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Synthesis of dummy-template molecularly imprinted polymer adsorbents for solid phase extraction of aminoglycosides antibiotics from environmental water samples
Talanta 208 (2020) 120385
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Synthesis of dummy-template molecularly imprinted polymer adsorbents for solid phase extraction of aminoglycosides antibiotics from environmental water samples
T
Zheng Zhanga, Xiaolin Caoa,∗, Ziping Zhanga, Jungang Yina, Dingnan Wangb, Yanan Xua, Wei Zhenga, Xinyi Lia, Qingsong Zhanga, Liwen Liua a b
College of Life Science, Yantai University, Yantai, 264005, China Aquatic Products Quality Inspection Center of Zhejiang Province, Hangzhou, 310023, China
ARTICLE INFO
ABSTRACT
Keywords: Dummy molecularly imprinted polymers Aminoglycoside antibiotics Solid phase extraction Water samples
A novel dummy molecularly imprinted polymers (DMIPs) for aminoglycoside antibiotics (AAs) was prepared for the first time by precipitation polymerization using raffinose as template molecule, methacrylic acid as functional monomer and trimethylolpropane triacrylate as cross-linker. The obtained DMIPs were characterized in detail, and their adsorbing and recognition performance were evaluated. The results showed that the DMIPs exhibited specific recognition towards six AAs with large adsorption capacity. The dummy molecularly imprinted solid phase extraction (DMISPE) conditions including elution/washing solutions and sample loading volumes were optimized. Under optimum conditions, a convenient and efficient method for the determination of AAs in environmental water samples based on DMISPE coupling with hydrophilic interaction-high performance liquid chromatography-tandem mass spectrometry was established. The developed method showed good linearity with correlation coefficients higher than 0.9921 for all the analytes and good recoveries (70.8–108.3%) with relative standard deviations from 2.6 to 11.4% spiked at three different concentration levels in water samples. The limit of detection (S/N = 3) ranged from 0.006 to 0.6 ng/mL. The results demonstrated good potential of DMIPs for sample pretreatment of trace AAs in environmental water samples.
1. Introduction Aminoglycoside antibiotics (AAs), composed of amino-modified sugars are an important group of antibiotics. Because of their desirable broad-spectrum antibacterial activity of both gram-positive and gramnegative infections, AAs have been widely used in animal husbandry and medical field to promote the growth of animals and treat bacterial infections of animals and humans [1,2]. As a result of medical discharge, incomplete elimination in sewage treatment plants, and extensive abuse in farming and aquaculture, substantial amounts of AAs have been released into the environment [3]. Considering their negative effects such as nephrotoxicity, ototoxicity, and neuromuscular blockade towards humans, and the potential risk of antibiotic resistance on ecosystems, many countries and organizations have established the maximum residue limits of AAs in different animal products [4,5]. AAs have also aroused growing attention in environmental water samples as emerging-easily water soluble pollutants. However, information about the occurrence of AAs in environmental samples is very limited. Thus, it
∗
is very necessary to determine the residual level of AAs in environmental samples. The most commonly methods for determination of AAs traditionally used high performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) because of its high sensitivity and stability. For addressing the great challenges resulted from high polarity of AAs in the sample pretreatment of HPLC-MS/MS detection, various sample preparing protocols including solid-phase extraction (SPE) [1,6], liquid extraction (LE) [7] and magnetic solid phase extraction [8] have been reported. However, the used enriching or purifying materials such as exchange resin [6,9], C18 [7] and Fe3O4@PVA [8] lack selectivity towards AAs and significant matrix effect existed when these packing materials or adsorbents were employed for sample preparation in the detection of AAs residue. Therefore, selective enriching materials are highly required for pretreatment of AAs samples. Molecularly imprinted polymers (MIPs) are artificially manufactural materials with specific recognition cavities for targets. The polymers are prepared by polymerization of proper ratio of functional monomer,
Corresponding author. E-mail address: [email protected] (X. Cao).
https://doi.org/10.1016/j.talanta.2019.120385 Received 17 July 2019; Received in revised form 18 September 2019; Accepted 19 September 2019 Available online 20 September 2019 0039-9140/ © 2019 Elsevier B.V. All rights reserved.
