Determination of Glyphosate and Four Pesticides and Their Metabolite Residues in Apples by Wayeal LCMS-TQ9200 Liquid Chromatography-Tandem Mass Spectrometry System

"Glyphosate, glufosinate, ethephon, and four other pesticides along with their metabolites, are widely used in crop cultivation due to their high efficiency and broad-spectrum herbicidal and ripening effects, making them important agricultural inputs for ensuring crop yield and improving farming efficiency."

"China has established maximum residue limits (MRLs) for glyphosate and other pesticides in plant-derived foods. Therefore, developing a sensitive, accurate, and efficient detection method is of great practical significance for ensuring the quality and safety of agricultural products, regulating agricultural production practices, and protecting consumer health."

"Currently, detection methods for glyphosate and three other pesticides along with their metabolites in plant-derived foods mainly include high-performance liquid chromatography (HPLC), gas chromatography (GC), and ion chromatography (IC)."

"Among these methods, HPLC is susceptible to matrix adsorption; GC requires derivatization of polar analytes such as glyphosate; and IC suffers from issues such as low separation efficiency and poor anti-interference capability. These limitations restrict their widespread application in residue detection across different types of plant-derived foods, including cereals, vegetables, and tea."

"GB 23200.122-2026 establishes a detection method for residues of glyphosate, three other pesticides, and their metabolites based on LC-MS/MS. By optimizing sample pretreatment conditions, chromatographic separation parameters, and mass spectrometric detection modes, this method aims to address the issues commonly encountered in traditional methods, such as high interference, low sensitivity, complex operation, and poor matrix adaptability. It enables rapid and accurate determination of residues of glyphosate and the four pesticides and their metabolites in plant-derived foods, thereby providing reliable technical support and standard references for quality control in agricultural production enterprises, routine testing in inspection and testing institutions, and supervisory inspection by regulatory authorities."

Keywords: Triple quadrupole LC-MS/MS system, Apple, Glyphosate.

1. Instrument and Reagents

1.1 Configuration List of LCMS/MS

Table 1 Instrument Configuration List

1.2 Reagents and Standards

Table 2 List of Reagents and Standards

1.3 Experiment Material and Auxiliary Equipment

Vortex mixer

One-hundred-thousandth balance

Centrifuge

NovaChrom NovaPre HLB Solid Phase Extraction Cartridge, 200mg/6mL

2. Experiment Method

2.1 Solution Preparation

2.1.1 Standard working solutions of glyphosate and four other pesticides and their metabolites: Accurately dilute the standard solution of glyphosate and four other pesticides and their metabolites to 1mg/L, then dilute with blank apple matrix to prepare a series of working curve concentrations of 5μg/L, 10μg/L, 20μg/L, 50μg/L, 100μg/L, 200μg/L, and 50μg/L.

2.2 Experiment Conditions

2.2.1 HPLC Conditions

Column: NovaChrom NovaIC A4-P Pesticide Residue Specialty Column, 4*150mm, 5μm

Mobile Phase: Phase A: Water; Phase B: 200mmol/L Ammonium Bicarbonate–0.05% Ammonia Solution

Flow Rate: 0.6mL/min

Column Temperature: 40°C

Injection Volume: 10µL

2.2.2 Mass Spectrometry Conditions

Table 3 Mass Spectrometry Ion Source Parameters

3. Experiment Result

3.1 Standards Chromatogram

The determination of glyphosate and four other pesticides and their metabolites in apple matrix was completed within 15 minutes, with good peak shapes, no peak tailing, and satisfactory compound responses, meeting the requirements of experimental analysis.

Fig 1 Chromatogram of Glyphosate and Four Other Pesticides and Their Metabolites in Apple Matrix (200μg/L)

3.2 Linear Range

The mixed standard solution of glyphosate and four other pesticides and their metabolites was serially diluted with blank apple matrix to prepare a series of concentrations for constructing the standard curve. The linear range for most compounds was 5–500μg/L. The deviation between the linear calibration results and the known concentrations was within the maximum permissible deviation. The linear correlation coefficients for all compounds were greater than 0.995, indicating good linearity.

Fig 2 Calibration Curves of Glyphosate, Four Other Pesticides and Their metabolites in apple Matrix

3.3 Limit of Quantitation (LOQ)

The signal-to-noise ratios for all compounds in this method at the LOQ of 5 μg/L exceed 10, complying with the standard sensitivity criteria.

Table 4 Mass Spectrometry Parameters for Compounds

3.4 Precision Test

An apple matrix spiked solution of glyphosate and four other pesticides and their metabolites at 10μg/L was injected six consecutive times to evaluate precision. The results are shown in the table below and meet the precision requirement of ≤10%.

Table 5 Compounds Precision

Fig 3 Chromatograms of Glyphosate and Four Other Pesticides and Their Metabolites in Apple Matrix (10μg/L)

3.5 Apple Sample Test

Commercial apple samples were pretreated following the standard procedure and analyzed. As shown in the figure below, no target compounds were detected.

Fig 4 Chromatograms of Glyphosate and Four Other Pesticides and Their Metabolites in Apple Samples

4. Conclusion

In this method, the Wayeal LCMS-TQ9200 liquid chromatography-tandem mass spectrometry system was used for the determination of residues of glyphosate and four other pesticides and their metabolites in apples. The experiment data demonstrate that the method provides good chromatographic peak shapes with no tailing, sensitivity that meets the requirements, linear correlation coefficients greater than 0.995 for all compounds, and precision within 10%. No system carryover was observed for high-concentration samples. These results indicate that the method, when equipped with the Wanyi Technology liquid chromatography-tandem mass spectrometry system, meets the routine qualitative and quantitative detection requirements for the target analytes.