• Application Note

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

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

  • Benjamin Wuyts
  • Euan Ross
  • Simon Hird
  • Dimple D. Shah
  • Waters Corporation

Abstract

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.

Introduction

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. 

Experimental

UPLC conditions

UPLC system:

ACQUITY UPLC H-Class Bio

Column:

Shodex HILICpak VT-50 2D 5 μm, 2 x 150 mm

Column temp.:

40 °C

Sample temp.

10 °C

Injection volume:

10 μL

Flow rate:

0.2 mL/min

Mobile phase A:

68: 12: 20 Water: 45 mM ammonium bicarbonate: acetonitrile

Mobile phase B:

50 mM Ammonium bicarbonate

Wash:

Acetonitrile

Purge:

80: 20 Water: methanol

Gradient

Time

%A

%B

0

100

0

6.5

55

45

18

0

100

21

0

100

21.01

100

0

24

100

0

MS conditions

MS system:

Xevo TQ-XS

Acquisition mode:

MRM

Ionization mode:

ESI–

Source temp.:

150 °C

Capillary voltage:

2.4 kV

Cone gas flow:

300 L/Hr

Desolvation temp.:

600 °C

Desolvation gas flow:

1000 L/Hr

Nebulizer:

7 bar

Table 1. MRM transitions and parameters for the anionic polar pesticides.

Compound

Transition

Cone Voltage (V)

Collision Energy (eV)

Glyphosate

167.85>62.85

167.85>80.85

30

16

15

AMPA

109.85>62.85

109.85>80.85

30

15

15

Glufosinate

179.9>62.85

179.9>84.85

30

25

16

Ethephon

142.85>106.8

142.85>78.8

20

10

15

Fosetyl-al

108.85>62.85

108.85>80.8

20

15

10

Phosphonic acid

80.8>78.8

80.8>62.8

20

14

12

Chlorate

82.8>66.8

84.8>68.9

25

15

15

Maleic hydrazide

110.85>81.85

110.85>54.9

20

15

Quantitative transition in bold.

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.

Results and Discussion

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. 

Figure 1. An example of chromatographic peak performance observed is shown for glyphosate, where significant peak tailing (A) was reduced by cleaning column and LC (B). Gaussian peak shape was maintained over long analytical runs using the inert ACQUITY UPLC H-Class Bio System.
Figure 2. Example of chromatographic separation of anionic pesticides, spiked to 0.01 mg/kg in beer, plotted relative to the most abundant intensity. Due to incurred residues phosphonic acid detected, the spiked analytes appear as low traces and so are offset. 

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.

Figure 3. Example of the chromatographic separation of anionic pesticides, spiked to 0.01 mg/kg in apple. The orange trace shows a full scan (RADAR) acquired throughout the full chromatographic method illustrating background contamination from matrix. 

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.

Figure 4. Example of calibration curve prepared  in beer over a range of  0.001 to 0.25 mg/kg, where all residuals are <15%. Inset shows a summary table with  the minimum R2 achieved for each analyte in all calibration curves, where all residuals were <25%.
Figure 5. Example of standard addition calibration, accurately quantifying and confirming the residue detection of phosphonic acid.

Table 2. LLOQs calculated (S/N>10) for each of the commodities.

Compound

Apple juic (mg/kg)

Tomato juice (mg/kg)

Beer (mg/kg)

Glyphosate

0.00004

0.0003

0.0001

AMPA

0.0003

0.0001

0.0001

Glufosinate

0.0001

0.0004

0.0002

Ethephon

0.0007

0.0025

0.0007

Fosetyl-al

0.0002

0.0008

0.0001

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.

Figure 6. Recoveries and precision (n=9) for each analyte in a selection of foods at A. 0.01 mg/kg; B. 0.05 mg/kg and C. 0.1 mg/kg. Residues >0.01 mg/kg were detected for maleic hydrazide, chlorate, and phosphonic acid and so are omitted from this data.

Conclusion

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.

References

  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.

720006070, August 2017

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