Fast and Online Determination of Five Avermectin ... - ACS Publications

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Fast and Online Determination of Five Avermectin Residues in Foodstuffs of Plant and Animal Origin Using Reusable Polymeric Monolithic Extractor Coupled with LC-MS/MS Xin Li,† Man-Man Wang,*,† Guo-Ying Zheng,† Lian-Feng Ai,‡ and Xue-Sheng Wang† †

School of Public Health, Hebei United University, Tangshan 063000, Hebei, China Hebei Entry-Exit Inspection and Quarantine Bureau, Shijiazhuang 050051, Hebei, China

‡

S Supporting Information *

ABSTRACT: A hydrophobic monolith (10 mm × 2.1 mm i.d.) was developed as a reusable online solid-phase extraction (SPE) sorbent coupled with LC-MS/MS for the rapid determination of five avermectin residues in foodstuffs of both plant and animal origin. The online SPE was achieved using a 10 mmol/L ammonium acetate solution as the loading solvent, and acetonitrile (MeCN) was selected for the washing step. After being transferred from the monolith into a C18 analytical column using MeCN, the analytes were analyzed by LC-MS/MS using MeCN/0.1% NH4OH (10:90, v/v) as the mobile phase. The detection limit was 2 μg/kg for five avermectins, and the recoveries in fresh pear, chili seed, bovine muscle, and milk ranged from 71.8% to 101.3% with relative standard deviations of less than 8.94%. The online SPE and determination were achieved within 15 min, and the monolithic extractor was reusable for more than 500 experiments. KEYWORDS: avermectins, on-line solid-phase extraction, monolithic column, animal origin food, plant origin food, LC-MS/MS

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INTRODUCTION Avermectins are a class of macrolide antibiotics used as veterinary drugs for food-producing animals and as plant protection agents because of their efficient broad-spectrum antiparasitic activity.1,2 The chemical structures of commercial avermectins, i.e., abamectin, 1, ivermectin, 2, doramectin, 3, eprinomectin, 4, and moxidectin, 5, are shown in Figure 1. However, the increasing and excessive use of these compounds may lead to the presence of residues in daily foodstuffs such as fruit, vegetables, edible tissues, and milk. Certainly long-term adverse effects can be related to allergic reactions and crossresistance to other antibiotics of similar structure or mechanisms of action. In addition, avermectins may be embryotoxic, and they can also cause rash, edema, diarrhea, and seizure in laboratory animals.3,4 To minimize the undesirable risks to public health associated with their consumption, maximum residue limits (MRLs) have been established by different countries. For instance, the European Union (EU) sets the MRLs for abamectin, ivermectin, doramectin, and eprinomectin at 20 μg/kg in animal muscles.5 For animal origin food, the tolerance levels recently recommended by the European Medicines Agency (EMEA) are also at low μg/kg levels (10−150 μg/kg) for widely used avermectins such as ivermectin and doramectin.6−9 In China, the MRLs for abamectin are 20 μg/kg in fruit and 10 μg/kg in vegetables.10 Consequently, the establishment of a fast and simple method for monitoring avermectins in both animal and plant products, especially in common foods such as milk, animal tissue, fruit, and vegetables, is important to protect humans from the disturbances caused by avermectins. However, the direct determination of these compounds in food matrices is usually difficult because of the complexity of the matrices and the extremely low concentrations of the © 2015 American Chemical Society

analytes. Therefore, sample pretreatment, which includes both the isolation and/or preconcentration of the compounds of interest from food matrices as well as making the analytes more suitable for separation and detection, remains an important part of obtaining accurate analyses. The pretreatment methods for avermectins in various food samples include liquid−liquid extraction,11−16 dispersive liquid−liquid microextraction,17 hollow fiber-supported liquid membrane extraction,18 and solid-phase extraction (SPE).19−21 Among these sample preparation methods, SPE is an attractive technique to handle food samples in terms of high sensitivity, less solvent, and high extraction capacity. Several sorbents, such as molecularly imprinted polymers (MIPs),22 C18/C8 packed particles,19,23 and aminopropyl packed particles,24 have been successfully used for the offline SPE of avermectins. However, offline SPE as a separate procedure prior to the final instrumental analysis has inherent disadvantages; it is time-consuming, the samples are easily contaminated, and it is a complex procedure with poor reproducibility. Online SPE is an effective method to overcome the aforementioned disadvantages. Moreover, these SPE materials suffer from poor reusability in the case of complicated samples. Thus, the development of reusable sorbents that can capture the target compounds from a large variety and number of samples for online SPE remains an important challenge. As an intriguing class of SPE materials, polymeric monolithic sorbents have attracted considerable attention for the pretreatment of compounds from environmental, food, and biological Received: Revised: Accepted: Published: 4096

February April 11, April 12, April 13,

7, 2015 2015 2015 2015 DOI: 10.1021/acs.jafc.5b00739 J. Agric. Food Chem. 2015, 63, 4096−4103

Article

Journal of Agricultural and Food Chemistry

Figure 1. Chemical structures of five avermectins of interest.

