Article pubs.acs.org/JAFC
Development and Validation of Ion Chromatography−Tandem Mass Spectrometry-Based Method for the Multiresidue Determination of Polar Ionic Pesticides in Food Stuart Adams,*,† Jonathan Guest,† Michael Dickinson,† Richard J. Fussell,§ Jonathan Beck,⊗ and Frans SchoutsenΘ †
Fera Science Ltd., Sand Hutton, York YO41 1LZ, United Kingdom Thermo Fisher Scientific, Hemel Hempstead, United Kingdom ⊗ Thermo Fisher Scientific, San Jose, California, United States Θ Special Solutions Center, Thermo Fisher Scientific, Dreieich, Germany §
ABSTRACT: An extraction method using acidified methanol based on the quick polar pesticide (QuPPe) method using suppressed ion chromatography coupled to mass spectrometry was developed and validated for the direct analysis of polar pesticides, without the need for derivatization or ion pairing, in cereals and grapes. The method was robust, and results for glyphosate, aminomethyl phosphonic acid (AMPA), N-acetyl-AMPA, glufosinate, 3-methylphosphinicopropionic acid (3MPPA), N-acetyl glufosinate, ethephon, chlorate, perchlorate, fosetyl aluminum, and phosphonic acid at three concentration levels (typically 0.01, 0.05, and 0.1 mg/kg) were compliant with SANTE/11945/2015 guideline method performance criteria. Cereal-based infant food proved to be a more challenging matrix and validated only for glyphosate, chlorate, and perchlorate at 0.005, 0.01, and 0.05 mg/kg. The developed method enables the multiresidue analysis of 12 ionic pesticides and relevant metabolites in a single analysis. Until now, the analysis of these compounds required several different single-residue methods using different chromatographic conditions. This multiresidue approach offers the possibility of more cost-effective and more efficient monitoring of polar ionic pesticides and contaminants that are of concern to food regulation bodies and consumers. KEYWORDS: glyphosate, chlorate, perchlorate, ion chromatography, mass spectrometry
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phthaldehyde (OPA) reagent.6 Recent advances in mixed-mode columns have enabled methods based on cation-exchange chromatography for the analysis of glyphosate in cereal-based crops.7 A review of methods for difficult pesticides in food was published by Raina-Fulton in 2014,8 including methods for the measurement of glyphosate, glufosinate, AMPA, and 3methylphosphinicopropionic acid (3-MPPA) by derivatization and hydrophilic interaction chromatography (HILIC) or ionpair separation prior to liquid chromatography−mass spectrometry (LC-MS) determination. Analysis of glyphosate and AMPA based on derivatization is more successful for water, but such methods are limited to glyphosate and glufosinate and some of their metabolites, and issues with matrix interferences in water have been reported by Ibanez et al.9 and Freuze et al.10 In our experience derivatization methods are not sufficiently robust for analysis across a wide range of food matrices. Hernandez et al.11 determined residues of fosetyl-aluminum (fosetyl-Al) in vegetables by LC-MS after the addition of tetrabutyl ammonium acetate as ion-pairing agent. Although derivatization and ion-pairing can provide some benefits, the methods are
INTRODUCTION The group of polar ionic pesticides include some of the most frequently used pesticides worldwide. Although these compounds result in residues in food and have been the subject of recent controversy, they have been infrequently monitored in food-testing programs. In the United States, for example, a report by the Government Accounting Office1 criticized the responsible government agencies (Environmental Protection Agency (EPA), Food and Drug Administration (FDA), and Department of Agriculture (USDA)) with respect to the lack of testing for glyphosate residues in food. The lack of testing is simply because of the analytical difficulties and higher costs associated with the single-residue methods that have been available until recently. Historically, pesticides such as glyphosate, glufosinate, and fosetyl were analyzed individually using specialist methods involving derivatization or ion