Separation of Vitamins Retinol Acetate, Ergocalciferol, or Cholecalciferol and Tocopherol Acetate Using Sequential Injection Chromatography

Abstract

A novel simple method for separation of vitamins A-acetate (all-trans retinol acetate), D₂ (ergocalciferol), or D₃ (cholecalciferol) and E-acetate (tocopherol acetate) using short monolithic column in the sequential injection chromatography system is described. Separation was carried out using FIAlab® 3000 system under following conditions: Onyx™ Monolithic C18 column (25 × 4.6 mm), mobile phase acetonitrile:methanol:H₂O 20:20:1 (v/v/v)), flow rate 0.9 mL min⁻¹, detection at 265 nm (D), 290 nm (E), and 325 nm (A). The method was validated with respect to peak asymmetry, resolution, number of theoretical plates, repeatability, linearity, precision, and accuracy. Analysis time was 5.5 min.

Keywords:

• Cholecalciferol
• Monolithic column
• Retinol-acetate
• Sequential injection chromatography
• Tocopherol-acetate

Acknowledgments

This paper was submitted as part of a Special Issue on Flow Injection Analysis.

The authors gratefully acknowledge the following financial support: H. S., P. K., P. Ch., D. Š., L. K., M. K., and P. S., the support of the Ministry of Education of the Czech Republic, MSM 0021620822. L. K., M. K., and D. S., the support of Ministry of Health, MZO 00179906. P. K., the support of the Grant Agency of the Charles University, No. 34609/2009. M. K. and P. K., the support of the SVV/2011/263 002.

Notes

Note: A = retinol-acetate; D = cholecalciferol; E = tocopherol-acetate; MP = mobile phase; t_R = retention time; ACN = acetonitrile; MeOH = methanol.

Note: HETP = height equivalent of the theoretical plate.

^a 6 repeated injections of standard solution.

^b Inter-day R.S.D. values were calculated for 6 repeated standard injections; three consecutive days.

^a Vitamin D₃.

^b Vitamin D₂ and A-acetate.

^c 6 sample preparations, three injections of each preparation.

^d 3 sample preparations, three injection of each preparation.