Isocratic rapid liquid chromatographic method for simultaneous determination of carotenoids, retinol, and tocopherols in human serum

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Vol. 877; no. 11; pp. 1077 - 1083
• Main Authors: Thibeault, Denis, Su, Haixiang, MacNamara, Elizabeth, Schipper, Hyman M.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 15.04.2009; Elsevier
• Subjects: Analysis; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Calibration; Carotenoids; Carotenoids - blood; Chromatography, High Pressure Liquid; Diode array detector; Fat-soluble vitamins; Fluorescence detector; Fundamental and applied biological sciences. Psychology; General pharmacology; HPLC; Humans; Medical sciences; Pharmacology. Drug treatments; Reference Standards; Retinol; Spectrometry, Fluorescence; Steel - chemistry; Tocopherols; Tocopherols - blood; Vitamin A - blood; Vitamins - blood; Fluorescence detector; Fat-soluble vitamins; Carotenoids; Tocopherols; Diode array detector; HPLC; Retinol; Human; Biological fluid; Chemical analysis; Vitamin; HPLC chromatography; Liquid chromatography; Isocratic condition; Rapid technique; Simultaneous measurement; Diode array; Carotenoid; Liposoluble compound
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2009.02.051

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 μm) 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 β-cryptoxanthin, cis–trans-lycopene, α-carotene, β-carotene, cis-β-carotene, retinol, δ-tocopherol, γ-tocopherol and α-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.