Nucleotide is the basic unit of genetic materials (DNA and RNA) and the precursor to synthesize nucleic acid. Because Infants are at the stage of rapid growth, their demand for nucleotide exceeds its production. However, breast milk can provide the required nucleotide for infants and young children. For those who were not breast fed, the use of formula with sufficient nucleotide can help them improve immunity and gastrointestinal conditions.
Table 1 Information of Analytes
Material and Reagents
Watsons water; HPLC grade of methanol;
HPLC grade of potassium dihydrogen phosphate; analytically pure of phosphoric acid and tetrabutylammonium hydrogen sulfate;
Cytosine nucleotide reference substance, adenine nucleotide reference substance, uracil nucleotide reference substance, hypoxanthine nucleotide reference substance and guanine nucleotide reference substance (provided by a dairy company);
Standard solution of 5 kinds of nucleotides mixed: weigh 10 mg standard CMP, AMP, UMP respectively and 5mg GMP, IMP ((+/- 0.1 mg)) respectively. Dissolve these standards to a certain volume of 100 mL with water, mix well.
Sample solution of infant formula milk powder: take 5.0 g milk powder sample, dissolve it with 20 mL heat water (50~60 °C) and cool the sample to ambient temperature. Adjust the pH to 4.1 with 10% acetic acid solution, transfer the mixture to a volumetric flask to the certain volume. Filter the solution through filter paper and 0.45μm membrane accordingly and the sample was ready for analysis.
HPLC Column: Venusil® C18 Plus, 5 μm, 120 Å, 4.6 × 250 mm;
Mobile phase*: transfer 1.360 g potassium dihydrogen phosphate and 0.4753 g tetrabutylammonium hydrogen sulfate into a 1000 mL volumetric flask and make up to the certain volume with water. Add 2.5 ml phosphoric acid and 40 mL methanol, mix well and sonicate for 10 min.
Detection: 254 nm;
Flow rate: 1.0 mL/min;
Column temperature: 30 °C;
Injection: 10 mL.
* Due to the existence of ion-pair reagent in mobile phase, the HPLC column need to be equilibrated with mobile phase for at least 90 min firstly and then run 4 syringe standards so as to stabilize the system.
Results and Discussion
Venusil® C18 Plus column was adopted to detect 5 kinds of nucleotides in infant formula milk powder in this application. The following result shows that the resolution of nucleotides and impurity in milk powder sample was over 1.5 and the recoveries were 97%~109%, so the result can meet the test demand.
Table 2 Performance of Venusil C18 plus in analysis of standard solution of 5 nucleotides mixed
Number of Theoretical Plates
Figure 1 Chromatogram of standard solution of 5 nucleotides mixed using Venusil® C18 Plus
Figure 2 Chromatogram of infant formula milk powder sample solution using Venusil® C18
Table 3 Performance of Venusil® C18 in analysis 5 kinds of nucleotides in infant formula milk
Figure 2, 3
Figure 3 Chromatogram of infant formula milk powder sample solution using Venusil® C18 (Magnification of vertical scale)
Table 4 Recoveries of spiked samples of 5 kinds of nucleotides in infant formula milk powder
Figure 4 Chromatogram of spiked sample in infant formula milk powder using Venusil® C18 Plus
A robust HPLC method to analysis 5 kinds of nucleotides in infant formula milk powder simultaneously was introduced in this paper. And Venusil® C18 Plus was adoped and excellent seperation were gained with high resolution to the impurities, together with good accuracy (% recoveries) in milk powder samples. The result shows that the Venusil® C18 Plus was suitable for the determination of nucleotides in infant formula milk powder, efficient and powerful.
Venusil® C18 Plus
5 μm, 120 Å, 4.6 × 250 mm
1.5 mL vials
Screw neck vials, 12 × 32 mm
Caps and Septa
Screw neck cap, center hole; red silicone/ white PTEE septa, slitted
Syringe Filter (Nylon)
Monofilm, 13 mm, 0.22 μm
Disposable Needle-Free injection systems
2 mL, 100/pk