Article Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX
pubs.acs.org/JAFC
Residue Analysis of 60 Pesticides in Red Swamp Crayfish Using QuEChERS with High-Performance Liquid Chromatography−Tandem Mass Spectrometry Shuangyu Song,† Kechen Zhu,† Lijun Han,*,† Yelena Sapozhnikova,‡ Zihao Zhang,† and Wei Yao† †
College of Science, China Agricultural University, Beijing 100193, People’s Republic of China Eastern Regional Research Center, Agricultural Research Service, United States Department of Agriculture, 600 East Mermaid Lane, Wyndmoor, Pennsylvania 19038; United States
‡
S Supporting Information *
ABSTRACT: In this study, a multi-residue analytical method using quick, easy, cheap, effective, rugged, and safe (QuEChERS) extraction and dispersive solid-phase extraction (d-SPE) cleanup, followed by high-performance liquid chromatography−tandem mass spectrometry (HPLC−MS/MS), was investigated for rapid determination of 60 pesticide residues in whole crayfish and crayfish meat. The final method used 10 mL of acetonitrile for extraction, 3 g of NaCl for partitioning, and 50 mg of primary secondary amine for d-SPE cleanup. The method was validated at three spiking levels (10, 50, and 100 ng/g) using triphenyl phosphate as an internal standard and both gradient and isocratic HPLC elution. Under gradient conditions, satisfactory recoveries (70−120%) and relative standard deviations of ≤20% were achieved for 83 and 88% of pesticides in whole crayfish and crayfish meat, respectively. Matrix effects were estimated using both gradient and isocratic HPLC elution. To our knowledge, this is the first study involving multi-residue analysis of HPLC-amenable pesticides in crayfish and mantis shrimp. The final method was successfully applied for analysis of 11 crayfish and mantis shrimp samples from markets in China, and propamocarb (95% purity. Individual standard stock solutions were prepared at 1 mg/mL in MeCN. A standard mixture solution of 60 analytes was prepared at 20 μg/mL in MeCN and used for fortification and preparation of calibration standard solutions. TPP IS solution was prepared at 10 μg/ mL in MeCN. All solutions were stored at −20 °C. Sample Collection and Pretreatment. Crayfish and mantis shrimp samples for method evaluation were purchased from Jingshen Seafood Market in Beijing, China. To test the optimized method, 11 market samples, including 6 crayfish and 5 mantis shrimps, were acquired, with 2 fresh crayfish samples purchased from Shandong and Jiangsu and 4 frozen crayfish samples purchased from Hubei and Jiangsu. A total of 4 fresh mantis shrimp samples were collected from Liaoning, Jiangsu, Shandong, and Zhejiang, and 1 cooked (salted) mantis shrimp sample was obtained from Hebei, China. All samples were frozen and kept at −20 °C overnight. After thawing, the heads and tails were removed and the samples were processed into crayfish meat. Then, both whole crayfish and crayfish meat samples were homogenized using a MJ-BL25B2 mill (Media, Ltd., Beijing, China). A test portion was taken for determination of residues of 60 pesticides. All of the samples were stored at −20 °C until analysis. Sample Preparation. Homogenized samples (10.0 g) were weighed into 50 mL centrifuge tubes. For fortification experiments, blank samples were spiked with an appropriate volume of standard solution mixtures and kept at room temperature for 15 min. The IS (TPP) was added to all samples to yield 100 ng/g before the extraction, except for the samples designated for matrix-matched calibration standards. Then, 10 mL of MeCN was added, and the tubes were vigorously vortexed for 10 min with a Targin VX-III multi-tube vortexer. After that, 3.0 g of NaCl was added, and the tubes were vortexed for 1 min thoroughly as before. Then, the tubes were centrifuged at 3800 rpm (2260 rcf) for 5 min. For d-SPE cleanup, 1 mL of supernatant was transferred into a 2 mL microcentrifuge tube with previously weighed 50 mg of PSA. The mixture was vortexed for 1 min and then centrifuged at 10 000 rpm (4472 rcf) for 1 min. The upper layer was filtered through a 0.22 μm nylon filter membrane syringe into a LC autosampler vial for HPLC−MS/MS analysis. As for matrix-matched calibration standards, standard solution mixtures and IS (TPP) were added prior to HPLC−MS/MS analysis. HPLC−MS/MS Conditions. Agilent 6410B triple quadrupole HPLC−MS/MS (Agilent Technologies, Santa Clara, CA, U.S.A.) was employed with a HPLC