Determination of Uridine 5'-Monophosphate in Food Nutrition Fortifiers by High Performance Liquid Chromatography

As a nucleotide, Uridine 5'-monophosphate (UMP) is a pivotal intermediate in nucleic acid metabolism within living organisms. It is the most abundant nucleotide in mammalian milk, and the dietary requirement for uridylate is significantly higher than for other nucleotides during the growth and development of offspring. In recent years, research has revealed that UMP not only participates in energy metabolism and RNA synthesis but also enhances intestinal barrier function by modulating cellular signaling pathways. Furthermore, it acts synergistically with fat-soluble vitamins to promote nutrient absorption and immune function. UMP is commonly added as a nutritional fortifier to infant formula and livestock/poultry feed, where its purity and concentration directly impact the nutritional value and safety of the final product.

Keywords: Food nutrition fortifier, UMP, Nucleotides, High performance liquid chromatography.

1. Instruments and Reagents

1.1 Configuration List of HPLC

Table 1 Configuration List of HPLC

1.2 Reagents and Standards

Table 2 Reagents and Standards List

1.3 Experiment Material and Auxiliary Equipment

Ultrasonic Cleaner

Vortex Mixer

2. Experiment Method and Conditions

Chromatography Column: Nova Element AQC18, 5μm 4.6x250mm.

3. Experiment Result

3.1 System Suitability Test

The system suitability test demonstrated that the target peak was well-shaped, with a high theoretical plate count and no interfering peaks in its vicinity, thus meeting all experimental requirements.

Fig 1 Chromatogram of the Uridine 5'-Monophosphate Standard Working Solution

Table 3 Test Results for the Uridine 5'-Monophosphate Standard Working Solution

3.2 Repeatability Testing

The standard working solution of Uridine 5'-Monophosphate (UMP) was injected six times. The relative standard deviation (RSD) of retention time was 0.085%, and that of peak area was 0.191%. Both values were below the specified limit of 2.0%, demonstrating compliance with the experimental requirements.

Fig 2 Chromatograms of the Uridine 5'-Monophosphate (UMP) Repeatability Test

Table 4 Repeatability Testing Results for Uridine 5'-Monophosphate (UMP)

3.3 Sample Test

The peak area of the 5'-uridine monophosphate standard working solution (20mg/L) was 322.525, while that of the test sample solution was 274.211. Based on the external standard method, the UMP content in the sample was determined to be 17mg/L.

Fig 3 Chromatogram of the Test Sample

Table 5 Sample Test Results

3.4 System Suitability Test for Related Substances

The test of the system suitability solution for related substances demonstrated that all compound peaks were well-shaped, with high theoretical plate numbers and resolution greater than 2.0, thus meeting the experimental requirements.

Fig 4 Chromatogram of the 5'-uridine Monophosphate (UMP) Standard Working Solution

Table 6 Test Results for the 5'-uridine Monophosphate (UMP) Standard Working Solution

3.5 Sample Reproducibility Test

After six consecutive injections of the system suitability solution, the retention time repeatability for 5'-UMP, uracil, and uridine was 0.031%, 0.058%, and 0.102%, respectively, while the peak area repeatability was 0.243%, 0.134%, and 0.134%, respectively, meeting the experimental requirements.

Fig 5 Chromatogram of Sample Reproducibility Test

Table 7 Test Results for Retention Time Repeatability of Related Substances

Table 8 Test Results for Peak Area Repeatability of Related Substances

3.6 Sensitivity Test for Related Substances

The signal-to-noise ratio of the 5'-UMP main peak in the sensitivity test solution for related substances is significantly greater than 3, meeting the experimental requirements.

Fig 6 Chromatogram of the Related Substances Sensitivity Test Solution

Table 9 Test Results of the Related Substances Sensitivity Solution

3.7 Testing of Related Substances Reference and Sample Solutions

The peak area of the reference solution (17 mg/L) was 275.856. The total peak area of impurities in the test solution, excluding 5'-uridine monophosphate, was 38.021. Calculation by the external standard method, the impurity content in the sample is 2.343mg/L.

Fig 7 Chromatogram of the Related Substances Reference Solution Test

Table 10 Test Results of the Related Substances Reference Solution

Fig 8 Chromatogram of the Related Substances Sample Solution Test

Table 11 Test Results for Related Sample Solution

4. Conclusion

In this experiment, with reference to "GB 1903.69-2024 National Food Safety Standard: Food Nutrition Enhancer 5'-Uridine Monophosphate," the analysis was conducted using a Wayeal LC3200 high-performance liquid chromatography system equipped with an ultraviolet detector. The experimental results show that the system suitability test demonstrated good peak shapes for all compounds, high theoretical plate numbers, and resolution above 2.0, meeting the experimental requirements. The retention time repeatability for the 5'-Uridine Monophosphate standard working solution and related substances was below 0.2%, and the peak area repeatability was below 0.3%, indicating good reproducibility. The sensitivity test solution for related substances showed a signal-to-noise ratio for the 5'-Uridine Monophosphate main peak significantly greater than 3, fulfilling the experimental requirements. All sample test results were normal. The above data meet the instrument requirements specified in the standard method.