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Evaluating Shikimic Acid as an Analyte Protectant for Pesticides in Food

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While using matrix-matched calibration standards can mitigate matrix effects and increase reporting accuracy, this approach is time-consuming, labor intensive, and sometimes limited by the availability of suitable blank matrix. The use of analyte protectants (AP) in gas chromatography is an intriguing alternative because it is much simpler in practice and can allow good results to be achieved with solvent-based calibration standards [1]. Analyte protectants, such as the sugar derivative shikimic acid, work by binding strongly to active sites in the inlet, which effectively blocks these sites from interacting with target compounds. This prevents analyte loss, improves transfer to the column, minimizes degradation in peak shape and response, and can potentially improve accuracy and reliability for low-level analysis.

Since using matrix-matched calibration standards is not feasible in all situations, a series of experiments was conducted to evaluate the effects of using an analyte protectant with solvent-based calibration standards on recovery and critical peak characteristics (area, symmetry, width, and tailing) for a variety of pesticides. The experiments were (1) recovery of 40 pesticides in kale samples; (2) response of captan in apple, strawberry, kale, grape, and celery samples; and (3) response of 40 pesticides in kale and strawberry samples. For all tests, the analyte protectant was shikimic acid (1 mg/mL), and 1 µL of it was co-injected with 1 µL of sample (for samples without analyte protectant, 1 µL of acetonitrile was co-injected).

Experiment 1: Recovery of 40 Pesticides in Kale

To determine if 1.0 mg/mL shikimic acid analyte protectant could increase pesticide recovery in matrix, kale samples were fortified at 10 and 50 ppb with a 40-compound QuEChERS performance standards kit (cat.# 31152). Quantification was done using matrix-matched calibration standards, solvent-based calibration standards with shikimic acid, and solvent-based calibration standards without shikimic acid. As shown in Figures 1 and 2, when using solvent standards, the analyte protectant did help more pesticides fall into the desired recovery range (70-120%), particularly at the higher spike level. However, matrix-matched calibration still outperformed solvent calibration with analyte protectant at both spike levels, and this was especially pronounced at the lower spike level.

Figure 1: Distribution of 40 Pesticides at 50 ppb in Kale Across Percent Recovery Brackets

Distribution of 40 pesticides at 50 ppb in kale across percent recovery brackets

 

Figure 2: Distribution of 40 Pesticides at 10 ppb in Kale Across Percent Recovery Brackets

Distribution of 40 pesticides at 10 ppb in kale across percent recovery brackets

 

Experiment 2: Response of Captan in Multiple Matrices

Captan is a commonly analyzed, yet challenging, GC-amenable pesticide, so it was chosen for a more detailed evaluation of the performance of shikimic acid as an analyte protectant. For this experiment, two sets of samples were prepared so that captan could be evaluated alone and when in the presence of other pesticides. A captan-only standard (cat.# 32583) was spiked at 10 ppm into solvent, apple, strawberry, kale, grape, and celery samples, and a multi-pesticide standard containing captan (cat.# 31152) was also spiked at 10 ppm into the same sample types. Samples were analyzed both with and without 1 µL sandwich injections of 1 mg/mL shikimic acid. Peak response was first visually compared in an XIC; then, area and other key peak characteristics were evaluated in detail using the MS/MS data.

Qualitatively, peak response (abundance) was greatly improved by the presence of shikimic acid analyte protectant when captan was in solvent (Figure 3); somewhat improved when it was in strawberry (Figure 4) or grape; and not improved when in kale, apple, or celery (data not shown for grape, kale, apple, and celery). No dramatic differences in peak shape were observed; however, the taller peaks tended to have narrower widths.

Figure 3: Shikimic acid analyte protectant considerably improved peak response for captan in solvent.

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GC_FS0589

 

Figure 4: For captan in strawberry, shikimic acid analyte protectant provided some improvement for peak response.

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GC_FS0590

 

For a statistical evaluation of the effect of analyte protectant on important peak characteristics, the peak area, symmetry, width, and tailing values provided by the TraceFinder 4.1 EFS software were compared in different combinations by t-test. The data sets for the comparisons were as follows:

  1. Captan/Solvent—captan spiked alone into solvent.
  2. Captan/Solvent/Samples—captan spiked alone into both solvent and all five sample matrices.
  3. Multi-pesticide/Solvent/Samples—mixed standard containing 40 pesticides, including captan, spiked into both solvent and all five sample matrices.
  4. All—all data from the above three data sets combined.

