Improved Multi-Analyte Method for the Underivatized Analysis of Anionic Pesticides in Food by LC-MS/MS

Expanding on previous work, this application note describes the quantitative determination of an extended suite of eight anionic polar pesticides, which are not amenable to conventional reverse-phase chromatography by LC-MS/MS. A hydrophilic interaction chromatography (HILIC)-based analytical column was used without the need for derivatization or specialty ion chromatography equipment.

Benefits

Application benefits include:

• Underivatized analysis of anionic polar pesticides in food
• Excellent sensitivity and precision
• Satisfactory chromatographic performance on routine LC-MS/MS system
• Accurate quantitation of residues in the absence of isotopically labeled internal standards.

Pesticides are used to protect crops from infestation by pests and plant diseases before and after harvest. They provide multiple benefits to consumers and producers alike. However, a possible consequence of pesticide use may be the presence of residues in food. Where pesticides  are approved for use, Maximum Residue Levels (MRLs) have been set at  the highest level of pesticide that the relevant regulatory body would expect to find in that crop when it has been treated in line with good agricultural practice. In the European Union (EU) a default MRL, equal to the limit of quantification (LOQ) achievable with analytical methods used for MRL enforcement, is applicable for pesticide/commodity combinations not explicitly mentioned in the MRL legislation.

Expanding on previous work,¹ this application note describes the quantitative determination of an extended suite of eight anionic polar pesticides, which are not amenable to conventional reverse-phase chromatography by LC-MS/MS. A hydrophilic interaction chromatography (HILIC)-based analytical column was used without the need for derivatization or specialty ion chromatography equipment. 

Sample extraction

Analytical standards, as detailed in Table 1, were purchased from Sigma Aldrich. All food samples (tomato juice, apple juice,  and beer) were purchased from local retail outlets. Samples and standards were prepared in accordance with the QuPPe v9 method.² All data were acquired and processed using MassLynx MS Software.

Various mobile phase compositions and gradients were evaluated, and MRM transitions were selected and conditions optimized. The conditions detailed in the Experimental section provided the best overall performance, in terms of sufficient retention, peak shape, practical run time, and for separation of the critical isobaric pairs: AMPA/fosetyl and fosetyl/phosphonic acid, as discussed in the EURL’s QuPPe method.

A common challenge encountered when analysing polar pesticides such as glyphosate is peak shape reproducibility. There are several factors that can affect peak shape such as matrix effects, secondary interactions with column stationary phases, and interactions with metal ions (e.g. calcium, manganese and iron). Tailing peaks, as shown in Figure 1A, are a frequent occurrence, observed in many previously published methods for these analytes.² The combination of the ACQUITY H-Class Bio and newly developed method described in this application note provided Gaussian peak shapes (shown in Figure 1B) for the tested matrices, thus increasing sensitivity and reproducibility. 

To evaluate the performance of the method, all food samples were spiked at three concentrations (0.01, 0.05, and 0.1 mg/kg) prior to extraction using the QuPPe method and replicate injections were made to provide information on sensitivity, accuracy, and precision of measurements and overall robustness. 

An example of the chromatographic performance and sensitivity observed from analysis of a beer sample spiked at 0.01 mg/kg  is shown in Figure 2. The orange trace in Figure 3 shows concurrent RADAR acquisition (ESI-, full scan m/z 50–300) for the chromatography of apple juice matrix over the entire LC run. This RADAR scan illustrates the complexity of the injected extract, even for this relatively simple commodity, where the y-axis for the RADAR full scan and MRMs are not linked due to the  significant intensity of co-extractives.

The response for all eight anionic polar pesticides were linear over the range investigated (0.001 to 0.25 mg/kg) with good residuals, even in the absence of isotopically labeled internal standards (Figure 4). Due to presence of incurred residues of  maleic hydrazide, chlorate, and phosphonic acid detected in some samples used as blanks, an offset on the X-axis for their  matrix-matched calibration graph was observed. The concentration of these incurred residues was determined in samples of apple juice, tomato juice, and beer by standard addition. An example is shown in Figure 5, where phosphonic acid was  quantified using the standard addition processing functionality within TargetLynx XS.

The method’s accuracy and precision were determined by analyzing the spikes (n=9). Excellent recovery and precision were observed for five of the eight analytes at all three concentrations (Figure 6), but the presence of incurred residues in the blanks limited the results for maleic hyrazide to 0.05 and 0.1 mg/kg in beer and prevented any results being reported for chlorate and phosphonic acid or maleic hyrazide in the other commodities.

This method has been developed for the direct analysis of a selection of anionic polar pesticides across a variety of foods. Utilizing routine LC and MS/MS technologies, a robustly sensitive method has been established  that achieves excellent levels of sensitivity, relative to the enforced MRLs.  In the absence of costly deuterated or isotopically labeled internal standards, accurate quantitation of residues in foods was readily achieved by standard addition, in compliance with SANTE guidelines 11495/2015.

1. B Wuyts, D Shah, E Ross, J Fox, E McCall. Highly Sensitive Analysis of Polar Pesticides in Food. Waters Technology Brief No. 720005822en. October, 2016.
2. M Anastassiades et al. Quick Method for the Analysis of Numerous Highly Polar Pesticides in Foods of Plant Origin via LC-MS/MS Involving Simultaneous Extraction with Methanol (QuPPe-Method). EURL-SRM. Version 9.2, October, 2016.