The vitamin E complex is easily quantified provided some precautions are observed during
the extraction of the biological tissues.
Extraction:
A- Extraction from fatty materials (liver,
adipose tissues, oil, food...)
When samples are too fatty, a saponification step is needed to remove the excess of fatty
acids before the HPLC analysis.
Saponification involves the heating of the sample in a strong alkaline medium,
thus reducing the amount of organic compounds extractabled from the sample.
Liberated fatty acids and alcohol will remain in the aqueous phase.
Up to 2 g of sample is saponified with 2 ml of KOH (60%, w/v), 2 ml ethanol, and
5 ml of ethanolic pyrogallol (6%, w/v). After a digestion time of 45 min at
70°C, the tubes are cooled and 15 ml of physiological saline are added. The
mixture is extracted with 15 ml of hexane/ethyl acetate (9/1, v/v). The upper
layer is evaporated and the dry residue is dissolved in a known volume of
hexane/isopropanol (99/1, v/v).
Solid phase extraction (SPE) may be optimized for the extraction of tocopherols
from vegetal oil before HPLC analysis (Grigoriadou D et al., Food Chem 2007,
105, 675). The study was shown to be useful in the official control of olive
oil.
B- Extraction from cellular membranes, cellular suspensions, blood...
When samples contain only few amounts of triacylglycerols, an extraction with apolar
solvents is suitable and the saponification step is not necessary.
Several methods are reliable.
When only vitamin E compounds are to be studied (tocopherols, tocotrienols) the rapid
extraction process given for vitamin A is
convenient. Vitamin A, carotenoids, and all apolar lipids may be also determined in this
type of extract.
When the sample is liquid (plasma, cell suspension), the method is slightly modified in
adding a different ethanol-SDS solution to the sample. At 1 ml of sample are added 1 ml of
2.5% sodium dodecyl sulfate in water and 2 ml ethanol. After vortexing 15-20 min in amber
tubes, lipids are extracted after the addition of 3 ml hexane and vortexing 5 min. After a
brief centrifugation, the upper hexane phase is evaporated and the residue is stored in a
small amount of methanol before HPLC analysis. If the final solution is not limpid
(because of triacylglycerols), the dry extract is re-dissolved in some µl of the mixture
chloroform/methanol (1/3).
An optimized procedure for the extraction of tocopherols and tocotrienols from
hazelnuts has been described (Amaral JS et al., Anal Sci 2005, 21, 1545).
The procedure is based on a simple solid-liquid extraction using ethanol and
hexane. A comparison of three method of extraction of vitamin E isomers from seeds and
nuts followed by HPLC and coulometric detection may be found by Delgado-Zamarreno
MM et al. (J Chromatogr A 2001, 935, 77). A large review of the different
extraction procedures of fat-soluble vitamins including vitamin E from human
fluids, foods and pharmaceutical preparations may be found by Luque-Garcia JL et
al. (J Chromatogr A 2001, 935, 3).
When a joint analysis of vitamin E, cholesterol and polar lipids (phospholipids) is
needed, all lipids are extracted according to the Folch's procedure or, if necessary, to
another procedure adapted to liquid samples.
For plasma or cell suspensions, we have described a reliable procedure which permits
quantitative analysis of vitamin E, cholesterol and phospholipid fatty acids in a tiny
biological sample (Leray C et al., J Chromatogr 1997,696, 33-42).
An alternative extraction procedure has been described which is simple and has a
good reproducibility and recovery for tocopherols as well as retinol in plasma
or serum (Taibi G et al., J chromatogr B 2002, 780, 261). A solid-phase
extraction of vitamin E as well as Vitamin A in animal feeds was shown to give
rapidly reproducible and precise results (Fedder R et al., J AOCS Int 2005,
88, 1579).
Analysis of tocopherols
Extraction : The cell suspension is
centrifuged and the cell pellet is resuspended in 0.8 ml of 25 mM EDTA pH 7 (to prevent
phospholipid degradation). Total lipids are extracted by vortexing 20 min after addition
of 1 ml chloroform, 1 ml methanol and 50 µl of 15 mM BHT, and again 5 min after further
additions of 1 ml chloroform and 1 ml NaCl to obtain two phases. After centrifugation, the
lower phase is washed if necessary with 2 ml of 1M NaCl and evaporated under nitrogen.
