Published:Journal of Chromatographic Science,
ISSN 0021-9665Volume
40, Number 2, February 2002, pp. 69-76
An Isocratic Liquid Chromatographic Method with Diode-Array Detection
for the Simultaneous Determination of a-Tocopherol,
Retinol, and Five Carotenoids in Human Serum
Sonia Gueguen[1], Bernard
Herbeth[1], Gérard Siest[1], and Pierre Leroy[2]
[1]Inserm U525, Centre de Médecine Préventive, 2 rue du Doyen Jacques
Parisot, 54500 Vandoeuvre-lès-Nancy, France and
[2]Thiols and Cellular Functions, Faculté de Pharmacie, Université
Henri Poincaré Nancy 1, 30 rue Lionnois, 54000 Nancy, France
An isocratic high-performance liquid chromatography (HPLC) method
for the simultaneous determination of a-tocopherol, retinol, and five carotenoids
(lutein–zeaxanthin, b-cryptoxanthin, lycopene, and a- and b-carotene) in
human serum is described. Serum samples are deproteinized with ethanol and extracted
once with n-hexane. Resulting extracts are injected onto a C18 reversed-phase
column eluted with methanol–acetonitrile–tetrahydrofuran (75:20:5,
v/v/v), and full elution of all the analytes is realized isocratically within
20 min. The detection is operated using three channels of a diode-array spectrophotometer
at 290, 325, and 450 nm for tocopherol, retinol, and the carotenoids, respectively.
An internal standard is used for each channel, which improves precision. The
choice of internal standards is discussed, as well as the extraction protocol
and the need for adding an antioxidant during the extraction and chromatographic
steps. The analytical recoveries for liposoluble vitamins and carotenoids are
more than 85%. Intra-assay relative standard deviation (RSD) values (n = 20)
for measured concentrations in serum range from 3.3% (retinol) to 9.5% (lycopene),
and interassay RSDs (n = 5) range from 3.8% (a-tocopherol)
to 13.7% (b-cryptoxanthin). The present method is
used to quantitate the cited vitamins in healthy subjects (n = 168) from ages
9 to 55 years old.
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