matrices.25−31 Polymeric monolithic materials possess obvious advantages including excellent biocompatibility, high permeability, and fast mass transfer, which are able to effectively extract trace target analytes from complicated food samples even at high flow velocities and under extreme pH conditions. Furthermore, organic polymer-based monoliths can be easily fabricated within the device, without the further packing steps required in the case of packed particulate adsorbents. Additionally, the porous structure and surface chemistry of the polymer are usually tunable and flexible. Therefore, several polymer-based monolithic columns have been shown to be promising tools for sample preparation.32−35 To further expand the application of monolithic sorbents, the present study focuses on the development of a robust and reusable polymeric monolithic sorbent capable of fast online SPE of five avermectins from both plant and animal origin food samples. In this work, a poly(butyl methacrylate-co-ethylene glycol dimethacrylate) (poly(BMA-co-EGDMA)) monolithic column was prepared and used as sorbent for online SPE of avermectins coupled with liquid chromatography tandem mass spectrometry (LC-MS/MS). The factors affecting the online SPE were studied in detail. A new method was established for the fast, automatic, and simultaneous determination of five avermectins in fresh pear, chili seed, bovine muscle, and milk samples.

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Waters Milli-Q system (Milford, MA, USA) and was used throughout all experiments. Butyl methacrylate (BMA) was purchased from Aladdin (Shanghai, China), and ethylene glycol dimethacrylate (EGDMA) was purchased from Alfa Aesar (Ward Hill, MA, USA). 1-Propanol and polyethylene glycol 400 were acquired from Tianjin Guangfu Fine Chemical Research Institute (Tianjin, China). Azobis(isobutyronitrile) (AIBN) was acquired from Tianjin Damao Chemical Reagent Factory (Tianjin, China) and was recrystallized with ethanol before use. HPLC-grade methanol (MeOH) and acetonitrile were provided by Fisher Scientific (Geel, Belgium). Ammonium acetate (NH4OAc), acetic acid, formic acid, acetone, and isopropyl alcohol were purchased from Dikma (Beijing, China). Ammonium hydroxide (NH4OH) was acquired from Chemical Reagent Factory (Shijiazhuang, China). Abamectin (97%), ivermectin (96%), doramectin (96%), eprinomectin (91%), and moxidectin (87%) were purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany). Stock standard solutions of the avermectins were separately prepared at a concentration of 100 μg/mL in MeOH and stored at 4 °C in the dark. Working mixture solutions were freshly prepared from the stock solutions by stepwise dilution with MeOH. All solvents and solutions for LC analysis were filtered through a 0.45 μm Millipore filter. Sample Preparation. The representative samples of plant origin (fresh pear and chili seed) and animal origin (bovine muscle and milk) were collected from supermarkets and local markets. Preliminary analysis showed that they were free of the analytes of interest. The samples including fresh pear, chili seed, and bovine muscle were first minced with a homogenizer, and the homogeneous samples were stored at −4 °C before use. Plant origin samples (fresh pear and chili seeds): 2.0 g of pear and chili seed samples were weighed into a 50 mL centrifuge tube. Then, 10.0 mL of MeCN and 2.0 g of NaCl were added, and the samples

MATERIALS AND METHODS

Reagents and Solutions. All chemicals and reagents used were at least of analytical grade or better. Purified water was obtained from a 4097

DOI: 10.1021/acs.jafc.5b00739 J. Agric. Food Chem. 2015, 63, 4096−4103

Article


Table 1. Experimental Conditions Used for Online Clean-up on the Poly(BMA-co-EGDMA) Monolithic Extractor and LC-MS/ MS Separation of the Avermectins from Various Samples loading pumpa

eluting pumpb

step

start (s)

duration (s)

function

flow (mL/min)

A%

1 2 3 4 5 6 7

0 60 150 200 440 570 690

60 90 50 240 130 120 210

loading washing eluting washing washing washing conditioning

0.5 0.5 0.5 0.5 0.5 0.5 0.5

100

B%

C%

100 100 100 100 100 100

function

flow (mL/min)

