A Rapid Miniaturized Residue Analytical Method for the Determination

Feb 19, 2014 - A Rapid Miniaturized Residue Analytical Method for the Determination of Zoxamide and Its Two Acid Metabolites in Ginseng Roots Using UP...
0 downloads 6 Views 1MB Size
Download PDF
Article pubs.acs.org/JAFC

A Rapid Miniaturized Residue Analytical Method for the Determination of Zoxamide and Its Two Acid Metabolites in Ginseng Roots Using UPLC-MS/MS Lynda V. Podhorniak* U.S. Environmental Protection Agency, Office of Pesticide Programs, Biological and Economic Analysis Division, Analytical Chemistry Branch, 701 Mapes Road, Fort George G. Meade, Maryland 20755-5350, United States S Supporting Information *

ABSTRACT: A miniaturized residue method was developed for the analysis of the fungicide zoxamide and its metabolites in dried ginseng root. The zoxamide metabolites, 3,5-dichloro-1,4-benzenedicarboxylic acid (DCBC) and 3,5-dichloro-4hydroxymethylbenzoic acid (DCHB), are small acid molecules that have not been previously extracted from the ginseng matrix with common multiresidue methods. The presented extraction method effectively and rapidly recovers both the zoxamide parent compound and its acid metabolites from fortified ginseng root. The metabolites are extracted with an alkaline glycine buffer and the aqueous ginseng mixture is partitioned with ethyl acetate. In addition, this method avoids the use of derivatization of the small acid molecules by using UPLC-MS/MS instrumental analysis. In a quantitative validation of the analytical method at three levels for zoxamide (0.007 (LOD), 0.02 (LOQ), and 0.2 mg/kg) and four levels (0.07 (LOD), 0.2 (LOQ), and 0.6 and 6 mg/kg) for both metabolites, acceptable method performances were achieved with recoveries ranging from 86 to 107% (at levels of LOQ and 3×, 10×, and 30× the LOQ) with 85% of the administered zoxamide dose was excreted, >94% of the administered dose of DCHB was eliminated unchanged in urine, and >92% of the administered dose of DCBC was recovered as unchanged in total excrement.5 Zoxamide is approved for use by potato growers to prevent disease. A residue analytical method was previously developed for potato crops to detect zoxamide and its metabolites, DCHB This article not subject to U.S. Copyright. Published 2014 by the American Chemical Society

Figure 1. Molecular structures of the parent pesticide, zoxamide; 3,5dichloro-4-hydroxymethylbenzoic acid (DCHB); and 3,5-dichloro-1,4benzenedicarboxylic acid (DCBC).

Special Issue: 50th North American Chemical Residue Workshop Received: Revised: Accepted: Published: 3702

December 2, 2013 February 10, 2014 February 12, 2014 February 19, 2014 dx.doi.org/10.1021/jf405403v | J. Agric. Food Chem. 2014, 62, 3702−3709

Journal of Agricultural and Food Chemistry

Article

to a final volume of 2.0 mL in acetonitrile for a final concentration 20 ng/mL zoxamide and 600 ng/mL DCHB and DCBC. A standard of zoxamide, used for the matrix-matched standards and for fortification of control ginseng extracts, was prepared by transferring 0.044 mL of the zoxamide intermediate standard (1.01 μg/mL) and diluting to a final volume of 2.0 mL in acetonitrile for a final concentration of 22 ng/mL. A 4 M NaOH solution was prepared by adding 10 mL of 10 M solution to a 25 mL volumetric flask and diluting to volume with Millipore water. The 1 M glycine buffer solution at pH 10 was prepared by dissolving 7.5 g of glycine with 60 mL of Millipore water in a 100 mL graduated cylinder, adding 3.5 mL of 4 M NaOH, and then diluting to 100 mL volume9−12 with Millipore water. Sample Preparation. A schematic of the extraction and cleanup procedure is shown in Figure 2. Dried, finely ground ginseng root (0.2