reverse-phase C18 column (50 mm × 2.1 mm × 3.5 μm particle size). The column temperature was 25 °C, and the injection volume was 5 μL. We evaluated the performance of isocratic versus gradient elution for separation of 60 selected pesticides. Isocratic elution is shorter than gradient elution, saving instrumental analysis time. Gradient elution was performed with MeCN as mobile phase A and 0.1% formic acid in ultrapure water as mobile phase B at the flow rate of 0.2 mL/min. The gradient elution program was 0.0−0.5 min/1.0% A, 2.0 min/30% A, 8.0 min/70% A, 11.0 min/99% A, 17.5 min/1.0% A, and 25 min/1.0% A, and the total run time was 25 min. Isocratic elution was carried out with the same mobile phases A and B by the ratio of 70:30 at the flow rate of 0.2 mL/ min, and the total run time was 11 min. For the isocratic elution, the HPLC column was washed using a gradient washing program after each sequence of 30 injections to avoid a possible occurrence of ghost peaks. Tandem mass spectrometry (MS/MS) was carried out in multiple reaction monitoring (MRM) mode with positive electrospray ionization (ESI+). The two most intense MRM transitions were selected and optimized for each pesticide. Table S1 of the Supporting Information lists MRM parameters and typical tR for 60 analytes.
study, aquaculture catfish, rainbow trout, and crayfish samples were extracted with a Soxhlet apparatus, and the extracts were cleaned by liquid−liquid extraction and solid-phase extraction (SPE). Sample preparation was time-consuming and required large quantities of solvents. As a quick, easy, cheap, effective, rugged, and safe sample preparation method, the QuEChERS approach has been widely used in multi-residue analysis of pesticides and other contaminants in food matrices, including aquatic species. For example, a QuEChERS sample preparation has been successfully applied for determination of multi-residues of pesticides in fish tissues with low-pressure gas chromatography−tandem mass spectrometry (LPGC−MS/MS)14 as well as GC−MS,9 and QuEChERS combined with a filter-in-vial dispersive solidphase extraction (d-SPE) method was applied in pesticide analysis in shrimps with both LPGC−MS/MS and highperformance liquid chromatography−tandem mass spectrometry (HPLC−MS/MS).15 A modified QuEChERS method was also reported in determination of 23 organochlorine pesticides and 10 polychlorinated biphenyls (PCBs) in crayfish with gas chromatography−tandem mass spectrometry (GC−MS/MS) coupled with electron ionization (EI) and positive chemical ionization−tandem mass spectrometry (PCI−MS/MS).16 The sample preparation involved extraction with acetonitrile (MeCN) acidified with 0.1% HAc and saturated with hexane and cleanup with d-SPE with primary secondary amine (PSA) and C18 sorbents. Published papers on pesticide residue analysis in crayfish to date focus on the analysis of gas chromatography (GC)amenable pesticides, and we were unable to find published studies on the multi-residue analysis of liquid chromatography (LC)-amenable pesticides. However, many pesticides that are commonly used in paddy rice and lotus cultivation are relatively polar and could only be analyzed by the LC method. Therefore, the purpose of this study was to develop a fast, easy, and efficient method for residual analysis of LC-amenable pesticides commonly used in paddy and lotus fields in crayfish. We selected 60 target analytes, including 15 insecticides, 21 herbicides, and 24 fungicides. The pesticides were selected on the basis of the following criteria: (1) registered on rice crops, (2) applied in ponds, bottomland, woodland, paddy, and lotus fields, and (3) LC amenable. Additionally, metabolites of trifloxystrobin (trifloxystrobin acid), clodinafop (clodinafoppropargyl), and fenoxaprop-p-ethyl (fenoxaprop-p) were included in the target list. Because crayfish can be processed and packed into whole crayfish or crayfish meat (also called shelled crayfish tail) in markets, we investigated the method for both whole crayfish and crayfish meat. QuEChERS sample preparation and HPLC−MS/MS were used and optimized for the determination of 60 analytes in both matrices. As a novel sorbent for d-SPE cleanup, multi-walled carbon nanotubes (MWCNTs) were also tested for cleanup of crayfish samples. The applicability of the method to a similar seafood species, mantis shrimp, and samples of crayfish and mantis shrimp from farm markets were also investigated.
