Complete solution for determining residues of 331 pesticides and their metabolites in plant-derived foods according to GB23200.121-2021, using Anyeep TQ9100.

On 2021 year 3 month 3 day, the National Health Commission, the Ministry of Agriculture and Rural Affairs, and the State Administration for Market Regulation jointly released GB 23200.121-2021, "Determination of Residues of 331 Pesticides and Their Metabolites in Plant-Derived Food by Liquid Chromatography-Tandem Mass Spectrometry," which officially took effect on 2021 year 9 month 3. This standard is China's first national standard using LC-MS/MS to detect the largest number of pesticide residues in a single injection. Compared with previous liquid chromatography-tandem mass spectrometry standards such as NY/T 761 and GB/T 20769, which suffered from complex sample preparation, limited applicable matrices, inability of some pesticides to meet MRL requirements due to insufficient limits of quantification, and poor overall applicability, the new standard introducesQuECHERSThe sample preparation method significantly simplifies the workflow, enabling a single protocol for both LC-MS/MS and GC-MS/MS analysis. The new standard applies to a wide range of plant-derived foods, including fruits, vegetables, grains, oilseeds, tea, nuts, spices, and edible fungi, providing comprehensive coverage for all plant-based food products.

This method follows GB23200.121-2021 for the pretreatment of plant-derived foods. Mass spectrometry was performed using an ESI source in positive/negative switching mode with dynamic MRM (DMRM) segment scanning for detection, and quantification was conducted via external standard calibration.

Instruments and Reagents

Anyeep TQ9100 Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry System

Methanol (mass spectrometry grade, Thermo Fisher Scientific, USA)

Formic acid (Pure MSCNW Company, Germany

Ammonium formate (Chromatographic gradeMerck KGaA, Darmstadt, Germany

Acetonitrile (Mass Spectrometry Grade, Thermo Fisher Scientific, USA)

Water (Ultra-pure Water)

Sodium Chloride (Pure Pesticide Residue, Shanghai Aladdin）

Magnesium Sulfate Anhydrous (Pure Pesticide Residues, Shanghai Anpel）

Sodium Citrate Dihydrate (Pure Pesticide Residue, ShanghaiMcLean

Disodium Citrate Sesquihydrate (Agro-residue Pure, ShanghaiMcLean

Sodium Acetate(Pesticide residues only，Shanghai ANPEL

Ceramic proton (Shanghai ANPEL，60105-370-B）

Public Service Announcement(Shanghai ANPEL, SBEQ-CA2401-10g）

GCB (40-120 μm, Shanghai ANPEL, GEEQ-072012）

Microporous filter membrane (0.2 μm,Shanghai ANPEL，50120965）

Information on 236 pesticide compounds

Liquid Chromatography Conditions

Mobile Phase A：0.01% formic acid in water with 2 mM ammonium formate

Mobile Phase B：0.01% formic acid-acetonitrile, containing 2 mM ammonium formate

Chromatography column：Waters ACQUITY UPLC BEH C18，2.1×100 mm, 1.7 μm

Flow Rate：0.3 mL/min

Column Temperature： 40 ℃

Injection volume：5 μL

The gradient elution program is shown in the table.2

Mass Spectrometry Conditions

Ionization Source：Electrospray Ionization Source (ESI ±)

Temperature： 550 ℃

Spray Voltage：5000 V

air curtain：35 psi

Vaporizer：60 psi

Auxiliary heating gas：60 psi

Crash： 12

Scan Mode：MRM

Sample Preparation

Vegetables, Fruits, Edible Fungi, and Sugar Crops

Weigh 10 g of sample (to the nearest 0.01 g) into a 50 mL plastic centrifuge tube. Add 10 mL acetonitrile and 1 ceramic beads, then vortex vigorously for 1 min. Add 4 g anhydrous magnesium sulfate, 1 g sodium chloride, 1 g sodium citrate, and 0.5 g disodium hydrogen citrate. Vortex vigorously for 1 min, then centrifuge at 4200 r/min for 5 min. Transfer 1 mL of the supernatant to a 2 mL plastic centrifuge tube containing 150 mg anhydrous magnesium sulfate and 25 mg PSA. For dark-colored samples, add 2.5 mg GCB to the tube, vortex to mix, and continue for 1 min. Centrifuge at 4200 r/min for 5 min, filter the supernatant through a microporous membrane, and proceed with analysis. For dried vegetables, fruits, and edible fungi, weigh 1 g of sample (to the nearest 0.0 g) into a 50 mL plastic centrifuge tube, add 9 mL water, vortex to mix, let stand for 30 min, and process as described above.

Grains, Oilseeds, and Nuts

Weigh 5 g of the sample (accurate to 0.01 g) into a 50 mL plastic centrifuge tube. Add 10 mL of water, vortex mix, and let stand for 30 min. Add 15 mL of acetonitrile-acetic acid solution and 1 ceramic beads, shake vigorously for 1 min. Then add 6 g of anhydrous magnesium sulfate and 1.5 g of sodium acetate, shake vigorously for 1 min, and centrifuge at 4200 r/min for 5 min. Transfer 1 mL of the supernatant into a 2 mL plastic centrifuge tube containing 150 mg of anhydrous magnesium sulfate, 50 mg of C8, and 50 mg of PSA. Vortex mix for 1 min. Centrifuge at 4200 r/min for 5 min, transfer the supernatant through a microporous filter, and proceed to analysis.

