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Detection of Nucleotides in infant formula milk powder

Application Introduction

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.

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Introduction

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

Abbreviation

Structure

Formula

Molecular Weigh

CAS

CMP

C9H14N3O8P

323.2

63-37-6

IMP

C10H13N4O8P

348.21

131-99-7

AMP

C10H14N5O7P

347.22

61-19-8

UMP

C9H13N2O9P

324.18

58-97-9

GMP

C10H14N5O8P

363.22

85-32-5

Experimental

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);

Sample Preparation

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.

Instrumentation

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

Analytes

Retention   Time

Number   of Theoretical Plates

Tailing   Factor

Resolution

Notes

CMP

3.660

15803

0.98

Figure 1

AMP

5.608

16882

0.97

13.46

GMP

11.061

18806

0.96

21.99

UMP

21.226

21178

0.98

22.40

IMP

26.011

20584

0.99

7.30

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

powder

Analytes

Retention/min

Resolution

Tailing Factor

Notes

CMP

3.659

0.99

Figure   2, 3

Impurity   1

4.342

5.73

-

AMP

5.605

8.48

0.94

Impurity   2

6.081

2.74

-

GMP

11.051

18.91

0.97

Impurity   3

19.841

20.47

-

UMP

21.223

2.57

0.95

IMP

26.015

7.30

0.99

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

Analytes

Retention/min

Spiked Concentrationug/mL

Recoveries%

Notes

CMP

3.660

12.2

108.4

Figure   4

AMP

5.605

7.6

101.8

GMP

11.050

9.2

97.7

UMP

21.213

14.8

106.3

IMP

26.028

11

101.9

Figure 4 Chromatogram of spiked sample in infant formula milk powder using Venusil® C18 Plus

Conclusion

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.

Ordering Information

Products

Specification

Cat.No

Venusil®   C18 Plus

5 μm, 120 Å, 4.6   × 250 mm

VPS952505-A

1.5 mL vials

Screw neck vials,   12 × 32 mm

AV1001-6

Caps and Septa

Screw neck cap,   center hole; red silicone/ white PTEE septa, slitted

AV2200-0

Syringe   Filter (Nylon)

Monofilm,   13 mm, 0.22 μm

AS021320

Disposable Needle-Free   injection systems

2 mL, 100/pk

LZSQ-2ML


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