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MATERIALS AND METHODS
Materials. Ultrapure water was prepared with an ABW-6000-U water purification system from Aquapro International Company LLC (Dover, DE, U.S.A.). High-performance liquid chromatography (HPLC)-grade MeCN and PSA were purchased from CNW Technologies GmbH (Duesseldorf, Germany). Analytical-reagentgrade formic acid, acetic acid, ammonium formate (NH4O2CH), and B
DOI: 10.1021/acs.jafc.7b05339 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry Method Validation. To evaluate trueness and precision of the method, the fortified recovery study of the 60 pesticides in the crayfish sample (including whole crayfish and crayfish meat) was conducted at three spiking levels, 10, 50, and 100 ng/g, with five replicates for each level. Matrix-matched calibration curves were prepared and applied for accurate quantification of the analytes. Fortified recoveries normalized to IS and relative standard deviations (RSDs) were calculated for each pesticide, and the results were evaluated in compliance with the Codex Alimentarius Commission (CAC) guideline CAC/GL 90-2017, which requires average recoveries of 70−120% with RSD of ≤20% for spiking level of >10 ng/g and RSD of ≤30% for spiking level of ≤10 ng/g. TPP was used as an IS for all pesticides, as an inexpensive alternative for isotopically labeled IS. The linearity of calibration curves for each analyte was studied by injecting calibration solutions at concentrations of 5, 10, 50, 100, and 200 ng/mL in both MeCN and matrix extracts. Matrix effects (MEs) were calculated by the slope differences using the following equation: ME (%) = 100% × (slopematrix/slopeMeCN − 1).17 The applicability of the method on a similar crustacean seafood, mantis shrimp, was investigated, and the fortification study on the mantis shrimp was also conducted at the same three spiking levels with three replicates per level. After that, market samples of crayfish and mantis shrimp were analyzed.
between MeCN and the aqueous layer in QuEChERS extraction.14,19 Recently, Gonzalez-Curbelo et al.20 demonstrated the efficiency of NH4O2CH as a phase-out salt, which is more volatile and helps to reduce salt deposition in the MS source. In our study, to compare the performance of NaCl and NH4O2CH, 3 g of NaCl and 7 g of NH4O2CH were tested for phase separation. The results (Figure S2a of the Supporting Information) showed that NaCl and NH4O2CH displayed a similar performance and 50−51 pesticides achieved acceptable recoveries in both whole crayfish and crayfish meat. The only difference was in the recoveries of trifloxystrobin acid that was 66% when using NH4O2CH and 80% when using NaCl in whole crayfish. We also compared NaCl with and without MgSO4 and did not observe any apparent differences (Figure S2b of the Supporting Information). In total, 50−51 pesticides had acceptable recoveries in whole crayfish and crayfish meat. Regardless of the presence of MgSO4, the only difference was in recoveries of nitenpyram in crayfish meat, which was 70% with NaCl. As part of a typical QuEChERS approach, d-SPE cleanup with different sorbents is used for the cleanup of different matrices. Traditional QuEChERS called for different amounts of PSA and C18 combined with an appropriate amount of anhydrous MgSO4 to gain better cleanup.21,22 Research showed that PSA effectively removes interferences, such as fatty acids, organic acids, pigments, etc., while C18 removes relatively nonpolar components, such as lipids and waxes.23 A zirconiumbased sorbent Z-Sep was successfully applied for the determination of 143 pesticide residues in fish based on sample extraction with QuEChERS14 and PSA and C18 for d-SPE cleanup.15 MWCNTs are novel nanotube material that have been successfully applied in the QuEChERS d-SPE cleanup procedure