Ion-pair high-performance liquid chromatography with diode array detection coupled to dual electrospray atmospheric pressure chemical ionization time-of-flight mass spectrometry for the determination of nucleotides in baby foods

A liquid chromatography with diode array detection coupled to dual electrospray atmospheric pressure chemical ionization time-of-flight mass spectrometry (HPLC/ESI-APCI-TOF-MS) method is described for the rapid determination of five monophosphate nucleotides (cytidine 5′-monophosphate, uridine 5′-monophosphate, adenosine 5′-monophosphate, inosine 5′-monophosphate and guanosine 5′-monophosphate) in baby foods. The method is based on the deproteinisation of foods and direct analysis of nucleotides by ion-pair HPLC using isocratic elution with a mobile phase of 5% (v/v) methanol and 95% (v/v) 0.1 M formate buffer (pH 5.5) containing 0.01 M N,N-dimethylhexylamine (DMHA) at a flow-rate of 0.7 mL min⁻¹. The HPLC was hyphenated with two different detection systems, photodiode-array (DAD) and ESI-APCI-TOF-MS in negative mode. The method was validated for linearity, detection and quantitation limits, selectivity, accuracy and precision. The recoveries obtained for spiked samples were satisfactory for all the analytes. The method was successfully applied to the analysis of nucleotides in different baby and/or functional food samples, as cereals, purees and dairy products. A study was also carried out on the stability of nucleotides in acidified dairy infant food with pasteurized yoghourt and follow-on formulae samples stored at room temperature and at 30 °C.