Tea and Spices

Weigh 2 g of the sample (to the nearest 0.01 g) into a 50 mL plastic centrifuge tube. Add 10 mL of water, vortex mix, and let stand for 30 min. Add 15 mL of acetonitrile-acetic acid solution and 1 ceramic beads, shake vigorously for 1 min. Then add 6 g of anhydrous magnesium sulfate and 1.5 g of sodium acetate, shake vigorously for 1 min, and centrifuge at 4200 r/min for 5 min. Transfer 1 mL of the supernatant to a 2 mL plastic centrifuge tube containing 150 mg of anhydrous magnesium sulfate, 50 mg of C18, 50 mg of PSA, and 25 mg of GCB. Vortex mix for 1 min. Centrifuge at 4200 r/min for 5 min, transfer the supernatant through a micro-pore filter membrane, and prepare for analysis.

Vegetable oil

Weigh 2 g of the sample (accurate to 0.01 g) into a 50 mL plastic centrifuge tube and add 5 mL of water. Add 10 mL acetonitrile and 1 ceramic beads, then vortex vigorously for 1 min. Add 4 g anhydrous magnesium sulfate, 1 g sodium chloride, 1 g sodium citrate, and 0.5 g disodium hydrogen citrate. Vortex again for 1 min, then centrifuge at 4200 r/min for 5 min. Transfer 1 mL of the supernatant to a 2 mL plastic centrifuge tube containing 150 mg anhydrous magnesium sulfate, 50 mg C18, and 50 mg PSA. Vortex for 1 min. Centrifuge at 4200 r/min for 5 min, filter the supernatant through a micro-pore membrane, and proceed with analysis.

Note,When determining sulfonylurea herbicides, cyclohexenone herbicides (clethodim, clethodim sulfone, clethodim sulfoxide, sethoxydim, tralkoxydim, quizalofop-P-ethyl), triazolopyrimidine sulfonamide herbicides (florasulam, pyroxsulam, triflusulfuron-methyl), fluensulfone, spirotetramat and its metabolites, mesotrione, benzobicyclon, bensulfuron-methyl, cyanosulfomethyldihydroisoxazole, CCIM metabolite of cyprodinil, and isoxaflutole-diketone in vegetables, fruits, edible fungi, sugar crops, and vegetable oils, reduce the PSA amount to 5 mg per mL of extract. For grains, oilseeds, and nuts, reduce it to 10 mg per mL of extract.

Experimental Results and Discussion

Development of a rapid quantitative screening method

TQMS Data Acquisition Software supports rapid creation of quantitative screening methods for multiple pesticide residues in plant-based matrices. In the software's method editor, navigate to the Mass Spectrometry Method interface, switch to DMRM scan mode, and import the pesticide residue method scan table (in .xls format) via right-click.（Includes compound name, retention time, ion pair, DP, and CE mass spectrometry parameters.）Achieve this effortlessly. No manual input of compound information is required, saving significant time and eliminating errors caused by manual entry. Enables high-throughput screening of multiple analytes in plant-based foods.

Method Sensitivity, Linearity, and Range

This method selected 4 types of plant-derived blank samples. After pretreatment according to 2.5, blank matrix solutions were obtained. A mixed standard solution was diluted stepwise with these matrices to prepare 8 calibration levels in the concentration range of 2–500 ng/mL, generating a calibration curve. Pesticide residues in 4 types of plant-derived samples were then quantified using this approach. Results showed that under matrix-matched calibration conditions, the linear correlation coefficient (R²) for all target pesticide components exceeded 0.99, indicating excellent linearity. The limit of quantification (LOQ) for all 236 pesticides met the requirements specified in GB 23200.121-2021 for 4 types of matrices, satisfying routine multi-residue analysis needs. At the LOQ level, all analyzed compounds produced high-quality spectra, enabling accurate quantitative and qualitative determination.

Method precision and recovery

Spiking recovery tests were conducted at the limit of quantification (LOQ) levels added to 4 matrix samples to evaluateAnyeep TQ100Accuracy and stability of pesticide residue testing for 236 types. Results show that recovery rates for 236 residues across 4 sample matrices ranged from 60% to 120%, with precision (CV) not exceeding 15%. Table 5 summarizes spiked recovery rates and precision results for selected compounds.

Summary

This article usesAnyeep TQ9100 Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry SystemA method was established for the determination of 236 pesticides and their metabolites in 4 plant-derived matrices (pear, cooking oil, rice, tea) using matrix-matched calibration curves. The results demonstrate that the method offers excellent sensitivity, stability, and linear range, making it suitable for routine pesticide residue testing in various plant-based foods.