pairing to overcome unwanted interactions during extraction and chromatographic separation. Methods for the analysis of glyphosate, (N-(phosphonomethyl) glycine, and its main metabolite, aminomethyl phosphonic acid (AMPA), include the use of derivatization with heptafluorobutanol and trifluoroacetic anhydride, followed by ion-exchange cleanup prior to GC-MS,2 using 9-fluoroenyl methyl chloroformate (FMOC) prior to liquid chromatography−mass spectrometry (LC-MS).3−5 More recently Piriyapittaya et al. reported the microscale membrane extraction of glyphosate and AMPA followed by determination using LCfluorescence detection with postcolumn derivatization using O© 2017 American Chemical Society
Special Issue: 53rd North American Chemical Residue Workshop Received: Revised: Accepted: Published: 7294
January 31, 2017 April 4, 2017 April 7, 2017 April 7, 2017 DOI: 10.1021/acs.jafc.7b00476 J. Agric. Food Chem. 2017, 65, 7294−7304
Article
Journal of Agricultural and Food Chemistry Table 1. MRL of Selected Pesticides in Selected Commodities compounds in residue definition
cereal (mg/kg)
grapes (table)(mg/kg)
infant food (mg/kg)
2 1/0.05/1, barley, oat, wheat
0.5 1
0.01 0.01
2
100
0.01
0.1
0.15 (wine grapes, 0.15 in force as of Jan 14, 2017)
0.01
glyphosate
20/20/10, barley, oat, wheat
0.5
0.01
perchlorate chlorate
0.1 0.01 (proposed 0.04)
0.1 0.01 (proposed 0.015)
0.02 0.01
clopyralid ethephon fosetyl-Al
fosetyl-Ala phosphonic acid
glufosinate
glufosinate-ammoniumb N-acetyl-glufosinatec 3-MPPAc
a
Sum of fosetyl, phosphonic acid, and their salts, expressed as fosetyl. bSum of glufosinate and its salts. cMPP and NAG expressed as glufosinate equivalents.
Figure 1. Chemical structures of polar pesticides.
developed by the European Reference Laboratory for Single Residue Methods.16 The method is based on acidic methanol extraction without cleanup. Although the method is capable of extracting a wide range of polar analytes, the extracts can contain high concentrations of matrix-co-extractives that contaminate the instruments. The overall approach also requires the use of several different chromatographic separations (including HILIC and nonsuppressed ion exchange chromatography) to determine all of the anionic analytes listed in the method. In this work we have explored the use of ion chromatography with postcolumn suppression of the eluent to analyze a higher number of anionic analytes in a single analysis of extracts prepared using the QuPPe method. Granby et al.17 and Andersen et al.18 used ion chromatography with postcolumn suppression coupled to MS for the analysis of glyphosate. In this project we further explored the possibilities for the analysis of multiple polar analytes using a modern ion chromatography
limited to one or two analytes and have not always proved to be robust for the analysis of food extracts as control of pH is a critical factor. Other polar/ionic compounds that are coming under closer scrutiny are chlorate and perchlorate, which have been detected at high frequency in food in recent years. Chlorate and perchlorate predominately appear as byproducts of biocides used for cleaning food preparation facilities.12,13 Chlorate is still currently considered as a pesticide (registered as sodium chlorate); new EU maximum residue levels (MRLs) have been set for both compounds with challenging limits of detection as low as 0.01 mg/kg for some commodities.14 Table 1 lists the current EU MRLs for polar compounds/pesticides included in this study. There are other potential options for the direct analysis of these compounds, such as HILIC,15 but suppressed ion chromatography has been our technique of choice. A new multiresidue approach for the extraction of polar analytes is the quick polar pesticides method (QuPPe) 7295
DOI: 10.1021/acs.jafc.7b00476 J. Agric. Food Chem. 2017, 65, 7294−7304
Article
Journal of Agricultural and Food Chemistry
