Determination of Acid Herbicides Using Modified QuEChERS with

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Determination of Acid Herbicides Using Modified QuEChERS with Fast Switching ESI+/ESI− LC-MS/MS Chris Sack, John Vonderbrink, Michael Smoker, and Robert E. Smith*

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U.S. Food and Drug Administration, 11510 West 80th Street, Lenexa, Kansas 66224, United States ABSTRACT: A method for the determination of 35 acid herbicides in food matrices was developed, validated, and implemented. It utilizes a modified QuEChERS extraction procedure coupled with quantitation by liquid chromatography tandem mass spectrometry (LC-MS/MS). The acid herbicides analyzed are all organic carboxylic acids, including the older chlorophenoxy acid herbicides such as 2,4-dichlorophenoxyacetic acid (2,4-D), dicamba, 4-chlorophenoxyacetic acid (4-CPA), quinclorac, and many of the newer imidazolinone herbicides such as imazethapyr and imazaquin. In the procedure, 10 mL of water is added to 5 g of sample and then extracted with 1% formic acid in acetonitrile for 1 min. The acetonitrile phase is salted out of the extract by adding sodium chloride and magnesium sulfate, followed by centrifugation. The acetonitrile is diluted 1:1 with water to enable quantitation by LC-MS/MS using fast switching between positive and negative electrospray ionization modes. The average recoveries for all the compounds except aminocyclopyrachlor were 95% with a precision of 8%. The method detection limits for all residues were less than 10 ng/g, and the correlation coefficients for the calibration curves was greater than 0.99 for all but two compounds tested. The method was used successfully for the quantitation of acid herbicides in the FDA’s total diet study. The procedure proved to be accurate, precise, linear, sensitive, and rugged. KEYWORDS: acid herbicides, imidazolinone herbicides, chlorophenoxy acid herbicides, QuEChERS, LC-MS/MS

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the newer AcHs use ESI+ or incorporate both ESI− and ESI+ in separate analytical segments.17−25 Recent advances in technology have significantly increased the speed at which LC-MS/MS instruments can switch between ESI+ and ESI−. This enables AcHs and other pesticides to be determined by both ionization modes in a single analytical determination.26−28 Several variations of the QuEChERS method29 have been investigated and evaluated for the extraction and cleanup of various AcHs from complex matrices such as foods, feeds, and waste.10,12−14,16,17,19,22,27,30−36 Attempts to extract the acid herbicides with nonacidic acetonitrile used in the original QuEChERS method and CEN 1566229,37 have generally resulted in lower recoveries due to the ionic nature of the acids at pH above 5.10,12,17,22,30−32,34 QuEChERS extraction using acidified acetonitrile10,16,17,22,35,38 performed better, although the buffered approach described in AOAC 2007.01 has generally resulted in lower, but erratic recoveries.38 Investigators have explored SPE and dispersive SPE (dSPE) cleanup procedures of nonbuffered acidified acetonitrile QuEChERS extracts with mixed success.10,13,14,27,36 Sorbents investigated include graphitized carbon black (GCB), primary secondary amine (PSA), C18, and alumina. In each case, the cleanup resulted in acceptable recoveries for some acid herbicides and/or erratic recoveries for others. One investigator that studied matrix effects of extracts after multiple cleanup procedures found that cleanup procedures generally did little to reduce matrix effects.34 Others have described a method for

INTRODUCTION Herbicides are widely used in the USA and around the world for weed control and as plant growth regulators for agricultural crops, lawns, and gardens. Active ingredients in herbicides account for more than all the other types of pesticides combined, being over 60% of the U.S. sales in 2007.1 Fourteen of the top 25 most commonly used pesticides in the US in 2007 are herbicides, and included among them is the acid herbicide (AcH) 2,4-dichlorophenoxyacetic acid (2,4-D). Seven of the top 10 active ingredients used in the home and garden sector are herbicides; five are AcHs including mecoprop, dicamba, triclopyr, pelargonic acid, and 2,4-D, the most commonly used pesticide in the nonagricultural sectors. Historically, the AcHs were determined by gas chromatography with element selective detectors such as electron capture. Because they are not volatile, they were derivatized to form volatile esters.2−4 Although sensitive, these methods were inefficient and unreliable. Quantitation by liquid chromatography tandem mass spectrometry (LC-MS/MS) is quickly replacing the GC technology, eliminating the need for the derivitization step. In a recent review of chlorophenoxy acid herbicide methods, LC-MS/MS was the predominant technology cited.5 LC-MS/MS methods for the analysis of the older and smaller phenoxy acid herbicides such as 2,4-D, 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), and dicamba used predominately negative electrospray ionization (ESI−) because the acid herbicides are most easily ionized as their conjugate base.6−16 However, the newer AcHs such as quinclorac, quizalofop, fluoroxypyr, and the imidazolinones, although easily ionized using ESI−, actually produce transitional ions that are detectable by LC-MS/MS at lower concentrations than when using positive electrospray ionization (ESI+). Therefore, many multiresidue methods for This article not subject to U.S. Copyright. Published 2015 by the American Chemical Society