D%

E%

conditioning eluting eluting eluting eluting eluting conditioning

0.5 0.5 0.5 0.5 0.5 0.5 0.5

10 10 10 95 95 95 10

90 90 90 5 5 5 90

a

The mobile phases for the loading pump: (A) 10 mmol/L NH4OAc solution, (B) MeCN, and (C) a mixture of MeCN, isopropyl alcohol, and acetone (1:1:1, v/v/v). bThe mobile phases for the eluting pump: (D) MeCN, (E) 0.1% NH4OH solution. synthesized poly(BMA-co-EGDMA) monolithic column (10 mm × 2.1 mm i.d.). The online SPE procedure can be divided into four general steps: loading, washing, transfer, and conditioning. The mobile phases used for the loading pump were (A) 10 mmol/L NH4OAc solution, (B) MeCN, and (C) the mixture of MeCN, isopropyl alsohol, and acetone (1:1:1, v/v/v). All of the parameters used for online cleanup on the poly(BMA-co-EGDMA) monolithic extractor and subsequent LC-MS/MS separation are listed in Table 1. In the loading step (step 1 in Table 1), 50 μL of the sample was injected into the poly(BMA-co-EGDMA) monolith. Then, during the washing step, the poly(BMA-co-EGDMA) monolithic column and the analytical column were not connected. The matrix components were washed out (waste) using solvent B, and the analytes were retained on the poly(BMA-co-EGDMA) monolith. For the transfer step (step 3 in Table 1), the poly(BMA-co-EGDMA) monolithic extractor was placed online with the LC-MS/MS system by switching the six-port valves, and the analytes were transferred from the poly(BMA-co-EGDMA) extractor into the analytical column. The last stage involved washing and conditioning of the poly(BMA-co-EGDMA) extractor (steps 4−7 in Table 1), while the analytes were being separated via LC-MS/MS. The poly(BMA-co-EGDMA) monolith was sequentially washed with solvents C and B to completely remove any residual contamination and to recondition the extractor for the next effective extraction. After that step, the entire online SPE procedure was completed. During the washing and conditioning steps, the poly(BMA-co-EGDMA) monolithic extractor and the analytical column were not connected. LC-MS/MS Separation. Analytical separation was achieved using a 100 mm × 4.6 mm, 3 μm Hypersil Gold C18 column (Thermo Fisher Scientific, Pittsburgh, PA, USA). The mobile phase for separation consisted of (D) MeCN and (E) 0.1% NH4OH solution. The parameters of LC-MS/MS separation (eluting pump) are shown in Table 1. The detection was performed by a triple-quadrupole Thermo Scientific TSQ Quantum Ultra mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) equipped with an atmospheric-pressure chemical ionization probe that was maintained at 350 °C. All analyses were performed in the negative ionization mode. The sheath and auxiliary gases were nitrogen at 30 and 5 arbitrary units, respectively. The system was operated in multiple reaction monitoring (MRM) mode with argon as the collision gas at a pressure of 1.5 mTorr. The ion-transfer tube was maintained at 320 °C. The scan time and width were 0.02 s and m/z 0.01, respectively. The data were processed using the LC Quan software version 2.6. The retention time and mass spectrometer parameters for each analyte are given in Table 2. The total analytical run time for this method, including both SPE and separation, was 15.0 min.

were blended with a homogenizer for 1 min. The samples were subsequently centrifuged at 0−4 °C for 5.0 min at 5000 rpm, and the supernatant was filtered through a 0.22 μm filter prior to the online extraction. Animal origin samples (milk and bovine muscle): First, 2.0 g of previously minced bovine muscle was weighed into a centrifuge tube of 50 mL. After addition of 8.0 mL of MeCN, the sample was blended with a homogenizer for 1 min. Subsequently, the samples were extracted with MeCN twice. For the milk samples, 2.0 g of the sample was weighed into a 50 mL centrifuge tube. Next, 8.0 mL of MeCN, 1.0 g of NaCl, and 2.0 g of MgSO4 were added. After being mixed with a vortex mixer for 2.0 min, the sample was centrifuged at 0−4 °C for 5.0 min at 5000 rpm. Subsequently, the supernatant was collected and the samples were extracted twice with MeCN. Finally, for the milk and bovine muscle samples, the supernatant was collected and diluted to 30.0 mL with MeCN. Three milliliters of each extraction solution was dried under flowing nitrogen gas at 40 °C. The residues were dissolved in 1.0 mL of MeCN and filtered through 0.22 μm filters prior to analysis. Spiked samples were prepared in the same way with spiking required amounts of avermectins. Fabrication of Poly(BMA-co-EGDMA) Monolithic Extractor. The poly(BMA-co-EGDMA) monolithic extractor was fabricated in a stainless steel chromatographic column (10 mm × 2.1 mm i.d.). Briefly, the polymerization mixture consisted of 0.5 mmol of BMA, 1.5 mmol of EGDMA, a mixture of 6.5 mmol of 1-propanol and 1.3 mmol of polyethylene glycol 400, and 4.0 mg of AIBN.36−38 After sonication for 30 min, the solution was introduced into the column with both ends sealed and placed vertically in a water bath. The polymerization was performed for 2−24 h at 55 °C, and the temperature was 55, 60, or 65 °C; the reaction time was fixed at 4 h. The resulting column was connected to the LC system and washed with MeOH to remove the porogen and monomeric residues. Since the poly(BMA-co-EGDMA) monolithic extractor is a typical hydrophobic column, we chose a neutral compound, toluene, as the marker analyte. The retention factor (k) of toluene of each monolithic extractor was calculated to evaluate the corresponding hydrophobicity. The retention factor k was calculated from the equation k = (tR − t0)/t0, where tR and t0 are the retention times of toluene and the void marker (MeOH), when MeOH/water (80:20, v/v) was used as the mobile phase. The scanning electron microscopy (SEM) images of the monoliths were obtained using an S-4800 scanning electron microscope (Hitachi, Tokyo, Japan). Online Sample Extraction Based on the Poly(BMA-coEGDMA) Monolithic Extractor. The online cleanup and separation was performed on an Aria TLX-2 system (Thermo Fisher Scientific, Franklin, MA, USA) equipped with a PAL autosampler (CTC Analytics, Zwingen, Switzerland) with a 200 μL sample injection loop, two high-pressure mixing quaternary pumps, one for online SPE (loading pump) and the other for analytical separation and elution (eluting pump), a multiple-column module, and a three-valve switching device unit with a six-port valve controlled by the Aria software, version 1.6.3. Automated SPE was achieved on the

