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Application of molecularly imprinted polymer based matrix solid phase dispersion for determination of fluoroquinolones, tetracyclines and sulfonamides in meat
Journal of Chromatography B 1065–1066 (2017) 104–111
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Journal of Chromatography B journal homepage: www.elsevier.com/locate/jchromb
Application of molecularly imprinted polymer based matrix solid phase dispersion for determination of fluoroquinolones, tetracyclines and sulfonamides in meat Geng Nan Wang, Lei Zhang, Yi Ping Song, Ju Xiang Liu, Jian Ping Wang
MARK
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College of Veterinary Medicine, Agricultural University of Hebei, Baoding, Hebei, 071000, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Molecularly imprinted polymer Matrix solid phase dispersion Ultra performance liquid chromatography Fluoroquinolones Sulfonamides Tetracyclines
In this study, a type of novel mixed-template molecularly imprinted polymer was synthesized that was able to recognize 8 fluoroquinolones, 8 sulfonamides and 4 tetracyclines simultaneously with recoveries higher than 92%. Then the polymer was used to develop a matrix solid phase dispersion method for simultaneous extraction of the 20 drugs in pork followed by determination with ultra performance liquid chromatography. During the experiments, the MMIP amount, washing solvent and elution solvent were optimized respectively. The limits of detection of this method for the 20 drugs in pork were in the range of 0.5–3.0 ng g−1, and the intra-day and inter-day recoveries from the fortified blank samples were in the range of 74.5%–102.7%. Therefore, this method could be used as a rapid, simple, specific and sensitive method for multi-determination of the residues of the three classes of drugs in meat.
1. Introduction The residues of veterinary drugs in foods of animal origin can pose many potential risks to the consumers. For example, the residues of fluoroquinolones (FQs) can induce the drug resistant pathogens [1], the residues of tetracyclines (TCs) can cause gastrointestinal disturbances and allergic reactions to the consumers [2], and the residues of sulfonamides (SAs) have potential carcinogenic effects on humans [3]. To ensure food safety and human health, China and the European Union have established different maximum residue limits (MRLs) for these drugs in meat, i.e. the sum of SAs, 100 ng g−1; FQs, 20–200 ng g−1; single or total amount of TCs, 100 ng g−1 [4,5]. Therefore, it is urgent to monitor their residues in foods of animal origin. The first thing for determination of residual veterinary drugs in foods of animal origin is to extract and purify the low level of analytes in the samples. By now, many sample preparation methods, such as solid phase extraction [6], solid-phase microextraction [7], stir bar sorptive extraction [8], QuEChERS [9], immunoaffinity chromatography [10], and dispersive solid phase extraction [11], have been reported for extraction and purification of FQs, SAs and TCs in various samples. However, the first step of these extraction methods is to transfer the analytes from the samples into various solvent phases, and then different purification procedures are performed. This meant that the solubilization procedure is a rate-limiting step.
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Matrix solid phase dispersion (MSPD) is a novel sample preparation method that was first developed in 1989 [12]. For a MSPD method, the solid sample, viscous sample or liquid sample is blended with a type of suitable dispersing sorbent, and then the sample-sorbent mixture is transferred into an empty syringe barrel. After the sample impurities are washed out with proper solvent, the analytes are eluted for analysis. This sample preparation method combines extraction and purification into one step that can be finished within several minutes, so this technique is simpler and more flexible than the commonly used sample preparation methods. Therefore, there have been many articles reporting the use of MSPD technique for determination of various analytes in different samples [13], including SAs [14–16], TCs [17] and FQs [18,19]. However, the usually used dispersing sorbents in the previous reports (C18, SiO2, HLB material, diatomite, N-propylethylenediamine, alumina, Florisil) are easily interfered by the sample impurities, and these sorbents may lead to competitive adsorption when one sample simultaneously contains other compounds besides the target analytes. Therefore, it is desirable to find a specific dispersing sorbent for MSPD technique. Over the past 10 years, molecularly imprinted polymer (MIP) has drawn the interests of many researchers due to its specific recognition ability. Therefore, MIP based materials have been widely used for the determination of different analytes in various samples [20], including TCs [21,22], FQs [23] and SAs [24–26]. In 2007, a novel sample
Corresponding author. E-mail address: [email protected] (J.P. Wang).
http://dx.doi.org/10.1016/j.jchromb.2017.09.034 Received 21 June 2017; Received in revised form 15 August 2017; Accepted 20 September 2017 Available online 21 September 2017 1570-0232/ © 2017 Elsevier B.V. All rights reserved.
Journal of Chromatography B 1065–1066 (2017) 104–111
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pretreatment method combining MIP and MSPD was first developed for extraction of FQs residues in egg and swine tissues [27]. Results showed that this technique avoided competitive adsorption and achieved highly selective purification effect, and the sorbent MIP showed better purification effect and higher recoveries than four tested sorbents (silica, C18, sand and Florisil). Thereafter, MIP-MSPD has been used for the determination of BPA [28], β-estradiol [29], steroids [30], clenbuterol [31], FQs [32–34] and Sudan dyes [35] in various samples. However, there has been no article reporting the use of MIP-MSPD method for the determination of SAs and TCs so far. The commonly synthesized MIP is only able to recognize a group of structurally similar analytes, so there has been no article reporting the use of MIP-MSPD method for multi-determination of different classes of veterinary drugs in foods of animal origin so far. In the present study, three molecules (pipemidic acid for FQs, sulfabenzamide for SAs, and chlortetracycline for TCs) were used to synthesize a novel mixed-template MIP (MMIP), and its recognition performance for FQs, SAs and TCs were studied. Then, a MMIP-MSPD method was developed for extraction and purification of the three classes of drugs in pork followed by determination with ultra performance liquid chromatography (UPLC).
2.3. Synthesis of MMIP particles The MMIP was synthesized according to our recent reports [22,23]. Briefly, the mixture of three templates (PA, SB and TC, 1 mmol of each template), the functional monomer MA (18 mmol) and the porogen chloroform (18 mL) were added into a glass bottle to be sonicated for 20 min, and kept at 4 °C overnight. Then, the cross-linker DGDMA (40 mmol) and the initiator AIBN (40 mg) were added. The bottle was filled with nitrogen for 10 min and sealed to be shaken in a 60 °C water bath for 24 h. The obtained MMIP particles were extracted on a Soxhlet apparatus for 72 h successively by using methanol/acetic acid (9:1, v/ v). Finally, the MMIP particles were dried at 110 °C for 2 h for the subsequent use. For comparison, the controlled non-imprinted polymer (NIP) was also synthesized as described above but without the three templates. The two types of polymers were all scanned by using scanning electron microscopy (SEM) (JSM-7500F, JEOL, Japan). Furthermore, the MMIP specificity for the 20 drugs was determined according to a previous report [24]. Briefly, 20 mg MMIP or NIP was added into 5 mL mixed standard solution (100 ng of each drug) to be stirred for 5 min. Then the supernatant was analyzed by UPLC. The partition coefficient (K) was calculated as: K = CA/CS (CA is the drug amount absorbed by MMIP or NIP, and CS is the drug amount in supernatant). The specificity was evaluated based on imprinting factor (IF): IF = KMMIP/KNIP (KMMIP and KNIP represent the K value of each drug from MMIP and NIP respectively).
