Determination of Phoxim Content in Rice by the Wayeal LCMS-TQ9200 Liquid Chromatography-Tandem Mass Spectrometry System

Phoxim, with the chemical formula C₁₂H₁₅N₂O₃PS, is a synthetic organophosphorus insecticide. It achieves its insecticidal effect by inhibiting the activity of cholinesterase in pests, disrupting the normal function of their nervous system, and preventing their regular physiological activities. Compared with other insecticides such as pyrethroids and carbamates, phoxim is characterized by a broad insecticidal spectrum, outstanding control efficacy against lepidopteran larvae, moderate pricing, and ease of application, making it widely favored by agricultural producers. In practical application, phoxim is prone to overuse or use beyond the recommended scope, leading to the widespread problem of pesticide residues in agricultural products. Pesticide residues can lead to numerous adverse effects. On one hand, they accumulate in the human body through the food chain, potentially causing acute poisoning, chronic damage, and other abnormal physiological reactions. On the other hand, they can enter the environment through farmland drainage, increasing the concentration of pesticide components in the environment and thereby indirectly impacting both the environment and human health. In particular, phoxim poses potential risks to sensitive populations such as children and pregnant women. If these individuals ingest excessive amounts of phoxiph or accumulate high levels of it in their bodies, it may lead to impaired organ function and adverse health effects. Therefore, the detection of phoxim pesticide residues in agricultural products has become an essential and critical component of food safety control.

This experiment was conducted in accordance with the national standard GB/T 20770-2008, "Determination of 486 Pesticides and Related Chemical Residues in Grains by Liquid Chromatography-Tandem Mass Spectrometry." The analysis of phoxim content in rice was performed using the Wayeal LCMS-TQ9200 liquid chromatography-tandem mass spectrometry system. This protocol is equipped with the Wayeal liquid chromatography-tandem mass spectrometry system, which meets the requirements for routine qualitative and quantitative detection of the test samples.

Keywords: Triple quadrupole mass spectrometry; Phoxim; Pesticide residues.

1. Instrument and Reagents

1.1 Instrument Configuration List

Table 1 Instrument Configuration List

1.2 Reagents and Standards

Table 2 Reagents and Standards

1.3 Experiment Material and Auxiliary Equipment

Vortex mixer;

High-speed centrifuge;

Analytical balance.

2. Experiment Method

2.1 Solution Preparation

2.1.1 Acetonitrile-Acetic Acid (99+1, v/v): Measure 10mL of acetic acid and add it to 990mL of acetonitrile, then mix thoroughly.

2.1.2 Ammonium Formate-Formic Acid Aqueous Solution (2mmol/L): Weigh 0.1261g of ammonium formate, dissolve it in and dilute to 1000mL with 0.01% formic acid aqueous solution, then shake well until homogeneous.

2.1.3 Ammonium Formate-Formic Acid in Methanol Solution (2 mmol/L): Weigh 0.1261g of ammonium formate, dissolve it in and dilute to 1000 mL with 0.01% formic acid in methanol solution, then shake well until homogeneous.

2.2 Sample Pretreatment

Weigh 5g of the test sample (accurate to ±0.01g) into a 50mL centrifuge tube. Add 10mL of water, vortex to mix thoroughly, and let stand for 30 min. Add 15mL of acetonitrile-acetic acid solution and one ceramic homogenizer, then shake vigorously for 1min. Subsequently, add 6g of anhydrous magnesium sulfate and 1.5g of sodium acetate, shake vigorously for another 1min, and then centrifuge at 4200 r/min for 5min. Quantitatively transfer the supernatant into a plastic centrifuge tube containing a dehydrating agent and purification materials (using 150mg of anhydrous magnesium sulfate, 50mg of C18, and 50mg of PSA per milliliter of extract). Centrifuge at 4200 r/min for 5min, then draw the supernatant and pass it through a microporous membrane filter for subsequent determination.

2.3 Experiment Conditions

2.3.1 Liquid Chromatography Conditions

Chromatography columns: C18 1.7 μm 2.1x50 mm；

Mobile phase: Phase A: Ammonium formate-formic acid in methanol solution (2mmol/L); Phase B: Ammonium formate-formic acid aqueous solution (2mmol/L).

Flow rate: 0.3mL/min;

Column temperature: 40 °C;

Injection volume: 5µL.

2.3.2 Mass Spectrometry Conditions

Table 3 Compound Mass Spectrometry Parameters

Note: The ion marked with * is the quantitative ion.

3. Experiment Result

3.1 Standards Chromatogram

The determination of phoxim was completed within 6.5 minutes. As shown in Figure 1, the compound peak exhibits good peak shape and satisfactory response, meeting the requirements of the experimental analysis.

Fig 1 Chromatogram of Phoxim

3.2 Linear Range

Take appropriate volumes of the phoxim standard solution and serially diluted with blank matrix to obtain concentrations of 10, 5, 2, 1, 0.5, 0.1, and 0.05ng/mL for the preparation of the calibration curve. The linear range was established from 0.05 to 10ng/mL. The deviation of the linear detection results from the known concentrations was within the maximum permissible deviation. The correlation coefficient (R²) was 0.99971, indicating an excellent linear relationship for the analyte.

Fig 2 Phoxim Standard Curve

3.3 Repeatability

Phoxim solutions at three concentrations (0.5, 2, and 10ng/mL) were injected six times consecutively. The results, as shown in the table below, indicate that the relative standard deviations (RSDs) for all data points at high, medium, and low concentrations of phoxim are within 5%, meeting the experimental requirements.

Table 4 Repeatability Test for Phoxim at High, Medium, and Low Concentrations

3.4 Spike Recovery

The phoxim standard solution was added to the sample to achieve a concentration of 4ng/mL, and the sample was then analyzed by LC-MS. The results are shown in Figure 3. The average value from six consecutive injections was 4.194ng/mL, with an RSD of 4.623% and a recovery rate of 104.85%, meeting the experimental requirements.

Fig 3 Chromatogram of Phoxim Spike Recovery

3.5 Blank Residual

After continuously injecting the 10ng/mL standard solution, a blank matrix sample was subsequently injected to assess and calculate any blank residual. The result are shown in Figure 4, indicates that no blank residual was detected.

Fig 4 Blank Chromatogram

3.6 Sample Test

Sample A was processed according to the pretreatment method described in this protocol, and the measured content of phoxim in Sample A was 9.552μg/kg.

Fig 5 Chromatogram of Phoxim in Sample A

4. Conclusion

This method utilizes the Wayeal LCMS-TQ9200 liquid chromatography-tandem mass spectrometry system for the determination of phoxim residues in plant-derived foods. 

The data demonstrate that this method produces good mass spectrometry peaks and no tailing. The sensitivity meets the experimental requirements, with an R² greater than 0.999. The repeatability at high, medium, and low concentrations is within 5%, and the spike recovery rate is 104.85%. No system carryover was observed after high-concentration samples. These results confirm that this method, equipped with the Wayeal liquid chromatography-tandem mass spectrometry system, satisfies the requirements for routine qualitative and quantitative analysis of the target analytes in test samples.