Purification : Tocopherols, cholesterol
and individual phospholipids are separated by one-dimensional TLC on Whatman LK5 silica
gel plates. Plates were previously washed by migration up to the top in
chloroform/methanol (1/1) in a special clean tank, dried and largely wetted with a
solution of 2.5% boric acid in ethanol (except the concentration zone). After draining and
dessication at 100°C (15 min), samples are applied as streaks in the concentration zone.
The solvent system used is the same we have described for phospholipids but with addition
of 10 mg/ml ascorbic acid (previously dissolved in water) and BHT (0.15 mg/ml) to protect
tocopherols and unsaturated fatty acids against oxidation. The plates are developed up to
1 cm below the top in a dark chamber (the glass tank is under a plastic box). After
drying, lipid spots are located under a UV lamp after a primulin spray (1 mg of primuline
powder in 100 ml of a mixture acetone/water, 80/20). Commercial standard are used to
locate the lipids of interest. Tocopherols and cholesterol have a quite similar migration
and are found near the solvent front (average RF: 0.87).
The spot is scraped off and tocopherols eluted by vortexing some minutes with 3 ml of a
mixture hexane/ter-butyl ether (92/8) or with pure ter-butyl ether if cholesterol must
also be determined. The solvent phases are evaporated under nitrogen and the residue,
dissolved in methanol, is stored in amber glass ware at -20°C.
HPLC : tocopherols (and cholesterol) are
separated on a column packed with 5 µm LiChrosorb RP18 (Merck) (120x4.6 mm), with
methanol/water (98/2) as the mobile phase, flow-rate: 1.5 ml/min.
Detection: tocopherols can be detected by light-scattering, spectrophotometry, fluorimetry
and electrochemistry (cholesterol can be detected by light scattering and
spectrophotometry). Tocopherol acetate (commonly used in food, dietetic and pharmaceutical
preparations) is not fluorescent and may be detected by UV photometry or by light-scattering.
As response factors are different for the different tocopherols with any detector system,
standard curves must be determined for each component.
With UV detection (292 nm for free tocopherols and 284 nm for tocopherol acetate), the
minimum detection limit is about 15-30 ng (35-70 pmol) per injection. With fluorescence
detection (excitation at 292 nm, emission at 327 nm) the minimum detection limit is about
0.5 ng (2-3 nmol) according to the instrument used. With the evaporative light-scattering
detector, responses are linear in the range 0.1-0.5 µg and the minimum detection
limits
are about 10-20 ng per injection. These observations were made with a DDL21 detector
(Eurosep Instruments) optimized with an air pressure set at 1 bar, an evaporation
temperature at 60°C and a high voltage at 700 V. Other instruments should be optimized
specifically.
d-tocopherol, rarely present in animal tissues, and tocopherol acetate are
often
used as internal standards to correct from losses during extraction and washings.
A typical chromatogram of a mixture of tocopherols obtained with a light-scattering
detector is given below.
Peaks
1: d-tocopherol, 2: g-tocopherol, 3: a-tocopherol, 4:
tocopherol acetate.
Time in minutes.
Several ways to perform sample pre-treatment and
analysis of a-tocopherol
may be found in the review by Ruperez FJ et al. (J Chromatogr A 2001, 935, 45).
That work includes referenced tables providing in-depth summaries of methodology
for the analysis of a-tocopherol
and related compounds in foods, pharmaceuticals, plants, animal tissues and
other matrices.
A comparison of chromatographic separation of tocopherols by reversed-phase,
normal-phase, and gas chromatography may be found by Pyka A et al. (J
Chromatogr A 2001, 935, 71).
Several methods have been developed
for simultaneously determining a-tocopherol
and cholesterol since they have both important dietary functions and furthermore
share similar chromatographic properties. With the HPLC system described above,
cholesterol eluted after about 9.5 min and is easily quantified with a
light-scattering detector.
Another chromatographic system has been described involving a silica column
eluted with an isocratic mobile phase made of isooctane/tetrahydrofuran (90/10,
v/v) (Cayuela JM et al., J Agric Food Chem 2003, 51, 1120). A
fluorescence detector was used to quantify tocopherols and a light-scattering
detector to quantify cholesterol. Fluorescence was preferably used since, in the
absence of previous purification of muscle extracts, this technique prevents
interferences from unknown non-fluorescent substances eluting near a-tocopherol.