to remove interferences in vegetable matrices for pesticide residue analysis. It demonstrated the effective removal of interferences as a result of its low particle size and high surface areas.24−27 To investigate efficient cleanup, different sorbent combinations were tested for d-SPE cleanup of crayfish samples: (A) no cleanup, (B) 50 mg of PSA, (C) 50 mg of PSA + 150 mg of anhydrous MgSO4, (D) 50 mg of PSA + 50 mg of C18 + 150 mg of anhydrous MgSO4, and (E) 50 mg of PSA + 5 mg of MWCNTs + 150 mg of anhydrous MgSO4. Fortified recoveries and RSDs were calculated and compared. The results showed that most of the analytes had similar recoveries using different sorbents; only some analytes showed lower (120%) recoveries as a result of either retaining on sorbents or matrix interferences. The recoveries and standard deviations (SDs) of 11 pesticides with significantly different results for different d-SPE approaches are presented in Figure 1. A comparison of 50 mg of PSA versus no cleanup showed very little difference, except that PSA retained pymetrozine, resulting in its low recovery in whole crayfish. In the case of crayfish meat, 50 mg of PSA worked better and RSD for dinotefuran was >20% without cleanup. In comparison to PSA only, PSA with MgSO 4 retained trifloxystrobin acid, nitenpyram, and penoxsulam, leading to low recoveries in whole crayfish. Additionally, florasulam and fenoxaprop-p were also retained in crayfish meat samples, resulting in fenoxaprop-p low recoveries (10%) when MgSO4 was used. While MgSO4 is added to remove residual water from QuEChERS extracts, it has been shown to enhance analyte retaining by PSA, which may explain the retaining of the above five analytes. The addition of C18 led to low recoveries of florasulam (120% and/or RSDs of >20% at the lowest spiking level (10 ng/g). The reason was mostly due to interferences in whole crayfish when using isocratic elution. The results for other analytes were similar to the results using gradient elution. Overall, isocratic elution saved a considerable instrumental run time; however, it led to higher recoveries and/or higher RSDs for 3−9 pesticides at a low spiking level probably as a result of interferences in matrices, which also negatively affected sensitivity for these analytes. Matrix Effects. MEs were tested under gradient and isocratic elution conditions, and the results were divided into “no MEs” (−20 < no MEs < 20%), “medium MEs” (20−50%), and “strong MEs” (MEs > ±50%). ME data listed in Table S5 of the Supporting Information and Figure 3 showed that, in isocratic elution conditions, more analytes had strong MEs and fewer analytes had no MEs than in gradient elution. For early eluting analytes (tR < 1 min), MEs were mostly < −50%, indicating high suppression. Figure 3 also showed that, in isocratic elution, more analytes showed matrix enhancement effects and less suppression effects than in gradient elution. This could be the reason for more analytes with strong matrix interferences causing recoveries of >120% or RSDs of >20% at a low spiking level, such as 10 ng/g. However, MEs were not significantly different between whole crayfish and crayfish meats. Typical MRM chromatograms of whole crayfish in gradient and isocratic elution for the blank control sample and medium-fortified sample was shown in Figure S3 of the Supporting Information. Method Application to Mantis Shrimp. Oratosquilla oratoria is known as mantis shrimp and is similar in composition and taste to crayfish seafood, which is widely consumed in Asia. Therefore, the applicability of the optimized method for crayfish was tested for mantis shrimp.
Figure 1. Percent recoveries and SDs (error bars) (100 ng/g; n = 3) for pesticides with significant differences using different cleanup sorbents for (a) whole crayfish and (b) crayfish meat.
