Introduction

Edible mushrooms are considered as a delicacy with high nutritive value and unique flavor, and they are also recognized as nutraceutical foods1,2. More importantly than all of that, it is accepted as healthy food with a good deal of medicinal functions and important positive health function3. They are more and more popular with consumers and have been regarded as ingredient of gourmet cuisine all over the world. Pleurotus ostreatus (oyster mushroom), Lentinus edodes (shiitake mushroom), Pleurotus eryngii (eryngii mushroom), Agaricus bisporus (crimini mushroom), Flammulina velutiper (enoki mushroom), and Hypsizygus marmoreus (bunashimeji mushroom) are the six of the most cultivated edible mushrooms. They are quite rich in protein, essential amino acids, fiber, chitin, vitamins and other substances4,5,6,7. These ingredients increase the value of these edible mushrooms. In China, edible mushroom production reached 7,868,782 ton in 2017. And the mushroom production accounted for 76.8% of the world2. And it is the most important producers and exporters of mushrooms. However, in recent years, the pests and diseases has become very serious with the expansion of cultivation scale of six edible mushrooms8. Sciarid flies and Cecid are the most important pests in mushroom throughout the world9. It was reported that these pests have caused significant economic losses in the mushroom industry8. Therefore, many insecticides were used on mushrooms to control these pests which can boost yields and reduce the economic losses. So, detailed investigations on pesticide residues in mushrooms are very important to reduce the use of pesticides and ensure food safety.

Many types of synthetic insecticides are frequently used to control pests in mushroom cultivation throughout the world10,11,12. Because pyrethroids has high level of effectiveness, broad spectrum of effects and low toxicity, it has been widely used in the production all over the world13,14. In 2015, pyrethroids was among the most important classes of insecticides in crop, and it accounted for 38% of the world insecticide market12. Therefore, it is not surprised that pyrethroid residues are frequently detected in different vegetables, fruits, soil and other matrices worldwide15. For example, Ding et al. reported that the highest concentration of pyrethroid residues in vegetables in Zhejiang province reached 330 μg kg−116. To prevent and control Sciarid and Cecid in the process of mushroom cultivation, pyrethroids are frequently used on mushrooms to boost yields. Therefore, determination of pyrethroid residues in mushrooms is important for food safety and normal foreign trade.

As far as we know, many analytical methods have been reported for the determination of pyrethroids in various matrices. Due to co-extraction of highly complex components, such as protein, sterols, essential amino acids, and polysaccharide, extracting pyrethroids residues from edible mushrooms is difficult2. In the extract, these components seriously interfere with the determination of the pyrethroids. Therefore, pretreatment technology is very important in the detection of pyrethroids residues in edible mushrooms17. Usually, a solid-liquid extraction followed by purification is a preliminary sample preparation for the determination of pesticide residues in edible mushrooms. The purification techniques, including solid-phase extraction (SPE), gel permeation chromatography (GPC), and matrix solid-phase dispersion, are the most commonly used techniques for pretreatment procedures18,19,20. However, these sample preparation methods are complicated and use larger amounts of organic solvents21,22. In recent years, the QuEChERS methodology has been develpoed as a very popular method to determine pesticide residues in all kinds of food matrices23,24,25,26,27,28,29. And there are some papers that applied QuEChERS for the extraction of pesticides from mushrooms and determination of residue used GC, GC-MS/MS30,31, or LC-MS/MS10,11. However, simultaneous determination of pyrethroid residues in mushrooms by GC-MS/MS has not been reported. Moreover, GC-MS/MS is becoming more and more popular in routine pesticide residue analysis because of fewer co-matrix effects resulting in sensitive identification and the reagents costing less compared with HPLC/UPLC-MS/MS32.

Therefore, the aim of this paper is focused on the investigation of a rapid and effective extraction procedure using a modified QuEChERS method for simultaneously analyzing pyrethroids in various edible mushrooms (oyster mushroom, shiitake mushroom, eryngii mushroom, crimini mushroom, enoki mushroom and bunashimeji mushroom). Various extraction solvents and cleanup sorbents were studied for optimizing the pretreatment method to obtain higher recoveries. The developed method was successfully used to analyze authentic samples.

Results and Discussion

Optimization of GC-MS/MS parameters

The pyrethroid insecticides involved were monitored in the full scanning mode in the range m/z 50–600 to describe its scanning mass spectrogram and retention time. Then, the multiple reaction monitoring (MRM) transitions were optimized. The purpose of selecting precursor ions was to achieve a compromise between selectivity (the highest m/z ion is preferred) and sensitivity (the highest abundance ion). The method was divided into as many time segments as possible to achieve the maximum signal for each compound. The MS/MS transition was selected according the highest response for the each target compound. The run time of these pesticides were completed in 16 min. All the parameters for precursor ions, product ion, corresponding collision energies, and other optimal conditions were determined and shown in Table 1.

Table 1 Details of the MS/MS parameters for analysis of the compounds.
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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.

Figure 1
figure 1

Effect of acetonitrile and n-hexane as extraction solvents for the target compounds in eryngii mushroom at 100 μg kg−1 level (n = 3).

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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 MgSO4, test B using 20 mg PSA + 30 mg C18 + 150 mg MgSO4, test C using 50 mg C18 + 150 mg MgSO4, 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 samples2,33,34. Particularly, the dSPE EMR is applied to remove the lipid35. 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 MgSO4 was ultimately selected as sorbent for oyster mushroom and eryngii mushroom extracts. 50 mg PSA + 150 mg MgSO4 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 MgSO4 purification.

Figure 2
figure 2

Effects of different sorbents for the targeted compounds in different matrix at 100 μg kg−1 level (n = 5).

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Matrix effects

The ionization of some pesticides may be significantly affected by the presence of substances, which are derived from samples36. 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 increased37. 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.

Table 2 Comparison of matrix-matched calibration and solvent calibration at 10–1000 μg kg−1.
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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 (R2 ≥ 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 literature31,38,39. 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.

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 RSDr of 1.0–8.4%, 81.5–103.6% with RSDr of 1.8–8.4%, 72.8–97.5% with RSDr of 1.4–7.0%, 81.4–102.2% with RSDr of 2.4–8.9%, 75.6–100.0% with RSDr of 2.4–9.0%, 75.0–103.3% with RSDr 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 RSDr (n = 5) and RSDR (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.

Table 3 Recoveries (n = 15, %), RSDra and RSDRb (%) for target compounds from different matrices at three spiked levels.
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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.

Table 4 Concentration levels of ten pyrethroid insecticides in edible mushroom samples from market in Anhui Province.
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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 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 (MgSO4) and sodium chloride (NaCl) were bought from Beijing Chemical and Reagent Company (Beijing, China). The sorbents of primary secondary amine (PSA) and octadecylsilane (C18) 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.

Figure 3
figure 3

Workflow of sample preparation.

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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: MgSO4). The tube was vortexed for 5 min and centrifuged for 3 min at RCF 2811 × g. Then, 1 mL upper layer (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 on three different days (n = 15 in total). However, the intra-day and inter-day relative standard deviation (RSD) were also investigated as the method precision. The linear regression equations of each target compound were achieved from the peak area ratios plotted against its respective concentrations (10–1000 μg kg−1). The linearity was presented as correlation coefficient (R2). The limit of detection (LOD) for each target compound was defined as the minimum spiking level that produced a chromatogram peak with signal-to-noise (peak to peak) ratio of 3, and the limit of quantification (LOQ) was defined as a signal-to-noise ratio of 1023.