2. Materials and methods 2.1. Reagents and chemicals Pipemidic acid (PA) and sulfabenzamide (SB) were purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany). The standards of enrofloxacin (ENR), sarafloxacin (SAR), ciprofloxacin(CIP), lomefloxacin (LOM), ofloxacin (OFL), pefloxacin (PEF), danofloxacin (DAN), marbofloxacin (MAR), sulfadiazine (SD), sulfadimidine (SM2), sulfamethoxypyridazine (SMP), sulfadimethoxine (SDM), sulfamonomethoxine (SMM), sulfamethoxazole (SMZ), sulfaquinoxaline (SQ), sulfachloropyridazine sodium (SCP) tetracycline (TC), oxytetracycline (OTC), chlortetracycline (CTC), and doxycycline (DC) were purchased from Sigma (St. Louis, MO, USA). Ethylene glycol dimethacrylate (EGDMA) was purchased from Aladdin Industrial Corporation (Shanghai, China). Methacrylic acid (MA) and 2,2-azobis (isobutyronitrile) (AIBN) were purchased from Kermer Chemical Company (Tianjin, China). Other chemical reagents were of analytical grade or better from Beijing Chemical Company (Beijing, China). Liquid chromatographic grade acetonitrile and methanol were purchased from Dikma (Richmond Hill, USA). Standard stock solutions of the 20 drugs were prepared with methanol respectively (100 μg mL−1), and these solutions were stable for four months at −20 °C. The working solutions of these drugs were diluted from the stock solutions with water (1–1000 ng mL−1), and these solutions were stable for one week at 4 °C.
2.4. Sample preparation with MMIP-MSPD An amount of 0.2 g homogenous pork sample was added into a mortar. At this stage, the mixed standards were fortified at different concentrations, and the mixture was ground with a pestle to obtain a homogenous sample. Then, 0.15 g MMIP was added, and the mixture was gently ground until a homogenous mixture was obtained (about 3 min). At the same time, an empty cartridge (100 mm × 9.9 mm, i.d.) with a lower frit was packed with 50 mg MMIP that was conditioned by washing with 1 mL methanol and 1 mL water in turn. Then the homogenous mixture was transferred into the cartridge, and compressed with a syringe plunger to create a compact column bed. The cartridge was washed with 3 mL methanol/water (2:8, v/v), and the solutions passed through the cartridge at a flow rate of 1 mL min−1 under the assistance of vacuum pump (about 3 min). Then, the analytes were eluted with 4 mL methanol/acetic acid (9:1, v/v) under the assistance of vacuum pump (about 4 min), and the eluate was evaporated to dry under gentle nitrogen. Finally, the dry residue was reconstituted in 200 μL water and filtered with a 0.22 μm membrane for UPLC analysis. During the experiments, the MMIP amount, washing solvent and eluting solvent were optimized respectively.
2.5. Method validation 2.2. UPLC conditions Some blank pork samples obtained from the controlled slaughterhouses were used to evaluate the method. The limits of detection (LODs) and the limits of quantification (LOQs) for the 20 analytes were calculated as the concentrations corresponding to peak/noise ratio (S/ N) of 3:1 and 10:1 respectively. Then the 20 drugs were fortified into the blank pork samples respectively at levels of 20–1000 ng g−1 for analysis. The intra-day recoveries (six repetitions for each fortification level in a single day) and the inter-day recoveries (duplicate injections for each fortification level on six successive days) were determined respectively. Finally, 70 pork samples purchased from several supermarkets in China were analyzed by the developed method.
UPLC system consisted of a ACQUITY H-CLASS liquid chromatography, a PDA detector and a BEH C18 column (2.1 × 50 mm, 1.7 μm) (Waters, USA). The mobile phase consisted of (A) 0.2% formic acid and (B) acetonitrile/methanol (1:1, v/v) with gradient elution at a flow rate of 0.4 mL min−1. The gradient elution program was: started with 10% (A), linearly increased to 12% (A) in 7.9 min, decreased to 1% (A) in 0.6 min, then increased to 12% (A) in 0.1 min, linearly increased to 65% (A) in 2.4 min, decreased to 10% (A) in 0.1 min, linearly increased to 50% (A) in 0.9 min, finally brought back to 10% (A) in 1.0 min and maintained for 1.0 min with a total running time of 14 min. The injection volume was 10 μL, and the detection wavelengths were 270, 279 and 289 nm.
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Table 1 Determination parameters of the MMIP-MSPD-UPLC method for the 20 drugs in pork (n = 5). Analyte
Linearity (ng mL−1)
r2
Intra-batch RSD (%)
Inter-batch RSD (%)
LOD (ng g−1)
LOQ (ng g−1)
Retention time (min)
Imprinting factor
SD SM2 TC MAR SMP OTC SCP OFL PEF SMM CIP SMZ LOM DAN ENR SAR CTC SDM DC SQ
5–1000 6–1000 2–1000 5–1000 5–1000 2–1000 6–1000 5–1000 5–1000 6–1000 6–1000 6–1000 6–1000 2–1000 2–1000 2–1000 2–1000 5–1000 2–1000 5–1000
0.9999 0.9997 0.9994 0.9997 0.9994 0.9992 0.9998 0.9998 0.9993 0.9996 0.9994 0.9991 0.9999 0.9998 0.9996 0.9997 0.9992 0.9995 0.9991 0.9997
3.1 1.7 2.2 2.1 2.7 3.4 2.0 2.8 2.6 2.6 3.1 2.7 1.7 2.8 2.0 2.1 1.7 1.9 2.6 3.4
3.2 2.4 2.8 2.8 3.0 2.8 2.7 3.0 3.8 3.2 3.6 3.4 2.9 2.7 3.0 2.9 3.2 3.1 2.4 3.2
2 3 1 3 2 1 3 2 2 3 3 3 3 1 1 1 0.5 2 0.5 2
5 6 2 5 5 2 6 5 5 6 6 6 6 2 2 2 1.5 5 1.5 5
1.939 4.926 5.182 5.894 6.147 7.17 7.977 8.181 8.376 8.568 9.336 10.16 10.542 10.63 10.676 10.811 10.926 11.236 11.49 11.556
6.2 4.2 6.5 6.9 5.7 5.2 5.3 7.6 5.1 8.3 5.6 6.1 5.0 8.4 9.1 6.7 6.8 7.2 4.9 8.8
3. Results and discussions
were used as the mixed-template in the present study to synthesize the MMIP for FQs, SAs and TCs. After the MMIP was obtained, three methods were used to characterize it. Firstly, the MMIP was subjected to SEM characterization. Results showed that the surface of MMIP was porous and rugged, but the surface of NIP was smooth and less porous (See Fig. S1 in Supplementary Material), indicating the templates were imprinted and the pores should be beneficial for capturing the analytes. Secondly, 50 mg MMIP or NIP particle was packed into an empty cartridge, and the 20 target analytes and several other competitors were loaded onto the cartridge respectively. Then the analytes were eluted for analysis. As shown in Fig. 2, the MMIP simultaneously captured 8 FQs, 8 SAs and 4 TCs with recoveries of 70%–90%, but showed very low recoveries to other competitors (< 7%). In comparison, the NIP showed comparably low recoveries to all of the tested analytes (5%–14%). This indicated that the MMIP captured FQs, SAs and TCs due to the specific molecular recognition. Thirdly, the imprinting factor (IF) for each analyte was calculated to verify the MMIP selectivity. As shown in Table 1, the MMIP showed IFs of 4.2-9.1 for the three classes of drugs. High IF value represented high recognition ability, so these results were consistent with the results shown in Fig. 2. This is the first paper reporting the synthesis of a type of MMIP material capable of simultaneous recognizing FQs, TCs and SAs. Then, this material was used as dispersing sorbent for development of the MSPD method.