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cross-linker and template molecule. After template molecule is removed from the MIPs, binding cavities with complementary shapes, sizes, and functionalities are exposed and available to selectively adsorb the targets [10–12]. Compared to other adsorbent, MIPs possess advantages of good selectivity, predetermined recognition ability, low production cost, chemical and mechanical stability [13–15] and have been widely used in SPE process for trace analytes in complex matrix [16,17]. Two main obstacles, however, existed in the preparation and application of traditional MIPs. Firstly, target analytes were directly used as molecular templates to prepare MIPs, consuming large amounts of standard products. Secondly, persistent template leakage during the sample preparation process often caused inaccurate quantification of trace analytes [18,19]. To overcome these problems, structural analogues of analytes (named dummy templates) were used to synthesize MIPs, which showed comparable recognition specificity for the analytes [20–22]. Despite of much progress, limited attention was paid on the preparation of MIPs and their application for detecting AAs residue. For example, only streptomycin was used as template to prepare MIPs [23] and recent several studies directly used expensive, commercially available SPE-aminoglycoside cartridge [24–26]. Considering the unique advantage of dummy template-MIPs (DMIPs), we explore here the preparation of DMIPs using raffinose (RAF) as dummy template and its application for the HPLC-MS/MS detection of AAs residue. RAF is composed of α-(1 → 6)-galactosides bound to sucrose at C6 of the glucose moiety, similar with the molecular structure of AAs. RAF have been usually used as food ingredients, widely obtained from legume seeds, vegetables and fruits, and easily dissolved in water and polar organic reagents [27,28]. To our best knowledge, there have been no reports about the use of DMIPs for simultaneous determination of AAs so far. In this paper, the synthesis of DMIPs and its application for the detection of AAs residue in environmental water samples was described for the first time. DMIPs was synthesized by precipitation polymerization method using the AAs-analogue RAF as dummy template, methacrylic acid as functional monomer, and trimethylolpropane trimethacrylate as cross-linker. The adsorption characteristics and selectivity of the DMIPs were investigated by binding experiments. The DMIPs was then used as the SPE adsorbent coupled with hydrophilic interaction-high performance liquid chromatography-tandem mass spectrometry (HILIC-HPLC-MS/MS) method for analysis of AAs from environmental water samples.
Table 1 The parameters of mass spectrometer of different compounds. Compound
Declustering potential (V)
Collision energy (eV)
Qualitative ion pair (m/z)
Quantitative ion pair (m/z)
STP
154 154 82 82 49 49 66 66 71 71 110 110 73 73 −104 −104
45 57 33 23 45 25 27 19 36 20 55 32 46 35 −47 −33
582.3 → 263.2 582.3 → 246.6 485.4 → 163.3 485.4 → 324.1 540.4 → 378.3 540.4 → 217.4 478.2 → 321.8 478.2 → 156.9 468.3 → 162.9 468.3 → 323.9 616.3 → 323.9 616.3 → 163.5 351.3 → 98.3 351.3 → 140.1 503.2 → 179.1 503.2 → 221.3
582.3 → 263.2
KAM APM GNT TOM PRM SPC RAF
485.4 → 163.3 540.4 → 378.3 478.2 → 321.8 468.3 → 162.9 616.3 → 323.9 351.3 → 98.3 503.2 → 179.1
500DB CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.) was also used. An HPLC system (ultraLC-100, AB Sciex, USA) coupled with a triple quadrupole mass spectrometer (AB-4000, USA) equipped with an electrospray ionization source was used to analyze samples. The detailed parameters of mass spectrometer were shown in Table 1. Chromatographic separation for AAs was carried out on a kinetex HILIC column (2.1 × 100 mm, id: 2.6 μm, phenomenex, America) at 35 o C. The injection volume was 5 μL. The mobile phase consisted of (A) 0.1% formic acid-10 mM ammonium acetate solution and (B) acetonitrile. The gradient elution procedure were as follows: 0–2 min, kept at 80% B; 2–3 min, from 80% to 5% B; 3–6 min, kept at 5% B; 6–6.1 min, changed from 5% to 80% B; 6.1–12 min, kept at 80% B. The flow rate was 0.3 mL/min. The chromatographic separation for RAF was performed on the same column and the mobile phase was under isocratic elution with 20% B solution from 0 to 6 min. The total ion chromatogram of RAF was showed in Fig. S1. 2.3. Synthesis of DMIPs and NIPs DMIPs was synthesized by precipitation polymerization method as follows: The template molecule RAF (0.l mmol) and the functional monomer MAA (1.4 mmol) were dissolved by 5.0 mL water in 100 mL roundbottomed flask, and shaken for 30 min to obtain pre-polymerization solution. Then, cross-linker TRIM (1.0 mmol), initiator AIBN (30 mg), porogen solvent methanol (20 ml) were added in the flask. After purging with nitrogen gas for 10 min, the flask was preceded at 60 oC for 24 h. The polymer particles were collected by centrifugation and eluted by soxhlet extraction with 10% acetic acid-methanol until no RAF could be detected. Finally, the particles were washed with methanol to remove residual acetic acid, dried under vacuum at 70 °C, and stored at room temperature. The non-molecularly imprinted polymers (NIPs) were synthesized following the same procedure but without the template molecule RAF.