Figure 2. Ion chromatograph−tandem mass spectrometer configuration. All standards were prepared in deionized water at an approximate stock concentration of 1 mg/mL and given a 1 month shelf life. Mixed standards were prepared (excluding fosetyl-Al and phosphonic acid) at 100, 10, and 1 μg/mL. Single solutions of phosphonic acid and fosetylAl were prepared at 100, 10, and 1 μg/mL. Separate single standards were prepared due to fosetyl-Al degrading to phosphonic acid. Commodities Selected. Three different types of matrices were selected for the validation of the method: oat flour to represent group 5 (high starch and/or protein content and low water and fat content) of the SANTE/11945/2015 document; grapes to represent group 2 (high acid and high water content); and infant food (creamy porridge) to represent group 6 (“difficult or unique commodities”). Sample Preparation. Samples of organic oat flour, grapes, and infant food (creamy porridge) were purchased from a retail outlet. No further preparation work was carried out on the oat flour, which was stored at room temperature. The grape sample was cryogenically milled and stored at −20 °C until required. The creamy porridge was reconstituted, using deionized water, in the proportions recommended on the label instructions and stored at −20 °C until required. QuPPe Sample Extraction. For all three sample types investigated, the QuPPe extraction method15 was used, but with additional steps to reduce the particulate matter in the extracts. Homogenized cereal (oat flour) samples (5 ± 0.05 g) were each weighed into 50 mL polypropylene centrifuge tubes. Samples were spiked with internal standards and native standards as appropriate and left to stand (10 min). Deionized water (9.5 mL) was added, followed by 10 mL of acidified methanol (1% formic acid). The extracts were mixed using a rotary shaker for 20 min. Samples were then were placed in a freezer at −20 °C for 10 min on a bed of dry ice. Afterward, the samples were centrifuged at 4500 rpm for 5 min at 4 °C. Once centrifuged, the falcon tubes containing the samples were placed on a bed of dry ice to keep the supernatant cold with the supernatant filtered through a mixed cellulose syringe filter (0.22 μm) as soon as possible after centrifugation. The final extract was diluted 10-fold with deionized water in a plastic 2 mL vial ready for determination using IC-MS/MS. Matrix-matched calibration standards were prepared by preparing the highest concentration calibration standard in matrix blank (spiked with internal standard after extraction) followed by serial dilution with the same matrix blank that had been spiked with internal standards after extraction. Matrix-matched calibration standards were prepared at 0.2, 0.1, 0.05, 0.025, 0.01, and 0.005 mg/kg (0.05, 0.025, 0.0125, 0.00625, 0.0025, and 0.00125 μg/mL before 1/10 dilution), for all compounds except phosphonic acid, which was at concentrations of 4, 2, 1, 0.5, 0.2, and 0.1 mg/kg (equivalent to 1, 0.5, 0.25, 0.125, 0.05, and 0.025 μg/mL before 1/10 dilution), and fosetyl-Al. Matrix-matched
system. Compared to these reports we were able to use a KOH mobile phase instead of carbonate mobile phases, electrolytic eluent generation, and electrolytic postcolumn suppressors in place of hollow membrane suppressors. All advancements that provide greater capacity for high concentrations of matrix and, thus, greater retention of analytes, improved the robustness and stability of retention times and improved peak shape and resolution. In addition, advancements in MS technology provided higher sensitivity, especially at low m/z values. The aim of this work was to investigate the suitability of using suppressed ion chromatography coupled to tandem mass spectrometry (IC-MS/MS) for the analysis of polar pesticides including glyphosate, AMPA, N-acetyl-AMPA, glufosinate, 3MPPA, ethephon, chlorate, perchlorate, fosetyl-Al, phosphonic acid, clopyralid, bialaphos, and cyanuric acid in a single injection and in diverse matrices. The chemical structures of the compounds of interest are illustrated in Figure 1. The extraction method used was the QuPPe-PO (QuPPe method for products of plant origin) method, version 9.2, published by the European Union Reference Laboratory for Single Residue Methods (CVUA Stuttgart), but with slight operational modifications for the matrices used.