Received: Revised: Accepted: Published: 9657

August 20, 2015 September 28, 2015 October 16, 2015 October 16, 2015 DOI: 10.1021/acs.jafc.5b04093 J. Agric. Food Chem. 2015, 63, 9657−9665

Journal of Agricultural and Food Chemistry

Article

Table 1. LC-MS/MS Parameters transition

Q1a

Q3b

RTc

DPd

EPe

CEf

CXPg

2,3,6-TBA 1 2,3,6-TBA 2 2,4,5-T 1 2,4,5-T 2 2,4,5-TB 1 2,4,5-TB 2 2,4,5-TBA 1 2,4,5-TBA 2 2,4-D 1 2,4-D 2 2,4-DB 1 2,4-DB 2 4-CPA 1 4-CPA 2 acifluorfen 1 acifluorfen 2 aminocyclopyrachlor 1 aminocyclopyrachlor 2 aminopyralid 1 aminopyralid 2 bromoxynil 1 bromoxynil 2 chloramben 1 chloramben 2 clopyralid 1 clopyralid 2 clopyralid 3 clopyralid ×1 clopyralid ×2 dalapon 1 dalapon 2 dicamba 1 dicamba 2 dicamba 3 dichlorprop 1 dichlorprop 2 diclofop 1 diclofop 2 diflufenzopyr 1 diflufenzopyr 2 diflufenzopyr 3 fluoroxypyr 1 fluoroxypyr 2 haloxyfop 1 haloxyfop 2 imazamethabenz 1 imazamethabenz 2 imazamox 1 imazamox 2 imazapic 1 imazapic 2 imazapyr 1 imazapyr 2 imazaquin 1 imazaquin 2 imazethapyr 1 imazethapyr 2 MCPA 1 MCPA 2 MCPB 1 MCPB 2

224.6 222.6 252.6 254.8 282.9 280.8 222.7 224.7 218.7 220.6 246.6 248.6 184.7 186.7 359.8 359.8 214 214 207 207 273.4 275.4 203.6 205.6 192 194 194 192 192 140.6 142.6 218.7 220.4 218.7 232.6 234.5 324.9 326.9 335.1 335.1 333 254.8 254.8 359.8 361.9 275.1 275.1 306.1 306.1 276.1 276.1 262.1 262.1 312.1 312.1 290.1 290.1 198.6 200.7 226.8 228.5

180.8 178.9 194.9 196.9 196.8 194.8 178.8 180.8 160.9 162.9 160.9 162.9 126.9 128.9 316 194.9 68 168 160.9 188.9 78.8 80.8 159.9 161.9 110 175.9 147.8 173.9 146 96.9 98.9 175 176.9 35 160.9 162.9 253 255 206.1 162.1 128 208.8 180.8 288.1 290 144.1 89.1 261.1 193 231.1 163.1 217 149 199 128.1 245.1 177.1 140.9 142.9 141 143

5.27 5.27 7.01 7.01 8.12 8.12 7.29 7.29 6.23 6.22 7.23 7.23 5.55 5.55 7.34 7.34 2.91 2.91 3.42 3.42 5.96 5.96 4.86 4.86 4.21 4.21 4.21 4.21 4.21 4.01 4.01 5.47 5.47 5.47 6.85 6.85 8.42 8.42 5.91 5.91 5.89 5.24 5.24 7.84 7.84 4.59 4.59 4.79 4.79 4.83 4.83 4.44 4.44 5.37 5.37 5.19 5.19 6.34 6.34 7.28 7.28

−30 −30 −35 −60 −35 −35 −35 −35 −20 −35 −20 −35 −15 −40 −25 −25 20 20 35 35 −25 −20 −35 −35 25 25 25 25 25 −50 −40 −45 −30 −45 −20 −20 −25 −25 50 50 −50 60 60 −40 −35 5 5 5 5 10 10 20 20 5 5 5 5 −20 −30 −30 −20

−10 −10 −10 −10 −10 −10 −10 −10 −10 −10 −10 −10 −10 −10 −10 −10 10 10 10 10 −10 −10 −10 −10 10 10 10 10 10 −10 −10 −10 −10 −10 −10 −10 −10 −10 10 10 −10 10 10 −10 −10 10 10 10 10 10 10 10 10 10 10 10 10 −10 −10 −10 −10

