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Retinol, a-tocopherol, Iycopene, and a- and /3-carotene can Materials and Methods
be simultaneously determined in human plasma by reversed-
Apparatus. The chromatographic instrumentation was a
phase liquid chromatography. Plasma-0.5 mL plus added Model 1090 LC System (Hewlett-Packard, Avondale, PA)’
internal standard, retinyl acetate-is deproteinized with 0.5 with a variable-wavelength diode-array detector, attached
mL of ethanol, then extracted with 1.0 mL of petroleum ether. to the Hewlett-Packard 3388 integration system and con-
The organic layer is removed and evaporated, the residue is trolled by a HP85B computer. For all analyses we used a 60
redissolved in 0.25 mL of ethanol, and 8-4 samples are x 4.6mm column of 3-nm Hypersil ODS (Hewlett-Packard),
injected into a 60 x 4.6 mm column of Hypersil ODS 3-.tm operated at 35#{176}C.
The mobile phase was methanol, the flow
particles at 35 #{176}C.
An isocratic methanol mobile phase, rate 0.9 mLlmin.
flow rate 0.9 mLimin, is used for the 9-mm run. Retinol and Reagents. Retinol, retinyl acetate, a-tocopherol, lycopene,
retinyl acetate are monitored at 305 nm, the tocopherols at a-carotene, and (3-carotene were all from Sigma Chemical
292 nm, and the carotenoids at 460 nm. Between-run CVs Co., St. Louis, MO. All solvents were “HPLC” grade.
were 3.1, 6.9, 6.1, and 6.5% for retinol, a-tocopherol, lyco- We dissolved the standards individually in chloroform.
pene, and (3-carotene, respectively. Small sample require- We then further diluted them in ethanol to obtain approxi-
ment, simplicity of extraction, short run time, and good mate final concentrations of 1 mg/L for retinol and the
reproducibility make this procedure ideal for clinical or re- carotenoids, 40 mg/L for a-tocopherol. The actual concentra-
search use. tions of the standards were determined from the absor-
bances and the absorptivities (in L mol’ cm’): 1780 at
AddItional Keyphrases: vitamin A vitamin E . nutrition 325 nm for retinol, 75.8 at 292 nm for a-tocopherol, 3450 at
effect of variousanticoagulants 472 nm for lycopene, 2800 at 444 nm for a-carotene, and
2396 at 465 nm for (3-carotene.
Clinical interest in evaluation of vitamin A andvitamin E Procedures. All procedures were carried out in a labora-
nutriture has increased in recent years, mainly owing to the tory equipped with yellow lights that did not includethe
possible roles of retinol (vitamin A), p-carotene, and a- ultraviolet end of the spectrum. This minimized light-
tocopherol (vitamin E) in decreasing the risk of cancer (1). induced degradation of the vitamins. We added0.5 mL of
These vitamins are also important in premature infants and ethanol that contained 0.6 g of retinyl acetate per milliliter
patients who are receiving long-term total parenteral nutri- to 0.5 mL of plasma in a 10 x 75 mm glass tube, vortex-
tion. Thus, a rapid, sensitive, relatively simple, and specific mixed the mixture for 1.0 mm, added 1.0 mL of petroleum
method for determination of these vitamins in plasma is ether (bp 36.6-56.6 #{176}C),
and vortex-mixed again for 1 mm.
desirable. Conventional colorimetric or fluorimetric meth- After centrifuging (600 x g, 5 mm) we transferred the
ods for determining retinol, carotene, or a-tocopherol (2-3) supernate to a separate tube with a Pasteur pipet and
are either nonspecific, time consuming, or insensitive. evaporated it to dryness in a 60#{176}C water bath, under a
Many of these problems are eliminated when these micro- stream of nitrogen. We then redissolved the extract in 0.25
nutrientsare determined by “high-performance” liquid mL of ethanol,2 vortex-mixed for 2.0 mm, then filtered the
chromatography (LC). Several LC procedures have already mixture through a Millipore ifiter (no. SFHOO4NS; Milli-
been described for determination of retinol anda-tocopherol, pore Corp., Bedford, MA) into injection vials. These were
either separately (4-6) or simultaneously (7-10). Additional placed in the autosampler and 84 was injected into the
procedures can separate lycopene, a-carotene, and /3-caro- chromatograph, which then was eluted as described above.
tene from other carotenoids (11-14). Nevertheless, an assay We programmed the diode array detector as follows: 0 to
that could resolve and quantify all of these compounds 1.6 mm at 305 ± 40 nm, to determine retinol and retinol
simultaneously would be highly desirable from the stand- acetate; from 1.6 mm to 2.5 mm at 290 ± 10 nm, to
point of clinical assessmentand studies of their metabolism. determine a- and $ocopherol; and from 2.5 to 9.0 mm at
Procedures recently described for determination of reti- 460 ± 25 mu, to determine the carotenoids. The reference
nol, a-tocopherol, p-carotene, a-carotene, and lycopene in a wavelength was 575 nm throughout the run. The baseline
single run (15, 16) involve either a multiple-solvent gradi- was automatically adjusted to zero with each wavelength
ent system or complex solvent mixtures and 15 to 30 mm of change. Four minutes after injection, the attenuation was
analysis times. Here we describe an isocratic LC procedure increased fourfold.
in which only methanol is used in the mobile phase. With it,
retinol, a-tocopherol, lycopene, a-carotene, and p-carotene
1 Mention of a trademark or proprietary product doesnot consti-
extracted from 0.5 mL of plasma can be resolved and
estimated in a single 9-mm run. tute a guaranteeor warranty by the U.S. Dept. of Agriculture, and
doesnot imply its approval to the exclusionof other products that
may alsobe suitable.
USDA, ARS, Grand Forks Human Nutrition Research Center, 2The sampleswere redisaolved in ethanol, becausethe fat-soluble
P.O. Box 7166,University Station, Grand Forks, ND 58202. vitamins and carotenoids are much more soluble in ethanol than in
ReceivedSeptember 30, 1985;acceptedJanuary 31, 1986. (more polar) methanol.