Very good and rapid separations of tocopherols were also obtained using
nano-HPLC with a capillary column (100
mm
ID) filled with 3
mm
RP18 particles (Fanali S et al., J Pharm Biomed Anal 2004, 34, 331).
Monolithic column were shown to improve the determination of vitamin E and A in
human serum in shortening the time of analysis four times in comparison with
using traditional particulate columns (Urbanek L et al., Anal Chim Acta 2006,
573, 267).
An example of simultaneous determination of a-tocopherol,
b-carotene
and retinol by HPLC with diode-array detection may be found in the work of
Gimeno E et al. (J Chromatogr B 2001, 758, 315) as well as in the work of
Casal S et al. (J Chromatogr B 2001, 763, 1).
The precise analysis of the carboxyethyl-hydroxychroman metabolites of
a-
and
g-tocopherol
found in plasma or urine is possible when using a combination of gas
chromatography with mass spectrometry (Galli F et al., Free Rad Biol Med
2002, 32, 333).
Analysis of tocotrienols and tocopherols
Several chromatographic HPLC methods
were published and analyses involved either normal or reversed phase columns.
Normal phase columns are more efficient to resolve all vitamin E vitamers (tocols)
as
they are found in foodstuffs.
One of the most reliable method for the simultaneous determination of
tocopherols and tocotrienols is given below (Panfili G et al., J Agric Food
Chem 2003, 51, 3940).
The vitamin E complex is extracted as described before for fatty
materials with a hot saponification to provide the highest vitamin E
recovery.
The chromatographic separation is achieved with a normal phase (Kromasil
Phenomenex Si column) eluted with hexane/ethyl acetate/acetic acid
(97.3/1.8/0.9, v/v) at a flow rate of 1.6 ml/min. Fluorometric detection is
performed at an excitation wavelength of 290 nm and an emission wavelength of
330 nm.Tocopherols may be obtained from Merck and totrienols may be purified
from barley extracts. The concentrations of the purified standards are
calculated using the extinction coefficients given in this
site.
A chromatogram of tocopherols and tocotrienols found in a barley sample is given below.
To improve the
stability and the reproducibility of the silica column used, the column was
flushed every eight injections with hexane/isopropanol (90/10, v/v).
Tocotrienols standards are available from commercial sources but give double
peaks due to the formation of racemic mixtures during synthesis. Thus, the
preparation of pure vitamers from natural sources (barley, indian corn, and
wheat) allows the formation of symmetrical peaks.
A base-line separation of all vitamers of tocopherol and tocotrienol has been
obtained by capillary electrochromatography with an octylsilica column and
acetonitrile/methanol as a mobile phase (Abidi SL et al., J Chromatogr A,
2001, 913, 379).
The determination of several geometrical isomers of tocotrienols was described
after derivatization into methyl ether and chromatography on a chiral phase.
Thus, a complete resolution of the eight isomers of a-tocotrienols
was easily achieved in a 30 min run (Drotleff
AM et al., J Chromatogr A 2001, 909, 215).
An efficient method to analyze tocopherols and tocotrienols, including
tocomonoenol, was described using a C30 silica stationary phase and a
fluorescence detector (Ng
MH et al., Lipids 2004, 39, 1031).
Analysis of tocopherolquinone and epoxytocopherolquinones
(Leray
C et al., J Lipid Res 1998, 39, 2099)
These compounds are analyzed simultaneously
with tocopherols or tocotrienols using an isocratic HPLC procedure
combined with post-column zinc reduction and a single electrode coulometric detection.
If pure compounds or clean extracts are analyzed, the monitoring of the elution may be
made at 262 nm for tocopherolquinone and 270 nm for epoxydes. The column and eluant are
similar to those used with UV or fluorescent detection of tocopherols but 5 mM ZnCl2, 2.5 mM Na acetate and 2.5 mM acetic acid are added
to the mobile phase. A solid-phase post-column reactor similar to that used for the HPLC
separation of vitamin K is used to reduce the quinone
compounds before their coulometric detection in the oxidative mode. One electrode was used
and set at a potential of + 600 mV. As for vitamin K, the mobile phase must be
continuously deaerated by nitrogen gas bubbling one hour before flowing and during the
run.
