Determination of Seven Parabens in Food by Liquid Chromatography

Parabens (p-hydroxybenzoates), as a class of highly effective and broad-spectrum preservatives, are widely used in the fields of food (such as soy sauce, vinegar, beverages, jam, etc.), cosmetics, and pharmaceuticals. Their antibacterial efficacy and safety directly impact product stability and consumer health. This experiment refers to the "GB 5009.31-2025 National Food Safety Standard — Determination of Parabens in Food", by using Wayeal LC3500 series high-performance liquid chromatography syste, equipped with a DAD detector for analysis.

Keywords: food additives; parabens; p-hydroxybenzoates; high-performance liquid chromatography.

1. Instruments and Reagents

1.1 Configuration list of High Performance Liquid Chromatography

Table 1 Table List of Instrument Configuration

1.2 Reagents and Standards

Table 2 Table List of Reagents and Standards

1.3 Experiment Material and Auxiliary Equipment

Analytical Balance

Ultrasonic Cleaner

Vortex Mixer

2. Experiment Method

2.1 Reagents Preparation

2.1.1 Mixed standard working solution of paraben compounds: Appropriately pipette the mixed standard of paraben compounds and dilute it with 30% methanol-water to prepare mixed standard working solutions with mass concentrations of 0.2mg/L, 0.5mg/L, 1.0mg/L, 2.0mg/L, 5.0mg/L, 10mg/L, 20mg/L, and 50mg/L, respectively.

2.1.2 Sample Pretreatment:

Weigh 5g (accurate to 0.01g) of the sample into a 50mL graduated centrifuge tube (carbonated beverages require ultrasonic degassing in an ultrasonic cleaner for 10 minutes before weighing). Add 30mL of methanol-water solution (3+7), vortex for 3 minutes, and sonicate for 20 minutes. Then, add methanol-water solution (3+7) to bring the total volume to 40mL. Centrifuge at 6000r/min for 3 minutes, filter the supernatant, and take the filtrate for purification. Activate the solid-phase extraction (SPE) column sequentially with 5mL of methanol and 5mL of water. Transfer the solution to be purified into the activated SPE column. Rinse the column sequentially with 5mL of water and 5mL of methanol-water solution (3+7), then elute with 6mL of methanol. Collect the eluate, dilute to 10mL with water, filter through a membrane, and inject into the high-performance liquid chromatograph for analysis.

3. Experiment Result

3.1 System Suitability Testing

Fig 1 Chromatogram of Standard Working Solution for Paraben Compounds

Table 3 Test Result of Paraben Compound Standard Working Solution

The system suitability test demonstrates that each chromatographic peak exhibits good shape, high theoretical plate numbers, no interference from surrounding impurity peaks, and resolution values all greater than 1.5, meeting the experimental requirements.

3.2 Repeatability Test

Fig 2 Repeatability Test Chromatogram of 1.0mg/L Paraben Compound (6 injections)

Table 4 Repeatability Test Result of 1.0mg/L Paraben Compound (6 injections)

The repeatability test showed that after six consecutive injections of the 1.0mg/L paraben compound standard working solution, the retention time repeatability was below 0.2%, and the peak area repeatability was below 1.0%, indicating good repeatability.

3.3 Linear Test

Fig 3 Chromatogram of Standard Curve Test

The linearity test demonstrates that within the range of 0.2–50mg/L, the mixed standard curves for the seven paraben compounds all exhibit linear correlation coefficients greater than 0.9999, indicating excellent linearity.

3.4 Accuracy Test

Fig 4 Accuracy Test Chromatogram

Table 5 Accuracy Test Results of Paraben Compounds

The accuracy test demonstrated that the recoveries of the seven paraben compounds in the spiked samples ranged from 97.6% to 104.8%, indicating good accuracy.

3.5 A Brand Beverage Test

Fig 5 Chromatogram of A Brand Beverage Test

Sample testing indicated that the seven paraben compounds were not detected in the beverage of a certain brand.

4. Conclusion

This experiment refers to the "GB 5009.31-2025 National Food Safety Standard — Determination of Parabens in Food", by using Wayeal LC3500 series high-performance liquid chromatography syste, equipped with a DAD detector for analysis. The experimental results demonstrate that in the system suitability test, each compound peak exhibits good shape and high theoretical plate numbers, meeting the experimental requirements. Six consecutive injections of the 1.0 mg/L paraben standard working solution showed retention time repeatability below 0.2% and peak area repeatability below 1.0%, indicating good repeatability. Within the range of 0.2–50 mg/L, the linear correlation coefficients all exceed 0.9999, indicating good linearity. The recoveries of the seven paraben compounds in the spiked samples ranged from 97.6% to 104.8%, indicating good accuracy. The recoveries of the seven paraben compounds in the spiked samples ranged from 97.6% to 104.8%, demonstrating good accuracy. The seven paraben compounds were not detected in the sample tests. All the above data meet the requirements for the instrument as specified in the standard method.