Determination By HPLC And Stability Of Nucleotides In Infant Formula

Nucleotides are primary units of nucleic acids and play an important role in human metabolism. Nucleotides have almost participated in every reaction of cell metabolism processes. The nucleotides synthesis may be not able to meet the requirement to these important organizational growths when infant grow rapidly. Adding nucleotides to infant formula powder have become a kind of trend, but the nucleotides detection method of national standard was not established in the current. Therefore, this article put forward a method which was used the RP-HPLC to determinate five free nucleotides and to study the nucleotides stability in infant milk powder. The main results were as follows.1. Determination of the 5 nucleotides in infant milk powder using RP-HPLCThe chromatographic conditions of five kinds of target nucleotides standard were optimized, including the selection of chromatographic column, the optimization of the mobile phase type and proportion, the p H, the type of ion pair reagent on the separation effect. The final separate conditions can separate the target nucleotides from the infant formula samples that were confirmed. The separation was performed on a Welch NCOT-C18(300×4.6mm, 5μm)column, wavelength was set at 254 nm, the mobile phase consisting of Phosphate(with tetrabutylammonium hydroxide solution, p H=3.3) and 5% methanol.The aims are sample extraction the total contents of five nucleotides and the degree of separation, According to the characteristics of infant formula, determinate the best pretreatment method in which Ultrasonic extraction time 10 min and 13% perchloric acid used for elimination of protein. Under the condition of the determination, it can be a very good separation between five kinds of nucleotides and impurity peak in sample.2. Method validationThe method feasibility through precision, linearity, limit of detection, limit of quantification and uncertainty studies to examine. The results showed that the method average recoveries were between 96% and 105%, the precision were below 0.95% and 3.33%, the five nucleotides concentration and their target peak area showed good linearity R2 ≥0.999 when their concentrations are in the range of 1.25-10.0μg/m L. The detection limits of cytidine,uridine, adenosine, guanosine and inosine 5-monophosphates were 0.06mg/100 g, 0.01mg/100 g,0.04mg/100 g, 0.02mg/100 g, and 0.01mg/100 g, respectively. Limits of quantification were 0.19mg/100 g, 0.04mg/100 g, 0.12mg/100 g, 0.07mg/100 g, and 0.05mg/100 g, respectively.The standard uncertainty was 5.55% and the expanded uncertainty was 11.1%. The method had good verification indicators.3. Nucleotides stability in shelf lifeIn order to research the influence on five nucleotides stability of infant formula powder in different storage time, this study detected the infant formula powder of between 0 and 6months, between 6 and 12 months. The results showed: after the samples at room temperature and dark conditions for 6 months and 12 months, the samples showed without any adverse reaction. The nucleotides of products had good quality stability and no obvious contents changes in shelf life.