Quantitatively, peak width was the only characteristic that was statistically improved (p<0.05) across all four data sets. Results showed that the use of shikimic acid as an analyte protectant did result in narrower peaks. Peak area and tailing were also significantly improved, but only in the All and Captan/Solvent/Sample data sets. There was no statistical difference for peak symmetry in any data set.

Table I: Evaluation of the Effect of Analyte Protectant on Captan Peak Characteristics (Statistically significant differences [p<0.05; 95% confidence level] are shown in bold.)

    Peak Area Symmetry Peak Width Tailing
All
Without AP (n=39) average 178137 92.02 1.32 1.34
SD 42461 5.70 0.06 0.18
RSD 24% 6% 5% 14%
With AP (n=29) average 216295 93.43 1.24 1.19
SD 55321 4.59 0.03 0.17
RSD 26% 5% 3% 14%
t-test 0.002 0.277 0.000 0.001
Multipesticide/Solvent/Samples
Without AP (n=9) average 222614 94.13 1.31 1.26
SD 47642 2.91 0.07 0.15
RSD 21% 3% 5% 12%
With AP (n=9) average 273640 94.42 1.26 1.16
SD 44426 4.10 0.03 0.11
RSD 16% 4% 2% 9%
t-test 0.032 0.863 0.040 0.130
Captan/Solvent/Samples
Without AP (n=30) average 164794 91.38 1.33 1.36
SD 30630 6.20 0.06 0.19
RSD 19% 7% 5% 14%
With AP (n=20) average 190490 92.98 1.23 1.20
SD 37687 4.83 0.03 0.19
RSD 20% 5% 3% 16%
t-test 0.016 0.336 0.000 0.004
Captan/Solvent
Without AP (n=5) average 195568 90.56 1.35 1.32
SD 51267 5.69 0.06 0.17
RSD 26% 6% 5% 13%
With AP (n=5) average 187051 90.32 1.23 1.35
SD 34072 5.41 0.05 0.21
RSD 18% 6% 4% 15%
t-test 0.339 0.335 0.002 0.657

 

Experiment 3: Response of 40 Pesticides in Kale and Strawberry

In a final experiment, the effect of shikimic acid analyte protectant on peak area for 40 pesticides was evaluated. For this assessment, multi-pesticide standard (cat.# 31152) was spiked at 10 ppm into solvent, kale, and strawberry samples, which were analyzed both with and without shikimic acid, and the area results were then compared.

In general, the addition of analyte protectants resulted in larger peak areas. When individual pesticides across all solvent and sample injections were evaluated by t-test, 21 out of 40 analytes had significantly improved areas. When separated by matrix, 14/40 were improved in solvent; 1/40 were improved in kale; and 0/40 were improved in strawberry. Overall, the data showed that the analyte protectant could increase peak area in solvent, but it did not offer much advantage for pesticides in matrix. Finally, the data sets were also evaluated by paired t-test using the averages from the triplicate runs for the whole set of pesticides. In this test, all data sets were found to be significantly different.

Table II: Evaluation of the Effect of Shikimic Acid Analyte Protectant on Average Peak Area for Pesticides in Solvent, Kale, and Strawberry (n = 3)

