concentration range be run at the same time in order to accomodate experimental
DETERMINATION
OF LU-TOCOPHEROL
407
variation due to specific parameters related to the thin-layer chromatography and oxidation conditions. The theoretical basis for the log-log relationship between the recorded “absorbance” and the amount of the compound in a spot remains unclear. The technique indirectly measures uv-absorbing properties of the oxidized tocopherol by monitoring decreased fluorescence at that location on the thin layer. The plate is illuminated from above by broad-band-pass illumination at 270 nm. The resulting fluorescence of the plate is monitored as transmitted light through the polymer support of the thin layer. This support also functions as a filter for uv irradiation, so that only photons from the fluorescing plate reach the photomultipliers. Thus, we are measuring a fluorescence intensity which decreases as a function of the thickness of the plate, which for most commercial plates is constant, as well as uvabsorbing properties of the oxidized tocopherol which competes for the radiation used for fluorescence excitation. Dark areas on the plate occur not because tocopherol quenches the fluorescent radiation but because it prevents excitation of the fluor. The log-log relationship is an excellent empirical description of these observations. As described, this technique permits rapid, accurate detection of l-pg quantities of cr-tocopherol. Instrumentation permits additional sensitivity; signal to noise ratio, however, decreases so that accurate detection of samples of less than 500 ng becomes di~cult. Quality of such data may be improved through use of a signal averaging device. In agreement with Nishiyama et al. (7), we found that recoveries of tocopherol were lower if the amount of lipid in a sample were large, e.g., when low cY-tocopherol content required processing of larger samples as in the case of diets deficient in a-tocopherol. The large quantity of fatty acids extracted with the nonsaponi~able lipids prevented effective chromatographic separation. ACKNOWLEDGMENTS The authors thank Ms. Teena Cochran for excellent technical assistance in obtaining the mass spectra of our samples and Dr. J. R. Vercellotti for consultation regarding the mass spectra.
REFERENCES 1. Bieri, J. B. (1969) in Lipid Chromatographic Analysis (Marinetti, G. V., ed.), Vol. 2, pp. 459-478, Dekker, New York. 2. Bunce, G. E., and Hess, J. L. (1976) f. N~rr. 106, 222-228. 3. Lovelady, H. G. (1973) J. Cfiromatogr. 85, 81-92. 4. Rao, M. K. G., and Perkins, E. G. (1972) J. Agr. Food Chem. 20, 240-245. 5. Fieser, L. F., and Fieser, M. (eds.) (1967) in Reagents for Organic Synthesis, Vol. 1, p. 393, Wiley, New York. 6. Elliot, W. H., and Wager, G. R. (1972) in Biochemical Applications of Mass Spectrometry(Wailer, G. R., ed.), pp. 510-513, Wiley Interscience, New York. 7. Nishiyama, J., E&son, E. C., Mizuno, G. R., and Chipault, J. R. (1976) J. Nurr. Sci. Vi~~~~~~~.
21, 355-361.