3.1. Optimization of UPLC conditions It is well known that UPLC can achieve excellent separation effect. For simultaneous separation of the 20 drugs belonging to three classes, the UPLC separation condition was examined for obtaining the optimal analytical time, peak shape and the maximum sensitivity. During the experiments, methanol, acetonitrile and their mixtures with different proportions of formic acid (0.1%-0.5%) were compared under different gradient elution procedures. Results showed that the mobile phase acetonitrile/methanol (1:1, v/v) plus 0.2% formic acid using the gradient program shown in Section 2.2 achieved the optimal performance. The retention times of the 20 drugs are shown in Table 1, and the chromatogram of the 20 standards is shown in Fig. 1A. For multi-detection of the three classes of drugs, the detection wavelength is very important. During the experiments, the PDA detector was operated in scan mode, and different wavelengths were compared. Results showed that the optimal detection wavelengths for FQs and SAs were around 280 nm, and the optimal detection wavelengths for TCs were around 280 and 350 nm. For multi-determination of the three classes of drugs, 270, 279 and 289 nm were selected as the detection wavelengths, and Fig. 1A was the chromatogram obtained at 270 nm.
3.2. Characterization of MMIP 3.3. Optimization of MMIP-MSPD In the previous reports, the MIPs for TCs [21,22], FQs [23,27,32–34] and SAs [24–26] were usually synthesized with a single molecule as the template, so those MIPs were only able to recognize a group of structurally similar analogs. For example, the CTC based MIP was able to capture four TCs with high recoveries but showed negligible recognition ability to SAs and FQs [22], whereas the norfloxacin based MIP only recognized four FQs but showed negligible recognition ability to SAs and TCs [23]. In four recent reports, two or five molecules were used as the mixed-templates to synthesize the MIPs, and the results showed that the MIPs’ recognition spectra were broadened [36–39]. Therefore, it is desirable to synthesize a MIP capable of simultaneous recognizing FQs, SAs and TCs. In the previous reports, different molecules were used as the templates to synthesize MIPs for FQs (OFL, SAR, norfloxacin, daidzein) and SAs (SMZ, SDM), but there has been no article reporting the use of PA and SB as the templates to synthesize MIPs for FQs and SAs so far. PA, SB, and CTC contain the common core structure of their respective classes of drugs, so the three molecules
3.3.1. MMIP amount For a MSPD method, the sorbent plays the parts of abrasive and bound solvent that can facilitate the interactions between the sorbent and the analytes, so the first thing for development of a MSPD method is to evaluate the sorbent amount. Furthermore, a suitable sample/ sorbent ratio can increase the interface area and reduce the sorbent consumption. In the present study, a miniaturized MSPD using small sample amount (0.2 g) was used to optimize the MMIP amount. During the experiments, 0.2 g pork sample was blended with 0.1, 0.15, 0.2, and 0.4 g MMIP particles respectively. As shown in Fig. 3, the recoveries of the 20 drugs reached balance when MMIP amount increased to 0.15 g, i.e. the sample/sorbent ratio of 4:3. For low sorbent amount (0.1 g), the sample component did not fully interact with MMIP particles, thus obtaining low recoveries. For high sorbent amounts (0.2 and 0.4 g), the sample/sorbent complexes were not blended to a homogenous mixture, and different amount of MMIP particles were left in the mortar, i.e. the 106
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Fig 1. Chromatograms of (A) FQs, TCs and SAs standards, (B) blank pork, and standards fortified blank pork treated by (C) MMIP, (D) C18 and (E) diatomaceous earth. (1 = SD, 2 = SM2, 3 = TC, 4 = MAR, 5 = SMP, 6 = OTC, 7 = SCP, 8 = OFL, 9 = PEF, 10 = SMM, 11 = CIP, 12 = SMZ, 13 = LOM, 14 = DAN, 15 = ENR, 16 = SAR, 17 = CTC, 18 = SDM, 19 = DC, 20 = SQ; 100 ng/mL (g)).
chromatograms showed that water and methanol/water (1:9, v/v) were not able to wash out most of the impurities, and methanol/water (2:8, v/v) achieved the best purification effect (Fig. 1B and C), so this solution was selected as the optimal washing solvent.
MMIP was excessive. Therefore, 0.15 g MMIP was used for the subsequent experiments. 3.3.2. Washing solvent For a MSPD procedure, it is necessary to find a suitable washing solvent that should wash out the sample impurities as fully as possible but show no influence on the analyte recovery. In this study, the sample/MMIP mixture was transferred into different cartridges followed by washing with several kinds of solvents. Results showed that the recoveries of the 20 drugs were comparable when using water and aqueous solvents containing 10% and 20% methanol (See Fig. S2 in Supplementary Material). When increasing methanol proportion to 50%, the analytes recoveries decreased because high portion of methanol was able to elute some analytes. However, the obtained
3.3.3. Eluting solvent For a MIP based extraction method, the most important thing is to find a suitable solvent to elute the absorbed analytes. In the previous reports, methanol, acetonitrile and their mixtures with different acids were usually used to elute FQs, TCs and SAs from MIP materials [21–27,32–34]. In this study, methanol, acetonitrile and their mixtures with acetic acid were used to optimize the eluting solvent. As shown in Fig. 4, the recoveries of the 20 drugs when using methanol/acetic acid (9:1, v/v) were generally higher than that when using other solvents, so 107
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MMIP
NIP AMO = amoxicillin CPZ = chlorpromazine CLP =clonazepam
80
60
40
20
0 SD
SM2
TC
MAR SMP OTC SCP OFL PEF SMM CIP SMZ LOM DAN ENR SAR CTC SDM
DC
SQ AMO CPZ CLP
Fig. 2. Recoveries of MMIP and NIP for the tested analytes (50 mg polymer, analyte amount 100 ng, eluting with methanol) (Recoveries were the mean values of three repetitions with standard deviations of 1.4%–4.3%).
MMIP 0.1g
90
MMIP 0.15g
MMIP 0.2g
MMIP 0.4g
80 70 60 50 40 30 20 10 0 Fig. 3. Recoveries of the 20 drugs when using different amounts of MMIP. (0.2 g sample, analyte100 ng, washing with water, eluting with methanol) (Recoveries were the mean values of three repetitions with standard deviations of 1.8%–4.7%).
120
100
SD
SM2
TC
MAR
SMP
OTC
SCP
OFL
PEF
SMM
CIP
SMZ
LOM
DAN
ENR
SAR
CTC
SDM
DC
SQ
80
60
40
20
0
methanol
acetonitrile
methanol/acetic acid
methanol/acetic acid
methanol/acetic acid
Fig. 4. Recoveries of the 20 drugs from MMIP-MSPD when using different eluting solvents. (0.2 g sample, 0.15 g MMIP, analyte 100 ng, washing with methanol/water (2:8, v/v)) (Recoveries were the mean values of three repetitions with standard deviations of 2.6%–4.0%).
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this solution was selected as the optimal elution solvent.
