Method development and validation of ten pyrethroid insecticides in edible mushrooms by Modified QuEChERS and gas chromatography-tandem mass spectrometry

A method for simultaneous determination of ten pyrethroid insecticides residues in edible mushrooms was developed. The samples were pretreated by a quick, easy, cheap, effective, rugged (QuEChERS) method. The ten pyrethroid insecticides were extracted from six kinds of edible mushrooms using acetonitrile and subsequently cleaned up by octadecylsilane (C18) or primary secondary amine (PSA). Instrumental analysis was completed in 16 min using gas chromatography-tandem mass spectrometry (GC-MS/MS). The overall average recoveries in the six kinds of edible mushrooms at three levels (10, 100 and 1000 μg kg−1) ranged from 72.8% to 103.6%. The intraday and interday relative standard deviations (RSD) were lower than 13.0%. The quantification limits were below 5.57 μg kg−1 in different matrices. The results demonstrated that the method is convenient for the quick detection of pyrethroid insecticides in edible mushrooms.

www.nature.com/scientificreports www.nature.com/scientificreports/ optimization of extraction solvents and clean up sorbents. The choice of suitable solvent and sorbent has huge influence on the recoveries. Therefore, the solvents and sorbents need to be optimized. Firstly, the extraction solvent was studied. Acetonitrile was frequently used for pesticide multi-residue analysis with advantages including, less co-extracted matrix components, higher recoveries, etc. 2,24,27,33 . Meanwhile, pyrethroid insecticides have high solubility in n-hexane at 20 °C. Therefore, the recoveries of acetonitrile and n-hexane as extraction solvents were compared. As shown in Fig. 1, taking eryngii mushroom as an example, the recoveries of the ten pyrethroid insecticides at a spiked level of 100 μg kg −1 using acetonitrile as the extraction solvent was significantly higher than that of n-hexane. Consequently, acetonitrile was selected as the extraction solvents in further study.
To achieve a satisfactory effect, we evaluated the different types of sorbents at a spiked level of 100 μg kg −1 . Test A was carried out using 50 mg PSA + 150 mg MgSO 4 , test B using 20 mg PSA + 30 mg C18 + 150 mg MgSO 4 , test C using 50 mg C18 + 150 mg MgSO 4 , and test D using an Enhanced Matrix Removal-Lipid (EMR-Lipid). Meanwhile, the four matrix standards were prepared from each dispersive solid phase extraction (dSPE) cleanup technique so that each (C18, PSA or PSA + C18, etc.) could be tested against standards with the same composition of matrix compounds. For the dSPE, PSA is mainly applied to adsorb various polar matrix components from non-polar samples like organic acids and pigments. Conversely, C18 is mainly used to remove non-polar and medium-polar compounds from the polar samples 2,33,34 . Particularly, the dSPE EMR is applied to remove the lipid 35 . As shown in Fig. 2, the recovery and RSD were both satisfied when the four different types of sorbents were used in the oyster mushroom. Nevertheless, when C as sorbent was used in shiitake mushroom, the recoveries of ten target compounds was satisfactory. Meanwhile, A as sorbent was used in bunashimeji mushroom, the recoveries was satisfactory. For the crimini mushroom and enoki mushroom, the recovery and RSD were both satisfied when sorbent A, sorbent B and sorbent D were used. PSA is relatively expensive than C18. Therefore, considering the efficacy and cost of each sorbent, 20 mg PSA + 30 mg C18 + 150 mg MgSO 4 was ultimately selected as sorbent for oyster mushroom and eryngii mushroom extracts. 50 mg PSA + 150 mg MgSO 4 was used as the sorbent for crimini mushroom, enoki mushroom and bunashimeji mushroom extracts, while for the shiitake mushroom requires used 50 mg C18 + 150 mg MgSO 4 purification.