−12 −12 −20 −18 −20 −16 −20 −16 −20 −20 −18 −18 −20 −20 −14 −38 29 23 29 19 −62 −60 −10 −12 49 16 30 17 31 −12 −12 −10 −10 −40 −18 −18 −22 −22 17 19 −34 21 31 −20 −20 45 83 29 35 27 37 27 35 39 63 29 37 −20 −20 −18 −18

−18 −18 −18 −18 −18 −18 −18 −18 −18 −18 −18 −18 −18 −18 −18 −18 15 15 15 15 −18 −18 −18 −18 15 15 15 15 15 −18 −18 −18 −18 −18 −18 −18 −18 −18 15 15 −18 15 15 −18 −18 15 15 15 15 15 15 15 15 15 15 15 15 −18 −18 −18 −18

9658

DOI: 10.1021/acs.jafc.5b04093 J. Agric. Food Chem. 2015, 63, 9657−9665


Article

Table 1. continued transition mecoprop 1 mecoprop 2 PCP 1 PCP 2 picloram 1 picloram 2 quinclorac 1 quinclorac 2 quizalofop 1 quizalofop 2 quizalofop 3 silvex 1 silvex 2 triclopyr 1 triclopyr 2

Q1a

Q3b

RTc

DPd

EPe

CEf

CXPg

212.7 214.6 264.4 262.4 242.9 240.9 241.9 241.9 345.1 343 347.1 266.6 266.6 255.4 253.3

141 143 35 35 196.9 194.9 223.9 161 299 271 301 194.8 158.9 197.9 195.8

6.9 6.9 9.45 9.45 4.52 4.52 5.33 5.33 7.76 7.76 7.76 7.64 7.64 6.69 6.69

−20 −30 −50 −60 20 20 20 20 5 −20 5 −30 −30 −35 −35

−10 −10 −10 −10 10 10 10 10 10 −10 10 −10 −10 −10 −10

−22 −24 −66 −68 31 31 23 53 25 −22 32 −18 −40 −18 −18

−18 −18 −18 −18 15 15 15 15 15 −18 15 −18 −18 −18 −18

a Q1: Precursor ion. bQ3: Product ion. cRT: Retention time. dDP: Declustering potential (V). eEP: Entrance potential (V). fCE: Collision energy (V). gCXP: Collision exit potential (V).

quantifying “very polar, non-QuEChERS-amenable pesticides in foods of plant origin”.39 In it, water is added to the dried sample, followed by homogenization and addition of acidified methanol, shaking, and thermal treatment (15 min at 80 °C) and centrifugation. The supernatant is analyzed by LC-MS/ MS.39 In this work, a method was developed and validated using an acidified QuEChERS approach without cleanup and combined with LC-MS/MS determination using fast switching between ESI+ and ESI− to analyze 35 different selected acid herbicides.

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Extraction and Cleanup. The samples (5.0 g) were placed in 50 mL centrifuge tubes, and 10 mL of deionized water was added. This was mixed for 10 min to allow for dry samples to soak up the water. [Alternative hydrolysis step: Add 300 μL of 5N NaOH to the sample (wetted as necessary) and mix; allow the sample to sit for 30 min, then add 300 μL of 5N H2SO4 and mix.] Then, 25.0 mL of extraction solvent (1% formic acid in acetonitrile) was added. For solid samples, two glass homogenizing beads were added. The cap was added to the tube, ensuring there are no leaks. This was shaken in a GenoGrinder at 1000 strokes/min for 1 min. Then, 6 g of anhydrous MgSO4 and 1.5 g of NaCl were added, followed by shaking in the GenoGrinder at 1000 strokes/min for 1 min. Next, the phases were separated by centrifugation for 10−20 min at 4500 rpm. The extracts were diluted with equal parts of deionized water, i.e., 250 μL of extract plus 250 μL of water. This mixture was vortexed for about 30 s. This was filtered through a 0.2−0.45 μm PTFE filter to obtain a final concentration of 0.1 g sample/mL. Validation. Eight TDS food items were selected for validation. Each item was analyzed once as a control and in duplicate at fortification levels listed in Table 2. Results for the method validation