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RESULTS AND DISCUSSION Characterization of the Poly(BMA-co-EGDMA) Monolithic Extractor. SPE sorbents based on hydrophobicity (reversed-phase retention) are most commonly used because of their versatility and ability to trap several compounds with weak polarity from a variety of food samples. Therefore, SPE 4098


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Journal of Agricultural and Food Chemistry Table 2. Retention Time, Precursor Ion, Product Ion, and Collision Energy of the Five Avermectins analyte

retention time (min)

precursor ion (m/z)

product ion (m/z)

collision energy (eV)

1

9.33

871.6

2

10.35

873.6

3

9.64

897.6

4

9.08

912.1

5

9.79

638.2

229.1 565.5a 229.1 567.4a 229.0 591.3a 269.8 565.4a 236.2 528.5a

44 27 37 25 35 27 33 29 29 23

a

Quantitative ion.

for avermectins is usually performed on a reversed-phase C18 or C8 column depending on their polarities.19,23 Thus, in the present experiment, since it is a widely accepted and convenient monomer, BMA was chosen to polymerize with EGDMA to prepare a miniature monolithic extractor with hydrophobicity provided by the butyl groups on the surface of the polymer skeletons. The monolithic extractor was fabricated via thermally initiated polymerization in the cartridge of a stainless steel column (10 mm × 2.1 mm i.d.). With the composition of the polymerization mixture fixed, the effects of the reaction time and reaction temperature on the performance of the extraction were investigated. The retention factor (k) of toluene on each monolith was calculated to evaluate the hydrophobicity of the extractors. The effects of reaction time over the range from 2 to 24 h and of the temperature in the range from 55 to 65 °C on k are shown in Figure 2A. No significant changes were observed when the reaction time exceeded 4 h and the temperature was higher than 55 °C. The procedure is easy, and the preparation conditions, including the reaction time and temperature, are controllable. A fast, robust and reproducible polymerization was established, offering the possibility for the facile fabrication of a hydrophobic monolithic sorbent. Thus, on the basis of these results, the monolithic column prepared at 55 °C for 4 h was chosen for further experiment. In addition, the mechanical stability of these monolithic columns is one of the most practical factors for SPE sorbent, especially for online operation. The relationship between the flow rate and the pressure in the synthesized columns was investigated when MeOH was used as the mobile phase. As shown in Figure 2B, a good linear (r = 0.999) dependence of the column pressure on

Figure 3. MRM chromatograms of the online SPE of the five avermectins with a 10 mmol/L NH4OAc solution as loading solution and MeCN as eluting solution.

the flow rate was obtained. The pressure drop was lower than 1.0 bar at a flow rate of 0.5 mL min−1, demonstrating the high permeability of the monolithic materials. The prepared monolith exhibited an adequate mechanical strength of up to 80 bar when it was operated in online mode. A SEM image of a poly(BMA-co-EGDMA) monolithic column further demonstrated the distinct continuous porous structure (Supporting Information, Figure S1). These results clearly indicate that the high mechanical strength and permeability allow the monolithic extractor to be available for online SPE, thus shortening the whole analysis time. Online SPE and LC-MS/MS. The online analysis procedure includes loading, washing, transfer, conditioning, and LC-MS/ MS separation. For the loading step, the loading solvent affects the efficiency of preconcentration and matrix elimination. The influence of the loading solvent was tested using 0.1% and 0.5% formic acid and 5 and 10 mmol/L NH4OAc solution. To

Figure 2. (A) Effect of the reaction time (h) and temperature (°C) on k. (B) Relationship between the flow rate and the back-pressure drop of the poly(BMA-co-EGDMA) monolithic extractor when MeOH was used as the mobile phase. 4099


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Figure 4. MRM mass chromatograms of blank samples and fortified samples: (A) chili seed, (B) fresh pear, (C) milk, and (D) bovine muscle.