Matrix effects.
The ionization of some pesticides may be significantly affected by the presence of substances, which are derived from samples 36 . In 1993, matrix effect was first explained by Erney and co-workers, and their study suggested that the response of one organic base decreased as the concentration of other bases increased 37 . The matrix effects can greatly affect the reproducibility and accuracy of the method. Thus, matrix effect was studied in edible mushrooms. Generally speaking, the matrix effect was ignored if the value was between −10% and 10%; the matrix effect was defined as suppression if the value was lower than −10%; the matrix effect was defined as enhancement if the value was higher than 10% 23,24 . As shown in Table 2, the matrix effects obviously enhance the response of the instrument in all matrices. And the slope ratios of matrix/n-hexane were in the range of 1.47-2.77. In order to eliminate the matrix effect and determine more accurate results for each target compound concentration in all samples, the matrix-matched calibration curves were selected to calibrate the GC-MS/MS system. Linearity, LOD, and LOQ. The calibration curves in different edible mushrooms matrices were shown in Table 2. The linearity for each target compound in each edible mushroom matrix was satisfactory (R 2 ≥ 0.9901 in all cases). The LODs of ten pyrethroid insecticides ranged from 0.015 to 1.67 μg kg −1 , and LOQs ranged from 0.051 to 5.57 μg kg −1 in original samples. These values were similar to the values of other pesitcides reported in the literature 31,38,39 . And the LOQs for ten target compounds were much lower than the maximum residue limit (MRLs) (100-1000 μg kg −1 ) recommended by the EU, Japan, USA and China. www.nature.com/scientificreports www.nature.com/scientificreports/ precision and accuracy. A recovery assay was performed to validate the performance of the proposed method. The blank samples of different matrices were spiked at three different concentrations (10, 100 and 1000 μg kg −1 ) and then determining them in quintuplicate. The method's precision was expressed as the RSD. As indicated in Table 3, mean recoveries of ten target compounds were in the acceptable ranges of 80.8-97.7% with RSD r of 1.0-8.4%, 81.5-103.6% with RSD r of 1.8-8.4%, 72.8-97.5% with RSD r of 1.4-7.0%, 81.4-102.2% with RSD r of 2.4-8.9%, 75.6-100.0% with RSD r of 2.4-9.0%, 75.0-103.3% with RSD r of 1.0-8.4% for oyster mushroom, shiitake mushroom, eryngii mushroom, crimini mushroom, enoki mushroom and bunashimeji mushroom, respectively. In general, the mean recoveries of ten target compounds were 72.8-103.6% in all matrices, and the RSD r (n = 5) and RSD R (n = 15) values ranged from 1.0% to 9.0% and 3.1% to 13.0%, respectively. For the statistical analysis, one-way analysis of variance (ANOVA) at 95% confidence limits was used to compare the interday and intraday assay recoveries, and there were no significant differences between the interday and intraday assays. Therefore, the results indicated that the extract method and GC-MS/MS analysis can obtain a satisfactory precision and accuracy for residue analysis of these ten pyrethroid insecticides in edible mushrooms.
Application to real samples. The proposed method was applied to monitor trace levels of each target compounds in real samples to demonstrate the effectiveness and applicability. These samples were purchased from markets in Anhui Province (China). A total of 90 samples (20 oyster mushroom samples, 20 shiitake mushroom samples, 10 eryngii mushroom samples, 20 crimini mushroom samples, 10 enoki mushroom samples, and 10 bunashimeji mushroom samples) were analyzed. As shown in Table 4, only two positive oyster mushroom samples and three positive crimini mushroom samples were detected containing cypermethrin in the range of 11-43 μg kg −1 . However, the presence of cypermethrin doesn't pose a threat to the consumer, because they are below the MRLs settled by EU (50 μg kg −1 for oyster mushroom and crimini mushroom), China (500 μg kg −1 for oyster mushroom and crimini mushroom) and Japan (50 μg kg −1 for crimini mushroom and 500 μg kg −1 for oyster mushroom). But the residual concentration of cypermethrin in some crimini mushroom samples is very close to MRL settled by EU and Japan. Therefore, detection of cypermethrin residues in mushrooms should be strengthened. However, ten pyrethroid insecticides were not found in most of tested samples.
In conclusion, in the present study, a simple, reliable and highly sensitive residue analytical method for the simultaneous determination of ten pyrethroid insecticides in six edible mushrooms using GC-MS/MS was  Continued developed. The results showed satisfactory validation parameters in the field of linearity, lower limits, accuracy, and precision. The LOQs were below MRLs recommended by EU, China and Japan in all mushroom matrices. The method has strong matrix effect, but it was successfully normalized using matrix-matched calibration. Therefore, this method may be a useful technique for monitoring pyrethroid insecticide residues in edible mushroom samples.

