Comprehensive LC-MS/MS Multi-Residue Pesticide Method for Food and Feed-Standardization With Quality Control Materials

To comply with strict regulations, hundreds of pesticide residues must be monitored using reliable analytical methods for their detection, quantification, and identification in many different food and feed commodities. One cost effective approach is to add more residues to a single method, but this requires generic sample preparation and the injection and analysis of more complex extracts. More residues can also mean that users spend more time data processing and reviewing results.

Herein we describe a LC-MS/MS multi-residue pesticide method for more than 200 residues using the ACQUITY™ Premier System, Xevo™ TQ-S micro Triple Quadrupole Mass Spectrometer and waters_connect™ for Quantitation Software to check compliance against commercially available quality control materials of strawberry purée, baby food, and animal feed.

The food and feed commodities were extracted using QuEChERS with the baby food and feed samples cleaned up using a simple and effective Oasis™ HLB pass through protocol. The 1 µL acetonitrile extract was injected without dilution using a post injector extension loop which significantly improved the peak shape of early eluting compounds and removed the need to manually reintegrate peaks.

Exception focused review in waters_connect for Quantitation Software highlighted results that fell outside SANTE tolerances defined by a ruleset, increasing the efficiency of data review.

Method performance was assessed using quality control (QC) materials with all measured values within the assigned range of the analytes, trueness was in the range of 100–130% and all calculated %RSDs less than 20%. The robustness of the method was demonstrated for animal feed extracts with peak response %RSDs all less than 9% for 250 injections.

Benefits

• Cost effective approach to monitoring multi-residue pesticides in food and feed using LC-MS/MS which met SANTE guidelines for method performance
• No dilution of final extract required as 1 µL injection of acetonitrile extract using a post injector extension loop improved early eluting peak shapes, removing the need for manual peak re-integrations
• Exception focused review in waters_connect for Quantitation Software increased the efficiency of data review

Plant protection products, commonly known as pesticides, are essential to the food and agricultural industries, helping to stabilize food supplies by controlling pests, weeds, and disease. Pesticide residues resulting from the use of plant protection products during planting, storage or production may pose a risk to public health or hinder trade due to rejections of imported consignments and product recalls.

Hundreds of pesticides are approved and routinely used for crop protection and regulations are in place for maximum residue levels (MRL), that are legally tolerated in food and feed when pesticides are applied correctly in accordance with Good Agricultural Practice. Excessive use of pesticides may result in residues that exceed the MRLs if the pre-harvest intervals and correct dosages are not adhered to by growers or illegal pesticides are used. Compliance with these MRLs is checked by competent authorities by monitoring for residues in food and feed samples. All food business operators must ensure compliance with the same requirements but also must consider brand protection too. Pesticides that have not been authorized for use on crops generally have a default MRL value of 0.01 mg/kg.

Hence, reliable analytical methods are needed for detection, quantification, and identification of hundreds of pesticide residues in many different commodities. A primary goal for all laboratories involved in pesticide residue testing is to accurately determine as many compounds as possible in the most cost-effective manner. By implementing multi-residue methods using Ultra Performance Liquid Chromatography Tandem Quadrupole Mass Spectrometry (UPLC™-MS/MS) systems, many labs have significantly extended their scope of analysis to meet the demand for testing. Nevertheless, multi-residue methods often employ QuEChERS for sample preparation and the injection of more complex extracts so the ability for systems to provide accurate and precise results over extended time periods is another important goal for these labs.

Herein we describe a QuEChERS extraction followed by determination with LC-MS/MS for multi-residue pesticide analysis using commercially available quality control materials of strawberry purée, baby food, and animal feed as proof of performance for this method approach.

Sample Description

Quality Control Materials (T19380QC strawberry purée, T09160QC cereal-based baby food, and T09151QC cereal-based animal feed) were obtained from FAPAS.

Pesticide standards (31971 LC Multiresidue Pesticide Standards Kit) were purchased from Restek and serial diluted with extracted blank matrix into the sample concentration calibration range 0.005–0.2 mg/kg.

Samples were extracted using a previously published QuEChERS CEN method with DisQue (p/n: 186006813).¹ In short, 10 g strawberry purée, 5 g baby food, and 2 g animal feed were taken through the method. Baby food and animal feed were subjected to a pass-through clean-up using Oasis HLB Plus Short (p/n: 186008887) cartridges. The pure acetonitrile extracts were injected without dilution using the bracketed calibration approach. Five replicates of each QC material were analyzed using this method.