evaluate the loading efficiency, the five extracted avermectins were eluted with MeCN and the chromatograms were compared. The most concentrated, sharp, and symmetric peaks with the maximum peak heights were obtained when 10 mmol/L NH4OAc was used as the loading solution (Figure 3), providing the most efficient extraction for target compounds. After the loading and washing steps, the extracted analytes were transferred from the poly(BMA-co-EGDMA) monolithic extractor into a C18 analytical column for LC-MS/MS separation. MeCN and MeOH were each evaluated for use as the eluting solvent. The results showed that both MeCN and MeOH gave an effective eluting for the extracted avermectins. However, the peaks with MeCN as eluting solvent were more symmetrical and less broadened (Figure 3). Therefore, the adsorbed compounds may be quickly transferred into the subsequent LC-MS/MS without diffusion and any loss of the

analytes. Considering peak broadening and tailoring in separation caused by the diffusion, MeCN was selected as the eluent. The LC-MS/MS parameters were optimized to ensure proper resolution and symmetry and adequate separation of the analytes. In this work, to enhance ionization of the avermectins in the MS source, a mixture of MeCN and a 0.1% NH4OH solution was found to be optimal for LC-MS/MS separation. The starting composition of MeCN for the gradient was investigated in the range 80−10%, and the final composition of MeCN was fixed at 95%. Baseline separation of five avermectins is achieved in all investigated mobile phases. However, different responses of the target compounds are found. Lower composition of MeCN may lead to better chromatographic resolutions and enhanced ionizations (Supporting Information, Figure S2). To reduce the retention time 4100


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performed using the poly(BMA-co-EGDMA)-based SPE method. Matrix Effect. The matrix effect was investigated by comparing the slopes of calibration curves obtained from a standard mixture of avermectins in solvent and the standards spiked with the samples. The matrix-matched calibration curves were constructed by spiking the avermectin standard solutions at seven concentration levels in the corresponding matrices. The solvent calibration curves were constructed in the same manner but without the addition of the matrix aliquots. For the bovine muscle sample, good linear relationships for five avermectins are achieved over the concentration range 0.5− 100.0 ng/mL with a correlation coefficient (r) value higher than 0.995, and the matrix/solvent slope ratio for each avermectin in the bovine muscle sample ranged from 0.93 to 1.07 (Supporting Information, Table S1). The signal enhancement or suppression effect was considered acceptable if the ratio of the slope was in the range 0.8 to 1.2. Slope ratios with values higher than 1.2 or lower than 0.8 indicate a distinct matrix effect. Additionally, the matrix/solvent slope ratios in pear, chili seed, and milk samples were in the range 0.95−1.05, showing no significant matrix effect presented in the analysis of these real samples. Specificity, Accuracy, and Precision. Figure 4 shows the MRM chromatograms of the blank samples and the corresponding blank samples spiked with 10 μg/kg of the standard avermectins. The results showed that the spiked samples were effectively purified by the poly(BMA-coEGDMA) extractor. Compared with the blank samples, no interference peaks are observed in the chromatograms of any of the spiked samples. The results were further confirmed by the recovery experiments. The recovery of the proposed method was assayed on spiked real samples at three levels of five avermectins in four kinds of matrices, and the precision was described on the basis of the intraday and interday relative standard deviations (RSDs). As shown in Table 3, the recoveries of six replicate extractions ranged from 71.8% to 101.3%. The intraday and interday RSDs were in the range 4.38−8.62% and 5.49−8.94%, respectively (Table 4). These results indicate the suitability and the satisfactory reproduci-

Table 3. Recoveries of the Five Avermectins in Various Foodstuffs Determined by the Online SPE and LC-MS/MS recovery % (n = 6) analyte

added (μg/kg)

fresh pear

chili seed

bovine muscle

milk

1

5 10 50 5 10 50 5 10 50 5 10 50 5 10 50

71.8 84.3 97.6 79.9 84.5 97.6 77.6 77.3 99.5 90.9 93.0 98.0 76.9 84.0 83.2

80.5 80.7 98.6 80.3 82.4 92.8 75.9 77.7 94.1 76.3 92.7 98.4 75.2 87.3 91.5

77.4 98.7 81.3 86.5 93.8 101.3 77.8 91.4 92.5 89.4 92.5 95.1 74.0 76.0 97.1

84.5 92.2 87.0 80.3 93.6 89.5 92.6 95.3 90.8 81.3 96.2 93.4 88.9 100.1 84.9

2

3

4

5

and improve the sensitivity of the avermectins, the mobile phase consisting of MeCN/0.1% NH4OH (10:90, v/v) was selected as starting mobile phase. The proposed approach enabled us to enrich the analytes online from the sample matrix and accomplished the analysis in a very short time of less than 15 min. The online SPE procedure reduced manual handling to a minimum and thus saved both manpower and solvents. In addition, the use of LCMS/MS ensured both highly sensitive and selective results. Validation of the Method. A very generic sample extraction method is necessary to extract the compounds from a broad range of complicated matrices. The monolithic materials have been demonstrated to be available for plant26 or animal origin27,32,34 samples. In this work, the foodstuffs of both plant and animal origins including pear, chili seed, bovine muscle, and milk were pretreated using the poly(BMA-coEGDMA) monolithic extractor. To evaluate the extraction efficiency and potential interfering compounds originating from the plant and animal origin matrices, blank samples, spiked samples, matrix effect, and recovery experiments were

