Simplified methodology to determine breast milk retinol concentrations

The impact of a nutritional intervention trial on vitamin A status can be evaluated by measuring the total vitamin A concentration in breast milk both before and after the intervention. Because breast milk contains a spectrum of retinyl esters as well as retinol and high lipid content, determination of total breast milk retinol routinely requires saponification with alcoholic potassium hydroxide. Retinol is then extracted with an organic solvent, usually hexanes, before HPLC analysis. Retinyl acetate, although commonly used as an internal standard, is not ideal because it can only be added after saponification and extraction and consequently, will only account for part of the total losses. A method has now been developed that uses 3,4-didehydroretinyl acetate (DRA) as an internal standard. DRA is an excellent choice as an internal standard for the following reasons: 1) DRA can be added to the breast milk before saponification and can be carried through the analysis as dehydroretinol (DR), 2) the percent recovery can be easily determined, and 3) DR is easily separated from retinol during HPLC analysis. The procedure, as described, typically gives a mean extraction efficiency of 80-90%. Moreover, the average coefficient of variation is <5% on the same sample run several times in the same day.