Table 2 Recoveries of FQs, TCs and SAs from fortified blank pork samples (n = 6). analyte
Added (ng g−1)
Inter-assay
3.4. MMIP-MSPD-UPLC
Intra-assay
Recovery (%)
CV (%)
Recovery (%)
CV (%)
SD
20 100 1000
81.2 91.8 97.3
3.4 6.2 8.8
83.4 86.7 92.0
5.3 5.4 8.6
SM2
20 100 1000
86.3 89.9 88.0
5.8 4.9 9.0
88.4 84.7 92.4
6.0 5.8 6.2
TC
20 100 1000
93.3 86.9 97.8
4.2 3.4 9.1
94.2 90.8 92.7
7.4 6.4 7.7
MAR
20 100 1000
74.5 93.8 95.3
5.1 6.0 7.4
79.6 88.9 93.2
5.8 5.3 5.9
SMP
20 100 1000
91.1 95.5 94.3
6.7 4.9 7.9
89.2 95.3 97.8
4.9 5.0 6.3
OTC
20 100 1000
87.0 93.9 96.3
5.2 6.3 5.2
91.3 88.6 94.2
7.2 4.6 5.7
SCP
20 100 1000
81.2 94.9 99.7
6.2 5.3 8.4
85.9 90.0 96.8
5.3 4.8 6.1
OFL
20 100 1000
85.3 89.7 98.1
6.8 6.7 8.7
90.1 90.9 94.7
6.7 8.1 6.0
PEF
20 100 1000
87.1 87.8 95.3
5.2 4.6 6.1
92.8 90.7 93.1
4.6 6.8 7.1
SMM
20 100 1000
95.9 97.5 89.0
3.8 5.8 7.7
97.3 90.9 94.8
6.7 5.8 6.6
CIP
20 100 1000
90.8 89.1 96.0
5.9 5.7 6.9
94.5 85.5 87.4
7.3 4.9 5.2
SMZ
20 100 1000
85.3 92.7 89.7
6.1 4.6 8.1
89.6 91.9 90.8
5.8 8.2 5.3
LOM
20 100 1000
93.4 89.0 96.8
3.7 5.8 6.5
90.4 91.8 97.4
6.2 5.8 7.3
DAN
20 100 1000
92.5 91.6 88.7
4.9 8.2 6.7
97.3 94.0 95.7
7.0 5.3 4.9
ENR
20 100 1000
89.2 96.9 90.8
6.4 7.2 6.1
87.4 91.4 94.6
4.9 5.8 8.0
SAR
20 100 1000
94.9 99.8 96.8
5.9 4.3 6.1
93.6 97.8 95.6
6.7 5.8 6.9
CTC
20 100 1000
91.2 96.2 100.3
4.5 8.7 6.2
94.1 88.5 98.4
7.4 6.3 5.8
SDM
20 100 1000
93.2 91.5 90.3
9.1 5.8 6.8
92.0 90.7 93.8
5.9 8.4 6.7
DC
20 100 1000
92.7 98.3 96.5
4.6 6.2 6.7
88.7 95.1 102.7
6.4 5.7 7.3
20 100 1000
84.1 86.3 88.6
6.4 5.9 7.8
89.6 81.3 91.3
8.0 6.0 8.6
SQ
During the experiments, the 20 drugs were diluted with the extracts of blank pork to prepare the matrix matched standards respectively, and these solutions were used to evaluate the performance of MMIPDSPD-UPLC method. The determination parameters of this method are shown in Table 1. As shown in Table 1, this method showed wide detection linearity range (2–1000 ng mL−1) and high sensitivity (LOD of 0.5–3 ng g−1 and LOQ of 1.5–6 ng g−1) for the 20 drugs. Then, the 20 drugs were fortified into the blank pork samples at concentrations of 20, 100 and 1000 ng g−1 to be extracted and analyzed respectively. The inter-assay recoveries were in the range of 74.5%–100.3%, the intraassay recoveries were in the range of 79.6%–102.7%, and the coefficients of variation (CV) were in the range of 3.4%–9.1% (Table 2). The chromatograms of blank pork and standards fortified blank pork illustrated that there was almost no interfering peak around the analytes peaks, indicating the extremely specific purification effect (Fig. 1B and C). Finally, the 70 real pork samples were analyzed by the method. Results showed that SM2 was detected in one sample (86 ng g−1), and SAR was detected in other two samples (11 and 43 ng g−1). Other samples were all determined as negative samples by this method. 3.5. Comparison with related methods This is the first study reporting the development of a mixed-template MIP based MSPD method for multi-determination of FQs, TCs and SAs in foods of animal origin. For comparison with related reports, the details of some previous reports about the use of MSPD method for determination of FQs, TCs and SAs are summarized in Table 3. As shown in Table 3, all these methods were able to detect low levels of FQs, TCs and SAs [14–19,27,32,34], but the recognition ability of those dispersing sorbents were worse than the present MMIP. General consideration, the method developed in the present study was better than the previously reported MSPD methods. To compare the extraction performance of the MMIP with common dispersing sorbents, C18 and diatomaceous earth were also used to perform MSPD procedure according to the previous reports [14,19]. The obtained chromatograms showed that the two sorbents were able to achieve the satisfactory purification effects (Fig. 1 D, E), but the recoveries of the 20 drugs from the two sorbents (70%–95%) were lower than that from MMIP (92%–99%, Fig. 5). Therefore, the MMIP based MSPD showed generally better performance than the two common dispersing sorbents. 4. Conclusion As a type of specific absorbent, MIP has been widely used for determination of veterinary drugs residues. In the present study, a novel mixed-template MIP capable of simultaneous capturing FQs, TCs and SAs was firstly synthesized, and a MSPD method was then developed and optimized to extract and purify 20 drugs in pork sample. Results showed that the MMIP-MSPD-UPLC method could be used as a rapid, specific and sensitive method to monitor the residues of FQs, TCs and SAs in animal derived foods. Acknowledgements The authors are grateful for the financial supports from Hebei Natural Science Foundation (C2011204021, C2015204049, C2016204039), Earmarked Fund for Hebei Lays Innovation Team of Modern Agro-industry Technology Research System (HBCT2013090202), and Shijiazhuang Technology Incubation Project (171550089A). 109
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Table 3 Comparison of the previous MSPD methods for FQs, TCs and SAs. Analytes
Sorbent
Detection method
LOD (ng mL−1 (g−1))
Sample
Ref.
7 SAs 14 SAs 13 SAs 3 TCs 3 FQs 2 FQs, 2 SAs and TC 5 FQs 2 FQs 8 FQs 8 FQs, 8 SAs and 4 TCs
diatomaceous earth hydrophilic lipophilic balance material C18 C18 C18 C18 OFL based MIP OFL based MIP daidzein based MIP mixed-template based MIP
HPLC-FLD LC–MS/MS LC–MS/MS CE-UV HPLC-DAD HPLC-DAD HPLC-FLD HPLC-DAD HPLC-FLD UPLC-PAD
1.33–2.47 2.3–16.4 0.75–3.0 0.0745–0.08 9–22 2–10 0.05–0.09 8–9 0.06–0.22 0.5–3
liver fish fish milk porcine tissues porcine tissues egg chicken fish pork
14 15 16 17 18 19 27 32 34 This study
MMIP
C18
diatomaceous earth
100
80
60
40
20
0 Fig. 5. Recoveries of the 20 drugs by using different dispersing sorbents. (Recoveries were the mean values of three repetitions with standard deviations of 1.9%–4.5%).