  All Solvent
Without AP With AP t-test Without AP With AP t-test
2-Phenylphenol 2.0E+06 2.5E+06 0.007 1.9E+06 2.4E+06 0.048
Acephate 6.5E+05 9.3E+05 0.002 6.6E+05 9.3E+05 0.006
Azinphos-methyl 4.5E+05 7.2E+05 0.004 4.0E+05 6.0E+05 0.055
BHC, gamma- 1.2E+06 1.4E+06 0.179 1.4E+06 1.4E+06 0.567
Bifenthrin 5.3E+06 5.9E+06 0.273 5.2E+06 5.9E+06 0.102
Captan 2.2E+05 2.7E+05 0.032 2.3E+05 2.8E+05 0.063
Carbaryl 5.5E+06 7.3E+06 0.004 5.0E+06 7.4E+06 0.007
Chlorothalonil 7.0E+05 8.7E+05 0.009 7.2E+05 9.2E+05 0.018
Chlorpyrifos 9.9E+05 1.1E+06 0.125 1.0E+06 1.2E+06 0.131
cis-Permethrin 7.5E+05 8.6E+05 0.243 8.0E+05 8.0E+05 0.974
Coumaphos 2.6E+05 3.0E+05 0.202 2.7E+05 2.8E+05 0.919
Cyprodinil 5.1E+05 6.1E+05 0.054 5.0E+05 6.1E+05 0.060
DDT, p,p'- 3.0E+06 3.5E+06 0.068 3.0E+06 3.7E+06 0.044
Deltamethrin 3.7E+05 4.2E+05 0.206 3.5E+05 4.1E+05 0.068
Diazinon 8.9E+05 1.0E+06 0.026 9.1E+05 1.1E+06 0.162
Dichlofluanid 1.8E+06 2.1E+06 0.088 1.8E+06 2.2E+06 0.143
Dichlorvos 1.8E+05 2.0E+05 0.341 1.5E+05 2.1E+05 0.265
Dicofol 1.7E+06 2.4E+06 0.002 1.6E+06 2.5E+06 0.027
Dimethoate 5.1E+05 6.5E+05 0.012 4.6E+05 6.6E+05 0.002
Endosulfan sulfate 4.4E+05 4.9E+05 0.231 4.7E+05 4.9E+05 0.556
Endrin 1.4E+05 1.7E+05 0.046 1.4E+05 1.8E+05 0.044
Fenhexamid 4.7E+05 6.1E+05 0.007 4.3E+05 6.1E+05 0.007
Fenpropathrin 1.8E+05 2.0E+05 0.334 2.0E+05 2.0E+05 0.965
Fenthion 1.7E+06 2.0E+06 0.044 1.7E+06 2.0E+06 0.132
Folpet 7.7E+05 9.9E+05 0.009 7.5E+05 1.0E+06 0.021
Imazalil 1.1E+05 1.5E+05 0.011 1.1E+05 1.4E+05 0.016
Iprodione 9.4E+04 1.2E+05 0.056 8.5E+04 1.2E+05 0.060
Malathion 1.5E+06 1.8E+06 0.013 1.4E+06 1.9E+06 0.025
Metalaxyl 4.4E+05 5.2E+05 0.036 4.5E+05 5.4E+05 0.100
Methamidophos 9.8E+04 1.1E+05 0.738 5.9E+04 4.8E+04 0.858
Methiocarb 1.4E+06 1.7E+06 0.036 1.4E+06 1.7E+06 0.093
Mevinphos 1.5E+06 1.6E+06 0.371 1.5E+06 1.5E+06 0.875
Myclobutanil 6.3E+05 7.7E+05 0.044 6.1E+05 7.6E+05 0.036
Omethoate 9.1E+05 1.3E+06 0.002 7.7E+05 1.3E+06 0.001
Phosalone 1.1E+05 1.4E+05 0.091 1.2E+05 1.3E+05 0.666
Pirimiphos methyl 6.9E+05 7.9E+05 0.099 6.9E+05 8.2E+05 0.069
Propargite 1.4E+05 1.8E+05 0.044 1.3E+05 1.7E+05 0.052
Thiabendazole 5.0E+06 6.5E+06 0.018 4.1E+06 5.5E+06 0.070
trans-Permethrin 8.6E+05 9.2E+05 0.530 9.3E+05 8.8E+05 0.708
Vinclozolin 2.5E+05 3.0E+05 0.025 2.5E+05 3.0E+05 0.075
Total significant     21     14
Paired test p value     0.000102     0.000349

 