The injection solvent and volume used in the method were both considered as the QuEChERs extraction methodology utilizes acetonitrile as the extraction solvent. With the expanding scope of the method to cover hundreds of pesticides, it has often been advised to dilute the final extract in an aqueous solution to improve chromatographic performance for the early eluting pesticides, such as methamidophos.

Peak shapes are compared in Figure 1 for the first four early eluting compounds at 1 µL with and without an extension loop installed and at 2 µL with it fitted. The pre-column extension loop provides sufficient mobile phase mixing to allow focusing of the early eluting pesticides with significant improvement in peak shape compared to the 1 µL injection without the loop fitted. This not only significantly improves the chromatographic peak shape but also reduces the time required to review data for these pesticides as no manual re-integration was required. In fact, the positive effect of the extension loop on peak shape could be observed up to omethoate, the sixth eluting compound at R_t=2.52 minutes. Although 2 µL injections provided similar peak shape improvements for acephate and beyond, the sensitivity of the Xevo TQ-S micro allowed for a 1 µL injection volume to be used for the method performance requirements to be met.

The described multi-residue method was evaluated in strawberry purée, cereal-based baby food, and cereal-based animal feed using FAPAS Quality Control Materials. Concentrations of all residues were calculated using waters_connect for Quantitation Software using exception focused review (XFR). With XFR, the method automatically flags out of specification QC, calibration, and ion ratios based on the ruleset that was loaded into the processing method allowing users to focus on the injections and analytes that need the most attention. In this instance, these criteria were based on the SANTE guidelines listed in Table 1.²

The method performance is shown in Figure 2 for each of the FAPAS QC materials against the assigned value with all observed values being within the assigned range of the analytes. The trueness of the detected pesticides was in the range of 100–130% compared to the assigned value with all calculated %RSDs under 20%.

Data was reviewed using waters_connect for Quantitation Software with the processing method used, directly generated from the acquisition method. Figure 3 shows the dashboard view for unknowns where the data assessment for the QC samples was completed. Positive results are shown at the top of the list, allowing users to focus their investigations on these residues efficiently. XFR was used to flag any problems with calibrations, QC samples, blanks, and ions ratios which significantly reduced the time on task reviewing the data set.

The last critical method parameter addressed in this work was the “up time” of the instrument to continue to analyze samples without excessive operator intervention. Figure 4 displays the peak area for Triadimefon in animal feed (with additional information on the other analytes in the legend) where the sample was repeatably injected into the LC-MS/MS system and the peak area plotted.

An LC-MS/MS multi-residue pesticide method for more than 200 residues using the ACQUITY Premier System, Xevo TQ-S micro Triple Quadrupole Mass Sspectrometer, and waters_connect for Quantitation Software was used to assess method performance against commercially available quality control materials of strawberry purée, baby food, and animal feed.

Complex food and feed commodities were extracted using QuEChERS with baby food, and animal feed samples cleaned up using a simple and effective Oasis HLB pass through protocol. The 1 µL acetonitrile extract was injected without dilution by using a post injector extension loop. The loop increased mobile phase mixing and significantly improved the peak shapes of early eluting compounds, such as methamidophos, which aided data processing and review of results, removing the need for any manual peak re-integrations.

Exception focused review in waters_connect for Quantitation Software was used to highlight results that fell outside SANTE tolerances, defined by a ruleset, increasing the efficiency of data review. The quality control materials were assessed using the Unknowns tab in this software, which significantly reduced the time taken for data review. The processing method was directly generated from the acquisition method reducing the possibility of transcription errors.

Method performance was assessed using FAPAS QC materials with all measured values being within the assigned range of the analytes, the trueness was in the range of 100–130% and with all calculated %RSDs less than 20%. The robustness of the method was established, with animal feed as the most complex matrix, with externally standardised peak responses %RSDs all less than 9% for 250 injections.

1. Shah, D, et al. Multi-residue Method for the Quantification of Pesticides in Fruits, Vegetables, Cereal, and Black Tea Using UPLC-MS/MS. Waters Application Note. 720006886. Revised February. 2021.
2. Document No. SANTE/11312/2021 V2. Guidance Document on Analytical Quality, Control, and Method Validation Procedures for Pesticides Residues Analysis in Food and Feed.