(>120%) in whole crayfish; however, in crayfish meat, C18 addition increased recoveries of florasulam and trifloxystrobin acid but led to >120% recoveries of nitenpyram and penoxsulam. MWCNT, a novel nanocarbon material, retained five analytes, carbendazim, thiabendazole, tricyclazole, pyrimethanil, and pymetrozine, in both whole crayfish and crayfish meat, which resulted in low recoveries (21−62%). These analytes were also found to be retained by MWCNTs in vegetables and fruits in some studies.28−30 Overall, Table S2 of the Supporting Information shows that 44−55 pesticides had satisfactory recoveries (70−120%) and RSDs of ≤20% using different d-SPE sorbent combinations and 50 mg of PSA worked the best among the five options, resulting in 53−55 pesticides with satisfactory recoveries and RSDs in whole crayfish and crayfish meat. As a result, 50 mg of PSA was used for validation experiments. Method Validation. Fortified recoveries, linearity, and MEs were evaluated to assess the trueness and precision of the method performance with both gradient and isocratic elution conditions. In gradient elution, each run was 25 min, whereas in isocratic elution, each run was only 11 min. Linearity was tested in the range of 5−200 ng/g (5, 10, 50, 100, and 200 ng/g) for 60 analytes in both whole crayfish and crayfish meat matrices. The results showed that R2 was 0.945− 1.000 for the majority of pesticides, with only four analytes, hymexazol, nicosulfuron, trifluralin, and chlorpyrifos, having lower R2 values (0.668−0.806) as a result of variability at low concentrations. Table S3 of the Supporting Information lists the lowest calibrated level (LCL), signal-to-noise (S/N) ratio, limit of detection (LOD), limit of quantitation (LOQ), and ion ratio for the selected pesticides. LCLs were the lowest matrixmatched calibration standards for which identification criteria for tR, MRMs, and their ratios were met. LODs and LOQs were calculated at 3 and 10, respectively. The S/N ratio was for D
DOI: 10.1021/acs.jafc.7b05339 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry
Table 1. Percent Recoveries and RSDs for Whole Crayfish and Crayfish Meat (n = 5), and Mantis Shrimp (n = 3) Using Gradient HPLC Elutiona
pesticide propamocarb hymexazol pymetrozine dinotefuran carbendazim nitenpyram thiabendazole methomyl pirimicarb thiamethoxam clothianidin imidacloprid acetamiprid tricyclazole nicosulfuron pyrimethanil prometryn carbofuran florasulam atrazine flutriafol metalaxyl-M isoproturon isoprocarb penoxsulam cyprodinil prochloraz pyrazosulfuron-ethyl azoxystrobin halosulfuron-methyl mefenacet epoxiconazole boscalid fenoxaprop-p tebuconazole clodinafop-propargyl hexaconazole isoprothiolane metolachlor triazophos trifloxystrobin acid propiconazole thifluzamide buprofezin carfentrazone-ethyl kresoxim-methyl picoxystrobin difenoconazole anilofos clodinafop trifluralin pyraclostrobine pretilachlor trifloxystrobin fenoxaprop-p-ethyl cyhalofop-butyl butachlor
whole crayfish
crayfish meat
mantis shrimp
% recoveries (% RSD)
% recoveries (% RSD)
% recoveries (% RSD)
10 ng/g 49 88 82 100 106 85 111 101 113 91 107 95 106 108 19 106 112 110 83 112 112 111 110 109 98 110 109 103 111 102 108 118 102 50 169 171 112 101 96 108 76 108 145 111 96 122 89 111 94 103 147 109 103 106 99 76 96