2. Experimental 2.1. Reagents and chemicals Raffinose (RAF), streptomycin sulfate (STP), kanamycin sulfate (KAM), apramycin sulfate (APM), gentamycin sulfate (GNT), tobramycin (TOM), paromomycin sulfate (PRM) and spectinomycin pentahydrate dihydrochloride (SPC) were purchased from Dr. Ehrenstorfer Gmbh (Augsburg, Germany). Methacrylic acid (MAA), trimethylolpropane triacrylate (TRIM), formic acid (FA, 98%) and ammonium acetate were bought from Sigma-Aldrich (St. Louis, MO, USA). The initiator 2,2-azobisisobutyronitrile (AIBN, > 98%) was supplied by the Reagent & H. V. Chemical Co., Ltd. (Shanghai, China). Ultrapure water was obtained from a Milli-Q system (France). Acetonitrile (ACN) and methanol (MeOH) were obtained from Tedia (Fairfield, OH, US). AAs standards were stored under refrigeration at 4 °C and used to prepare working standard solutions. All other reagents were of analytical grade.
2.4. Molecularly imprinted solid phase extraction 30 mg of synthesized DMIPs particles were packed into a 3 mL SPE cartridge (Agilent). The columns were sequentially conditioned with 3 mL methanol and 3 mL ultra-pure water. Then, 50 mL water sample was loaded through the SPE column and 3 mL of ultra-pure water (washing solvent) passed through the column with a flow rate of 1 mL/ min. After the column became dried, 3 mL of 1% FA-H2O was used to elute AAs. This elute solution was directly analyzed by HPLC-MS/MS.
2.2. Apparatus and LC-MS/MS conditions Fourier transform infrared spectroscopy (FT-IR) was measured with PerkinElmer spectrum 100. Scanning electron microscopy (SEM) images were carried out on a JSM-6300 SEM instrument (Japan). KQ2
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2.5. Binding experiments
So, different molar ratios of RAF to MAA (1:6, 1:10, 1:14) and RAF to TRIM (1:10,1:20,1:50) were investigated (Table 2) and results showed that DMIPs prepared using a ratio of 1:14:10 possessed the optimum adsorption capacity towards different AAs molecules. Therefore, the molar ratio of 1:14:10 was selected as the synthetic condition in the following study.
To evaluate the adsorbing performance of DMIPs, the sized and washed particles (10.0 mg) were mixed with 2.0 mL KAM solution of concentrations ranging from 10 to 2000 μg/mL in a 5 mL eppendorf tube. The mixture was shaken at 25 °C for 2 h. After centrifugation, the supernatants were analyzed by HPLC-MS/MS to quantify the concentration of free KAM.
3.2. Characterization of DMIPs Chemical composition of the synthesized DMIPs and NIPs were characterized with FT-IR (Fig. 2A). Absorption peaks of 998 cm−1 and 966 cm−1 assigned to glycosidic bonds were found both in RAF (curve a) and DMIPs (curve b), and NIPs synthesized using the same procedure but without the template molecule RAF did not show these two absorption peaks (curve c). After washing with HAc-MeOH, the intensity of 998 cm−1 and 966 cm−1 in DMIPs was disappeared (curve d), indicating the removal of template molecule RAF. Also, absorptions of 1732 cm−1 and 1259 cm−1 resulted from carboxyl groups in the functional monomer MAA existed in all the three polymers (curve b, c and d). The SEM image of DMIPs and NIPs were shown in Fig. 2B. DMIPs were uniform spherical morphology with about 1.0 μm in diameter, while NIPs exhibited irregular bulk shapes. Taken together, these results demonstrated that DMIPs could be successfully prepared using RAF as a template molecule.
2.6. Sample pretreatment Environmental water samples were obtained from rivers or reservoir located in the city of Yantai. The samples were filtered through a 0.22 μm membrane and loaded through DMIPs cartridge as described above. 3. Results and discussion 3.1. Synthesis of DMIPs for AAs Appropriate dummy template molecules could offer an effective way to circumvent the template leakage problem associated with the preparation and application of traditional MIPs. In this study, RAF was selected as a dummy template to synthesize MIPs for enriching AAs. The chemical structures of RAF and AAs are shown in Fig. 1. The spatial locations of three or four six-membered ring structure in AAs are similar with RAF compound. Also, the abundant OH groups existing in RAF could provide hydrogen bonding interaction with COOH groups in MAA. During the polymerization process of DMIPs, the molar ratio of template, functional monomer and crosslinker is the main factor influencing the morphology and final adsorption capacity of polymers.
3.3. Binding study of DMIPs and scatchard analysis Equilibrium absorption and scatchard analysis experiments were used to evaluate the binding properties of the synthesized RAF-DMIPs. Firstly, DMIPs particles (10 mg) were mixed with 2 mL of 20 μg/ mL KAM water solution at different periods of shaking time (0.5–4 h) to
Fig. 1. The structure of raffinose and representative molecular structures of AAs. 3
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Table 2 Adsorption capacity of dummy molecularly imprinted polymers with different molar ratios. Analyte
STP KAM APM GNT TOM PRM
Template molecule：Functional monomer：Cross-linking agent 1:6:10
1:10:10
1:14:10
1:6:20
1:10:20
1:14:20
1:6:50
1:10:50
1:14:50
50 130 172 176 156 176
80 176 182 190 174 182
110 190 196 198 190 196
80 140 180 184 176 184
84 150 182 192 178 188
80 164 186 194 182 188
70 120 164 174 146 174
70 130 172 180 160 182
76 144 178 188 170 186
Note: the unit of adsorbing data was μg g−1.