Table 4. Precisions (RSDs) of the Five Avermectins in Foodstuffs Determined by the Online SPE and LC-MS/MS RSD % (n = 6) fresh pear

chili seed

bovine muscle

milk

analyte

added (μg/kg)

intraday

interday

intraday

interday

intraday

interday

intraday

interday

1

5 10 50 5 10 50 5 10 50 5 10 50 5 10 50

7.43 7.44 5.03 7.36 7.11 6.22 7.69 7.36 6.54 7.35 6.80 7.12 6.41 5.97 5.68

8.06 7.76 6.51 7.97 7.65 6.87 8.01 7.89 7.08 8.11 7.67 7.79 8.51 6.09 5.78

7.68 6.42 6.63 7.56 6.88 5.90 7.84 7.21 6.03 8.16 8.06 7.92 7.25 6.69 6.02

8.20 7.26 7.18 7.94 7.25 6.13 8.25 8.07 6.99 8.52 7.64 7.98 7.99 7.36 6.58

7.84 6.36 6.49 6.61 6.49 5.81 8.62 8.04 5.76 7.10 6.70 5.01 7.54 7.60 6.56

8.30 8.14 7.47 7.36 6.61 6.41 8.94 7.96 7.22 7.63 6.78 5.73 7.45 7.57 6.81

7.98 7.18 4.48 6.76 6.88 4.84 6.86 6.49 6.09 7.39 7.10 5.79 7.98 7.56 4.38

8.13 8.07 6.46 7.15 7.25 5.49 7.68 6.94 6.16 7.44 7.75 6.73 8.27 7.38 6.82

2

3

4

5

4101



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bility of the developed method for the determination of five avermectins in four matrices of both plant and animal origin. LODs and LOQs. The limit of detection (LOD) was determined as the concentration that produced a peak with a height 3 times the level of the baseline noise, and the limit of quantitation (LOQ) was determined as the sample concentration that produced a peak 10 times greater than the baseline noise. The LODs and the LOQs of the proposed method were 2 μg/kg and 5 μg/kg for each analyte, which showed that the present method is sufficiently sensitive to measure avermectins at the MRLs6−10 in various matrices. Reusability and Reproducibility of the Poly(BMA-coEGDMA) Monolithic Extractors. Traditional commercial SPE sorbents, such as C18, C8, and Al2O3 particles, are disposable. The reusability and reproducibility of the poly(BMA-co-EGDMA) monolithic sorbent trap were studied. Under the optimized conditions, at least 500 injections of pear sample were performed. Moreover, poly(BMA-co-EGDMA) monolithic extractors prepared from various batches were also used. No significant changes were observed in the extraction efficiency. The column-to-column reproducibility was assessed by calculating the RSDs of the extracted amount of abamectin while performing the online SPE. The intrabatch and interbatch RSDs were in the range 4.6−6.3% and 6.7−9.3%, respectively, indicating that the proposed sample preparation method is robust and practical. Assay of Avermectins in Real Samples. The feasibility of the developed poly(BMA-co-EGDMA) monolithic column based on the online SPE method was further demonstrated for the analysis of real samples. Among the assayed samples from a local market, abamectin was detected in one bovine muscle sample with a concentration of 5.9 ± 0.6 μg/kg and ivermectin was detected in two chili seed samples with concentrations of 7.9 ± 0.9 and 8.2 ± 0.9 μg/kg. The present study focused on the development of a robust, simple, and practical method that is capable of quickly and simultaneously extracting and determining five avermectins in samples of both plant and animal origin. Although the poly(BMA-co-EGDMA) monolithic extractor gave better or comparable recovery and detection limits compared with other reported adsorbents,15−21 this method gave more advantages by reducing analysis time and improving reusability of the sorbent. An offline cleaning and preconcentration process using a conventional SPE required several steps and took more than 2 h, while the time for online SPE and analysis of this work was only 15 min. In addition, the cost of the determination was also significantly reduced because the poly(BMA-co-EGDMA) monolithic extractor could be reused more than 500 times without any loss of extraction efficiency, even for different varieties of food samples. Since this kind of hydrophobic monolithic extractor could be easily obtained and showed good availability, we believe that the method was able to deal with a wide range of compounds with weak polarity from complicated samples.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +86-315-3725730. Fax: +86-315-2592281. E-mail: [email protected]. Funding

This work was supported by the National Natural Science Foundation of China (No. 21305028), the Natural Science Foundation of Hebei Province, China (No. B2013209238), the Research Foundation of Education Bureau of Hebei Province, China (No. Q2012155), and the Research Foundation from General Administration of Quality Supervision, Inspection and Quarantine of China (Nos. 2013IK154 and 2014IK092). Notes

The authors declare no competing financial interest.