Appendix A. Supplementary data [11]
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jchromb.2017.09.034.
[12]
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Journal of Chromatography B journal homepage: www.elsevier.com/locate/jchromb
Application of molecularly imprinted polymer based matrix solid phase dispersion for determination of fluoroquinolones, tetracyclines and sulfonamides in meat Geng Nan Wang, Lei Zhang, Yi Ping Song, Ju Xiang Liu, Jian Ping Wang
MARK
⁎
College of Veterinary Medicine, Agricultural University of Hebei, Baoding, Hebei, 071000, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Molecularly imprinted polymer Matrix solid phase dispersion Ultra performance liquid chromatography Fluoroquinolones Sulfonamides Tetracyclines
In this study, a type of novel mixed-template molecularly imprinted polymer was synthesized that was able to recognize 8 fluoroquinolones, 8 sulfonamides and 4 tetracyclines simultaneously with recoveries higher than 92%. Then the polymer was used to develop a matrix solid phase dispersion method for simultaneous extraction of the 20 drugs in pork followed by determination with ultra performance liquid chromatography. During the experiments, the MMIP amount, washing solvent and elution solvent were optimized respectively. The limits of detection of this method for the 20 drugs in pork were in the range of 0.5–3.0 ng g−1, and the intra-day and inter-day recoveries from the fortified blank samples were in the range of 74.5%–102.7%. Therefore, this method could be used as a rapid, simple, specific and sensitive method for multi-determination of the residues of the three classes of drugs in meat.
1. Introduction The residues of veterinary drugs in foods of animal origin can pose many potential risks to the consumers. For example, the residues of fluoroquinolones (FQs) can induce the drug resistant pathogens [1], the residues of tetracyclines (TCs) can cause gastrointestinal disturbances and allergic reactions to the consumers [2], and the residues of sulfonamides (SAs) have potential carcinogenic effects on humans [3]. To ensure food safety and human health, China and the European Union have established different maximum residue limits (MRLs) for these drugs in meat, i.e. the sum of SAs, 100 ng g−1; FQs, 20–200 ng g−1; single or total amount of TCs, 100 ng g−1 [4,5]. Therefore, it is urgent to monitor their residues in foods of animal origin. The first thing for determination of residual veterinary drugs in foods of animal origin is to extract and purify the low level of analytes in the samples. By now, many sample preparation methods, such as solid phase extraction [6], solid-phase microextraction [7], stir bar sorptive extraction [8], QuEChERS [9], immunoaffinity chromatography [10], and dispersive solid phase extraction [11], have been reported for extraction and purification of FQs, SAs and TCs in various samples. However, the first step of these extraction methods is to transfer the analytes from the samples into various solvent phases, and then different purification procedures are performed. This meant that the solubilization procedure is a rate-limiting step.
⁎
Matrix solid phase dispersion (MSPD) is a novel sample preparation method that was first developed in 1989 [12]. For a MSPD method, the solid sample, viscous sample or liquid sample is blended with a type of suitable dispersing sorbent, and then the sample-sorbent mixture is transferred into an empty syringe barrel. After the sample impurities are washed out with proper solvent, the analytes are eluted for analysis. This sample preparation method combines extraction and purification into one step that can be finished within several minutes, so this technique is simpler and more flexible than the commonly used sample preparation methods. Therefore, there have been many articles reporting the use of MSPD technique for determination of various analytes in different samples [13], including SAs [14–16], TCs [17] and FQs [18,19]. However, the usually used dispersing sorbents in the previous reports (C18, SiO2, HLB material, diatomite, N-propylethylenediamine, alumina, Florisil) are easily interfered by the sample impurities, and these sorbents may lead to competitive adsorption when one sample simultaneously contains other compounds besides the target analytes. Therefore, it is desirable to find a specific dispersing sorbent for MSPD technique. Over the past 10 years, molecularly imprinted polymer (MIP) has drawn the interests of many researchers due to its specific recognition ability. Therefore, MIP based materials have been widely used for the determination of different analytes in various samples [20], including TCs [21,22], FQs [23] and SAs [24–26]. In 2007, a novel sample
Corresponding author. E-mail address: [email protected] (J.P. Wang).
http://dx.doi.org/10.1016/j.jchromb.2017.09.034 Received 21 June 2017; Received in revised form 15 August 2017; Accepted 20 September 2017 Available online 21 September 2017 1570-0232/ © 2017 Elsevier B.V. All rights reserved.
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pretreatment method combining MIP and MSPD was first developed for extraction of FQs residues in egg and swine tissues [27]. Results showed that this technique avoided competitive adsorption and achieved highly selective purification effect, and the sorbent MIP showed better purification effect and higher recoveries than four tested sorbents (silica, C18, sand and Florisil). Thereafter, MIP-MSPD has been used for the determination of BPA [28], β-estradiol [29], steroids [30], clenbuterol [31], FQs [32–34] and Sudan dyes [35] in various samples. However, there has been no article reporting the use of MIP-MSPD method for the determination of SAs and TCs so far. The commonly synthesized MIP is only able to recognize a group of structurally similar analytes, so there has been no article reporting the use of MIP-MSPD method for multi-determination of different classes of veterinary drugs in foods of animal origin so far. In the present study, three molecules (pipemidic acid for FQs, sulfabenzamide for SAs, and chlortetracycline for TCs) were used to synthesize a novel mixed-template MIP (MMIP), and its recognition performance for FQs, SAs and TCs were studied. Then, a MMIP-MSPD method was developed for extraction and purification of the three classes of drugs in pork followed by determination with ultra performance liquid chromatography (UPLC).
2.3. Synthesis of MMIP particles The MMIP was synthesized according to our recent reports [22,23]. Briefly, the mixture of three templates (PA, SB and TC, 1 mmol of each template), the functional monomer MA (18 mmol) and the porogen chloroform (18 mL) were added into a glass bottle to be sonicated for 20 min, and kept at 4 °C overnight. Then, the cross-linker DGDMA (40 mmol) and the initiator AIBN (40 mg) were added. The bottle was filled with nitrogen for 10 min and sealed to be shaken in a 60 °C water bath for 24 h. The obtained MMIP particles were extracted on a Soxhlet apparatus for 72 h successively by using methanol/acetic acid (9:1, v/ v). Finally, the MMIP particles were dried at 110 °C for 2 h for the subsequent use. For comparison, the controlled non-imprinted polymer (NIP) was also synthesized as described above but without the three templates. The two types of polymers were all scanned by using scanning electron microscopy (SEM) (JSM-7500F, JEOL, Japan). Furthermore, the MMIP specificity for the 20 drugs was determined according to a previous report [24]. Briefly, 20 mg MMIP or NIP was added into 5 mL mixed standard solution (100 ng of each drug) to be stirred for 5 min. Then the supernatant was analyzed by UPLC. The partition coefficient (K) was calculated as: K = CA/CS (CA is the drug amount absorbed by MMIP or NIP, and CS is the drug amount in supernatant). The specificity was evaluated based on imprinting factor (IF): IF = KMMIP/KNIP (KMMIP and KNIP represent the K value of each drug from MMIP and NIP respectively).
