Journal
JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES
Volume 877, Issue 11-12, Pages 1077-1083Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.jchromb.2009.02.051
Keywords
Fat-soluble vitamins; Retinol; Carotenoids; Tocopherols; HPLC; Diode array detector; Fluorescence detector
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An improved isocratic and rapid HPLC method was developed for the measurement of carotenoids, retinol and tocopherols in human serum. Vitamins were extracted with hexane. Mobile phase consisted of a mixture acetonitrile:methylene chloride: methanol with 20 mM ammonium acetate. This method used a small bead size (3 mu m) Spherisorb ODS2 column with titane frits. Diode array and fluorescence detectors were used respectively for the detection of carotenoids and retinol/tocopherols. Chromatographic separation was complete in 13 min for beta-cryptoxanthin, cis-trans-lycopene, alpha-carotene, alpha-carotene, cis-beta-carotene, retinol, delta-tocopherol, gamma-tocopherol and alpha-tocopherol. Echinenone and tocol were employed as internal standards for diode array and fluorescence detection. Accuracy was validated using standard reference material (SRM) 968C. Intra-assay and inter-assay precision were respectively 0.2-7.3% and 3.6-12.6%. Sensitivity was verified using the ICH recommendations and the limit of detection (LOD) obtained was sufficient for routine clinical application. (C) 2009 Elsevier B.V. All rights reserved.
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