Abstract: A method for the determination of five free nucleotides in infant formula was established. The analytes were extracted by ethylene diamine tetraacetic acid and sodium chloride solution, and purified by strong anion exchange (SAX) solid-phase extraction. An Atlantis T₃ reverse-phase column was applied to separate the five free nucleotides. A high performance liquid chromatography (HPLC) system equipped with a diode array detector was applied to detect the target compound, and a series of external standards were used for quantification. Good linearities (r≥0.999) were observed, with the range of 0.5-10 mg/L for the five free nucleotides. When the addition levels were 0.05-0.50 g/kg, the recoveries were between 91.1% and 112%, and the precisions were between 2.3%-4.7%. The limits of detection (LODs) and the limits of quantification (LOQs) for the five free nucleotides ranged from 0.0010 to 0.0015 g/kg and from 0.0030 to 0.0045 g/kg. In conclusion, the proposed method has several merits. First, the high accuracy and precision indicated that the quality control sample had a relative deviation of less than 10% to the median. Second, perfect anti-disturbance was observed for the complex goat milk powder. Thus, this method can provide technical support for supervision departments and guarantees healthy development of the dairy industry.

Key words: high performance liquid chromatography (HPLC), solid phase extraction (SPE), nucleotides, infant formula milk powder