2. Materials and methods 2.1. Reagents and chemicals Pipemidic acid (PA) and sulfabenzamide (SB) were purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany). The standards of enrofloxacin (ENR), sarafloxacin (SAR), ciprofloxacin(CIP), lomefloxacin (LOM), ofloxacin (OFL), pefloxacin (PEF), danofloxacin (DAN), marbofloxacin (MAR), sulfadiazine (SD), sulfadimidine (SM2), sulfamethoxypyridazine (SMP), sulfadimethoxine (SDM), sulfamonomethoxine (SMM), sulfamethoxazole (SMZ), sulfaquinoxaline (SQ), sulfachloropyridazine sodium (SCP) tetracycline (TC), oxytetracycline (OTC), chlortetracycline (CTC), and doxycycline (DC) were purchased from Sigma (St. Louis, MO, USA). Ethylene glycol dimethacrylate (EGDMA) was purchased from Aladdin Industrial Corporation (Shanghai, China). Methacrylic acid (MA) and 2,2-azobis (isobutyronitrile) (AIBN) were purchased from Kermer Chemical Company (Tianjin, China). Other chemical reagents were of analytical grade or better from Beijing Chemical Company (Beijing, China). Liquid chromatographic grade acetonitrile and methanol were purchased from Dikma (Richmond Hill, USA). Standard stock solutions of the 20 drugs were prepared with methanol respectively (100 μg mL−1), and these solutions were stable for four months at −20 °C. The working solutions of these drugs were diluted from the stock solutions with water (1–1000 ng mL−1), and these solutions were stable for one week at 4 °C.
2.4. Sample preparation with MMIP-MSPD An amount of 0.2 g homogenous pork sample was added into a mortar. At this stage, the mixed standards were fortified at different concentrations, and the mixture was ground with a pestle to obtain a homogenous sample. Then, 0.15 g MMIP was added, and the mixture was gently ground until a homogenous mixture was obtained (about 3 min). At the same time, an empty cartridge (100 mm × 9.9 mm, i.d.) with a lower frit was packed with 50 mg MMIP that was conditioned by washing with 1 mL methanol and 1 mL water in turn. Then the homogenous mixture was transferred into the cartridge, and compressed with a syringe plunger to create a compact column bed. The cartridge was washed with 3 mL methanol/water (2:8, v/v), and the solutions passed through the cartridge at a flow rate of 1 mL min−1 under the assistance of vacuum pump (about 3 min). Then, the analytes were eluted with 4 mL methanol/acetic acid (9:1, v/v) under the assistance of vacuum pump (about 4 min), and the eluate was evaporated to dry under gentle nitrogen. Finally, the dry residue was reconstituted in 200 μL water and filtered with a 0.22 μm membrane for UPLC analysis. During the experiments, the MMIP amount, washing solvent and eluting solvent were optimized respectively.
2.5. Method validation 2.2. UPLC conditions Some blank pork samples obtained from the controlled slaughterhouses were used to evaluate the method. The limits of detection (LODs) and the limits of quantification (LOQs) for the 20 analytes were calculated as the concentrations corresponding to peak/noise ratio (S/ N) of 3:1 and 10:1 respectively. Then the 20 drugs were fortified into the blank pork samples respectively at levels of 20–1000 ng g−1 for analysis. The intra-day recoveries (six repetitions for each fortification level in a single day) and the inter-day recoveries (duplicate injections for each fortification level on six successive days) were determined respectively. Finally, 70 pork samples purchased from several supermarkets in China were analyzed by the developed method.
UPLC system consisted of a ACQUITY H-CLASS liquid chromatography, a PDA detector and a BEH C18 column (2.1 × 50 mm, 1.7 μm) (Waters, USA). The mobile phase consisted of (A) 0.2% formic acid and (B) acetonitrile/methanol (1:1, v/v) with gradient elution at a flow rate of 0.4 mL min−1. The gradient elution program was: started with 10% (A), linearly increased to 12% (A) in 7.9 min, decreased to 1% (A) in 0.6 min, then increased to 12% (A) in 0.1 min, linearly increased to 65% (A) in 2.4 min, decreased to 10% (A) in 0.1 min, linearly increased to 50% (A) in 0.9 min, finally brought back to 10% (A) in 1.0 min and maintained for 1.0 min with a total running time of 14 min. The injection volume was 10 μL, and the detection wavelengths were 270, 279 and 289 nm.
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Table 1 Determination parameters of the MMIP-MSPD-UPLC method for the 20 drugs in pork (n = 5). Analyte
Linearity (ng mL−1)
r2
Intra-batch RSD (%)
Inter-batch RSD (%)
LOD (ng g−1)
LOQ (ng g−1)
Retention time (min)
Imprinting factor
SD SM2 TC MAR SMP OTC SCP OFL PEF SMM CIP SMZ LOM DAN ENR SAR CTC SDM DC SQ
5–1000 6–1000 2–1000 5–1000 5–1000 2–1000 6–1000 5–1000 5–1000 6–1000 6–1000 6–1000 6–1000 2–1000 2–1000 2–1000 2–1000 5–1000 2–1000 5–1000
0.9999 0.9997 0.9994 0.9997 0.9994 0.9992 0.9998 0.9998 0.9993 0.9996 0.9994 0.9991 0.9999 0.9998 0.9996 0.9997 0.9992 0.9995 0.9991 0.9997
3.1 1.7 2.2 2.1 2.7 3.4 2.0 2.8 2.6 2.6 3.1 2.7 1.7 2.8 2.0 2.1 1.7 1.9 2.6 3.4
3.2 2.4 2.8 2.8 3.0 2.8 2.7 3.0 3.8 3.2 3.6 3.4 2.9 2.7 3.0 2.9 3.2 3.1 2.4 3.2
2 3 1 3 2 1 3 2 2 3 3 3 3 1 1 1 0.5 2 0.5 2
5 6 2 5 5 2 6 5 5 6 6 6 6 2 2 2 1.5 5 1.5 5
1.939 4.926 5.182 5.894 6.147 7.17 7.977 8.181 8.376 8.568 9.336 10.16 10.542 10.63 10.676 10.811 10.926 11.236 11.49 11.556
6.2 4.2 6.5 6.9 5.7 5.2 5.3 7.6 5.1 8.3 5.6 6.1 5.0 8.4 9.1 6.7 6.8 7.2 4.9 8.8
3. Results and discussions
were used as the mixed-template in the present study to synthesize the MMIP for FQs, SAs and TCs. After the MMIP was obtained, three methods were used to characterize it. Firstly, the MMIP was subjected to SEM characterization. Results showed that the surface of MMIP was porous and rugged, but the surface of NIP was smooth and less porous (See Fig. S1 in Supplementary Material), indicating the templates were imprinted and the pores should be beneficial for capturing the analytes. Secondly, 50 mg MMIP or NIP particle was packed into an empty cartridge, and the 20 target analytes and several other competitors were loaded onto the cartridge respectively. Then the analytes were eluted for analysis. As shown in Fig. 2, the MMIP simultaneously captured 8 FQs, 8 SAs and 4 TCs with recoveries of 70%–90%, but showed very low recoveries to other competitors (< 7%). In comparison, the NIP showed comparably low recoveries to all of the tested analytes (5%–14%). This indicated that the MMIP captured FQs, SAs and TCs due to the specific molecular recognition. Thirdly, the imprinting factor (IF) for each analyte was calculated to verify the MMIP selectivity. As shown in Table 1, the MMIP showed IFs of 4.2-9.1 for the three classes of drugs. High IF value represented high recognition ability, so these results were consistent with the results shown in Fig. 2. This is the first paper reporting the synthesis of a type of MMIP material capable of simultaneous recognizing FQs, TCs and SAs. Then, this material was used as dispersing sorbent for development of the MSPD method.