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MATERIALS AND METHODS
Reagents and Chemicals. An Elga Purelab Ultra (Veolia Water Technologies UK, High Wycombe, UK) was used to provide deionized water with a purity of 18.2 MΩ. Formic acid (98−100% Certified AR), methanol (HPLC grade), acetonitrile (Optima UHPLC/MS grade), and Kinesis Mixed Cellulose syringe filters (30 mm, 0.22 μm) were purchased from Fisher Scientific (Loughborough, UK). Glufosinate ammonium, N-acetyl-glufosinate, 3-methylphosphinicopropionic acid (3-MPPA), glyphosate, aminomethyl phosphonic acid (AMPA), phosphonic acid (neat reference standard materials), and ethephon-D4 (solution, 100 ng/μL) were purchased from QMx (Essex, UK). Ethephon, fosetyl-aluminum, cyanuric acid, clopyralid, and cyanuric acid-13C3 were purchased from Sigma-Aldrich (Dorset, UK) as neat materials with chlorate and perchlorate as stock solutions at 1000 μg/mL. Bialaphos, glufosinate-d3 hydrochloride, N-acetylglufosinate-d3 disodium salt, 3-methylphosphinicopropionic acid-d3 sodium salt, and 13C215N glyphosate were purchased from Toronto Research Chemicals (Toronto, Canada). N-Acetyl-AMPA was purchased from Carbosynth Limited (Compton, UK). 7296
DOI: 10.1021/acs.jafc.7b00476 J. Agric. Food Chem. 2017, 65, 7294−7304
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Journal of Agricultural and Food Chemistry standards for fosetyl-Al were prepared separately at 4, 2, 1, 0.5, 0.2, and 0.1 mg/kg. Homogenized grape samples (10 ± 0.1 g) were weighed into a 50 mL polypropylene centrifuge tube. Samples were spiked with internal standard and native standards as appropriate and left to stand for 10 min. Deionized water (2 mL) was added, followed by 10 mL of acidified methanol (1% formic acid). The sample was then placed on a rotary shaker for 20 min. Afterward, the samples were centrifuged at 4500 rpm for 5 min. Supernatant was then filtered through a mixed cellulose syringe filter (0.22 μm). The final extract was diluted 10-fold with deionized water and an aliquot transferred to a plastic 2 mL vial ready for IC-MS/MS analysis. Plasticware was used throughout to avoid adsorption of the analytes onto glass surfaces. Matrix-matched calibration standards were prepared by making the top calibration standard in the matrix blank (spiked with internal standard after extraction) and then serial dilution with blank that had been spiked with internal standards after extraction. The following concentrations were used to calibrate the IC-MS system: 0.2, 0.1, 0.05, 0.025, 0.01, and 0.005 mg/kg (0.1, 0. 05, 0.025, 0.0125, 0.005, and 0.0025 μg/mL before 1/10 dilution), where phosphonic acid was at concentrations of 1, 0.5, 0.25, 0.125, 0.05, and 0.025 mg/kg (0.5, 0.25, 0.125, 0.00625, 0.0025, and 0.0125 μg/mL before 1/10 dilution). Separate matrix-matched standards for fosetyl aluminum only were prepared at concentrations of 4, 2, 1, 0.5, 0.2, and 0.1 mg/kg and by serial dilution of the top calibration standard prepared in matrix. For infant food samples the same method was followed as for grapes with the exception that only 1 mL of deionized water was added. Matrix-matched calibration standards were prepared at the following levels: 0.1, 0.05, 0.025, 0.0.1, 0.005, and 0.0025 mg/kg (0.05, 0.025, 0.0125, 0.005, 0.0025, and 0.00125 μg/mL before 1/10 dilution). Matrix-matched standards for fosetyl aluminum only were prepared separately at 0.1, 0.05, 0.025, 0.01, 0.005, and 0.0025 mg/kg. IC-MS/MS Analysis. The IC-MS/MS analysis was performed using a Thermo Scientific Dionex ICS-5000+ Reagent Free HPIC system (Sunnyvale, CA, USA) coupled to a Thermo Scientific TSQ Quantiva triple-quadrupole mass spectrometer (San