The comparison of HPLC and spectrophotometric method for cholesterol determination


  • Lukáš Kolarič Slovak University of Technology in Bratislava, Faculty of Chemical and Food Technology, Institute of Food Science and Nutrition, Department of Food Technology, Radlinského 9, 812 37 Bratislava, Slovakia, Tel.: +421 2 593 25 555
  • Peter Šimko Slovak University of Technology in Bratislava, Faculty of Chemical and Food Technology, Institute of Food Science and Nutrition, Department of Food Technology, Radlinského 9, 812 37 Bratislava, Slovakia, Tel.: +421 2 593 25 558



cholesterol, HPLC, spectrophotometry, analysis, milk


The present study was carried out to compare two different analytical methods (HPLC and spectrophotometric) for determination of cholesterol content in milk while cholesterol in food is important not only for the nutritional value setting of foods but also due to the validation of a fast, reliable and economical method for studying the possible mechanism of its reduction. Spectrophotometric determination of cholesterol content was based on the Liebermann-Burchard (LB) reaction among cholesterol, ethyl acetate, acetic anhydride, plus concentrated H2SO4 and measuring absorbance of formed color at 625 nm. HPLC method was performed by column chromatography on reverse phase C18 with DAD detection at 205 nm. The methods were applied to the milk sample. The achieved LOD and LOQ for HPLC were 2.13 and 6.45, respectively, while for spectrophotometric method were 12.55 and 38.04 The difference between cholesterol content determined by both methods was statistically insignificant at p <0.05. Therefore, it can be concluded that both methods are suitable for determination of cholesterol content in milk, however, HPLC method exhibited higher sensitivity and lower limits of detection or quantification, respectively.


Download data is not yet available.


Adu, J. K., Amengor, C. D. K., Kabiri, N., Orman, E., Patamia, S. A. G., Okrah, B. K. 2019. Validation of a Simple and Robust Liebermann-Burchard Colorimetric Method for the Assay of Cholesterol in Selected Milk Products in Ghana. International Journal of Food Science, vol. 2019, p. 1-7.

Ahn, J. H., Jeong, I. S., Kwak, B. M., Leem, D., Yoon, T., Yoon, Ch., Jeong, J., Park, J. M., Kim, J. M. 2012. Rapid determination of cholesterol in milk containing emulsified foods. Food Chemistry, vol. 135, no. 4, p. 2411-2417.

Albuquerque, T. G., Oliveira, M. B. P. P., Sanches-Silva, A., Costa, H. S. 2016. Cholesterol determination in foods: Comparison between high performance and ultra-high performance liquid chromatography. Food Chemistry, vol. 193, p. 18-25.

Atinafu, D. G., Bedemo, B. 2011. Estimation of total free fatty acid and cholesterol content in some commercial edible oils in Ethiopia, Bahir DAR. Journal of Cereals and Oil seeds, vol. 2, no. 6, p. 71-76.

Bauer, L. C., Santana, D. A., Macedo, M. S., Torres, A. G., de Souza, N. E., Simionato, I. 2014. Method Validation for Simultaneous Determination of Cholesterol and Cholesterol Oxides in Milk by RP-HPLC-DAD. Journal of the Brazilian Chemical Society, vol. 25, no. 1, p. 161-168.

Borkovcová, I., Janoušková, E., Dračková, M., Janštová, B., Vorlová, L. 2009. Determination of Sterols in Diary Products and Vegetable Fats by HPLC and GC Methods. Czech Journal of Food Sciences, vol. 27, no. 1, p. 217-219.

Burke, R. W., Diamondstone, B. I., Velapoldi, R. A., Menis, O. 1974. Mechanisms of the Liebermann-Burchard and Zak Color Reactions for Cholesterol. Clinical Chemistry, vol. 20, no. 7, p. 794-801.

Dinh, T. T. N., Thompson, L. D., Galyean, M. L., Brooks, J. Ch., Patterson, Y., Boylan, L. M. 2011. Cholesterol Content and Methods for Cholesterol Determination in Meat and Poultry. Comprehensive Reviews in Food Science and Food Safety, vol. 10, p. 269-289.

Eckschlager, K., Horsák, I., Kodejš, Z. 1980. Vyhodnocování analytických výsledků a metod (Evaluation of analytical results and methods). 1st ed. PRAHA ČESKÁ REPUBLIKA : Státní nakladatelství technické literatúry, 224 p. (In Czech language)

Essaka, D. C. 2007. Reversed-Phase HPLC Determination of Cholesterol in Food Items. Electronic Theses and Dissertations. Available at:

Faye, B., Bengoumi, M., Al-Masaud, A., Konuspayeva, G. 2015. Comparative milk and serum cholesterol content in dairy cow and camel. Journal of King Saud University – Science, vol. 25, no. 2, p. 168-175.

Fletouris, D. J., Botsoglou, N. A., Psomas, I. E., Mantis, A. I. 1998. Rapid Determination of Cholesterol in Milk and Milk Products by Direct Saponification and Capillary Gas Chromatography. Journal of Dairy Science, vol. 81, no. 11, p. 2833-2840.

Kenny, A. P. 1952. The Determination of Cholesterol by the Liebermann-Burchard Reaction. The Biochemical Journal, vol. 52, no. 4, p. 611-619.

Kim, E., Goldberg, M. 1969. Serum Cholesterol Assay Using a Stable Liebermann-Burchard Reagent. Clinical Chemistry, vol. 15, no. 12. p. 1171-1179.

Li, L. H., Dutkiewicz, P., Huang, Y. C., Zhou, H. B., Hsu, C. C. 2019. Analytical methods for cholesterol quantification. Journal of Food and Drug Analysis, vol. 27, no. 2, p. 375-386.

Manzi, P., Di Costanzo, M. G., Mattera, M. 2013. Updating Nutritional Data and Evaluation of Technological Parameters of Italian Milk. Foods, vol. 2, no. 2, p. 254-273.

Oh, H. I., Shin, T. S., Chang, E. J. 2001. Determination of Cholesterol in Milk and Dairy Products by High Performance Liquid Chromatography. Asian-Australian Journal of Animal Sciences, vol. 14, no. 10, p. 1465-1469.

Osman, H., Chin, Y. K. 2006. Comparative Sensitivities of Cholesterol Analysis Using GC, HPLC and Spectrophotometric Methods. The Malaysian Journal of Analytical Sciences, vol. 10 , no. 2, p. 205-210.

Ramalho, H. M. M., Casal, S., Oliveira, M. B. P. P. 2011. Total Cholesterol and Desmosterol Contents in Raw, UHT, Infant Formula Powder and Human Milks Determined by a New Fast Micro-HPLC Method. Food Analytical Methods, vol. 4, no. 3, p. 424-430.

Ribeiro, T. M., Brandäo, P. R. G. 2017. Development and Validation of Graphitic Carbon Analysis of Graphite Ore Samples. Tecnologia em Metalurgia Materiais e Mineração, vol. 14, no. 2, p. 183-189.

Sperry, W. M., Brand, F. C. 1943. The Colorimetric Determination of Cholesterol. The Journal of Biological Chemistry, vol. 150, p. 315-324.

Xiong, Q., Wilson, W. K., Pang, J. 2007. The Liebermann-Burchard Reaction: Sulfonation, Desaturation, and Rearrangment of Cholesterol in Acid. Lipids, vol. 42, no. 1, p. 87-96.



How to Cite

Kolarič, L., & Šimko, P. (2020). The comparison of HPLC and spectrophotometric method for cholesterol determination . Potravinarstvo Slovak Journal of Food Sciences, 14, 118–124.