Rapid sample preparation procedure for determination of retinol and α-tocopherol in human breast milk.

The liposoluble vitamins (retinol and α-tocopherol) concentration in human breast milk is of a cardinal knowledge especially for nutrition of prematurely born. It enables the feeding optimization of these important micronutrients for preterm infants. The novel rapid liquid-liquid extraction procedure for human breast milk investigation was developed and validated according to FDA guidelines. The recovery of retinol was 82-90% measured at three concentration levels 1.0, 2.5 and 5.0 μmol/L, for α-tocopherol 92-109% at concentration levels 2.5, 5.0 and 10.0 μmol/L. The repeatability of extraction procedure expressed as relative standard deviation was 3.26% for retinol and 4.79% for α-tocopherol. Developed extraction procedure was applied on 120 human breast milk samples. The separation of vitamins was completed using advantages of a monolithic column which accomplished demands of acceleration made by modern bio-analytical HPLC methodology. The analytes of interest were detected by diode-array detector at wavelengths 325 nm for retinol and 290 nm for α-tocopherol.