Development of chromatographic control methods for identification and quantification of cow’s milk lactoferrin in lactoferrin substances

I. V. Spichak¹, M. V. Tikhanina², E. T. Zhilyakova¹, N. V. Avtina¹, V. E. Gulyaeva¹, D. A. Fadeeva^1
1Belgorod State National Research University, ul. Pobedy, 85, Belgorod, 308015, Russian Federation
²«VIC-Animal Health», ul. Berezovaya, 1/16, pgt. Severnyi, Belgorodskii r-n, Belgorodskaya obl., 308519, Russian Federation

Abstract. Lactoferrin is a bioactive multifunctional milk protein. The possibility of its production from lactic acid whey, which serves as a waste in the production of dairy products and is not recycled, is of interest. Effective use of such raw materials requires reliable analytical methods for monitoring the lactoferrin content. The purpose of the studies was to develop and validate a method for its quantification. In the process of developing the method, we compared the methods of high performance liquid chromatography (HPLC) and ultra-high performance liquid chromatography (UHPLC), octyl and butylsilanol reverse phase chromatographic columns, and optimized the composition of the mobile phase. The work was performed using chromatographs Waters Alliance with the DAD-detector and Waters Acquity H-Class with the PDA-detector. A gradient of mobile phases (phase A – 0.03% solution of trifluoroacetic acid in acetonitrile, phase B – 0.05% solution of trifluoroacetic acid in water) using a chromatographic column YMC-Triart BIO C 4/S-1.9um/30nm, 75x2.1 mml.D was recognized as an optimal option. The method was validated for the following characteristics: specificity (selectivity), linearity/analytical range (correlation coefficient 0.9994), lowest quantification limit, precision, and correctness (accuracy) (RSD 1.28%). The analytical range of the method was 0.349–200 ug/mL. According to the results of its approbation, the lactoferrin content in three commercially available preparations was lower than that declared by the manufacturers. The technique is applicable to control the lactoferrin content in the development of laboratory, pilot, and industrial technology for the production of this protein from whey, as well as to control the quality of the final product.

Keywords: lactoferrin; ultra-high performance liquid chromatography; quantification; validation.

Author Details: I. V. Spichak, D. Sc. (Pharm.), prof.; M. V. Tikhanina, research fellow; E. T. Zhilyakova, D. Sc. (Pharm.), head of department; N. V. Avtina, Cand. Sc. (Pharm.), assoc. prof.; V. E. Gulyaeva, post graduate student; D. A. Fadeeva, Cand. Sc. (Pharm.), assoc. prof.

For citation: Spichak IV, Tikhanina MV, Zhilyakova ET, et al. [Development of chromatographic control methods for identification and quantification of cow’s milk lactoferrin in lactoferrin substances] Dostizheniya nauki i tekhniki APK. 2021;35(8):57-62. Russian. doi: 10.53859/02352451_2021_35_8_57.