and DCBC.6 The metabolites, however, are not well recovered from the ginseng matrix using this method. Zoxamide, the parent compound, is well recovered from ginseng root by a modification of the commonly used multiresidue method known as the QuEChERS-based (for quick, easy, cheap, effective, rugged, safe) approach,7 which has gained international popularity for its extraction efficiency and ease of use. However, the small acid metabolites of zoxamide (DCHB molecular weight 221.04 and DCBC molecular weight 235.02) were not well recovered from ginseng root through the aforementioned QuEChERS multiresidue approach during the initial recovery trials in this work. Residue analytical methods are required by the EPA to determine the parent compound and metabolites of significance to enforce tolerances.8 The residue analytical method presented here is a miniaturized extraction that effectively recovered the small acid metabolites DCHB and DCBC as well as the zoxamide parent molecule and uses liquid chromatography coupled with tandem quadrupole mass spectrometry (UPLC-MS/MS). This method rivals the QuEChERS-based approach for its ease of use and rapid sample preparation time.

■

MATERIALS AND METHODS

Materials and Standards Preparation. Zoxamide and the zoxamide metabolite DCHB analytical standards, of 99.1 and 99.82% purity, respectively, were provided by the U.S. EPA National Pesticide Standard Repository. The zoxamide metabolite DCBC, of 98.8% purity, was provided by the U.S. Department of Agriculture InterRegional Research Project No. 4 (IR-4 Project, also referred to as the Minor Crop Pest Management Program). American ginseng root samples, untreated with pesticides, dried for 2 weeks at 35 °C, and finely homogenized using a Wiley mill, were also provided by the IR-4 Project. HPLC grade acetonitrile, ethyl acetate, toluene, and sodium hydroxide solution 40% were purchased from Fisher Scientific (Pittsburgh, PA, USA). Hydrochloric acid 37% ACS Reagent, reagent plus grade ≥99 glycine, and ammonium hydroxide solution 28−30% were purchased from Sigma-Aldrich (St. Louis, MO, USA). Supelclean ENVI-Carb SPE tubes, 0.5 g, 6 mL, were purchased from Supelco Analytical (Bellefonte, PA, USA). pH indicator strips (pH 0−2.5 and 0−14) were purchased from colorpHast (Gibbstown, NJ, USA). Eppendorf variable-volume (10−100 μL, 100−1000 μL) pipets were from Eppendorf AG (Hamburg, Germany), calibrated and certified. Separate stock solution standards of zoxamide and metabolite DCBC were prepared by dissolving approximately 25 mg in 25.0 mL of acetonitrile for approximately 1.0 mg/mL standard concentrations. Intermediate dilutions were prepared by transferring 1.0 mL of the stock solutions to separate 100 mL volumetric flasks and diluting to volume with acetonitrile for concentrations of 10.1 μg/mL zoxamide and 10.9 μg/mL DCBC. The metabolite DCHB standard was prepared by dissolving approximately 10 mg in 25.0 mL of acetonitrile for a stock standard concentration of approximately 0.4 mg/mL. An intermediate dilution was prepared by transferring 2.5 mL of DCHB stock standard to a separate 100 mL volumetric flask and diluting to volume with acetonitrile for a concentration of 10.6 μg/mL. A second intermediate dilution (1.01 μg/mL) of zoxamide standard was prepared by transferring 0.5 mL of the 10.1 μg/mL standard and diluting to a final volume of 5.0 mL in acetonitrile. A mixed solvent standard, used to prepare the matrix-matched standards for quantitation and for fortification of untreated control ginseng extracts, was prepared by transferring 0.1 mL of the metabolite intermediate standards (10 μg/mL) and diluting to a final volume of 2.0 mL in acetonitrile for final concentrations of 532 ng/mL DCHB and 545 ng/ mL DCBC. A second mixed standard, also used for the matrixmatched standards for quantitation and for fortification of control ginseng extracts, was prepared by transferring 0.040 mL of the zoxamide intermediate standard (1.01 μg/mL) and 0.113 and 0.110 mL of the metabolite intermediate standards (10 μg/mL) and diluting