Below, a chromatogram of products of a-tocopherol oxidation is shown
:
Peaks 1: tocopherolhydroquinone, 2: tocopherol quinone epoxyde, 3:
tocopherolquinone, 4: g-tocopherol and 5: a-tocopherol.
A similar procedure was used not only for vitamin E
and its direct oxidation products but also for the addition products of a-tocopherol
with peroxyl radicals derived from cholesteryl ester hydroperoxides and
phosphatidylcholine hydroperoxides in plasma (Yamauchi
R et al., Lipids 2002, 37, 515).
The determination of
a-tocopherol
and tocopherolquinone may also
be effected by HPLC with electrochemical detection (Kanazawa
H et al., Chem Pharm Bull 2000, 48, 1462).
A direct and efficient procedure of tocopherol analysis in vegetable oils was
described using differential pulse voltammetry (Diaz TG et al., Anal Chim
Acta 2004, 511, 231).
The fluorescence detection of
tocopherolquinone following
derivatization by a photoreactor (excitation wavelength : 254 nm) has been shown
to be an efficient alternative to the derivatization with a zinc reduction
column (Pollok D et al., J Chromatogr A 2004, 1056, 257).
Preparation of tocopherolquinone
(Schudel P et al Helv Chim Acta 1963, 46, 333)
This compound can be prepared easily by Fe+++
oxidation in methanol solution.
To a solution of tocopherol in diethyl ether (1 g in 10 ml), add 2.5 ml of a solution
made by dissolving FeCl3 (1.2 g in 15 ml methanol/water, 50/50, v/v). After 30 min
agitation, the mixture is centrifuged and the lower phase is removed. Repeat this process
4 times and wash extensively the ether phase with water (8-10 times).
Evaporate the ether phase and dissolve in ethanol or ether.
Determine the exact concentration of a solution in measuring the absorbance at 260 nm
of a mother solution (about 1 mg/ml ethanol) diluted 100 times. The molar extinction
coefficient is taken as 19500 (260 nm).
The purity of the product is verified by HPLC as for tocopherols by injecting 10 µl
of a tocopherolquinone solution (1 mg/ml ethanol) and recording the absorbance at 260 nm.
Preparation of tocopherolquinone epoxyde
(Liebler C et al Biochemistry 1992, 31, 8278)
Analysis of 5-nitro-g-tocopherol
Extraction : the
same procedures as for tocopherols
HPLC : nitro-g-tocopherol
were separated from tocopherols on a deactivated octadecyl silane column
(15 x 0.46 cm Supelcosil LC-18-DB, 3 mm
particle size) eluted isocratically with 95/5 (v/v) methanol/0.5 M lithium
acetate (pH 4.75) at a flow rate of 1.3 ml/min.
A coulometric detection was performed quantitatively at +500 mV (downstream
electrode) after elimination of interfering substances at +300 mV (upstream
electrode).
Below, a chromatogram of products of 5-nitro-g-tocopherol
(peak 3) and the two main tocopherols (peak 1 : g-tocopherol,
peak 2 :
a-tocopherol.
The detection of nitro-g-tocopherol
was linear over more than four orders of magnitudes from 0.1 pmol to 0.3 nmol,
the detection limit being about 10 fmol (Christen S et al., J Lipid Res 2002,
43, 1978).
The isolation and quantification of 5-nitro-g-tocopherol
and the other reaction products of a-tocopherol
with nitric oxide was done by HPLC and UV-visible detection but the detection by
atmospheric pressure chemical ionization was shown to be more selective for
these compounds (Nagata Y et al., J Chromatogr A 2004, 1036, 177).
Preparation of of 5-nitro-g-tocopherol
This compound was synthesized by the
nitrous acid method.
An ethanolic solution of g-tocopherol
(1 mg/ml) was acidified with 0.04 vol glacial acetic acid and nitration induced
by the addition of 0.6 vol of a 2% sodium nitrite solution. After 2 min, the
reaction was stopped with 0.4 vol of 20% potassium hydroxide. Two volumes of
water were added and the crude product was extracted onto hexane and was
purified by HPLC using 100% methanol as the eluent. The peak containing the pure
product was collected after detection at 410 nm (extinction coefficient : 1,976
M-1 cm-1).