Materials and Methods
Reagents and chemicals. Insecticide analytical standards were supplied from the National Institute of Metrology (Beijing, China) and were of more than 98% purity. Chromatography grade acetonitrile and n-hexane were achieved from Honeywell International Inc. (New Jersey, USA). The anhydrous magnesium sulfate (MgSO 4 ) and sodium chloride (NaCl) were bought from Beijing Chemical and Reagent Company (Beijing, China). The sorbents of primary secondary amine (PSA) and octadecylsilane (C 18 ) were bought from Agela Technologies Inc.
(Beijing, China), and Agilent Bond Elut dSPE EMR-Lipid was also bought from Agela Technologies Inc. Stocks solutions (1000 mg L −1 ) of each insecticide standard were prepared in n-hexane. A mixed stock standard solution of 100 mg L −1 containing bifenthrin, fenpropathrin, cyhalothrin, permethrin, cyfluthrin, cypermethrin, flucythrinate, tau-fluvalinate, fenvalerate, and deltamethyrin was prepared by mixing ten stock solutions in equal volume. Subsequently, several standard solutions (10, 50, 100, 200, 500, and 1000 μg L −1 ) were prepared from the mixed stock solution by serial dilution with n-hexane. The matrix-matched standard solutions (10, 50, 100, 200, 500, and 1000 μg L −1 ) were similarly prepared by adding the blank sample extracts (oyster mushroom, shiitake mushroom, eryngii mushroom, crimini mushroom, enoki mushroom and bunashimeji mushroom) to each serially diluted standard solution. For the preparation of matrix-matched standard, the method was that appropriate volumes of work standard solution was firstly dried under nitrogen and then redissolved by 1 mL blank sample extract. All solutions were stored at −20 °C in the dark.
Instruments and chromatographic conditions. All sample analyses used an Agilent intuvo 9000 gas chromatograph coupled with a 7000D triple quadrupole mass spectrometer. Separations were performed using Agilent Technologies Capillary Column HP-5MS phenylmethyl siloxane fused-silica capillary analytical column (30 m length × 0.25 mm i.d. × 0.25 μm film thickness). A helium (purity 99.99%) was employed as carrier gas and the flow rate was 1.0 mL min −1 . The temperature of the injection port was 280 °C. The column temperature was initially at 70 °C for 1 min, increased to 120 °C at the rate of 40 °C min −1 , and increased to 200 °C at the rate of 30 °C min −1 , then increased to 240 °C at the rate of 10 °C min −1 , and then increased to 300 °C at the rate of 20 °C min −1 , and holding for 3.7 min. A volume of 1 μL was injected in the splitless mode.
The mass spectrometer was performed in electron ionization mode with an ionizing energy of 70 eV. The electron multiplier voltage was 1300 V. The transfer line, manifold and ionization source temperatures were 280, 40 and 250 °C, respectively. A solvent delay was 8 min. The mass spectrometer mode was set at multiple reaction monitoring (MRM) to collect data. The concrete MS/MS parameters for all the analytes listed in Table 1.
Sample preparation. Figure 3 shows the workflow of the sample preparation procedure. For the cleanup procedure, the 20 mg PSA and 30 mg C18 were selected to clean up the oyster mushroom and eryngii mushroom; the 50 mg PSA was used to purify the crimini mushroom, enoki mushroom and bunashimeji mushroom, and 50 mg C18 was used to clean up the shiitake mushroom.
For the Agilent Bond Elut EMR-Lipid clean up, the extract procedure is the same as above. 5 mL Milli-Q water was added into EMR-Lipid tube to activate the sorbent. Then, 5 mL upper layer (acetonitrile) was added into the tube. The tube was vortexed for 5 min and then centrifuged for 5 min at relative centrifugal force (RCF) 3913 × g. Subsequently, 5 mL upper layer was transferred into the EMR-Polish tube that containing 2 g salt (1:4 NaCl: MgSO 4 ). The tube was vortexed for 5 min and centrifuged for 3 min at RCF 2811 × g. Then, 1 mL upper layer www.nature.com/scientificreports www.nature.com/scientificreports/ (acetonitrile) was reduced to dryness under a gentle stream of nitrogen at room temperature. The residue was reconstituted in 1 mL n-hexane and was filtered with 0.22-μm filters for GC-MS/MS analysis.

Method validation.
The developed method was validated by fortifying blank mushroom samples at three different levels (10, 100, and 1000 μg kg −1 ). Recovery assays were performed to determine the accuracy and precision of the method. For determination of the accuracy, five replicates of each fortification level were prepared  Table 3. Recoveries (n = 15, %), RSDr a and RSD R b (%) for target compounds from different matrices at three spiked levels. The recovery is the mean recovery. a Intra-day (n = 5). b Inter-day (n = 15).  Table 4. Concentration levels of ten pyrethroid insecticides in edible mushroom samples from market in Anhui Province. a Number of positive sample (positive sample ratio). b The result of positive samples.