Determination of Multiple Phosphates in Food by Wayeal Ion Chromatograph

This application note introduces the determination of multiple phosphates in foods according to Method 2 of GB 5009.256-2025 using Wayeal's ion chromatography system.

Excessive intake of phosphates may disrupt the body's calcium-phosphorus metabolism, leading to impaired mineral absorption or other health problems. Accurate detection helps enterprises scientifically control additive usage, balance food quality and safety, and provides data support for optimizing formulations.

Keywords: Ion chromatography, anions, food.

1. Instrument and Regents

1.1 Ion Chromatograph Configuration List

Table 1 Instrument Configuration List

1.2 Reagents and Standards

Table 2 List of Reagents and Standards

1.3 Experiment Material and Auxiliary Equipment

Canned syringe-type hydrophilic filter (0.45μm)

Injector (20mL)

2. Experiment Method

2.1 Sample Pretreatment

Weigh 1-2g (accurate to 0.001g) of the sample into a 50mL colorimetric tube. Add 22.5mL of 100mmol/L sodium hydroxide solution. Vortex mix for 1 minute, then ultrasonically extract at 80°C for 30 minutes, shaking every 5 minutes to ensure complete dispersion of the sample. After cooling to room temperature, dilute to the 50mL with ultrapure water and mix thoroughly. Transfer the entire solution to a 50mL centrifuge tube and centrifuge at 8,000 r/min for 5 minutes. Measure 5mL of the supernatant into another 50mL colorimetric tube, add 1mL of 30% nitric acid, and vortex to mix. Place the tube in a water bath at 90 °C ± 5 °C and heat for 60 minutes. After heating, remove the tube and cool it to room temperature in a cold-water bath. Finally, dilute to the mark with water and mix well.

Pipette 2mL of the solution into a 10mL centrifuge tube, then dilute to the mark with ultrapure water. Place the tube in a refrigerated centrifuge and centrifuge at 4 °C and 8,000 r/min for 5 minutes. Collect the supernatant and pass it through a 0.45μm filter membrane. Then, take an appropriate volume of the filtrate for instrumental analysis.

Note: In the original standard method, 45mL of 50mmol/L sodium hydroxide solution is added separately. For this test, to facilitate volumetric adjustment due to the larger spiked amount, the extraction solution was modified to 22.5mL of 100mmol/L sodium hydroxide solution added separately. This modification does not affect the validity of the experimental results.

2.2 Experiment Conditions

Table 3 Anions Test Conditions

3. Experiment Result

3.1 Standard Chromatography

The determination of multiple phosphates in food according to Method 2 of GB 5009.256-2025 was completed within 30 minutes. The test results demonstrated good linearity and linear repeatability, excellent limits of detection and quantification, satisfactory precision, strong sample repeatability, reliable sample parallelism, consistent spiked-sample repeatability, and excellent recovery rates. All performance indicators meet the requirements specified in GB 5009.256-2025 for the determination of multiple phosphates in food.

Overlap Chromatogram of Standard Curve

3.2 Linear Range

Take an appropriate amount of the standard solution and dilute to prepare the calibration curve. The deviation between the linear detection results and the known concentrations was less than the maximum allowable deviation, with an R-value greater than 0.999, indicating excellent linearity for each component.

Table 4 Table of Linear Range for Phosphate Ion

Linearity Results for Phosphate Ion

3.3 Linearity Repeatability Test

Repeatability Test Chromatograms for Standard Curve Low-Value Point S1 with 7 Consecutive Injections

Repeatability Test Chromatograms for Standard Curve Medium-Value Point S4 with 7 Consecutive Injections

Repeatability Test Chromatograms for Standard Curve High-Value Point S7 with 7 Consecutive Injections

Repeatability Test Data for Calibration Curve

3.4 LOD Test

LOD Test Chromatogram

LOD Test Data

3.5 Precision Test

Overlap Chromatograms of Two Independent Tests

Precision Test Data

3.6 Blank Sample Test Chromatogram

Blank Sample Test Chromatogram

Blank Sample Test Data

3.7 Sample Parallelism and Repeatability Test Chromatograms

Overlay Chromatograms of Frozen Shrimp Parallel Sample 1 (8 Injections)

Overlap Chromatograms of Frozen Shrimp Parallel Sample 2 (8 Injections)

Frozen Shrimp Test Data

3.8 Sample Spiking and Spike Repeatability Test

Overlap Chromatogram of 50% Spiked Frozen Shrimp (8 Injections)

Repeatability Test Data for 50% Spike Level Frozen Shrimp

Overlap Chromatograms of Frozen Shrimp with 100% Spike Level (8 Injections)

Repeatability Test Data for Frozen Shrimp with 100% Spike Level

Overlap Chromatograms of Frozen Shrimp with 150% Spike Level (8 Injections)

Reproducibility Test Data for Frozen Shrimp with 150% Spike Level

Spiking Test Data for Frozen Shrimp

Based on the data provided, the chromatographic peaks demonstrate good shape, and the linear correlation coefficients are all greater than 0.999. The determination of phosphates in food was carried out using NovaChrom’s hydrogen-hydroxide chromatographic column (HS-9A-PP, 4 × 250mm, 9μm), with reference to the second method specified in GB5009.256-2025 for the determination of multiple phosphates in food. The experimental results demonstrate that the second method exhibits excellent linearity and linear reproducibility. It also shows outstanding detection limit (0.003g/kg), quantification limit (0.009g/kg), and precision. The method demonstrates good repeatability, parallelism for samples, and excellent spike recovery rates. Both the detection limit and quantification limit are well below the standard requirements of 0.05g/kg and 0.1g/kg, respectively. The RSD of the peak areas for sample repeatability ranged from 0.515% to 0.934%. The absolute difference between the two independent determinations obtained under repeatability conditions for the sample was 0.37% of the arithmetic mean. Under precision testing, the absolute difference between the two independent determinations obtained under repeatability conditions was 0.06% of the arithmetic mean, which does not exceed the standard requirement of 15%. The recovery rates ranged from 92.57% to 102.15%. This method complies with the requirements of GB5009.256-2025 for the determination of multiple phosphates in food.

4. Conclusion

This analysis was conducted using Wayeal ion chromatograph to determine multiple phosphates in food according to Method 2 of GB5009.256-2025. The method enables accurate quantification of phosphate content in food, verifying its compliance with national standards (such as GB 5009.256-2025). This helps prevent enterprises from exceeding permissible additive limits and supports the integrity of food safety regulatory systems.