Abstract: A method for the determination of the contents and composition of total nucleotides in breast milk and cow milk by high performance liquid chromatography (HPLC) was established. The total nucleotides detected included free nucleotides, free nucleosides, nucleoside polymers and nucleoside adducts. The nucleosides in breast milk were released by enzymatic hydrolysis, purified by polyacrylamide gel (Affi-Gel 601) as a solid phase extraction medium, detected by reverse-phase-high performance liquid chromatography (RP-HPLC) and quantified by an internal standard method. The results were calculated as nucleotides. According to the characteristics of breast milk, a high-temperature inactivation step was introduced into sample pretreatment before the detection of nucleotides in breast milk. The proposed method was systematically validated. The results showed that this method had good linearity with a correlation coefficient (r2) higher than 0.999. The limit of detection (LOD) and the limit of quantification (LOQ) for all nucleotides in breast milk were 0.18–0.45 and 0.60–1.51 mg/L, respectively. The recoveries of total nucleotides in breast milk samples spiked at three concentration levels ranged from 99% to 108%, with relative standard deviations (RSDs) 0.8%–7.8% for six replicate determinations in three consecutive days, suggesting good accuracy and precision. The HPLC method was used to evaluate the composition and contents of total nucleotides in breast milk and five types of commercial cow milk in order to verify its applicability. The sensitivity, accuracy, and precision of this method can meet the requirements for the determination of natural nucleotides in breast milk and cow milk. This study should provide a more scientific and accurate method for the determination of nucleotides in massive samples of breast milk and cow milk in the future.

Key words: total nucleotides; content and composition; high performance liquid chromatography; breast milk; cow milk