MATERIALS AND METHODS

Analytical Standards. Mixed pesticide standards containing 32 of 35 compounds analyzed in the study (all at concentrations of 100 μg/ mL in methanol) were from Absolute Standards, Inc. (Hamden, CT). Neat standards of the remaining four compounds (aminocyclopyrachlor, imazamethabenz, 2,3,6-trichlorobenzoic acid (2,3,6-TBA), and 2,4,5-trichlorobenzoic acid (2,4,5-TBA)) were provided by the U.S. EPA (Fort Meade, MD). They were combined to make a 500 ng/mL stock solution containing all 35 compounds. The 500 ng/mL solution was further diluted in acetonitrile to make the fortification standards, while methanol was used for the injection standards. The calibration standard mixture containing all 35 compounds was diluted to 5 ng/mL with HPLC water (1:1) at the time of injection. LC-MS/MS Analyses. LC-MS/MS analyses were done using an AB Sciex 5500 QTrap (Framingham, MA), equipped with a Shimadzu Prominence XR HPLC (Kyoto, Japan) using scheduled multiple reaction monitoring (MRM) and rapid switching between ESI+ and ESI−. The switching time was 50 ms. The MRM detection window was 40 s, with a 5.0 ms pause. The target scan time was 0.25 s. The resolution was 1 Da. The ionization voltage was 4500 V. The source temperature was 450 °C. The gas flow for nebulizers 1 and 2 were 50 psi. The MS transition parameters are listed in Table 1. Analytes were separated at 40 °C on an Agilent Eclipse Plus C18, 1.8 μm, 4.6 mm × 75 mm column (Santa Clara, CA). A gradient elution was used that started with 1:9 CH3OH (v/v) and increased linearly to 7:3 CH3OH (v/v) from 0 to 3 min. This was then increased linearly to 95:5 CH3OH (v/v) from 3 to 8 min and held at this from 8 to 12 min. Then, it was decreased linearly to 1:9 CH3OH (v/v) from 12.0 to 12.5 min and kept at this from 12.5 to 14.5 min. The flow rate was 500 μL/ min and the injection volume was 5 μL. Samples. The samples that were analyzed were taken from the FDA total diet study (TDS), in which foods are prepared for consumption and subsequently comminuted using blenders and food processors. Validation items included white rice, cream of wheat, corn flakes, whole milk, 2% milk, apple, lettuce, and okra.

Table 2. Fortification Levels sample milk, whole milk, 2% white rice cream of wheat corn flakes apples iceberg lettuce okra

fortification levels (ng/g) 5, 10, 5, 10, 5, 10, 5, 10, 5, 10, 20 20 20

50 20, 50, 100 20, 50, 100, 200 50 50

were conducted for each individual transition that was analyzed. Average responses of the 5 ng/mL calibration standard for each analysis were used to calculate the concentrations and percentage of nominal concentration of the analytes using the external standard calibration method. Individual recoveries were calculated as the net recovery after subtracting the contribution from control samples. The linear range of each compound was evaluated by finding the lowest and highest levels over the fortification range that demonstrated consistent and reliable recoveries. Mean recoveries were simple averages calculated from all levels within the linear range of each compound. The relative standard deviation (RSD) was calculated from the standard deviation (SD) of the recoveries that fell within the linear range. The method uncertainty (MU) was two times the RSD. 9659



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Linearity was calculated as the coefficient of determination (r2) using the recoveries within the linear range. The method detection limit (MDL) was calculated as the three times the standard deviation of the calculated concentrations of the 5 ng/g spikes. The selectivity of the method was evaluated by examining the method blanks and control matrices. Validation acceptability criteria are: specificity, none in blank; recovery, 70−130%; relative standard deviation (RSD), ≤25%; linearity, correlation coefficient ≥0.99; minimum detection level (MDL), ≤10; initial calibration verification (ICV), 75−125%.