Figure 2. Flowchart of the method for the analysis of zoxamide and its metabolites (DCHB and DCBC) in dried ginseng powder. g) was weighed into a screw-capped polypropylene 15 mL centrifuge tube. Glycine buffer (1 M), pH 10 (2 mL), was added and vigorously vortexed for 30 s. Millipore water (2 mL) was added and shaken vigorously by hand for 1 min. Both 1 N HCl (0.8 mL) and 12 N HCl (0.2 mL) were added to the aqueous extract and vigorously shaken for 1 min. Four milliliters of ethyl acetate was added and the centrifuge tube vigorously shaken for 1 min and centrifuged (Jouan Inc., 3703

dx.doi.org/10.1021/jf405403v | J. Agric. Food Chem. 2014, 62, 3702−3709

Journal of Agricultural and Food Chemistry

Article

Table 1. Analyte Name, Retention Time, Primary and Confirmatory Ion Transitions, and Molecular Formulas of Zoxamide and Its Metabolites compd

mol formula

mol wt

retention time (min)

LOD (ng/g)

primary and confirmatory ion transitions (P/C ratio %)

zoxamide DCHB DCBC

C14H16Cl3NO2 C8H6Cl2O3 C8H4Cl2O4

336.64 221.04 235.02

5.45 1.14 0.5

6.7 71 73

336, 186.9, 158.8 (2.2) 218.8, 174.8, 144.8 (2.05) 232.8, 188.8, 152.8 (2.6), 108.8 (1.9)

Fortification Studies. For all fortification studies, various amounts of spiking standards prepared in acetonitrile were added to 0.2 g test portions of dried control ginseng root in centrifuge tubes. The tubes containing fortified ginseng were vigorously vortexed to distribute the analytes through the ginseng test portions, and the tubes were uncapped to allow the acetonitrile solvent to evaporate prior to sample extraction. For quantification by external calibration, matrix-matched standards were used. Matrix-matched standards were prepared by extracting control ginseng samples (as described above under Sample Preparation including GCB cleanup) and then fortifying the dried extracts with the acetonitrile standards and adjusting the final volume to 0.5 mL with acetonitrile. For LOD and LOQ fortification studies of DCHB and DCBC, appropriate amounts of a mixed spiking standard containing DCHB (532 μg/mL) and DCBC (545 μg/mL) were added to 0.2 g of ginseng for final concentrations of 0.071 and 0.216 μg/g for DCHB and 0.073 and 0.221 μg/g for DCBC. The matrix-matched standards were prepared at concentration levels of 14.4, 28.7, 86.2, and 172.5 ng/mL for DCHB and 14.7, 29.4, 88.3, and 176.5 ng/mL for DCBC. For the LOD fortification study of zoxamide, an appropriate amount of a standard of zoxamide (22.4 ng/mL) was added to 0.2 g of ginseng to a final concentration of 0.007 μg/g. The matrix-matched standards were prepared at concentration levels of 0.605, 1.21, and 3.63 ng/mL for zoxamide. For the fortification studies of the LOQ of zoxamide and 3× LOQ of DCHB and DCBC, a mixed spiking standard prepared in acetonitrile containing zoxamide (20 ng/mL), DCHB (600 ng/mL), and DCBC (600 ng/mL) was added to 0.2 g of ginseng to final concentrations of 0.020 μg/g (zoxamide) and 0.6 μg/g (DCHB and DCBC). The matrix-matched standards were prepared at concentration levels of 1.18, 3.53, 7.07, and 14.1 ng/mL for zoxamide and 35, 105, 210, and 420 ng/mL for DCHB and DCBC. For the fortification studies of 10× LOQ of zoxamide and 30× LOQ of DCHB and DCBC, various amounts of the intermediate dilutions of 1.01 and 10 μg/mL were added directly to 0.2 g of ginseng to final concentrations of 0.2 μg/g (zoxamide) and 6.0 μg/g (DCHB and DCBC). The matrix-matched standards were prepared at concentration levels of 1.18, 3.53, 7.07, 14.1, 28.3, and 35.3 ng/mL for zoxamide and 35, 105, 210, 420, 830, and 1045 ng/mL for DCHB and DCBC. Statistics and Calculations. Averages and standard deviations from fortification studies were determined using Microsoft Excel 2003. Linear regressions, correlation coefficients, and analyte concentrations from fortification studies for the UPLC-MS/MS were calculated with the Waters Target Lynx/Mass Lynx software version 4.1 using the sum of peak area responses of the primary ion transition and the confirmatory ion transition.

