Determination of retinol palmitate and tocopherol acetate in lipid microcapsules by HPLC/UV

An original method for the quantitative determination of retinol palmitate (RP) and tocopherol acetate (TA) in lipid microcapsules using reverse-phase high-performance liquid chromatography with ultraviolet detection (HPLC-UV) in isocratic elution mode is proposed. The method includes sample preparation of lipid microcapsules, HPLC separation of microcapsule components TA and RP, as well as metrological support for the quantitative determination of vitamins. Sample preparation of microcapsules included the following stages: dissolution of microcapsules by heating, cooling of the solution, centrifugation and filtration. The proposed sample preparation procedure provides the extraction degree no less than 98.5% for TA and 99.6% for RP, respectively. Optimal HPLC-UV chromatography conditions for isocratic elution were selected. The optimal wavelength of 288 ± 2 nm determined from spectrophotometric measurements provides the joint determination of RP and TA. The effect of mobile phase (MP) composition on chromatographic parameters (retention time, peak area, resolution, peak width, peak asymmetry coefficient) for standard TA and RP samples is considered. The best values for these parameters were determined from the peaks obtained when using the mobile phase acetonitrile: isopropyl alcohol: 1,2-dichloroethane (55:40:5). Calibration dependences of the peak areas on the concentration of TA from 0 to 5% and RP from 0 to 10% were constructed; the correlation coefficients were 0.9981 and 0.9976, respectively. The accuracy of the method for the quantitative determination of fat-soluble vitamins (TA and RP) in lipid microcapsules was assessed in spike recovery tests. The method can be recommended for quality control laboratories to determine the quantitative content of fat-soluble vitamins such as retinol palmitate and tocopherol acetate in the pharmaceutical, cosmetic and food industries.

1. Figueiredo J. A., Silva C. R. P., Souza Oliveira M. F., et al. Microencapsulation by spray chilling in the food industry: Opportunities, challenges, and innovations / Trends Food Sci. Technol. 2022. Vol. 120. P. 274 – 287. DOI: 10.1016/j.tifs.2021.12.026

2. Dhakal S. P., He J. Microencapsulation of vitamins in food applications to prevent losses in processing and storage: A review / Food Res. Int. 2020. Vol. 16. P. 137 – 180. DOI: 10.1016/j.foodres.2020.109326

3. Jadoon S., Waseem A., Yaqoob M., Nabi A. Flow injection spectrophotometric determination of vitamin E in pharmaceuticals, milk powder and blood serum using potassium ferricyanide — Fe (III) detection system / Chinese Chem. Lett. 2010. Vol. 21. N 6. P. 712 – 715. DOI: 10.1016/j.cclet.2009.11.013

4. Qi F. F., Tao L. M., Dai Y. M., el al. Optimization and application of high-throughput supported liquid extraction for simultaneous determination of carotenoids and fat-soluble vitamins in serum / J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2021. Vol. 1173. 122672. DOI: 10.1016/j.jchromb.2021.122672

5. Khan A., Khan M. I., Iqbal Z., et al. An optimized and validated RP-HPLC/UV detection method for simultaneous determination of all-trans-Retinol (Vitamin A) and α-Tocopherol (Vitamin E) in human serum: Comparison of different particulate reversed-phase HPLC columns / J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2010. Vol. 878. N 25. P. 2339 – 2347. DOI: 10.1016/j.jchromb.2010.07.009

6. Craft N. E., Furr H. C. Chapter 2 — Methods for assessment of Vitamin A (Retinoids) and carotenoids / Laboratory Assessment of Vitamin Status / D. Harrington, Ed. — Elsevier Inc., 2019. P. 21 – 47. DOI: 10.1016/B978-0-12-813050-6.00002-4

7. de la Cruz Quiroz R., Chotyakul N., Saraiva J. A., et al. Retention of Ascorbic Acid, Retinol, β-Carotene, and α-Tocopherol in Milk Subjected to Pressure-Assisted Thermal Processing (PATP) / Food Eng. Rev. 2020. Vol. 13. P. 634 – 641. DOI: 10.1007/s12393-020-09242-z

8. State Standard GOST R 54635–2011. Functional food products. Method for determining vitamin A. — Moscow: Standartinform, 2013. — 15 p. [in Russian].

9. OFS.1.2.3.0017.15. Methods for the quantitative determination of vitamins / State Pharmacopoeia of Russian Federation XIV Edition [in Russian].

10. Temova Rakusa Z., Srecnik E., Roskar R. Novel HPLC-UV method for simultaneous determination of fat-soluble vitamins and coenzyme Q10 in medicines and supplements / Acta Chim. Slov. 2017. Vol. 64. P. 523 – 529. DOI: 10.17344/acsi.2016.2856

11. Interstate Standard GOST 32043–2012. Premixes. Methods for determining vitamins A, D, E. — Moscow: Standartinform, 2012. — 16 p. [in Russian].

12. State Standard GOST R 54950–2012 (ISO 14565:2000) Animal feed. Determination of vitamin A content by high-performance liquid chromatography. — Moscow: Standartinform, 2012. — 15 p. [in Russian].

13. State Standard GOST EN 12822–2014. Food products. Determination of vitamin E content (alpha-, beta-, gamma- and delta-tocopherols) by high-performance liquid chromatography. — Moscow: Standartinform. 2014. — 25 p. [in Russian].

14. GOST R 54949–2012 (ISO 6867:2000). Animal feed. Determination of vitamin E content by high-performance liquid chromatography. —- Moscow: Standartinform, 2012. — 11 p. [in Russian].