Fig. 2. (A):FT-IR spectra of RAF (a), DMIPs (b), NIPs (c) and DMIPs with template removing (d); (B) Scanning electron micrographs of DMIPs (a) and NIPs (b).
Fig. 3. (A) Adsorption isotherms of RAF-DMIPs and NIPs for KAM; (B) Scatchard plot analysis of RAF-DMIPs and NIPs. 4
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Fig. 4. Effect of different elution solvents (A), washing solvents (B) and sample loading volumes (C) on recoveries (%) of STP, KAM, APM, GNT, TOM, PRM.
obtain the saturation adsorption time by dynamic absorption experiment (Fig. S2). A saturation adsorption time of 2 h was finally set. Subsequently, the DMIPs particles and NIPs (10.0 mg) were mixed with kanamycin (2.0 mL) from 10 μg/mL to 2000 μg/mL for 2 h, respectively. Fig. 3A showed the adsorption isotherms of KAM on DMIPs and NIPs adsorbents. In spite of adsorption capacity of DMIPs and NIPs both improving with the increasing concentrations of KAM, the adsorption amount of DMIPs (128 mg/g) displayed an obviously higher capacity than that of NIPs (80 mg/g) when the initial concentration was 800 mg/ L. This may be due to the predetermined specific binding sites selectively binding to kanamycin molecules, while NIPs having no selective sites. Moreover, the subsequent Scatchard analysis of the polymers was evaluated. The Scatchard equation is:
Q/Ce = (( Qmax
on MAA and AAs. In this experiment, 5 mL water samples spiked with 5 μg/mL of each AAs was loaded into the pre-conditioned cartridge (3 mL of methanol, 3 mL ultra-pure water). Then, the cartridges were followed by eluting with FA-H2O (0.5%, 1%, 2% v/v), FA-ACN (10:90, 20:80, 30:70), respectively. The recoveries improving with the increasing concentration of FA in CAN and reached a maximum while 3 mL 1% FA-H2O was used (Fig. 4A). 1% and 2% FA-H2O witnessed no significant changes. Therefore, 3 mL 1% FA-H2O was selected as the elution solution. 3.4.2. Washing solvents During the sample loading process, the retention of AAs was mainly attributed to the specific imprinting interaction between analytes and the cavity of DMIPs. Also, there was no-specific interaction due to the inter-molecular interactions. In order to achieve the optimized imprinting selectivity and remove non-target molecules, the washing solution including 3 mL of H2O, ACN-H2O (20:80, 50:50, 80:20 v/v), and ACN were used for washing, respectively. As shown in Fig. 4B, the recoveries of STP, KAM, APM, GNT, TOM and PRM were all close to 100% when both H2O and ACN were performed. Considering the consumption of organic reagents, 3 mL of H2O was finally selected as the washing solution.
Q)) /KD
where Q was the amount of KAM adsorbed to polymers, Ce was the free KAM concentration at equilibrium, KD was the equilibrium dissociation constant and Qmax was the maximum number of binding sites. From Fig. 3B, two different straight lines were obtained, inferring that the binding sites in DMIPs were heterogeneous. The linear regression equation for the left part and right part was Q/Ce = −91.043x +1954.6 (R2 = 0.89) and Q/Ce = −2.9141x+779.68 (R2 = 0.88), respectively. The calculated Qmax were 23.46 mg/g and 246.96 mg/g, furtherly indicating the good adsorbing capacity towards KAM.
3.4.3. Sample loading pH and volume The pH value of samples is an important factor influencing the recognition capacity of DMIPs and the existing forms of analytes. In our study, sample loading pH was optimized by percolating water samples with different pH values from 3.0 to 11.0 adjusted by aqueous ammonia and FA (Fig. 4C). The recoveries of AAs was higher than 85.0% in the pH 5.0, 7.0 and decreased substantially with too acid or too alkaline environment due to possible ionization of AAs and interfering hydrogen bond between MAA and AAs. Considering the easy operation and maximum recovery, sample loading pH was set at 7. In order to obtain the optimum extraction and enriching efficiency, different loading volumes of spiked water sample were also evaluated. 5, 10, 20, 30, 50, 70, 90, 100 mL samples spiked with 5 μg/mL of each
3.4. Optimization of the DMISPE procedure 30 mg of RAF-DMIPs was packed into 3 mL SPE cartridges by dry method. In order to obtain the maximum recovery, the adsorption and elution conditions including elution/washing solutions, sample loading volumes were optimized. 3.4.1. Elution condition The optimum composition of elution solvent could desorb the analytes from the adsorbent to the utmost extent. The mainly interaction between the synthesized DMIPs and analytes was hydrogen bond based 5
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AAs and even after 15 times recycling use of the cartridge, the recoveries of AAs were more than 92% (Fig. 6). Thus, the DMIPs could provide excellent recycling capacity and guarantee the accuracy of six trace AAs due to the predetermined recognition cavities, chemical and mechanical stability. 3.7. Method validation Fig. S3 presented the extracted ion chromatograms of AAs solutions. A series of aqueous solutions containing each of STP, KAM from 10 to 200 ng/mL, and APM, GNT, TOM and PRM from 50 to 1000 ng/mL were prepared for calibration curve. Analysis was performed with five replicates for each level. Linearity, correlation coefficient (r2), limit of detections (LODs) and relative standard deviations (RSDs) were carefully studied (Table 3). The results showed all the analytes exhibited good linearity with r2 larger than 0.9921. The LODs (S/N = 3) were from 0.006 ng/mL to 0.6 ng/mL. A repeatability study (the spiked samples of 10 ng/mL for each of the AAs) was also carried out by performing five parallel experiments, and RSDs ranged from 5.3% to 9.3%. Those parameters indicated that the developed method had high sensitivity and good repeatability.