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REFERENCES

(1) Martin, D.; Wolfgang, R.; Lucija, K.; Jemma, K.; Vesna, C. F. Recent developments in the analysis of avermectin and milbemycin residues in food safety and the environment. Curr. Pharm. Biotechnol. 2012, 13, 936−951. (2) Cabrera, J. A.; Menjivar, R. D.; Dababat, A. F. A.; Sikora, R. A. Properties and nematicide performance of avermectins. J. Phytopathol. 2013, 161, 65−69. (3) Macdonald, N.; Gledhill, A. Potential impact of ABCB1 (pglycoprotein) polymorphismson avermectin toxicity in humans. Arch. Toxicol. 2007, 81, 553−563. (4) Sung, Y. F.; Huang, C. T.; Fan, C. K.; Lin, C. H.; Lin, S. P. Avermectin intoxication with coma, myoclonus, and polyneuropathy. Clin. Toxicol. 2009, 47, 686−688. (5) EC Regulation (EU) No. 37/2010 of 22 December 2010 on pharmacologically active substances and their classification regarding maximum residue limits in foodstuffs of animal origin. Off. J. Eur. Union 2010, L15, 1−17. (6) European Medicines Agency, EMEA, Summary opinion of the committee for medicinal products for veterinary use on the establishment of MRL, doramectin, EMEA/CVMP/283233/2006, 21 July, 2006. (7) European Medicines Agency, EMEA, Committee for medicinal products for veterinary use, ivermectin, EMEA/CVMP/915/04FINAL, November 2004. (8) European Medicines Agency, EMEA, Committee for medicinal products for veterinary use, emamectin, EMEA/CVMP/863/03FINAL, February 2003. (9) European Medicines Agency, EMEA, Committee for medicinal products for veterinary use, abamectin, EMEA/CVMP/838/02FINAL, May 2002. (10) National Standards of the People’s Republic of China. National food safety standard maximum residue limits for 85 pesticides in food. GB 28260-2011. (11) Wei, X. X.; Dou, C. Y.; Duan, Y. S.; Ma, Y. S. Determination of avermectin residues in dried chili by HPLC with fluorescence detection. J. Food Saf. Qual. 2013, 1298, 35−43. (12) Giannetti, L.; Giorgi, A.; Necci, F.; Ferretti, G.; Buiarelli, F.; Neri, B. Validation study on avermectine residues in foodstuffs. Anal. Chim. Acta 2011, 700, 11−13. (13) Rübensam, G.; Barreto, F.; Hoff, B. R.; Kist, L. T.; Pizzolato, M. T. A liquid-liquid extraction procedure followed by a low temperature purification step for the analysis of macrocyclic lactones in milk by liquid chromatography-tandem mass spectrometry and fluorescence detection. Anal. Chim. Acta 2011, 705, 24−29. (14) Tao, Y. F.; Yu, G.; Chen, D. M.; Pan, Y. H.; Liu, Z. L.; Wei, H. M.; Peng, D. P.; Huang, L. L.; Wang, Y. L.; Yuan, Z. H. Determination of 17 macrolide antibiotics and avermectins residues in meat with accelerated solvent extraction by liquid chromatography-tandem mass spectrometry. J. Chromatogr. B 2012, 897, 64−71. (15) Rübensam, G.; Barreto, F.; Hoff, R. B.; Pizzolato, T. M. Determination of avermectin and milbemycin residues in bovine

ASSOCIATED CONTENT

S Supporting Information *

Detailed description of the SEM image of a poly(BMA-coEGDMA) monolithic extractor prepared at 55 °C for 4 h, ×5000; response of the avermectins with different starting compositions of the mobile phase, and standard curves of avermectins in solvent and matrix and their slope ratios. This material is available free of charge via the Internet at http:// pubs.acs.org. 4102


Article


(32) Romanovskaya, A.; Sarin, L. P.; Bamford, D. H.; Poranen, M. M. High-throughput purification of double-stranded RNA molecules using convective interaction media monolithic anion exchange columns. J. Chromatogr. A 2013, 1278, 54−60. (33) Javanbakht, M.; Moein, M. M.; Akbari-adergani, B. On-line clean-up and determination of tramadol in human plasma and urine samples using molecularly imprinted monolithic column coupling with HPLC. J. Chromatogr. B 2012, 911, 49−54. (34) Hu, X. Z.; Wang, J. X.; Feng, Y. Q. Determination of benzimidazole residues in edible animal food by polymer monolith microextraction combined with liquid chromatography-mass spectrometry. J. Agric. Food Chem. 2010, 58, 112−119. (35) Rogeberg, M.; Malerod, H.; Roberg-Larsen, H.; Aass, C.; Wilson, S. R. On-line solid phase extraction-liquid chromatography, with emphasis on modern bioanalysis and miniaturized systems. J. Pharm. Biomed. Anal. 2014, 87, 12−129. (36) Liu, W.; Qi, J. R.; Yan, L. N.; Jia, Q.; Yu, C. Application of poly(butyl methacrylate-co-ethylene glycol dimethacrylate) monolith microextraction coupled with high performance liquid chromatography to the determination of polycyclic aromatic hydrocarbons in smoked meat products. J. Chromatogr. B 2011, 879, 3012−3016. (37) Lin, S. L.; Wu, Y. R.; Lin, T. Y.; Fuh, M. R. Preparation and evaluation of 1,6-hexanediol ethoxylate diacrylate-based alkyl methacrylate monolithic capillary column for separating small molecules. J. Chromatogr. A 2013, 1298, 35−43. (38) Liu, Y.; Wang, M. M.; Ai, L. F.; Zhang, C. K.; Li, X.; Wang, X. S. Determination of Sudan dyes in chili pepper powder by online solidphase extraction with a butyl methacrylate monolithic column coupled to liquid chromatography with tandem mass spectrometry. J. Sep. Sci. 2014, 37, 1648−1655.