Winchester, VA, USA) for 5 min at 2500 rpm at ambient temperature. The ethyl acetate layer (2 mL) was transferred to a second 15 mL centrifuge tube. Ethyl acetate (4 mL) was added to the first tube containing the ginseng and hand shaken again vigorously for 1 min and centrifuged for 5 min at 2500 rpm. The ethyl acetate layer (5−6 mL) was removed and added to the second tube (sum 7−7.5 mL) and vigorously vortexed for 10 s. A portion of the ethyl acetate extract (3.5 mL) was transferred to a third 15 mL tube and concentrated to dryness with nitrogen evaporation (N-EVAP, Organomation Associates, Inc., Berlin, MA, USA). Acetonitrile (1 mL) was added to the centrifuge tube, sonicated for 10 s, and vortexed for 10−15 s. A GCB SPE cartridge was prerinsed with acetonitrile to waste. The extract (1 mL) was transferred to the GCB and collected, without vacuum, in a 15 mL tube labeled fraction 1. Acetonitrile (5 mL) was added to the GCB and collected in the same tube. A solution of 30% toluene/70% acetonitrile (5 mL) was added to the GCB and collected in a second tube labeled fraction 2. Both tubes were concentrated to dryness using nitrogen evaporation. Acetonitrile (0.5 mL) was added to both tubes, vortexed for 15 s, sonicated for 15 s, and centrifuged for 5 min at 2500 rpm. The extracts were transferred to LC autosampler vials for UPLCMS/MS analysis. Sample preparation time for a set of 6−10 samples is 45 min or less. UPLC-MS/MS Analysis. A Waters Acquity ultraperformance liquid chromatograph (UPLC) was equipped with a Quattro Premier tandem quadrupole mass spectrometer and an Acquity HSS-T3 column (100 mm × 2.1 mm) of 1.8 μm particle size, all supplied by Waters (Milford, MA, USA), with a void volume of 0.24 mL13 and a dwell volume of 0.090 mL. Reverse phase operating conditions were as follows: an injection volume of 5 μL at ambient column temperature; a flow rate of 0.3 mL/min using a mobile phase of acetonitrile, H2O, and ammonium hydroxide prepared as two separate solutions: solvent A, 100% H2O and 1 mM ammonium hydroxide (500 mL of H2O + 34 μL of NH4OH); solvent B, 100% ACN and 3.5 mM ammonium hydroxide (500 mL of H2O + 120 μL of NH4OH). The initial conditions were set at 100% solvent A for 2 min using a 0.3 mL/min flow rate; a linear gradient was programmed within 2 min to 10 + 90 (solvent A + solvent B), followed by a 2 min linear gradient to 30 + 70 (solvent A + solvent B) continuing at 0.3 mL/min, and a step back to 100% solvent A for a 3 min equilibration for a total analysis time of 10 min. The mass spectrometer was operated by switching between positive (ESI+) and negative (ESI−) electrospray ionization (interchannel delay of 0.020 s) in the multiple reagent monitoring (MRM) mode. The ion source temperature was 130 °C and the desolvation temperature 450 °C. The cone gas flow (high-purity compressed nitrogen) was 50 L/h, and the desolvation gas flow was 800 L/h of nitrogen gas. The collision cell gas was ultrahigh-purity argon with a flow of 0.3 mL/min. Two precursor/product ion transitions were monitored for each compound, and the sum of the areas of both the target ion and the confirmatory ion were used as the total response for quantitation. Analytes were further confirmed when MRM ion transition ratios were within ±20% (absolute) of the ratios in standards. The MRM ion transitions, cone voltages, and collision energies used were as follows: for zoxamide (ESI+), 336.0 → 158.80 (30 V, 40 eV) and 336.0 → 186.9 (30 V, 20 eV); for DCHB (ESI−), 218.8 → 144.6 (25 V, 14 eV) and 218.8 → 174.8 (25 V, 10 eV); for DCBC (ESI−), 232.8 → 108.8 (10 V, 21 eV), 232.8 → 152.8 (10 V,14 eV), and 232.8 → 188.8 (10 V, 9 eV) (see Table 1). Because an external standard was used for quantitation, the retention times of the compound of interest in the standard and the same compound in the sample were ±0.1 min.