Jose, CA, US). The ion chromatography separation column was a Thermo Scientific Dionex IonPac AS19-4 μm (2 × 250 mm, 4 μm particle size) with a guard column Dionex IonPac AG19-4 μm (2 × 50 mm) maintained at 30 °C. The eluent flow rate was 0.35 mL/min with a gradient from 5 mM KOH (aq) to 20 mM KOH (aq) at 8 min, then to 60 mM KOH (aq) at 12 min, held at 60 mM KOH (aq) until 22 min, and back to 5 mM KOH (aq) at 22.1 min, with a cycle time of 26 min. The KOH eluent was neutralized using a Dionex AERS 500e 2 mm electrolytically regenerated suppressor (Thermo Scientific). The injection volume was 100 μL of the extract diluted 10-fold with water. Figure 2 shows the system configuration. The TSQ Quantiva was tuned using an extended mass range solution (Thermo Fisher Scientific P/N 88340) with the following ions in negative mode: m/z 69, 113, 302, 602, 1033, 2233, and 2833. Multiple-reaction monitoring (MRM) acquisition was conducted in the ESI negative mode. Table 2 lists the details for the MS method. The vaporizer temperature was set to 250 °C and the ion transfer tube temperature to 350 °C. The following recommended values were set for gas flows: sheath gas, 42; auxiliary gas, 12; and sweep gas, 1, with the spray voltage set to −3000 V. Thermo Scientific TraceFinder 3.2 software was used for instrument control and data acquisition. Data Analysis. TraceFinder 4.0 was used for data analysis. Glyphosate, glufosinate, 3-MPPA, N-acetyl-glufosinate, chlorate, perchlorate, ethephon, and cyanuric acid were all internally standardized using labeled parent compounds. AMPA and N-acetyl-AMPA were internally standardized against N-acetyl-glufosinate-d3 as this eluted closely to these compounds in the chromatographic run. Bialaphos, phosphonic acid, and fosetyl-Al were not internally standardized. The validation results are presented with recoveries calculated using internally standardized external calibration. Calibration criteria were set at an R2 value of ≥0.95 and residuals for the calibration graph within ±20%. Data generated from calibration graphs that do not meet these criteria are identified in the relevant tables.
Table 2. Information on MS/MS Transitions (Quantification Transitions in Bold) retention time (min)
precursor (m/z)
product (m/z)
collision energy (V)
fosetyl-Al
5.8
109.1 109.1 109.1
62.9 78.9 80.9
32 24 13
clopyralid
8.7
190.0 192.0
145.8 147.9
10 10
chlorate
9.8
IS-chlorate
9.8
83.1 83.1 85.1 89.1
66.9 50.9 68.9 70.9
22 33 22 22
bialaphos
10.5
322.2 322.2 322.2
88.0 94.0 133.9
30 31 31
glufosinate
12.2
IS-glufosinate
12.2
180.1 180.1 183.1
62.9 136.0 62.9
39 18 39
AMPA
11.7
110.1 110.1
80.9 78.9
13 31
N-acetyl-AMPA
11.7
152.1 152.1 152.1
62.9 78.9 110.0
31 33 11
N-acetylglufosinate
11.7
IS-N-acetylglufosinate
11.7
222.2 222.2 222.2 225.2
62.9 133.9 136.0 62.9
49 21 23 49
3-MPPA
12.2
IS-3-MPPA
12.2
151.1 151.1 154.1
62.9 132.9 62.9
35 12 35
phosphonic acid
12.4
81.1 81.1
62.9 78.8
31 12
ethephon
12.6
IS-ethephon
12.6
143.1 143.1 147.1
106.9 78.9 110.9
10 21 10
glyphosate
15.1
IS-glyphosate
15.1
168.0 168.0 171.0
62.9 78.9 62.9
25 40 25
cyanuric acid
15.5
IS-cyanuric acid
15.5
128.0 128.0 131.0
42.0 85.0 43.0
28 13 28
perchlorate
19.3
IS-perchlorate
20.0
99.0 99.0 99.0 101.0 107.0
82.9 66.9 50.9 84.9 88.9
27 39 47 28 27
compound
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DOI: 10.1021/acs.jafc.7b00476 J. Agric. Food Chem. 2017, 65, 7294−7304
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Journal of Agricultural and Food Chemistry
Figure 3. Effect of solvent composition on the peak shape of glufosinate. Matrix effects were calculated as per eq 1 for all validation batches using data not subject to internal standard correction.