specificity of the transitions used requires these two compounds to be confirmed by an orthogonal technique. Fourteen of the 35 AcHs analyzed demonstrated more sensitive transitions using ESI+ including the six imidazolinones (imazamethabenz, imazamox, imazapic, imazapyr, imazaquin, imazethapyr), the three dichloropicolinic acids (aminopyralid, clopyralid, picloram), and quinclorac, fluoroxypyr, diflufenzopyr, quizalofop, and aminocyclopyrachlor. All of these compounds contain nitrogen, which serves to stabilize the positive ions. The sensitivity of the ESI+ transitions were generally 1−2 orders of magnitude more sensitive than the ESI− compounds. Interestingly, quizalofop and diflufenzopyr had ESI− transitions that were as sensitive as the ESI+ transitions; therefore, one of the ESI− transitions was included in the method as a third confirmational response. Once the optimal MS/MS transitional parameters for each compound were determined, the general MS/MS parameters of target scan time and ion spray voltage were studied and optimized. The individual transition responses were compared for ion spray voltages set at 4500 and 3000 V. While the average responses for the ESI+ transitions increased at 3000 V, the ESI− transitions responses were reduced slightly. Because the sensitivity of the ESI− transitions was weaker, the ion spray voltage of 4500 was selected. The target scan times for the ESI+ and ESI− were adjusted to the 500 × 500, 100 × 100, 200 × 200, 100 × 500, 100 × 900, and 200 × 200 ms, respectively, and the average responses of all transitions were determined. The best sensitivity was found when the target scan times for both ESI+ and ESI− experiments were set to 200 ms. Interestingly, time differentials favoring the ESI− transitions did not affect their responses. Selected MS/MS parameters for all compounds were combined into two experiments, one for ESI− and the second for ESI+. The experiments were analyzed consecutively using the AB Sciex scheduled MRM mode to minimize total cycle time. Next, the LC parameters were optimized. The ODS chromatographic column was chosen because of its excellent retention capacity, the 1.8 μm particle size, and its unique pressure-reducing dimensions (4.6 mm × 75 mm). Because the ESI± parameter studies indicated that the ESI− AcHs were significantly less sensitive than the ESI+ AcHs, optimization of the HPLC parameters focused on the latter. Initial attempts to chromatograph the AcHs using nonacidified and formic acid/ ammonium formate buffers in methanol/water resulted in poor chromatography and unacceptable sensitivity for many compounds. Formic acid (0.1%) proved to be the modifier resulting in the best sensitivity and chromatography. The lower pH of the buffered formic acid prevents the dissociation of the acid in the AcHs and ionization of residual silanols in the stationary phase, improving the chromatographic peak shapes. A brief study was conducted to compare acetonitrile vs methanol as organic modifiers. The ESI− AcH responses were determined in mobile phases made of 0.1% formic acid acetonitrile/water vs methanol/water using a simple gradient to elute them in less than 10 min. Although the peak height responses of a few compound transitions exhibited slight loss in sensitivity when using the methanol, most exhibited higher responses (up to 500%) than when using acetonitrile; the average increase in signal of all transitions was 44%. Methanol’s ability to enhance ESI ionization due its protic nature is wellknown.20,27 Once the mobile phase composition was selected, the retention was optimized so the earliest eluting compound,

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RESULTS AND DISCUSSION LC-MS/MS Method. The LC-MS/MS method enables acid herbicides to be quantified directly without the need for any derivitization and requiring minimal cleanup. The method was developed for the acidic herbicides using a QuEChERs method that was modified slightly by the adding 1% formic acid to the acetonitrile extraction solvent. The combined QuEChERS with LC-MS/MS determination was successfully validated per the FDA validation guideline, “Guidelines for the Validation of Chemical Methods for the FDA Foods Program”.40 The first step in method development was to optimize the LC-MS/MS parameters. So, the MRM parameters were optimized by infusing standard solutions containing 100−500 ng/mL of each individual AcH in 1:1 methanol:water (v/v) containing 1% formic acid at a flow rate of 10 μL/min using both ESI− and ESI+. The most abundant precursor/product ion combinations that were selected for each compound are listed in Table 1. Most of the older chlorinated acid herbicides were ionized using only ESI−. This included the phenoxy acids (2,4-D, 2,4DB, 2,4,5-T, 2,4,5-TB, 4-chlorophenoxyacetic acid (4-CPA), dichlorprop, diclofop, haloxyfop, 2-methyl-4-chlorophenoxyacetic acid (MCPA), 4-(4-chloro-o-tolyoxy)butyric acid (MCPB), mecoprop, silvex), the benzoic acids (dicamba, 2,4,5-TBA, 2,3,6-TBA, chloramben), pyridyloxy acid (triclopyr), and the aliphatic acid (dalapon). Only one significantly sensitive product ion could be produced for each of these compounds, so the chlorinated isotopic precursors with their corresponding product ions were used in the method.28 Some exceptions to this were found. Acifluorfen is a larger chlorinated benzoic acid for which multiple product ions were generated from the shared precursor ion. Quizalofop is a chlorinated phenoxy acid for which two isotopic precursor/ product ions in ESI+ were generated. However, an equally abundant ion was also produced using ESI−. Therefore, all three were included in the method. Diflufenzopyr is the only nonhalogenated compound with a strong product ion using ESI−. The remaining two compounds ionized using ESI−, bromoxynil and pentachlorophenol, are also the only two compounds included in the method that are not carboxylic acids. Neither of these compounds produced a significant product ion other than their corresponding isotopic halogen ions. Difficulties with ESI of pentachlorophenol have been reported by other investigators who found that the molecular ion was very difficult to fragment.41 They found that increasing collision energies up to 70 eV resulted in an acute signal decrease for both the molecular ion and fragment.41 Of four investigations for analysis of bromoxynil6,22,34,42 using LC-MS/ MS, only one was reported using a nonbromide ion transition of m/z 273.9 to 194.0 Da; however, this transition was found to be over 50 times less sensitive than the bromide ion transitions optimized for this work. Although the validation data for both bromoxynil and pentachlorophenol were excellent, the limited 9660