■

RESULTS AND DISCUSSION Extraction Procedure. Recoveries of >90% have been reported for the parent molecule, zoxamide, from ginseng using an acetonitrile extraction of the QuEChERS approach,2 but the acid metabolites are not well recovered from this matrix. Although a solution of 2% sodium bicarbonate in acetonitrile effectively extracts the metabolites from potato matrices,6 this solution yielded recoveries of only 30−40% for the acid metabolites in ginseng during the initial recovery trials in this work. To effectively extract the metabolites of zoxamide from ginseng, several different aqueous solutions of various pH values, including glycine buffer solutions,9−12 a sodium 3704

dx.doi.org/10.1021/jf405403v | J. Agric. Food Chem. 2014, 62, 3702−3709

Journal of Agricultural and Food Chemistry

Article

Table 2. Recoveries (Percent) of Zoxamide and Metabolites DCHB and DCBC Extracted from Dried Ginseng Powder Fortified at Three and Four Different Levels, Respectively zoxamide

DCHB

DCBC

validation level (replicates)

spike level (μg/g)

% mean recovery

% RSD

spike level (μg/g)

% mean recovery

% RSD

spike level (μg/g)

% mean recovery

% RSD

LOD (3) LOQ (5) 10× LOQ (3) 3× LOQ (5) 30× LOQ (3)

0.007 0.020 0.202

124 95 96

15 4 5

0.071 0.216

103 106

10 11

0.073 0.221

113 107

4 15

0.600 6.0

86 96

12 6

0.600 6.0

95 105

12 10

Figure 3. LC-MS/MS of ginseng root fortified with zoxamide at the LOQ of 0.020 ppm.

centrifugation, zoxamide recoveries were improved to >90% and the extraction efficiency of the metabolites remained the same. A 3.5 mL aliquot of the 7.5 mL ethyl acetate extract is taken through cleanup for instrumental analysis. The remaining 3.5 mL portion of ethyl acetate extract is reserved for a potential second analysis if necessary. A simple solid phase extraction (SPE) cleanup method using graphitized carbon black (GCB) was used for the complete recovery of the analytes. Two fractions were collected through the GCB and injected separately. The first, 5 mL of acetonitrile, captures approximately 30% of the zoxamide analyte. The second fraction of 5 mL of toluene/acetonitrile, 30:70, captures the remaining 70% zoxamide and the metabolites DCHB and DCBC. Two fractions are used because the zoxamide response is affected by matrix coextractants and more coextractants are present in the second, toluene, fraction. To calculate zoxamide recovery, the levels of zoxamide found in the first and second fractions are summed. The advantages of the GCB cleanup are simplicity and speed. The final acetonitrile extract is centrifuged prior to instrumental analysis. Alternatively, filtration with syringe filters should be tested for suitability for the full recovery of the analytes. Method Validation. Zoxamide and its metabolites, DCHB and DCBC, were analyzed using UPLC-MS/MS. Retention times, quantitation, confirmation, and regression coefficients of zoxamide were determined in the electrospray positive (ESI+) mode and DCHB and DCBC were determined in the electrospray negative (ESI−) mode. The extraction and