  Kale Strawberry
Without AP With AP t-test Without AP With AP t-test
2-Phenylphenol 2.0E+06 2.6E+06 0.077 2.2E+06 2.4E+06 0.549
Acephate 6.7E+05 1.0E+06 0.078 6.1E+05 8.2E+05 0.270
Azinphos-methyl 5.0E+05 9.0E+05 0.020 4.5E+05 6.5E+05 0.304
BHC, gamma- 1.1E+06 1.4E+06 0.202 1.2E+06 1.3E+06 0.679
Bifenthrin 5.2E+06 6.1E+06 0.465 5.5E+06 5.6E+06 0.943
Captan 2.1E+05 2.9E+05 0.096 2.3E+05 2.5E+05 0.732
Carbaryl 5.5E+06 7.6E+06 0.083 6.1E+06 7.0E+06 0.556
Chlorothalonil 6.5E+05 8.7E+05 0.097 7.4E+05 8.3E+05 0.571
Chlorpyrifos 9.5E+05 1.1E+06 0.284 1.0E+06 1.1E+06 0.787
cis-Permethrin 7.0E+05 9.3E+05 0.177 7.5E+05 8.4E+05 0.716
Coumaphos 2.6E+05 3.5E+05 0.132 2.6E+05 2.9E+05 0.697
Cyprodinil 4.8E+05 6.0E+05 0.191 5.6E+05 6.1E+05 0.696
DDT, p,p'- 2.9E+06 3.7E+06 0.238 3.1E+06 3.3E+06 0.771
Deltamethrin 3.6E+05 4.4E+05 0.330 3.9E+05 4.1E+05 0.858
Diazinon 8.7E+05 1.0E+06 0.228 8.8E+05 1.0E+06 0.415
Dichlofluanid 1.7E+06 2.0E+06 0.266 1.8E+06 2.0E+06 0.766
Dichlorvos 1.8E+05 1.8E+05 0.944 2.1E+05 2.0E+05 0.952
Dicofol 1.6E+06 2.5E+06 0.053 1.8E+06 2.2E+06 0.502
Dimethoate 5.2E+05 6.9E+05 0.167 5.4E+05 6.0E+05 0.647
Endosulfan sulfate 4.3E+05 5.4E+05 0.204 4.3E+05 4.5E+05 0.888
Endrin 1.4E+05 1.7E+05 0.299 1.4E+05 1.5E+05 0.730
Fenhexamid 4.9E+05 6.6E+05 0.115 4.8E+05 5.5E+05 0.520
Fenpropathrin 1.7E+05 2.1E+05 0.340 1.7E+05 2.0E+05 0.613
Fenthion 1.6E+06 2.1E+06 0.178 1.8E+06 2.0E+06 0.631
Folpet 7.5E+05 1.0E+06 0.119 8.2E+05 9.5E+05 0.543
Imazalil 1.2E+05 1.7E+05 0.066 1.1E+05 1.4E+05 0.472
Iprodione 9.4E+04 1.3E+05 0.180 1.0E+05 1.1E+05 0.867
Malathion 1.4E+06 1.8E+06 0.213 1.5E+06 1.8E+06 0.478
Metalaxyl 4.2E+05 5.1E+05 0.264 4.4E+05 5.1E+05 0.502
Methamidophos 1.3E+05 1.7E+05 0.743 1.1E+05 1.3E+05 0.809
Methiocarb 1.3E+06 1.8E+06 0.208 1.4E+06 1.7E+06 0.535
Mevinphos 1.5E+06 1.8E+06 0.391 1.5E+06 1.6E+06 0.784
Myclobutanil 6.4E+05 8.2E+05 0.219 6.4E+05 7.1E+05 0.661
Omethoate 1.0E+06 1.6E+06 0.057 9.3E+05 1.2E+06 0.341
Phosalone 1.1E+05 1.5E+05 0.106 1.1E+05 1.2E+05 0.601
Pirimiphos methyl 6.6E+05 8.1E+05 0.247 7.1E+05 7.4E+05 0.870
Propargite 1.5E+05 2.0E+05 0.289 1.5E+05 1.8E+05 0.536
Thiabendazole 4.9E+06 6.6E+06 0.087 5.9E+06 7.3E+06 0.287
trans-Permethrin 7.9E+05 9.4E+05 0.463 8.5E+05 9.4E+05 0.722
Vinclozolin 2.4E+05 2.9E+05 0.212 2.6E+05 3.1E+05 0.432
Total significant     1     0
Paired test p value     3.02E-05     0.000435


Conclusions

This evaluation of shikimic acid as an analyte protectant demonstrated that it can provide benefits for some pesticides when using solvent standards for calibration. Matrix-matched calibration provided better overall results, but the use of shikimic acid can improve some chromatographic parameters when matrix-matched calibration is not feasible.

References

  1. R. Rodríguez-Ramos, S.J. Lehotay, N. Michlig, B. Socas-Rodríguez, M. Rodríguez-Delgado, Critical review and re-assessment of analyte protectants in gas chromatography, J. Chromatogr. A 1632 (2020) 461596. https://www.sciencedirect.com/science/article/pii/S0021967320308700
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