(4) (11) (9) (14) (5) (16) (9) (9) (3) (5) (13) (7) (7) (7) (4) (5) (4) (7) (4) (3) (4) (5) (2) (7) (4) (5) (7) (8) (6) (15) (4) (3) (9) (18) (139) (133) (4) (13) (14) (4) (6) (6) (32) (4) (18) (46) (12) (4) (13) (12) (85) (8) (9) (9) (12) (4) (10)
50 ng/g
100 ng/g
105 99 109 106 87 115 91 89 99 91 88 87 101 84 17 102 99 96 83 101 98 98 96 104 89 110 82 86 81 84 99 101 95 34 83 82 97 99 92 99 111 95 130 98 98 107 96 91 100 94 50 103 91 93 94 67 88
85 (7) 100 (12) 83 (8) 87 (9) 112 (7) 105 (7) 109 (7) 106 (9) 113 (4) 110 (6) 103 (7) 97 (8) 84 (6) 98 (6) 15 (1) 111 (6) 100 (7) 95 (8) 83 (5) 101 (5) 100 (5) 106 (6) 99 (5) 98 (6) 87 (7) 103 (3) 108 (2) 88 (6) 108 (7) 88 (9) 107 (6) 105 (7) 103 (10) 38 (4) 99 (10) 92 (9) 101 (8) 109 (6) 105 (7) 107 (6) 99 (8) 103 (6) 90 (6) 104 (6) 101 (6) 111 (6) 106 (8) 106 (6) 100 (8) 90 (8) 89 (29) 106 (6) 98 (10) 112 (6) 103 (6) 112 (30) 100 (8)
(4) (13) (4) (13) (9) (2) (5) (4) (3) (6) (10) (7) (5) (4) (1) (4) (3) (5) (3) (3) (3) (4) (1) (4) (3) (2) (13) (4) (4) (6) (3) (4) (6) (6) (7) (8) (4) (5) (9) (3) (4) (3) (15) (4) (9) (11) (7) (3) (9) (3) (20) (6) (4) (8) (3) (34) (6)
10 ng/g 85 103 82 92 89 83 97 90 112 100 96 104 106 111 21 109 109 105 95 101 112 109 105 105 93 109 100 101 110 100 107 114 103 55 101 101 109 110 164 112 80 113 93 109 92 200 102 102 98 107 84 104 103 103 108 364 120
(7) (27) (7) (12) (10) (7) (7) (10) (3) (11) (18) (10) (8) (2) (7) (5) (8) (6) (10) (6) (4) (6) (8) (8) (16) (4) (8) (9) (6) (13) (3) (5) (7) (10) (6) (13) (6) (6) (22) (4) (9) (2) (18) (4) (19) (51) (12) (3) (11) (8) (34) (10) (10) (16) (10) (138) (28) E
50 ng/g 74 101 76 87 93 86 91 108 102 87 100 95 99 90 18 103 104 97 88 103 105 100 104 96 80 105 97 91 105 102 103 106 110 34 105 113 104 112 96 115 77 105 97 104 108 112 114 106 108 101 136 111 112 114 115 305 107
(2) (8) (3) (8) (4) (8) (11) (5) (5) (4) (5) (6) (5) (5) (2) (5) (5) (1) (6) (6) (4) (4) (5) (8) (6) (5) (5) (6) (4) (8) (3) (5) (4) (12) (3) (6) (4) (3) (13) (2) (5) (4) (12) (3) (5) (6) (3) (5) (4) (7) (60) (6) (6) (3) (5) (168) (10)
100 ng/g
10 ng/g
50 ng/g
100 ng/g
76 91 79 84 95 81 92 94 101 90 109 104 100 91 20 98 101 102 82 101 103 103 102 104 84 97 100 89 97 94 101 102 104 41 103 102 101 107 107 104 77 106 87 101 102 110 104 105 108 93 88 104 109 110 107 114 106
47 99 105 103 107 58 94 89 96 104 62 84 74 88 17 102 93 95 71 105 88 101 92 91 93 93 105 87 103 95 67 98 114 45 106 105 95 103 106 89 66 93 118 89 77 77 82 98 110 59
(6) (11) (12) (17) (12) (9) (3) (8) (6) (4) (12) (8) (5) (5) (4) (3) (6) (2) (2) (2) (3) (3) (7) (14) (8) (2) (7) (4) (6) (2) (5) (0) (3) (2) (18) (18) (5) (7) (7) (3) (8) (3) (33) (2) (14) (20) (8) (5) (4) (4) b 88 (4) 85 (3) 101 (1) 75 (2) 35 (22) 79 (4)
70 95 89 99 71 118 116 119 76 116 118 74 78 120 26 75 78 76 119 76 75 82 78 74 119 79 88 70 70 119 119 77 76 77 74 87 71 72 84 76 96 73 111 74 116 75 79 76 74 99
75 81 100 79 90 70 86 84 89 84 88 85 91 82 15 90 90 89 72 91 89 91 91 91 83 90 114 83 94 82 76 92 90 94 92 94 88 89 87 88 71 90 76 89 79 87 91 93 91 113
(4) (12) (4) (9) (9) (6) (8) (7) (7) (6) (4) (7) (3) (6) (1) (7) (7) (5) (5) (7) (8) (7) (6) (8) (3) (7) (5) (9) (8) (11) (7) (7) (7) (3) (7) (14) (6) (6) (10) (8) (4) (8) (9) (8) (3) (6) (5) (7) (7) (9) (28) (5) (6) (5) (4) (30) (21)