hydroxide solution, and a neutral acetonitrile/water solution, were evaluated. The results of these trials revealed that a glycine buffer solution (pH 10) effectively recovered both the parent molecule, zoxamide, and the metabolites DCBC and DCHB from the ginseng matrix with average recoveries >95 and >86%, respectively (Table 2). The 2% sodium bicarbonate/acetonitrile method, which was developed for the extraction of the metabolites from potato,6 yields poor recoveries of the metabolites from ginseng root and also required derivatization with diazomethane to methylate the metabolites to their methyl esters prior to GC determination. In the method presented here for ginseng root, derivatization with diazomethane is not required. The purified ginseng extract is directly injected into the LC-MS/MS without derivatization. Specifically, in this method, 0.2 g of ground, homogenized ginseng matrix is rendered alkaline with glycine buffer (pH 10) to extract the parent and metabolite molecules and then diluted with water. The solution is acidified with 1 N HCl to neutralize the acid metabolites and partitioned into ethyl acetate in preparation for LC analysis without the need for derivatization. Initially during the development of the method, the aqueous ginseng extracts were centrifuged prior to partitioning with ethyl acetate. When the aqueous extracts were centrifuged and decanted from the ginseng solids and then partitioned with ethyl acetate, the recoveries of the metabolites DCHB and DCBC were acceptable, but the extraction efficiency of the parent molecule, zoxamide, resulted in low recoveries (30%). By partitioning the water/buffer−ginseng mixture with ethyl acetate in the presence of the ginseng solids, followed then by 3705

dx.doi.org/10.1021/jf405403v | J. Agric. Food Chem. 2014, 62, 3702−3709

Journal of Agricultural and Food Chemistry

Article

Figure 4. LC-MS/MS of ginseng root control in ES+ at the retention time and transitions of zoxamide.

Figure 5. LC-MS/MS of ginseng root fortified with metabolite DCHB at the LOQ of 0.200 ppm.

Figures 3, 5, and 7 show chromatograms of ginseng root extract fortified at the LOQ of 0.020 μg/g for zoxamide and 0.2 μg/g for DCHB and DCBC metabolites. The peak shapes are consistent and Gaussian with baseline separations. No significant background interfering peaks are observed in the control ginseng chromatograms shown in Figures 4, 6, and 8. Instrumental Analysis. Weak acids, such as acetic acid and formic acid, are frequently used mobile phase modifiers that increase ionization of many analytes using positive ESI.14 However, neither formic acid nor acetic acid improved the response of the acid metabolites, DCHB and DCBC, in the ESI− mode. The neutral salts, ammonium formate and ammonium acetate, which are also commonly used modifiers in ESI+ mode, also did not result in improved ionization of the zoxamide metabolites in ESI− mode. The volatile base,

instrumental analysis method was validated at three different fortification levels for zoxamide including replicates at the limit of detection (LOD), the limit of quantitation (LOQ), and 10× the LOQ. Four different levels were evaluated for the studied metabolites, DCBC and DCHB, including the limit of detection (LOD), the limit of quantitation (LOQ), and 3× and 10× the LOQ. The LOD was defined by the fortification level with a signal-to-noise of a minimum of 3:1, which was determined by the Mass Lynx software and is the standard deviation of the noise data in the monitored range, for both the primary and confirmatory ion transitions; the LOQ was defined by 3× the LOD. Table 2 lists the results of method validation. In general, the regression coefficients r2 for the matrix-matched calibration curves of each analyte were as follows: zoxamide ranged from 0.986 to 0.998; DCHB, r2 > 0.995; and DCBC, r2 > 0.988. 3706

dx.doi.org/10.1021/jf405403v | J. Agric. Food Chem. 2014, 62, 3702−3709

Journal of Agricultural and Food Chemistry

Article

Figure 6. LC-MS/MS of ginseng root control in ES− at the retention time and transitions of DCHB.

