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Fluorometric determination of retinol in human tear fluid using high-performance liquid chromatography

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Author: Speek, A.J. · Agtmaal, E.J. van · Saowakontha, S. · Schreurs, W.H.P. · Haeringen, N.J. van
Institution: Centraal Instituut voor Voedingsonderzoek TNO
Source:Current Eye Research, 11, 5, 841-845
Identifier: 230190
Keywords: Retinol · Drug analysis · Drug determination · Drug tissue level · Fluorometry · High performance liquid chromatography · Human · Human cell · Methodology · Normal human · Tear · Visual system · Chromatography, High Pressure Liquid · Drug Stability · Evaluation Studies · Fluorometry · Human · Support, Non-U.S. Gov't · Tears · Vitamin A · Biology · Biochemistry · Medicine · Geneeskunde


A fast and sensitive high-performance liquid chromatographic (HPLC) method for the determination of vitamin A (all-trans retinol) in 50 ??l human tear fluid is described. After deproteinization with ethanol and extraction with n-hexane, retinol is separated from the extract on a straight-phase HPLC column and detected fluorometrically. Vitamin A can be determined in concentrations as low as 0.4 ng/ml. A single analysis can be completed in 12 min while the analysis of a series of 60 samples takes about 8 h. The within-assay and between-assay coefficients of variation were 3.1 and 4.2% resp. The between-assay analytical recovery of retinol added to tear fluid was 97.4 ?? 6.0% (mean ?? SD). Retinol was determined in tear fluid of nine adult volunteers. The amounts observed ranged from <0.4-10.6 ng/ml. Chemicals/CAS: retinol, 68-26-8, 82445-97-4; Vitamin A, 11103-57-4