Fig. 5. The recoveries of DMIPs for extraction of different AAs.
3.8. Analysis of environmental water samples The established SPE cartridge based on DMIPs adsorbents was evaluated for its practical application for extracting AAs from environmental water samples (tap water, lake water and irrigation water samples). The results were listed in Tables 4 and 2 ng/mL of STP was detected in lake water samples. The typical residual ion chromatogram of STP was showed in Fig. S4. Different types of environmental water samples were spiked at three different concentration levels (2, 4 and 20 ng/mL for STP and KAM, and 10, 20 and 100 ng/mL for TOM, GNT, APM and PRM, respectively). The recoveries fell into 70.8%–108.3% and RSDs were from 2.6% to 11.4%.
Fig. 6. The recoveries of AAs with different recycling times.
AAs was loaded into DMIPs cartridge, respectively. The recoveries of six AAs remained almost unchanged (higher than 95%) when at 5, 10, 20, 30, 50 mL samples (Fig. 4D). In spite of the slightly decreasing recoveries with subsequent volume, considering sample loading time consuming, a volume of 50 mL was conducted in the real sample process.
3.9. Comparison with other methods The developed DMISPE-HILIC-HPLC-MS/MS method was compared with previously represented literatures for determining AAs in different matrices (Table S1). In spite of less number of detected AAs in our developed method, the other parameters (LOQs, recovery, simplicity, amount of sample) were better than traditional adsorbents (C18, exchange resin) [7,9,29], and the traditional method usually consisted of tedious process with two consecutive pretreatment method (e.g. C18-weak cation exchange) [9,29]. It was noteworthy that this method showed several advantages of less packed adsorbent, larger loading sample, low cost, and high sensitivity in comparison with other MISPE method, especially superior to the reported synthetic MIPs (AAs as template) [23].
3.5. Specific recognition As we could see from Fig. 1, the molecule structure of SPC was similar with RAF, but much different from other AAs. In order to evaluate the specific recognition ability of DMIPs, the water samples spiked at 5 μg/mL of seven AAs were treated by DMIPs-based cartridges. The recovery of structural analogue-SPC was compared with other six AAs in Fig. 5 and the recovery (51%) of SPC was obviously lower than STP、 KAM、APM、GNT、TOM and PRM (94.7%, 98.0%, 99.0%, 98.5%, 229 95.0%, 99.0%), indicating that DMIPs possessed excellent class-specific recognition capacity towards six targeted AAs.
