Loss of vitamin A in long-term stored, frozen sera

Vitamin A (retinol) was measured by a high performance liquid chromatography (HPLC) method in human serum samples stored frozen at -20 degrees C for 2-6 yr. In 40% of the sample, both vitamin A and the internal standard, vitamin A acetate (retinyl acetate) which was added at the time of assay, were destroyed. Controlled studies of each phase of the assay showed that the vitamin A began to degrade during the extraction step immediately after ethanol was added to the serum. Vitamin E (alpha-tocopherol) and beta-carotene also degraded concurrently with vitamin A. Vitamin A may be lost because of free radical oxidation after the vitamin is released from its serum binding protein (retinol-binding protein), following the addition of ethanol to the serum sample. The loss of vitamin A is eliminated completely if ascorbic acid (0.1% w/v) is added to the ethanol before it is used in the preassay extraction.