Figure 7. LC-MS/MS of ginseng root fortified with metabolite DCBC at the LOQ of 0.200 ppm.

were from 95 to 124% for zoxamide, from 86 to 106% for DCHB, and from 95 to 113% for DCBC. Matrix Effects. The use of ginseng matrix-matched standards for quantitation instead of solvent standards was necessary because of pronounced matrix effects from ginseng extracts. Although there were no significant matrix interferences, the presence of the ginseng matrix reduced the signal by approximately 3× for DCHB, 4× for DCBC, and 6× for zoxamide. Although recoveries at the LOD of DCHB and DCBC were generally consistent using ginseng matrix standards, recoveries of zoxamide at the LOD of 0.007 μg/g were less consistent. The challenge of the ginseng matrix was the efficient extraction of the metabolites of the parent molecule zoxamide. The miniaturized extraction presented here effectively recov-

ammonium hydroxide, resulted in the ionization of the small metabolite molecules DCBC and DCHB in the negative-ion mode ESI. A low concentration of 1 mM ammonium hydroxide in the aqueous mobile phase was sufficient for ionization in the ESI− mode, and higher molar concentrations did not improve ionization or detection limits. Fortification Studies. The recoveries from fortification studies of zoxamide and the metabolites DCHB and DCBC, evaluated by UPLC-MS/MS and based on external calibration using ginseng root matched standards, are shown in Table 2. The dried, powdered ginseng was fortified with zoxamide to levels of 0.007, 0.020, and 0.202 μg/g; DCHB to levels of 0.071, 0.216, 0.600, and 6.0 μg/g; and DCBC to levels of 0.073, 0.221, 0.600, and 6.00 μg/g. The mean recovery ranges for zoxamide and the metabolites at the aforementioned levels 3707

dx.doi.org/10.1021/jf405403v | J. Agric. Food Chem. 2014, 62, 3702−3709

Journal of Agricultural and Food Chemistry

Article

Figure 8. LC-MS/MS of ginseng root control in ES− at the retention time and transitions of DCBC. (3) 49110 Federal Register/Vol. 66, No. 187/Wednesday, Sept 26, 2001/Rules and Regulations, Environmental Protection Agency 40 CFR Part 180 [OPP-301176; FRL-6803-7] RIN 2070-AB78 Zoxamide 3,5-dichloro-N-(3-chloro-1-ethyl-1-methyl-2-oxopropyl)-4-methylbenzamide; Pesticide Tolerance; http://www.gpo.gov/fdsys/pkg/FR2001-09-26/pdf/01-23640.pdf. (4) European Commission Health and Consumer Protection Directorate-General Directorate E − Food Safety: plant health, animal health and welfare, international questions E1 − Plant health Zoxamide SANCO/10297/2003-Final. 4 February 2004 Appendix II End Points and Related Information 1. Toxicology and metabolism, 5 Aug 2003; p 9; http://ec.europa.eu/food/plant/protection/ evaluation/newactive/zoxamide.pdf. (5) National Library of Medicine HSDB Database TOXNET Toxicology Data Network; http://toxnet.nlm.nih.gov/cgi-bin/sis/ search/a?dbs+hsdb:@term+@DOCNO+7278. (6) Ipin Guo, I.; Kurilla, K.; Hofmann, C. Tolerance Enforcement Method for Parent RH-7281 and Its Two Acid Metabolites, RH-1452 and RH-1455, in Potato Peel Waste: Lab Project Number: 34-00-49, 2000; unpublished study prepared by Rohm and Haas Company; 85 pp; http://www.epa.gov/pesticides/methods/rammethods/2001_ 009M.pdf. (7) Wong, J.; Hao, C.; Zhang, K.; Yang, P.; Banerjee, K.; Hayward, D.; Iftakhar, I.; Schreiber, A.; Tech, K.; Sack, C.; Smoker, M.; Chen, X.; Utture, S. C.; Oulkar, D. P. Development and interlaboratory validation of a QuEChERS-based liquid chromatography-tandem mass spectrometry method for multiresidue pesticide analysis. J. Agric. Food Chem. 2010, 58, 5897−5903. (8) U.S. Environmental Protection Agency, Prevention, Pesticides and Toxic Substances (7101) EPA 712-C-96-174, Aug 1996; Residue Chemistry Test Guidelines OCSPP 860.1340, (b) (1) p 1, Residue Analytical Method; http://fedbbs.access.gpo.gov. (9) Identification of Poisons and Toxins by GC/MS Food Safety and Inspection Service, Office of Public Health Science. U.S. Department of Agriculture Food Safety and Inspection Service, Office of Public Health Science, May 5, 2013; http://www.fsis.usda.gov/horses/CLGTOX1.pdf. (10) Crockett, D.; Lorrie Lin, L.; Mulligan, K. SOP No: FERNCHE.0006.00, USFDA, ORA Forensic Chemistry Center, 2010; pp 1− 21; unpublished.