4. Conclusion
3.6. Recycling times of DMIPs
In this study, the selective dummy molecularly imprinted polymers for AAs was successfully synthesized using raffinose as dummy template. The obtained DMIPs exhibited uniform sizes and high selective adsorption capacity to six AAs, suitable for SPE adsorbents, avoiding
Furthermore, the recycling capacity of DMIPs cartridge were investigated by loading 50 mL water samples spiked with 5 μg/mL of each Table 3 The parameters of the developed method for the six AAs. Neonicotinoid
Linear range (ng/mL)
Regression equation
r2
LODs (ng/mL)
LOQs (ng/mL)
RSD (%) (n = 5)
STP KAM APM GNT TOM PRM
10–200 10–200 50–1000 50–1000 50–1000 50–1000
y y y y y y
0.9969 0.9921 0.9958 0.9971 0.9954 0.9970
0.006 0.25 0.5 0.6 0.6 0.6
0.02 0.8 1.5 1.8 1.8 1.8
7.6 5.3 6.2 9.3 7.2 6.8
= = = = = =
−11200 + 1890x −3080 + 366x −3640 + 89.1x −10100 + 276x −77900 + 241x −810 + 23.7x
6
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Table 4 The recoveries of the six AAs from tap water, lake water and irrigation water samples. Analyte
Spiked (ng/mL)
Tap water (ng/mL) Found (ng/mL)
STP
KAM
APM
GNT
TOM
PRM
0 2 4 20 0 2 4 20 0 10 20 100 0 10 20 100 0 10 20 100 0 10 20 100
nd 1.83 3.93 20.3 nd 1.94 3.8 20.7 nd 10.54 20.72 96.5 nd 9.34 21.2 94.5 nd 9.45 19.86 101.9 nd 9.31 21.24 88.6
Lake water (ng/mL) R (%)
RSD (%)
91.5 98.2 101.3
8.3 7.6 4.9
96.9 95.2 103.5
4.2 5.7 9.7
105.4 103.6 96.5
6.3 8.2 5.6
93.4 106.3 94.5
5.7 11.4 8.1
94.5 99.3 101.9
4.7 6.2 7.1
93.1 106.2 88.6
8.2 7.6 3.7
Found (ng/mL) 2.0 1.89 3.75 19.04 nd 1.95 4.08 19.9 nd 9.52 19.74 99.3 nd 10.25 19.46 99.2 nd 10.35 19.48 105.4 nd 10.83 20.44 98.6
Irrigation water (ng/mL) R (%)
RSD (%)
94.4 93.6 95.2
4.3 3.6 8.2
97.6 102.1 99.4
8.6 4.2 7.5
95.2 98.7 99.3
5.2 4.7 4.8
102.5 97.3 99.2
8.3 4.1 5.6
103.5 97.4 105.4
5.4 3.9 8.1
108.3 102.2 98.6
7.2 8.3 4.7
Found (ng/mL) nd 1.52 2.84 14.4 nd 1.98 3.82 16.5 nd 10.15 19.66 87.9 nd 9.92 20.54 98.6 nd 9.95 19.88 99.8 nd 9.56 19.84 94.4
R (%)
RSD (%)
76.4 70.8 71.6
7.2 4.1 3.5
99.2 95.4 81.1
7.3 9.6 5.1
101.5 98.3 87.9
8.2 4.5 7.7
99.2 102.7 98.6
5.2 6.9 8.4
99.5 99.7 99.8
6.3 10.5 8.6
95.6 99.2 94.4
4.1 2.6 5.4
Note: nd means not found.
the defect of template leakage in the sample pretreatment. The developed method based on DMIPs cartridges coupled with HILIC-HPLC-MS/ MS showed good recoveries, reproducibility and high sensitivity for extraction of STP, KAM, APM, GNT, TOM and PRM from environmental water samples. The DMIPs adsorbents possess great potential application for detecting residual AAs in other matrix.
[8] [9]
Acknowledgements [10]
This work was supported by the Natural Science Foundation of Shandong Province, China (ZR2019BCE091) and the National Natural Science Foundation of China (Contact No. 21305121)
[11] [12]
Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.talanta.2019.120385.
[13] [14]
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Synthesis of dummy-template molecularly imprinted polymer adsorbents for solid phase extraction of aminoglycosides antibiotics from environmental water samples
T
Zheng Zhanga, Xiaolin Caoa,∗, Ziping Zhanga, Jungang Yina, Dingnan Wangb, Yanan Xua, Wei Zhenga, Xinyi Lia, Qingsong Zhanga, Liwen Liua a b
College of Life Science, Yantai University, Yantai, 264005, China Aquatic Products Quality Inspection Center of Zhejiang Province, Hangzhou, 310023, China
ARTICLE INFO
ABSTRACT
Keywords: Dummy molecularly imprinted polymers Aminoglycoside antibiotics Solid phase extraction Water samples
A novel dummy molecularly imprinted polymers (DMIPs) for aminoglycoside antibiotics (AAs) was prepared for the first time by precipitation polymerization using raffinose as template molecule, methacrylic acid as functional monomer and trimethylolpropane triacrylate as cross-linker. The obtained DMIPs were characterized in detail, and their adsorbing and recognition performance were evaluated. The results showed that the DMIPs exhibited specific recognition towards six AAs with large adsorption capacity. The dummy molecularly imprinted solid phase extraction (DMISPE) conditions including elution/washing solutions and sample loading volumes were optimized. Under optimum conditions, a convenient and efficient method for the determination of AAs in environmental water samples based on DMISPE coupling with hydrophilic interaction-high performance liquid chromatography-tandem mass spectrometry was established. The developed method showed good linearity with correlation coefficients higher than 0.9921 for all the analytes and good recoveries (70.8–108.3%) with relative standard deviations from 2.6 to 11.4% spiked at three different concentration levels in water samples. The limit of detection (S/N = 3) ranged from 0.006 to 0.6 ng/mL. The results demonstrated good potential of DMIPs for sample pretreatment of trace AAs in environmental water samples.