muscle by liquid chromatography-tandem mass spectrometry and fluorescence detection using solvent extraction and low temperature cleanup. Food Contr. 2013, 29, 55−60. (16) Sannino, A. Determination of three natural pesticides in processed fruit and vegetables using high-performance liquid chromatography/tandem mass spectrometry. Rapid Commun. Mass Spectrom. 2007, 21, 2079−2086. (17) Campillo, N.; Viñas, P.; Férez-Melgarejo, G.; HernándezCórdoba, M. Dispersive liquid-liquid microextraction for the determination of macrocyclic lactones in milk by liquid chromatography with diode array detection and atmospheric pressure chemical ionization ion-trap tandem mass spectrometry. J. Chromatogr. A 2013, 1282, 20−26. (18) Raich-Montiu, J.; Krogh, K. A.; Granados, M.; Jönsson, J. A.; Halling-Sørensen, B. Determination of ivermectin and transformation products in environmental waters using hollow fiber-supported liquid membrane extraction and liquid chromatography-mass spectrometry/ mass spectrometry. J. Chromatogr. A 2008, 1187, 275−280. (19) Berendsen, B. J. A.; Mulder, P. P. J.; Rhijn, V. H. (J.) A. The derivatisation of avermectins and milbemycins in milk: New insights and improvement of the procedure. Anal. Chim. Acta 2007, 585, 126− 133. (20) Cerkvenik-Flajs, V.; Milčinski, L.; Süssinger, A.; Hodošcě k, L.; Danaher, M.; Antonić, J. Trace analysis of endectocides in milk by high performance liquid chromatography with fluorescence detection. Anal. Chim. Acta 2010, 663, 165−171. (21) Durden, D. A. Positive and negative electrospray LC-MS-MS methods for quantitation of the antiparasitic endectocide drugs, abamectin, doramectin, emamectin, eprinomectin, ivermectin, moxidectin and selamectin in milk. J. Chromatogr. B 2007, 850, 134−146. (22) Tom, L. A.; Foster, N. Development of a molecularly imprinted polymer for the analysis of avermectin. Anal. Chim. Acta 2010, 680, 79−85. (23) Wang, F. M.; Chen, J. H.; Cheng, H. Y.; Tang, Z. X.; Zhang, G.; Niu, Z. Y.; Pang, S. P.; Wang, X. R.; Lee, F. S. C. Multi-residue method for the confirmation of four avermectin residues in food products of animal origin by ultra-performance liquid chromatography-tandem mass spectrometry. Food Addit. Contam. Part A 2011, 28, 627−639. (24) Krogh, K. A.; Björklund, E.; Loeffler, D.; Fink, G.; HallingSørensen, B.; Ternes, T. A. Development of an analytical method to determine avermectins in water, sediments and soils using liquid chromatography-tandem mass spectrometry. J. Chromatogr. A 2008, 1211, 60−69. (25) Namera, A.; Nakamoto, A.; Saito, T.; Miyazaki, S. Monolith as a new sample preparation material: Recent devices and applications. J. Sep. Sci. 2011, 34, 901−924. (26) Jandera, P. Advances in the development of organic polymer monolithic columns and their applications in food analysis-A review. J. Chromatogr. A 2013, 1313, 37−53. (27) Wei, F.; Feng, Y. Q. Methods of sample preparation for determination of veterinary residues in food matrices by porous monolith microextraction-based techniques. Anal. Methods 2011, 3, 1246−1256. (28) Arrua, R. D.; Causon, T. J.; Hilder, E. F. Recent developments and future possibilities for polymer monoliths in separation science. Analyst 2012, 137, 5179−5189. (29) Zheng, M. M.; Gong, R.; Zhao, X.; Feng, Y. Q. Selective sample pretreatment by molecularly imprinted polymer monolith for the analysis of fluoroquinolones from milk samples. J. Chromatogr. A 2010, 1217, 2075−2081. (30) Chienthavorn, O.; Ramnut, N.; Subprasert, P.; Sasook, A.; Insuan, W. Effective and reusable monolith capillary trap of nitrosamine extraction by superheated water from Frankfurter sausage. J. Agric. Food Chem. 2014, 62, 1240−1246. (31) Regueiro, J.; Á lvarez, G.; Mauriz, A.; Blanco, J. High throughput analysis of amnesic shellfish poisoning toxins in bivalve molluscs by dispersive solid-phase extraction and high-performance liquid chromatography using a monolithic column. Food Chem. 2011, 127, 1884−1891. 4103


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