15. Timofeev N. P. Content of vitamins in leaves of Leuzea carthamoides (against ecdysteroids) / New and non-traditional plants and prospects for their use. Vol. 3. — Moscow: VNIISSOK, 2017. P. 72 – 74 [in Russian].

16. Vostokov V. M., Kartashev V. R. Chromatographic control of the biochemical activity of fat-soluble vitamins (A, D, E) in food and feed products / Izv. Vuzov. Ser. Khimiya Khim. Tekhnol. 2006. Vol. 49. N 4. P. 115 – 118 [in Russian].

17. Belozertseva E. A., Finkelshtein E. I., Eremin S. V., Shvets V. I. A new approach to HPLC analysis of liquid vitamin-containing dosage forms / Vestn. MITKhT im. M. V. Lomonosova. 2012. Vol. 7. N 2. P. 58 – 64 [in Russian].

18. Samuseva K. R. Control of the content of fat-soluble vitamins in cosmetic creams / Proc. of VIII Int. Scientific-practical Conference «Youth. Intelligence. Initiative». Vitebsk, 2020. P. 97 – 98 [in Russian].

19. Trineeva O. V., Safonova E. F., Slivkin A. I. Determination of vitamins A and E in a thin layer when present together / Vestn. VGU. Ser. Khim. Biol. Farm. 2015. Vol. 3. P. 129 – 134 [in Russian].

20. Romanov O. E., Budinov S. V., Shtykov S. N. Thin layer chromatography of vitamins A and E on silica gel / Sorb. Khromatogr. Prots. 2014. Vol. 14. N 4. P. 563 – 571 [in Russian].

21. State Pharmacopoeia of the USSR. XI edition. — Moscow: Meditsyna, 1987. — 324 p. [in Russian].

22. Fadeikina I. N., Peunkova E. S., Zuev B. K. Determination of vitamin E (α-tocopherol acetate) on the surface of human skin by IR Fourier-transform spectrometry and study of some aspects of its transdermal transfer / J. Anal, Chem. 2021. Vol. 76. N 2. P. 191 – 195 [in Russian]. DOI: 10.1134/S1061934821020088

23. Finkelshtein E. I., Rudakova I. P., Samylina I. A. Methods for the quantitative determination of vitamins in dosage forms / Farmatsiya. 2007. Vol. 4. P. 45 – 48 [in Russian].

24. State Pharmacopoeia of the Russian Federation. XIV edition. — Moscow, 2018. — 704 p. [in Russian].

25. Mikheeva E. V., Anisimova L. S. Physicochemical methods for determining vitamin E in various objects (review) / Industr. Lab. Mater. Diagn. 2005. Vol. 71. N 2. P. 3 – 9 [in Russian].

26. Kulneva Yu. Yu. Optimization of determination of fat-soluble vitamins / Proc. of VI (63rd) Annual Scientific and Practical Conference «University science for the region» of the North Caucasus Federal University. — Stavropol, 2018. P. 383 – 387 [in Russian].

27. Kazakevich Y. V., LoBrutto R., Eds. HPLC for Pharmaceutical Scientists. — John Wiley & Sons Inc., 2007. — 1104 p. DOI: 10.1002/0470087951

28. Gogal’ová Z., Kahle V., Šesták J. An improved design of the fused silica capillary flow cell for absorbance detection in microcolumn liquid chromatography / Anal. Chim. Acta. 2023. Vol. 1238. 340637. DOI: 10.1016/j.aca.2022.340637

29. Alekseeva A. S., Shemeryankina T. B., Lyakina M. N., et al. Analysis of Vitamin A in Multivitamin products / Vedom. Nauch. Tsentra Ékspert. Sredstv Med. Prim. 2021. Vol. 11. N 3. P. 174 – 184 [in Russian]. DOI: 10.30895/1991-2919-2021-11-174-184

30. Kostarnoi A. V., Golubitskii G. B., Basova E. M., et al. High-performance liquid chromatography in the analysis of multicomponent / J. Anal. Chem. 2008. Vol. 63. N 6. P. 516 – 529. DOI: 10.1134/S1061934808060026

31. State Standard GOST R 54634–2011. Functional food products. Method for determining vitamin E. — Moscow: Standartinform, 2013. — 15 p. [in Russian].

32. Rimkus G. G., Schubert M., Morgan D., Jungjohann S. Rapid direct analysis of retinyl palmitate (vitamin A) in fortified vegetable oils by HPLC-FLD / Food Addit. Contam.: Part A. 2022. Vol. 39. N 1. P. 24 – 34. DOI: 10.1080/19440049.2021.1977854

33. Vorontsova O. S., Markova K. V. A method for determining vitamin A (retinol) in food products using high-performance liquid chromatography / Proc. of the International Scientific and Practical Conference «Health and the Environment». — Minsk, 2016. P. 227 – 229 [in Russian].

34. Vorobyova O. A. Features of sample preparation during RP-HPLC analysis of tocols in pumpkin seed oil / Fund. Prikl. Issl. Probl. Result. 2016. Vol. 24. P. 147 – 151 [in Russian].

35. Rudakov O. B., Khorokhordina E. A., Rudakova L. V., Groshev E. N. Acetonitrile is a unique solvent for liquid chromatography and extraction / Vestn. VGU. Ser. Khim. Biol. Farm. 2015. Vol. 3. P. 41 – 46 [in Russian].

36. Sadek P. C. The HPLC solvent guide. — NY: Wiley, 1996. — 346 p.

37. Filimonov V. N., Zamuruev O. V. Multiparametric optimization of the composition of a three-component mobile phase for separating a mixture of fat-soluble vitamins by normal-phase high-performance liquid chromatography / Russ. J. Phys. Chem. A. 2001. Vol. 75. N 12. P. 2046 – 2052.