3.1. Optimization of UPLC conditions It is well known that UPLC can achieve excellent separation effect. For simultaneous separation of the 20 drugs belonging to three classes, the UPLC separation condition was examined for obtaining the optimal analytical time, peak shape and the maximum sensitivity. During the experiments, methanol, acetonitrile and their mixtures with different proportions of formic acid (0.1%-0.5%) were compared under different gradient elution procedures. Results showed that the mobile phase acetonitrile/methanol (1:1, v/v) plus 0.2% formic acid using the gradient program shown in Section 2.2 achieved the optimal performance. The retention times of the 20 drugs are shown in Table 1, and the chromatogram of the 20 standards is shown in Fig. 1A. For multi-detection of the three classes of drugs, the detection wavelength is very important. During the experiments, the PDA detector was operated in scan mode, and different wavelengths were compared. Results showed that the optimal detection wavelengths for FQs and SAs were around 280 nm, and the optimal detection wavelengths for TCs were around 280 and 350 nm. For multi-determination of the three classes of drugs, 270, 279 and 289 nm were selected as the detection wavelengths, and Fig. 1A was the chromatogram obtained at 270 nm.
3.2. Characterization of MMIP 3.3. Optimization of MMIP-MSPD In the previous reports, the MIPs for TCs [21,22], FQs [23,27,32–34] and SAs [24–26] were usually synthesized with a single molecule as the template, so those MIPs were only able to recognize a group of structurally similar analogs. For example, the CTC based MIP was able to capture four TCs with high recoveries but showed negligible recognition ability to SAs and FQs [22], whereas the norfloxacin based MIP only recognized four FQs but showed negligible recognition ability to SAs and TCs [23]. In four recent reports, two or five molecules were used as the mixed-templates to synthesize the MIPs, and the results showed that the MIPs’ recognition spectra were broadened [36–39]. Therefore, it is desirable to synthesize a MIP capable of simultaneous recognizing FQs, SAs and TCs. In the previous reports, different molecules were used as the templates to synthesize MIPs for FQs (OFL, SAR, norfloxacin, daidzein) and SAs (SMZ, SDM), but there has been no article reporting the use of PA and SB as the templates to synthesize MIPs for FQs and SAs so far. PA, SB, and CTC contain the common core structure of their respective classes of drugs, so the three molecules
3.3.1. MMIP amount For a MSPD method, the sorbent plays the parts of abrasive and bound solvent that can facilitate the interactions between the sorbent and the analytes, so the first thing for development of a MSPD method is to evaluate the sorbent amount. Furthermore, a suitable sample/ sorbent ratio can increase the interface area and reduce the sorbent consumption. In the present study, a miniaturized MSPD using small sample amount (0.2 g) was used to optimize the MMIP amount. During the experiments, 0.2 g pork sample was blended with 0.1, 0.15, 0.2, and 0.4 g MMIP particles respectively. As shown in Fig. 3, the recoveries of the 20 drugs reached balance when MMIP amount increased to 0.15 g, i.e. the sample/sorbent ratio of 4:3. For low sorbent amount (0.1 g), the sample component did not fully interact with MMIP particles, thus obtaining low recoveries. For high sorbent amounts (0.2 and 0.4 g), the sample/sorbent complexes were not blended to a homogenous mixture, and different amount of MMIP particles were left in the mortar, i.e. the 106
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Fig 1. Chromatograms of (A) FQs, TCs and SAs standards, (B) blank pork, and standards fortified blank pork treated by (C) MMIP, (D) C18 and (E) diatomaceous earth. (1 = SD, 2 = SM2, 3 = TC, 4 = MAR, 5 = SMP, 6 = OTC, 7 = SCP, 8 = OFL, 9 = PEF, 10 = SMM, 11 = CIP, 12 = SMZ, 13 = LOM, 14 = DAN, 15 = ENR, 16 = SAR, 17 = CTC, 18 = SDM, 19 = DC, 20 = SQ; 100 ng/mL (g)).
chromatograms showed that water and methanol/water (1:9, v/v) were not able to wash out most of the impurities, and methanol/water (2:8, v/v) achieved the best purification effect (Fig. 1B and C), so this solution was selected as the optimal washing solvent.
MMIP was excessive. Therefore, 0.15 g MMIP was used for the subsequent experiments. 3.3.2. Washing solvent For a MSPD procedure, it is necessary to find a suitable washing solvent that should wash out the sample impurities as fully as possible but show no influence on the analyte recovery. In this study, the sample/MMIP mixture was transferred into different cartridges followed by washing with several kinds of solvents. Results showed that the recoveries of the 20 drugs were comparable when using water and aqueous solvents containing 10% and 20% methanol (See Fig. S2 in Supplementary Material). When increasing methanol proportion to 50%, the analytes recoveries decreased because high portion of methanol was able to elute some analytes. However, the obtained
3.3.3. Eluting solvent For a MIP based extraction method, the most important thing is to find a suitable solvent to elute the absorbed analytes. In the previous reports, methanol, acetonitrile and their mixtures with different acids were usually used to elute FQs, TCs and SAs from MIP materials [21–27,32–34]. In this study, methanol, acetonitrile and their mixtures with acetic acid were used to optimize the eluting solvent. As shown in Fig. 4, the recoveries of the 20 drugs when using methanol/acetic acid (9:1, v/v) were generally higher than that when using other solvents, so 107
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MMIP
NIP AMO = amoxicillin CPZ = chlorpromazine CLP =clonazepam
80
60
40
20
0 SD
SM2
TC
MAR SMP OTC SCP OFL PEF SMM CIP SMZ LOM DAN ENR SAR CTC SDM
DC
SQ AMO CPZ CLP
Fig. 2. Recoveries of MMIP and NIP for the tested analytes (50 mg polymer, analyte amount 100 ng, eluting with methanol) (Recoveries were the mean values of three repetitions with standard deviations of 1.4%–4.3%).
MMIP 0.1g
90
MMIP 0.15g
MMIP 0.2g
MMIP 0.4g
80 70 60 50 40 30 20 10 0 Fig. 3. Recoveries of the 20 drugs when using different amounts of MMIP. (0.2 g sample, analyte100 ng, washing with water, eluting with methanol) (Recoveries were the mean values of three repetitions with standard deviations of 1.8%–4.7%).
120
100
SD
SM2
TC
MAR
SMP
OTC
SCP
OFL
PEF
SMM
CIP
SMZ
LOM
DAN
ENR
SAR
CTC
SDM
DC
SQ
80
60
40
20
0
methanol
acetonitrile
methanol/acetic acid
methanol/acetic acid
methanol/acetic acid
Fig. 4. Recoveries of the 20 drugs from MMIP-MSPD when using different eluting solvents. (0.2 g sample, 0.15 g MMIP, analyte 100 ng, washing with methanol/water (2:8, v/v)) (Recoveries were the mean values of three repetitions with standard deviations of 2.6%–4.0%).
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this solution was selected as the optimal elution solvent.