transitions were optimized using the automatic optimization function in the TraceFinder software. At least two transitions were selected for each analyte to allow identification of the analytes across the concentration range used in the validation study. The addition of an organic solvent modifier, after the conductivity detector (inline after the suppressor) and before the MS, was investigated as a possible aid to desolvation within the ion source. The use of methanol was quickly discounted due to the high viscosity (0.55 cP at 20 °C), which would significantly increase the backpressure on the IC electrolytic suppressor. Acetonitrile proved to be the most suitable solvent due to its lower viscosity, (0.36 cP at 20 °C). The optimum flow rate was found to be 0.2 mL/min, giving a total flow into the source of 0.55 mL/min, which is acceptable. The analyte responses were improved by a factor of 2.8−6.3 as shown in Table 3, which clearly demonstrate the benefits of using a postsuppressor modifier. This additional flow of acetonitrile was sufficiently low to ensure the backpressure on the suppressor was below 150 psi. The optimum temperatures for the vaporization and ion transfer tube were investigated, and 300 and 250 °C gave the best responses for the analytes of interest. The optimum electrospray spray voltage was −3000 s. Validation Results: Cereal (Oat Flour). The cereal matrix was validated at three concentrations, 0.01, 0.05, and 0.1 mg/kg (fosetyl-Al and phosphonic acid were validated at 0.2, 1, and 2 mg/kg to reflect the significantly higher EU MRL). Sample chromatograms for the analytes in the method are displayed in
single suppression/enhancement (SSE) (%) = gradient matrix‐matched standards/gradient neat solvent standards × 100
(1)
The SANTE/11945/2015 Guidance document on analytical quality control and method validation procedures for pesticide residues in food and feed19 was used to verify that the method was fit for purpose.
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RESULTS AND DISCUSSION Ion Chromatography Optimization. A series of IC separation methods were investigated using the AS19-4 μm column with the starting method based on an already established method used at Fera.20 The starting conditions were altered to increase the retention of fosetyl aluminum on the system with the method selected giving sufficient retention of fosetyl-Al (at just over 2 column void volumes) and allowing for the partial separation of analytes during the 10−12 min region of the chromatogram. To maintain acceptable chromatography, it was necessary to dilute the extracts 10fold with water prior to the injection of 100 μL. This approach caused less distortion of the peak shape for glufosinate compared to an injection of 10 μL of extract without dilution as shown in Figure 3. Optimization of Mass Spectrometer Method Settings. The analytes were infused individually into the TSQ Quantiva triple-quadrupole MS system. The parent ion to product ion 7298
DOI: 10.1021/acs.jafc.7b00476 J. Agric. Food Chem. 2017, 65, 7294−7304
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Journal of Agricultural and Food Chemistry
Glufosinate and the two relevant metabolites, 3-MPPA and N-acetyl-glufosinate, gave acceptable results for the relative recoveries, with all three analytes giving recoveries >80%, and all of the RSDs were ≤10%. Similar results were observed for the non-corrected recoveries except for glufosinate at the 0.01 mg/kg spike concentration level, where the recovery average was 42% with a very high RSD of 31%. All identification criteria passed for both ion ratios and retention times. All analytes demonstrated acceptable retention on column. Validation Results: Grapes. The grape matrix was validated at three concentrations, 0.01, 0.05, and 0.1 mg/kg (fosetyl-Al and phosphonic acid were validated at 0.1, 0.5, and 1 mg/kg to reflect the significantly higher EU MRLs). The results of the validation are listed in Table 4. All of the