SIMULTANEOUS DETERMINATION OF VITAMIN A AND E IN INFANT MILK FORMULAS USING SEMI-MICRO LIQUID–LIQUID EXTRACTION FOLLOWED BY HPLC-UV

Abstract

An efficient and inexpensive semi-micro liquid–liquid extraction method has been developed for simultaneous clean-up and extraction of vitamin A and vitamin E in infant milk formula samples. After saponification of milk samples, extraction procedure was done in a 5-mL volumetric flask. 4.5 mL of saponified sample solution was added to the volumetric flask, and then 300 µL of organic solvent was added slowly to the surface of the saponified solution in the neck of the flask by a gas tight micro syringe. The mixture was agitated during the extraction time. After the extraction time a prescribed amount of organic phase was withdrawn, evaporated and reconstituted with methanol and finally was injected into the HPLC system for analysis. The proposed method provided good reproducibility (3.9–5.1%), good linearity (0.006–80 µg mL⁻¹ for vitamin A and 0.1–400 µg mL⁻¹for vitamin E), low detection limits (0.003 µg mL⁻¹ for vitamin A and 0.08 µg mL⁻¹ for vitamin E), and was a simple, low cost, efficient, and easy handling technique for determination of vitamin A and E according to the results obtained. Furthermore, using monolithic column has the advantage of decreasing the total time of analysis.

Keywords:

• high-performance liquid chromatography with ultraviolet detection
• monolithic column
• saponification
• semi-micro liquid–liquid extraction
• vitamin A
• vitamin E

ACKNOWLEDGMENT

The authors gratefully acknowledge the financial support of this research by Ferdowsi University of Mashhad and also Nutricia-Multi Nutrition.

Notes

^a Concentration unit for vitamin A and E is µg mL⁻¹.

^b Concentration unit for vitamin A and E is pg per 50 µL injection.