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

A fast and sensitive high-performance liquid chromatographic (HPLC) method for the determination of vitamin A (all-trans retinol) in 50 microliter 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 less than 0.4 - 10.6 ng/ml.