calibration graphs that were internally and externally standardized pass the calibration criteria. For all analytes both the non-corrected and corrected results show acceptable recoveries across all concentrations when both internally and externally standardized. RSDs were all below 20% with the exception of AMPA at 0.01 mg/kg, which gave recoveries outside specification when corrected, and when noncorrected gave a high RSD of 22%. For the grape validation all analytes passed the identification criteria in both ion ratio comparisons with the matrix standards and retention time. The retention time stability was similar to those observed in the oats validation batch. Figure 6 shows acceptable retention time stability for chlorate, glyphosate, and perchlorate in the grape validation. Validation Results: Infant Food (Creamy Porridge). The infant food matrix was validated at three concentrations, 0.005, 0.01, and 0.05 mg/kg. The results of the infant food validation are listed in Table 5. All of the calibration graphs for glyphosate and perchlorate that were internally and externally standardized pass the calibration criteria. Many of the analytes of interest were not detected using this approach. Some analytes were readily detectable with sufficient signal-to-noise in the matrix-matched calibration standards but not in the recovery samples, indicating that the analytes were not extracted using the QuPPe method. The decision to apply the standard QuPPe method and not evaluate the QuPPe for products of animal origin (QuPPe-AO)21 was based on problems observed at Fera in the past using the QuPPe-AO to determine glyphosate in milk products, where the glyphosate was not recovered following the cleanup listed in the method (dSPE using octadecylsilane (ODS)). The results for glyphosate show a pattern similar to those obtained for cereal with low non-corrected recoveries but acceptable relative recoveries with both sets of results giving RSDs of ≤12%. Glyphosate passes all of the identification criteria for both ion ratio and retention time identification, with Figure 7 showing acceptable retention time stability throughout the validation. The results for perchlorate have been corrected to account for an incurred residue of perchlorate at the 0.002 mg/kg level, calculated using standard addition (hence, the concentration levels have been listed as 0.007, 0.012, and 0.052 mg/kg). Perchlorate passes all of the identification criteria for both ion ratio and retention time identification, with Figure 7 showing acceptable retention time stability throughout the validation. Chlorate was also readily detected in all spikes, but there was a high incurred residue present in the sample at 0.038 mg/kg (calculated by standard addition), which is significantly higher than the current MRL of 0.01 mg/kg. This highlights one of the
Table 3. Organic Modifier (Post-suppressor) Effect on Polar Pesticide Responsea
compound glyphosate AMPA N-acetylAMPA glufosinate 3-MPPA N-acetylglufosinate perchlorate chlorate ethephon clopyralid fosetyl-Al phosphonic acid cyanuric acid
av peak area (n = 4) with post-suppressor MeCN
response increase (MeCN/no MeCN)
307,755 96,480 497,987
1,294,126 463,516 1,854,753
4.2 4.8 3.7
92,423 726,123 118,348
337,068 2,837,665 426,449
3.6 3.9 3.6
3,435,072 727,635 195,581 458,844 414,228 336,819
14,781,008 3,333,338 803,967 1,304,068 2,594,673 1,401,104
4.3 4.6 4.1 2.8 6.3 4.2
13,645
68,229
5.0
av peak area (n = 4) without post-suppressor MeCN
All evaluations were undertaken using a 20 μL injection volume of a 0.1 μg/mL solvent standard. a
Figure 4 and show that the method gives acceptable peak shapes for the analytes included in the method. The results of the cereal validation are listed in Table 4. All of the calibration graphs that were internally standardized pass the calibration criteria, but without the use of internal standard correction, the following analyte calibration graphs fail either on residuals tolerance or R2 value of