ered the acid metabolites 3,5-dichloro-4-hydroxymethyl benzoic acid and 3,5-dichloro-1,4-benzenedicarboxylic acid at a LOQ of 0.2 ppm as well as the parent, zoxamide, at a LOQ of 0.02 ppm from fortified ginseng root samples. The method uses a pH 10 1 M glycine buffer and also uses a simple graphitized carbon black cleanup and UPLC-MS/MS for quantitation. Unlike other single-analyte or specialized methods, this extraction method is simple and rapid. This analytical method is suitable for the EPA approved use of the fungicide zoxamide for ginseng root crops.

■

ASSOCIATED CONTENT

S Supporting Information *

Additional information about the dried control ginseng samples. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We thank the USDA IR-4 Project, especially Johannes Corley and Dan Kunkel, for providing ground ginseng roots for blanks used to prepare the matrix-matched standards and to fortify spiked samples and for providing analytical standards.

■

REFERENCES

(1) Method Validation and Quality Control Procedures for Pesticide Residues Analysis in Food and Feed, Document No. SANCO/12495/ 2011; pp 13, 24. (2) Wong, J.; Hennessy, M.; Hayward, D.; Krynitsky, A.; Cassias, I.; Schenck, F. Analysis of organophosphorus pesticides in dried ground ginseng root by capillary gas chromatography-mass spectrometry and -flame photometric detection. J. Agric. Food Chem. 2007, 55, 1117− 1128. 3708

dx.doi.org/10.1021/jf405403v | J. Agric. Food Chem. 2014, 62, 3702−3709

Journal of Agricultural and Food Chemistry

Article

(11) Mohan, C. Buffers: A Guide for the Preparation and Use of Buffers in Biological Systems; EMD Biosciences: Darmstadt, Germany, 2003, p 21. (12) Mulligan, K. J. General Method to Examine Foods and Beverages for Volatile and Semi-volatile Contaminants Using Acidic and Basic Extraction with Capillary gas Chromatography − Mass Spectrometry. U.S. FDA SOP T021(004); Elexnet −FERN/FERN Methods and SOPs/Documents/Chemical/Interim Methods, 2006. (13) http://www.chiralizer.com/colvol.htm. (14) Wu, Z.; Gao, W.; Phelps, M. A.; Wu, D.; Miller, D. D.; Dalton, J. T. Favorable effects of weak acids on negative-ion electrospray ionization mass spectrometry. Anal. Chem. 2004, 76 (3), 839−847.

3709

dx.doi.org/10.1021/jf405403v | J. Agric. Food Chem. 2014, 62, 3702−3709