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Figure 1. Extracted ion chromatogram of 35 acid herbicides in standard. Imazamethabenz isomers integrated together in inset.

dilute the sample and reduce matrix effects (ion suppression). The sample was further diluted 1:1 with water to reduces chromatographic strength of extract and remove lipophilic coextractants by precipitation. Several investigators include a hydrolysis step prior to the acid solvent extraction of the AcHs;9,12,43−46 this was also the case for several state lab procedures reviewed. Many AcHs are formulated and applied as esters of the acid, and the hydrolysis step releases the free acid from the conjugate. Anastassiades suggests also that hydrolysis can free AcHs that have partially bound through esterification to components of the matrix such as commercial coating waxes or natural components in raw agricultural products.45 A common hydrolysis method is the addition of 300 μL of 5N NaOH to the sample (wetted as necessary) prior to extraction, allowing the sample to sit for 30 min, followed by acidification with 300 μL of 5N H2SO4. A quick study was conducted to confirm the effectiveness of this hydrolysis procedure on a sample of shredded wheat breakfast cereal. Average recovery of the 35 AcHs fortified at 50 ng/g was 89%, excluding partial recoveries of aminocyclopyrachlor. The same sample was fortified with 2,4-D ethyl ester at 50 ng/g, yielding a similar recovery of 85%. Having verified the effectiveness of the hydrolysis step for the analysis of the AcH esters and demonstrated that recoveries of the free acid AcHs were not adversely affected, the decision was made to exclude it from the validated method because only the nonesterified AcHs were evaluated and extraction efficiency studies were not conducted. Next, the method was validated. Summary results for the validation are reported in Table 3. Figure 2 contains chromatograms of quinclorac in the standard, rice control with 5 ng/g incurred residue, and rice fortified at 10 ng/g. Analysis of recoveries by matrix showed no correlation between them. Only aminocyclopyrachlor failed the majority of the validation criteria, mostly due to very low recoveries that ranged from less than 20% to as high as 40%. Thirty-two of the remaining 34 AcHs evaluated met all the criteria specified in the validation protocol. Average recovery of 34 AcHs is 95%, ranging from 87 to 107%. Method uncertainties (MUs) ranged from 8 to 36%; only 2,4,5-TB had an MU exceeding 30%. No false positive residue responses were found in the method

aminocyclopyrachlor, and the latest eluting compound, pentachlorophenol (PCP), eluted within 3−10 min. Because of the relatively few compounds analyzed during the run, overcrowding in the elution profile was not an issue for the MS/MS detection. Figure 1 shows an extracted ion chromatogram of the standard containing 35 AcHs. Note that peak due to imazamethabenz appears to be distorted because imazamethabenz is actually a mixture of two partially resolved structural isomers: 6-[(RS)-4-isopropyl-4-methyl-5-oxo-2-imidazolin-2yl]-m-toluic acid and 2-[(RS)-4-isopropyl-4-methyl-5-oxo-2imidazolin-2-yl]-p-toluic acid. Forcing the instrument to integrate them together simplified the determination and quantitation of imazamethabenz with no loss of method performance. Next, the QuEChERs method was developed. A simple study was conducted to compare acidified vs nonacidified ACN extraction of the selected AcHs. First, 10 g of dry breakfast cereal was fortified at 10 ng/g and extracted with 20 mL of ACN and 1% formic acid in ACN. The extract was then diluted 5× in mobile phase and determined using matrix matched standards. An average recovery for the acidified ACN was 105% compared to 63% for ACN alone, a finding consistent with other investigators.10,14,17,22,27,32,33,35 During the initial attempts to combine the acidified ACN extraction with the QuEChERS salt-out step, a problem was discovered with the dry breakfast cereals. The addition of 10 mL of water to 10 g of sample was insufficient to thoroughly wet the sample. By reducing the sample weight to 5 g, the problem was corrected for most matrices. For a couple of very dry breakfast cereals (oat ring and crisped rice cereals) even though the sample matrix was sufficiently moistened, the sample plus added water resulted in a doughy clump that was difficult to break up. Because the amount of QuEChERS salts (6 g of magnesium sulfate + 1.5 g of sodium chloride) have a capacity to remove over 15 mL of water from the ACN extract, a few extra mL of water (up to 5 mL) can be added to extremely dry samples to create a more fluid slurry that extracts easily. Because of the excellent sensitivity of the LC-MS/MS determination, the ACN volume was increased to 25 mL to 9661