1. Introduction Aminoglycoside antibiotics (AAs), composed of amino-modified sugars are an important group of antibiotics. Because of their desirable broad-spectrum antibacterial activity of both gram-positive and gramnegative infections, AAs have been widely used in animal husbandry and medical field to promote the growth of animals and treat bacterial infections of animals and humans [1,2]. As a result of medical discharge, incomplete elimination in sewage treatment plants, and extensive abuse in farming and aquaculture, substantial amounts of AAs have been released into the environment [3]. Considering their negative effects such as nephrotoxicity, ototoxicity, and neuromuscular blockade towards humans, and the potential risk of antibiotic resistance on ecosystems, many countries and organizations have established the maximum residue limits of AAs in different animal products [4,5]. AAs have also aroused growing attention in environmental water samples as emerging-easily water soluble pollutants. However, information about the occurrence of AAs in environmental samples is very limited. Thus, it
∗
is very necessary to determine the residual level of AAs in environmental samples. The most commonly methods for determination of AAs traditionally used high performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) because of its high sensitivity and stability. For addressing the great challenges resulted from high polarity of AAs in the sample pretreatment of HPLC-MS/MS detection, various sample preparing protocols including solid-phase extraction (SPE) [1,6], liquid extraction (LE) [7] and magnetic solid phase extraction [8] have been reported. However, the used enriching or purifying materials such as exchange resin [6,9], C18 [7] and Fe3O4@PVA [8] lack selectivity towards AAs and significant matrix effect existed when these packing materials or adsorbents were employed for sample preparation in the detection of AAs residue. Therefore, selective enriching materials are highly required for pretreatment of AAs samples. Molecularly imprinted polymers (MIPs) are artificially manufactural materials with specific recognition cavities for targets. The polymers are prepared by polymerization of proper ratio of functional monomer,
Corresponding author. E-mail address: [email protected] (X. Cao).
https://doi.org/10.1016/j.talanta.2019.120385 Received 17 July 2019; Received in revised form 18 September 2019; Accepted 19 September 2019 Available online 20 September 2019 0039-9140/ © 2019 Elsevier B.V. All rights reserved.
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cross-linker and template molecule. After template molecule is removed from the MIPs, binding cavities with complementary shapes, sizes, and functionalities are exposed and available to selectively adsorb the targets [10–12]. Compared to other adsorbent, MIPs possess advantages of good selectivity, predetermined recognition ability, low production cost, chemical and mechanical stability [13–15] and have been widely used in SPE process for trace analytes in complex matrix [16,17]. Two main obstacles, however, existed in the preparation and application of traditional MIPs. Firstly, target analytes were directly used as molecular templates to prepare MIPs, consuming large amounts of standard products. Secondly, persistent template leakage during the sample preparation process often caused inaccurate quantification of trace analytes [18,19]. To overcome these problems, structural analogues of analytes (named dummy templates) were used to synthesize MIPs, which showed comparable recognition specificity for the analytes [20–22]. Despite of much progress, limited attention was paid on the preparation of MIPs and their application for detecting AAs residue. For example, only streptomycin was used as template to prepare MIPs [23] and recent several studies directly used expensive, commercially available SPE-aminoglycoside cartridge [24–26]. Considering the unique advantage of dummy template-MIPs (DMIPs), we explore here the preparation of DMIPs using raffinose (RAF) as dummy template and its application for the HPLC-MS/MS detection of AAs residue. RAF is composed of α-(1 → 6)-galactosides bound to sucrose at C6 of the glucose moiety, similar with the molecular structure of AAs. RAF have been usually used as food ingredients, widely obtained from legume seeds, vegetables and fruits, and easily dissolved in water and polar organic reagents [27,28]. To our best knowledge, there have been no reports about the use of DMIPs for simultaneous determination of AAs so far. In this paper, the synthesis of DMIPs and its application for the detection of AAs residue in environmental water samples was described for the first time. DMIPs was synthesized by precipitation polymerization method using the AAs-analogue RAF as dummy template, methacrylic acid as functional monomer, and trimethylolpropane trimethacrylate as cross-linker. The adsorption characteristics and selectivity of the DMIPs were investigated by binding experiments. The DMIPs was then used as the SPE adsorbent coupled with hydrophilic interaction-high performance liquid chromatography-tandem mass spectrometry (HILIC-HPLC-MS/MS) method for analysis of AAs from environmental water samples.
