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

► The LLE procedure for human breast milk investigation was developed and validated. ► Extracted vitamins, retinol and α-tocopherol, were separated by HPLC. ► Developed extraction procedure was applied on 120 human breast milk samples. 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 5.0μmol/L, for α-tocopherol 92–109% at concentration levels 2.5, 5.0 and 10.0μmol/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 325nm for retinol and 290nm for α-tocopherol.