(6) (16) (15) (17) (3) (7) (4) (13) (1) (5) (7) (1) (2) (4) (4) (2) (0) (1) (1) (2) (1) (3) (1) (1) (8) (2) (10) (2) (3) (2) (3) (2) (1) (6) (6) (3) (2) (2) (5) (1) (5) (2) (34) (0) (15) (6) (4) (3) (3) (3) b 75 (4) 71 (2) 70 (2) 119 (6) 86 (35) 118 (3)
(8) (11) (12) (2) (2) (1) (2) (1) (1) (5) (7) (2) (3) (1) (1) (4) (1) (1) (2) (0) (1) (1) (1) (2) (6) (3) (11) (1) (6) (6) (3) (2) (5) (1) (1) (1) (3) (4) (8) (3) (0) (2) (18) (3) (11) (9) (4) (2) (5) (4) b 91 (2) 83 (4) 93 (4) 75 (9) 78 (20) 84 (2)
DOI: 10.1021/acs.jafc.7b05339 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry Table 1. continued whole crayfish
crayfish meat
% recoveries (% RSD) pesticide bentazone chlorpyrifos pyridaben a
mantis shrimp
% recoveries (% RSD)
% recoveries (% RSD)
10 ng/g
50 ng/g
100 ng/g
10 ng/g
50 ng/g
100 ng/g
10 ng/g
50 ng/g
100 ng/g
85 (23) b 104 (7)
91 (10) b 85 (4)
99 (12) 114 (50) 110 (6)
107 (18) 96 (12) 104 (10)
107 (10) 113 (5) 105 (7)
105 (9) 108 (5) 98 (6)
95 (6) b 77 (11)
76 (4) b 116 (4)
96 (5) b 82 (3)
Recoveries of 120% and RSDs of >20% (RSDs of >30% for 10 ng/g spiking level) are in bold. bLow sensitivity.
Figure 2. Average recoveries (n = 5) and SDs (error bars) for pesticides with unsatisfactory recoveries (120%) and RSDs of >20% for whole crayfish and crayfish meat.
difenoconazole (8 ng/g) was found in the cooked (salted) mantis shrimp from Dandong, Liaoning, China. Both residues measured in our study do not have an established MRL in crayfish or shrimp. Difenoconazole has a MRL of 50 ng/g in meat (cattle, hog, and sheep) for U.S.A. and EU, and concentrations measured in our study were below this value. In conclusion, a simple, fast, efficient, and reliable residue analysis method for crayfish (including whole crayfish and crayfish meat) using modified QuEChERS sample preparation and HPLC−MS/MS detection was investigated. The method was simple and used MeCN for extraction, NaCl for phase separation, and 50 mg of PSA for d-SPE cleanup. Both gradient elution (25 min) and isocratic elution (11 min) were evaluated for the HPLC analysis. A fortified study at three spiking levels showed that, in gradient elution conditions, 50 (83%) and 53 (88%) analytes had 70−120% recoveries with RSDs of ≤20% in whole crayfish and crayfish meat, respectively, whereas in isocratic elution, 42 (70%) and 51 (85%) analytes had satisfactory results, respectively. Although isocratic elution saved time, it caused higher recoveries (>120%) and/or high RSDs at the lowest spiking level (10 ng/g) for some analytes, especially for early eluting analytes in whole crayfish. Therefore,
Method validation was carried out on mantis shrimp only in gradient elution conditions. As listed in Table 1, 51 pesticides (85%) had satisfactory recoveries (70−120%) and RSDs of 50%) in different conditions.
the applicability of the isocratic elution method depends upon MRLs of these analytes in crayfish in the future. If the MRLs are higher than 10 ng/g, the isocratic method could be reevaluated at higher spiking levels and used to save instrumental analysis time. The investigated method was applicable to detect these target pesticides in mantis shrimp with gradient elution, and 51 (85%) analytes had satisfactory results, which was similar to the results in crayfish samples. The validated method was also applied to detect 60 pesticides in 6 crayfish samples and 5 mantis shrimp samples from the market, and propamocarb (