Table 2 Recoveries of FQs, TCs and SAs from fortified blank pork samples (n = 6). analyte
Added (ng g−1)
Inter-assay
3.4. MMIP-MSPD-UPLC
Intra-assay
Recovery (%)
CV (%)
Recovery (%)
CV (%)
SD
20 100 1000
81.2 91.8 97.3
3.4 6.2 8.8
83.4 86.7 92.0
5.3 5.4 8.6
SM2
20 100 1000
86.3 89.9 88.0
5.8 4.9 9.0
88.4 84.7 92.4
6.0 5.8 6.2
TC
20 100 1000
93.3 86.9 97.8
4.2 3.4 9.1
94.2 90.8 92.7
7.4 6.4 7.7
MAR
20 100 1000
74.5 93.8 95.3
5.1 6.0 7.4
79.6 88.9 93.2
5.8 5.3 5.9
SMP
20 100 1000
91.1 95.5 94.3
6.7 4.9 7.9
89.2 95.3 97.8
4.9 5.0 6.3
OTC
20 100 1000
87.0 93.9 96.3
5.2 6.3 5.2
91.3 88.6 94.2
7.2 4.6 5.7
SCP
20 100 1000
81.2 94.9 99.7
6.2 5.3 8.4
85.9 90.0 96.8
5.3 4.8 6.1
OFL
20 100 1000
85.3 89.7 98.1
6.8 6.7 8.7
90.1 90.9 94.7
6.7 8.1 6.0
PEF
20 100 1000
87.1 87.8 95.3
5.2 4.6 6.1
92.8 90.7 93.1
4.6 6.8 7.1
SMM
20 100 1000
95.9 97.5 89.0
3.8 5.8 7.7
97.3 90.9 94.8
6.7 5.8 6.6
CIP
20 100 1000
90.8 89.1 96.0
5.9 5.7 6.9
94.5 85.5 87.4
7.3 4.9 5.2
SMZ
20 100 1000
85.3 92.7 89.7
6.1 4.6 8.1
89.6 91.9 90.8
5.8 8.2 5.3
LOM
20 100 1000
93.4 89.0 96.8
3.7 5.8 6.5
90.4 91.8 97.4
6.2 5.8 7.3
DAN
20 100 1000
92.5 91.6 88.7
4.9 8.2 6.7
97.3 94.0 95.7
7.0 5.3 4.9
ENR
20 100 1000
89.2 96.9 90.8
6.4 7.2 6.1
87.4 91.4 94.6
4.9 5.8 8.0
SAR
20 100 1000
94.9 99.8 96.8
5.9 4.3 6.1
93.6 97.8 95.6
6.7 5.8 6.9
CTC
20 100 1000
91.2 96.2 100.3
4.5 8.7 6.2
94.1 88.5 98.4
7.4 6.3 5.8
SDM
20 100 1000
93.2 91.5 90.3
9.1 5.8 6.8
92.0 90.7 93.8
5.9 8.4 6.7
DC
20 100 1000
92.7 98.3 96.5
4.6 6.2 6.7
88.7 95.1 102.7
6.4 5.7 7.3
20 100 1000
84.1 86.3 88.6
6.4 5.9 7.8
89.6 81.3 91.3
8.0 6.0 8.6
SQ
During the experiments, the 20 drugs were diluted with the extracts of blank pork to prepare the matrix matched standards respectively, and these solutions were used to evaluate the performance of MMIPDSPD-UPLC method. The determination parameters of this method are shown in Table 1. As shown in Table 1, this method showed wide detection linearity range (2–1000 ng mL−1) and high sensitivity (LOD of 0.5–3 ng g−1 and LOQ of 1.5–6 ng g−1) for the 20 drugs. Then, the 20 drugs were fortified into the blank pork samples at concentrations of 20, 100 and 1000 ng g−1 to be extracted and analyzed respectively. The inter-assay recoveries were in the range of 74.5%–100.3%, the intraassay recoveries were in the range of 79.6%–102.7%, and the coefficients of variation (CV) were in the range of 3.4%–9.1% (Table 2). The chromatograms of blank pork and standards fortified blank pork illustrated that there was almost no interfering peak around the analytes peaks, indicating the extremely specific purification effect (Fig. 1B and C). Finally, the 70 real pork samples were analyzed by the method. Results showed that SM2 was detected in one sample (86 ng g−1), and SAR was detected in other two samples (11 and 43 ng g−1). Other samples were all determined as negative samples by this method. 3.5. Comparison with related methods This is the first study reporting the development of a mixed-template MIP based MSPD method for multi-determination of FQs, TCs and SAs in foods of animal origin. For comparison with related reports, the details of some previous reports about the use of MSPD method for determination of FQs, TCs and SAs are summarized in Table 3. As shown in Table 3, all these methods were able to detect low levels of FQs, TCs and SAs [14–19,27,32,34], but the recognition ability of those dispersing sorbents were worse than the present MMIP. General consideration, the method developed in the present study was better than the previously reported MSPD methods. To compare the extraction performance of the MMIP with common dispersing sorbents, C18 and diatomaceous earth were also used to perform MSPD procedure according to the previous reports [14,19]. The obtained chromatograms showed that the two sorbents were able to achieve the satisfactory purification effects (Fig. 1 D, E), but the recoveries of the 20 drugs from the two sorbents (70%–95%) were lower than that from MMIP (92%–99%, Fig. 5). Therefore, the MMIP based MSPD showed generally better performance than the two common dispersing sorbents. 4. Conclusion As a type of specific absorbent, MIP has been widely used for determination of veterinary drugs residues. In the present study, a novel mixed-template MIP capable of simultaneous capturing FQs, TCs and SAs was firstly synthesized, and a MSPD method was then developed and optimized to extract and purify 20 drugs in pork sample. Results showed that the MMIP-MSPD-UPLC method could be used as a rapid, specific and sensitive method to monitor the residues of FQs, TCs and SAs in animal derived foods. Acknowledgements The authors are grateful for the financial supports from Hebei Natural Science Foundation (C2011204021, C2015204049, C2016204039), Earmarked Fund for Hebei Lays Innovation Team of Modern Agro-industry Technology Research System (HBCT2013090202), and Shijiazhuang Technology Incubation Project (171550089A). 109
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Table 3 Comparison of the previous MSPD methods for FQs, TCs and SAs. Analytes
Sorbent
Detection method
LOD (ng mL−1 (g−1))
Sample
Ref.
7 SAs 14 SAs 13 SAs 3 TCs 3 FQs 2 FQs, 2 SAs and TC 5 FQs 2 FQs 8 FQs 8 FQs, 8 SAs and 4 TCs
diatomaceous earth hydrophilic lipophilic balance material C18 C18 C18 C18 OFL based MIP OFL based MIP daidzein based MIP mixed-template based MIP
HPLC-FLD LC–MS/MS LC–MS/MS CE-UV HPLC-DAD HPLC-DAD HPLC-FLD HPLC-DAD HPLC-FLD UPLC-PAD
1.33–2.47 2.3–16.4 0.75–3.0 0.0745–0.08 9–22 2–10 0.05–0.09 8–9 0.06–0.22 0.5–3
liver fish fish milk porcine tissues porcine tissues egg chicken fish pork
14 15 16 17 18 19 27 32 34 This study
MMIP
C18
diatomaceous earth
100
80
60
40
20
0 Fig. 5. Recoveries of the 20 drugs by using different dispersing sorbents. (Recoveries were the mean values of three repetitions with standard deviations of 1.9%–4.5%).
Appendix A. Supplementary data [11]
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jchromb.2017.09.034.
[12]
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