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Poor recovery of aminocyclopyrachlor was not unexpected, as reported by Nanita et al.,44 who found the compound is not well extracted using the QuEChERS procedure. They attributed the low recoveries to the salting out step of the QuEChERS method and the high polarity and aqueous solubility of aminocyclopyrachlor, i.e., the majority of the compound remains in the aqueous layer. Because the LC-MS/MS determination procedure was acceptable, the method can be used qualitatively for screening purposes for this herbicide. For all other compounds, the method is found acceptable for the quantitative determination of the selected acid herbicides. Findings in Market Baskets. After the method was validated it was used in the TDS program for the analysis of 64 selected items per market basket collection. Three TDS market baskets were analyzed; Table 4 summarizes the findings for

Table 3. Validation Results acid herbicide

mean recovery

RSD

MU

linearity

MDL

2,3,6-TBA 2,4,5-T 2,4,5-TB 2,4,5-TBA 2,4-D 2,4-DB 4-CPA acifluorfen aminocyclopyrachlor aminopyralid bromoxynil chloramben clopyralid dalapon dicamba dichlorprop diclofop diflufenzopyr fluoroxypyr haloxyfop imazamethabenz imazamox imazapic imazapyr imazaquin imazethapyr MCPA MCPB mecoprop pentachlorophenol picloram quinclorac quizalofop silvex triclopyr

98 96 107 98 100 99 100 99 18 92 98 101 87 104 98 99 96 92 94 101 94 94 94 93 97 96 98 102 99 98 92 99 96 100 101

13 6 18 12 6 7 5 6 37 9 6 15 12 15 13 5 5 4 7 5 6 6 8 6 4 4 5 7 5 6 10 7 6 4 8

26 12 36 23 12 14 10 12 73 18 12 30 25 30 27 10 11 8 14 11 12 12 17 11 8 8 10 15 10 11 21 15 11 8 16

0.995 0.998 0.979 0.997 0.999 0.999 0.998 1.000 0.971 0.998 0.998 0.995 0.998 0.978 0.993 0.999 0.999 0.999 0.998 0.999 0.999 0.999 0.999 0.999 0.999 0.999 0.999 0.998 0.999 0.999 0.997 0.999 0.998 0.999 0.998

1.4 0.9 1.1 1.2 2.6 1.6 0.8 0.9 1.6 3.1 0.9 4.5 2.7 1.5 2.5 0.9 0.9 0.8 1.2 0.7 0.6 0.9 0.6 0.6 0.6 0.6 0.7 1.1 0.7 0.8 7.0 0.7 0.9 1.1 1.3

Table 4. Residue Findings (ng/kg) in Three Market Baskets freqa

min

max

2,4-D

25

0.3

8.7

clopyralid

17

0.4

39.1

imazamox

14

0.1

5.2

quinclorac triclopyr imazethapyr 4-CPA dicamba acifluorfen haloxyfop imazapic imazapyr

10 6 6 3 2 2 1 1 1

0.1 0.2 0.1 0.9 3 0.2 0.2 0.2 0.4

28 0.4 0.3 23 11 2 7.8 0.2 0.4

acid herbicide

examples of products in which they were found grain products: rice, wheat, rye, corn; vegetables: brassica, okra, celery; citrus fruits: orange, grapefruit; peanut products: peanuts, peanut butter; white beans; sunflower seeds grain products: oats, wheat, rye, corn, rice; vegetables: beets, cabbage grain products: wheat, corn, oats, rice; peas products; sunflower seeds grain products: rice, wheat, corn rice products; peanuts rice products; peas, peanut products; popcorn grain products: wheat, corn rice products peanut butter crisped rice cereal crisped rice cereal

a

Number of items in which residues were found in the 64 products that were analyzed from three TDS market baskets.

each pesticide. Of the 171 residues found, only 22 were above the default regulatory limit of 10 ng/g. As expected 2,4-D was the most frequently found acid herbicide, occurring 44 times in all products analyzed and in 25 of the 65 different items. The residue levels of all 2,4-D findings were below 10 ng/g. Residue levels of the second most frequently found pesticide (clopyralid, with 43 residues in 17 different items) were much higher, averaging over 9 ng/g and ranging from 0.4 to almost 40 ng/g. All clopyralid residue levels were within EPA tolerances listed for the products. Low levels of quinclorac, triclopyr, and acifluorfen were found almost entirely in rice

blanks, and all residues found in the control samples were verified by reanalysis, demonstrating the selectivity of the procedure. The highest MDL of 7 ng/g was determined for picloram, which exhibited the least sensitivity during the LCMS/MS determination. MDLs for all other compounds were less than 5 ng/g. Two compounds, dalapon and 2,4,5-TB, met all the criteria with the exception of the linearity requirement. The coefficient of correlation for both (0.978) was just below the limit of 0.99. The LC-MS/MS determination of both of these compounds was minimally sensitive.

