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Breast milk is a main source of fat-soluble vitamins for newborns and it is needful to monitor the nutritional status prior to its application. In this work a novel, high-throughput and low-cost method for monitoring of retinol and alpha-tocopherol in breast milk was developed, validated and compared with reference method using monolithic column. For this purpose five various porous shell and monolithic columns were tested on the basis of relationship between HETP and linear mobile phase velocity, analysis time and consumption of solvents. Finally the core-shell analytical column Kinetex C18 (2.6 μm, 100 Å, 100×4.6 mm) was chosen as the best and optimal values of flow rate, injection volume and temperature of analysis were established. The detection of retinol and alpha-tocopherol was carried out at 325 and 295 nm, respectively by diode array detector. The LOD 0.004 μmol/L and 0.078 μmol/L, the LOQ 0.012 μmol/L and 0.182 μmol/L for retinol and alpha-tocopherol, respectively were calculated. The validation data showed good linearity, repeatability of retention time with RSD 0.22% and 0.12%, repeatability of peak area with RSD 6.94% and 1.75%, recovery 114.1-116.3% and 99.0-108.6% for retinol and alpha-tocopherol, respectively. Moreover, the newly developed method substantially decreased the solvent consumption by about 263 mL per 100 samples with the total time of analysis 1.75 min in comparison with analysis time 1.80 of the reference method., (Copyright © 2013 Elsevier B.V. All rights reserved.)

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., (Copyright © 2012 Elsevier B.V. All rights reserved.)