Development of an ultra high performance liquid chromatography method for the separation and determination of nucleotides and nucleosides in extracts from infant milk formulas and human milk samples

Abstract

Chromatographic methods developed to date allow the separation of nucleosides and nucleotides mixtures, but the resolution is not always complete, and analysis times are usually long. Therefore, this study aimed to develop a method for separating and determining these two groups of compounds in a short time and with good selectivity using the reversed-phase ultra-high performance liquid chromatography. The type of stationary phase and pH of the mobile phase had the most significant impact on the separation. The developed method allowed to separate and quantify a mixture of eleven compounds in 14 min. It was successfully applied to the analysis of milk extracts and appeared to be accurate, simple, and rapid. The LOQ values were in the 0.05–0.16 μg/ml range with high linearity (R2 higher than 0.998). The inter-day precision did not exceed 3.3 %, while Intra-day was in the range of 2.3–4.6 %. The procedure for extracting nucleosides and nucleotides from milk samples using protein precipitation and liquid-liquid extraction was reproducible, easy, and inexpensive, with analyte recovery between 69 % and 73 %.