Development and Application of an HILIC-MS/MS Method for the Quantitation of Nucleotides in Infant Formula

A method for the quantitation of nucleotides (adenosine 5'-monophosphate (AMP), guanosine 5'-monophosphate (GMP), uridine 5'-monophosphate (UMP), cytidine 5'-monophosphate (CMP), and inosine 5'-monophosphate (IMP)) in infant formula was developed by hydrophilic interaction chromatography with tandem mass spectrometry (HILIC-MS/MS). The internal standards used (AMP-C-13(10), N-15(5); GMP-C-13(10), N-15(5); UMP-C-13(9), N-15(2); CMP-C-13(9), N-15(3)) were prepared with centrifugal ultrafiltration (CUF). Data acquisition was achieved by using multiple reaction monitoring (MRM) of product ions of protonated molecules of the five nucleotides generated by the positive-ion ESI. HILIC conditions were performed with 30 mmol/L ammonium formate in water (pH 2.5, adjusted with formic acid) and methanol. The LOD and LOQ were 5-10 mu g/mL and 10-30 mu g/mL for standard solution, respectively. Recovery for intra- and interday assays ranged from 98.1 to 108.9% (RSD: 0.7-5.4%) spiked with three concentration levels (5, 25, and 250 mu g/g powder infant formula). This method could be applied for the determination of nucleotides in infant formula samples. The detected concentrations of five nucleotides ranged from not detected (n.d.) to 278 mu g/g powder infant formula. The total nucleotide level ranged from n.d. to 600.2 mu g/g powder infant formula.