Figure 2. Extracted ion chromatograms of quinclorac: (A) standard 50 ng/mL; (B) rice control sample containing 5 ng/g incurred residue; (C) rice control fortified at 10 ng/g. 9662



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products, consistent with the most prevalent use of those herbicides. Concentrations of triclopyr and quinclorac below 1 ng/g in peanuts in corn products, respectively, would most likely be due to unavoidable low level environmental contamination. Four of the seven imidazolinones analyzed by the procedure were found at very low levels. Imazamox was the most frequently reported, with 27 residues in 14 different products all at 5 ng/g and lower. Imazamox is exempted from tolerance requirements when used properly. Imazethapyr was found in rice products and peas, both of which are allowed uses. The remaining two imidazolinones (imazapyr and imazapic) were both found at concentrations below 1 ng/g in a sample of crisped rice cereal; no US tolerance is listed for either of these compounds in rice, the primary ingredient. However, crisped rice cereal also contains sugar which is derived from sources for which tolerances of imazapic and imazapyr are listed. A few trace level dicamba residues were found in corn and wheat grain products, consistent with previous findings. One additional herbicide, haloxyfop, was found in two of the TDS peanut butter samples, both at less than 10 ng/g. The presence of haloxyfop is unexplained, as there are no allowed uses for it in the U.S. and very limited usage worldwide. One interesting finding was the presence of 4-CPA in all peanut and peanut butter samples at violative levels ranging from 9 to 23 ng/g. 4-CPA has only one allowed use, i.e., as a plant growth regulator on mung beans during germination. The likely route of 4-CPA contamination in peanut products is as a degradant of the ubiquitous 2,4-D, which was also found in all peanut product samples. 4-CPA has consistently been shown to be a degradation product in extensive studies of the environmental fate of 2,4-D.47−51 EPA evaluation of the chronic toxicity of 4-CPA did not deem it a serious health risk. Because of the unexpected findings of 4-CPA residues in peanut products, an additional study of peanuts was conducted. Two samples of organic raw peanuts and one sample of roasted peanuts were collected from local grocery stores and analyzed. As can be seen in the Table 5, both 4-CPA and 2,4-D were found in each sample.

Table 6. Incurred Residue Levels (ng/kg) Using Hydrolysis vs No Hydrolysis item

4-CPA

2,4-D

shelled blanched raw peanuts shelled raw Spanish peanuts roasted peanuts in the shell

10 4 0.4

7 1 1

hydrolysisa

no hydrolysisa

2,4-D 4-CPA 2,4-D 4-CPA imazamox 2,4-D quinclorac imazamox 2,4-D clopyralid imazamox clopyralid clopyralid imazamox 2,4-D imazamox 2,4-D clopyralid 2,4-D

4.6 19 4.4 33 1.6 72 13 0.4 2.6 12 0.4 4 51 0.3 4.4 0.3 4.1 30 8.1

3.7 17 4 26 1.2 31 25 0.5 2.7 7 0.4 2.6 12 0.4 4.1 0.3 3.6 10 3.8

peanut butter peanuts sunflower seeds rice white bread

cornbread wheat bread

cracked wheat bread

orange a

Residue levels significantly different are in bold.

(72/31 in sunflower seed and 8.1/3.8 in orange); the 2,4-D residue levels were not significantly different in the other five items. No significant differences were seen in the residue levels of 4-CPA in two items (peanuts and peanut butter) and imazamox in five items (sunflower seeds, white bread, cornbread, wheat bread, and cracked wheat bread). Only quinclorac in rice had significantly lower residue levels (13 vs 25 ng/g) using the hydrolysis step. Recoveries of quinclorac were not significantly different between the two methods.

■

AUTHOR INFORMATION

Corresponding Author

*Phone: 913-752-2127. Fax: 913-752-2122. E-mail: Robert. [email protected].

Table 5. Concentraions (ng/g) of 4-CPA and 2,4-D Found in Different Peanuts product

acid herbicide

Notes

This work should not be taken as reflecting FDA policy or regulations. The authors declare no competing financial interest.

■

REFERENCES

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Comparison of Incurred Residue Levels Using the Alternative Hydrolysis Step. Selected TDS items found to contain residues using the method without hydrolysis were concomitantly analyzed using the alternate procedure that includes the hydrolysis step, and the results are listed in Table 6. As can be seen in the data, residues levels were comparable for most residue/item combinations; however, some some differences are notable. The incurred clopyralid residue levels of all items analyzed were significantly higher using the hydrolysis step ranging from 50% (4.0/2.6) in cornbread to over 400% (51/12) in wheat bread. Interestingly, the ratio between the multiple clopyralid transition responses demonstrated much better agreement between sample and standards when the hydrolysis step was used. Only two of the seven items containing 2,4-D had significantly higher levels using hydrolysis 9663



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