Analysis of Advantame in Food by High Performance Liquid Chromatography

Analysis of Advantame in Food Additives

This experiment was conducted with reference to "GB 1886.377-2024 National Food Safety Standard - Food Additive Advantame", by using Wayeal's LC3200 Series High-Performance Liquid Chromatography (HPLC) system equipped with UV detector for analysis. The experimental results demonstrated excellent system suitability, with well peak shape for both benzoic acid and advantame. There are on other peaks around the target peaks, and the high theoretical plate numbers confirmed compliance with all test requirements. The linear correlation coefficient exceeded 0.999. The experiment showed excellent repeatability, with retention time relative standard deviations (RSD) of 0.027% for benzoic acid and 0.049% for advantame, and peak area RSDs of 0.184% and 0.133%, respectively. The theoretical detection limits are 0.066mg/L for benzoic acid and 0.113mg/L for advantame. No target analytes were detected in the sample testing. All analytical data met the instrumental performance requirements specified in the pharmacopoeial methodology.

Keywords: Advantame, high performance liquid chromatography, UV detector.

1. Experiment Method

1.1 Instrument Configuration

Table 1 Configuration List of HPLC System

1.2 Experiment Materials and Auxiliary Equipment

Acetonitrile: Chromatographic grade

Benzoic acid standard

Advantame standard

1.3 Test Conditions

Table 2 HPLC Analysis Conditions

1.4 Solution Preparation

1.4.1 Internal Standard Solution

Accurately weigh 40mg of benzoic acid, dissolve in a water-acetonitrile mixed solvent, and precisely dilute to 50mL.

1.4.2 Standard Solution

Accurately weigh 40mg of advantame, dissolve in a water-acetonitrile mixed solvent, and dilute precisely to 50mL to prepare a 0.8mg/mL standard stock solution. Precisely pipette 8mL, 9mL, 10mL, 11mL, and 12mL of the standard stock solution into five separate volumetric flasks. Add 5mL of internal standard solution to each flask, then dilute to 50mL with water-acetonitrile mixed solvent to prepare standard working solutions at concentrations of 0.128mg/mL, 0.144mg/mL, 0.160mg/mL, 0.176mg/mL, and 0.192mg/mL for calibration curve construction.

1.4.3 Sample Preparation

Accurately weigh 40mg of the test sample, dissolve in a water-acetonitrile mixed solvent, and precisely dilute to 50mL. Pipette 10mL of the prepared solution into a volumetric flask, add 5mL of the internal standard solution, and dilute to 50mL with a water-acetonitrile mixed solvent. Filter the solution through a membrane filter for testing.

2. Results and Discussion

2.1 System Suitability

Figure 1 System Suitability Test Chromatogram

Table 3 System Suitability Test Data

Note: As the above table showed, the system suitability test showed excellent peak shapes for both benzoic acid and advantame, with tailing factors below 1.2. The resolution exceeded 1.5, and the high theoretical plate numbers fully met the analytical method requirements.

2.2 Calibration Curve Test

Fig 2 Test Report of Advantame Calibration Curve

Noted: The experimental results demonstrated excellent linearity for advantame quantification, with a correlation coefficient (R²) exceeding 0.999, which satisfying experiment requirements.

2.3 Repeatability Testing

Fig 3 Six-Injection Repeatability Test Chromatogram

Table 4 Benzoic Acid Repeatability Test Data (6 Injections)

Table 5 Advantame Repeatability Test Data (6 Injections)

Note: From the data in the above table, the retention time repeatability of benzoic acid and advantame is 0.027% and 0.049%, and the peak area repeatability is 0.184% and 0.133%, respectively, which shows good repeatability.

2.4 Detection Limit Testing

Fig. 4 Chromatogram of the 0.8mg/L Benzoic Acid and 1.28mg/L Advantame Standard Solution

Table 6 Test Results for the 0.8mg/L Benzoic Acid and 1.28mg/L Advantame Standard Solution containing

Note: According to the standard sample test data in the table above, with a benzoic acid concentration of 0.8mg/L and an advantame concentration of 1.28mg/L, the theoretical detection limits for benzoic acid and advantame, calculated based on a 3-fold signal-to-noise ratio, are 0.066mg/L and 0.113mg/L, respectively.

2.5 A Brand Beverage Testing

Fig 5 Chromatogram of A Brand Beverage

Note: No advantame was detected in the tested branded beverage.

3. Conclusion

This experiment was conducted in accordance with the “GB 1886.377-2024 National Food Safety Standard - Food Additive Advantame" by using Wayeal LC3200 high-performance liquid chromatography system, equipped with a UV detector for analysis. The experimental results demonstrated excellent system suitability, with well peak shape for both benzoic acid and advantame. There are on other peaks around the target peaks, and the high theoretical plate numbers confirmed compliance with all test requirements. The linear correlation coefficient exceeded 0.999. The experiment showed excellent repeatability, with retention time relative standard deviations (RSD) of 0.027% for benzoic acid and 0.049% for advantame, and peak area RSDs of 0.184% and 0.133%, respectively. The theoretical detection limits are 0.066mg/L for benzoic acid and 0.113mg/L for advantame. No target analytes were detected in the sample testing. All analytical data met the instrumental performance requirements specified in the pharmacopoeial methodology.