Table 1 The parameters of mass spectrometer of different compounds. Compound
Declustering potential (V)
Collision energy (eV)
Qualitative ion pair (m/z)
Quantitative ion pair (m/z)
STP
154 154 82 82 49 49 66 66 71 71 110 110 73 73 −104 −104
45 57 33 23 45 25 27 19 36 20 55 32 46 35 −47 −33
582.3 → 263.2 582.3 → 246.6 485.4 → 163.3 485.4 → 324.1 540.4 → 378.3 540.4 → 217.4 478.2 → 321.8 478.2 → 156.9 468.3 → 162.9 468.3 → 323.9 616.3 → 323.9 616.3 → 163.5 351.3 → 98.3 351.3 → 140.1 503.2 → 179.1 503.2 → 221.3
582.3 → 263.2
KAM APM GNT TOM PRM SPC RAF
485.4 → 163.3 540.4 → 378.3 478.2 → 321.8 468.3 → 162.9 616.3 → 323.9 351.3 → 98.3 503.2 → 179.1
500DB CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.) was also used. An HPLC system (ultraLC-100, AB Sciex, USA) coupled with a triple quadrupole mass spectrometer (AB-4000, USA) equipped with an electrospray ionization source was used to analyze samples. The detailed parameters of mass spectrometer were shown in Table 1. Chromatographic separation for AAs was carried out on a kinetex HILIC column (2.1 × 100 mm, id: 2.6 μm, phenomenex, America) at 35 o C. The injection volume was 5 μL. The mobile phase consisted of (A) 0.1% formic acid-10 mM ammonium acetate solution and (B) acetonitrile. The gradient elution procedure were as follows: 0–2 min, kept at 80% B; 2–3 min, from 80% to 5% B; 3–6 min, kept at 5% B; 6–6.1 min, changed from 5% to 80% B; 6.1–12 min, kept at 80% B. The flow rate was 0.3 mL/min. The chromatographic separation for RAF was performed on the same column and the mobile phase was under isocratic elution with 20% B solution from 0 to 6 min. The total ion chromatogram of RAF was showed in Fig. S1. 2.3. Synthesis of DMIPs and NIPs DMIPs was synthesized by precipitation polymerization method as follows: The template molecule RAF (0.l mmol) and the functional monomer MAA (1.4 mmol) were dissolved by 5.0 mL water in 100 mL roundbottomed flask, and shaken for 30 min to obtain pre-polymerization solution. Then, cross-linker TRIM (1.0 mmol), initiator AIBN (30 mg), porogen solvent methanol (20 ml) were added in the flask. After purging with nitrogen gas for 10 min, the flask was preceded at 60 oC for 24 h. The polymer particles were collected by centrifugation and eluted by soxhlet extraction with 10% acetic acid-methanol until no RAF could be detected. Finally, the particles were washed with methanol to remove residual acetic acid, dried under vacuum at 70 °C, and stored at room temperature. The non-molecularly imprinted polymers (NIPs) were synthesized following the same procedure but without the template molecule RAF.
2. Experimental 2.1. Reagents and chemicals Raffinose (RAF), streptomycin sulfate (STP), kanamycin sulfate (KAM), apramycin sulfate (APM), gentamycin sulfate (GNT), tobramycin (TOM), paromomycin sulfate (PRM) and spectinomycin pentahydrate dihydrochloride (SPC) were purchased from Dr. Ehrenstorfer Gmbh (Augsburg, Germany). Methacrylic acid (MAA), trimethylolpropane triacrylate (TRIM), formic acid (FA, 98%) and ammonium acetate were bought from Sigma-Aldrich (St. Louis, MO, USA). The initiator 2,2-azobisisobutyronitrile (AIBN, > 98%) was supplied by the Reagent & H. V. Chemical Co., Ltd. (Shanghai, China). Ultrapure water was obtained from a Milli-Q system (France). Acetonitrile (ACN) and methanol (MeOH) were obtained from Tedia (Fairfield, OH, US). AAs standards were stored under refrigeration at 4 °C and used to prepare working standard solutions. All other reagents were of analytical grade.
2.4. Molecularly imprinted solid phase extraction 30 mg of synthesized DMIPs particles were packed into a 3 mL SPE cartridge (Agilent). The columns were sequentially conditioned with 3 mL methanol and 3 mL ultra-pure water. Then, 50 mL water sample was loaded through the SPE column and 3 mL of ultra-pure water (washing solvent) passed through the column with a flow rate of 1 mL/ min. After the column became dried, 3 mL of 1% FA-H2O was used to elute AAs. This elute solution was directly analyzed by HPLC-MS/MS.
2.2. Apparatus and LC-MS/MS conditions Fourier transform infrared spectroscopy (FT-IR) was measured with PerkinElmer spectrum 100. Scanning electron microscopy (SEM) images were carried out on a JSM-6300 SEM instrument (Japan). KQ2
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2.5. Binding experiments
So, different molar ratios of RAF to MAA (1:6, 1:10, 1:14) and RAF to TRIM (1:10,1:20,1:50) were investigated (Table 2) and results showed that DMIPs prepared using a ratio of 1:14:10 possessed the optimum adsorption capacity towards different AAs molecules. Therefore, the molar ratio of 1:14:10 was selected as the synthetic condition in the following study.
To evaluate the adsorbing performance of DMIPs, the sized and washed particles (10.0 mg) were mixed with 2.0 mL KAM solution of concentrations ranging from 10 to 2000 μg/mL in a 5 mL eppendorf tube. The mixture was shaken at 25 °C for 2 h. After centrifugation, the supernatants were analyzed by HPLC-MS/MS to quantify the concentration of free KAM.
3.2. Characterization of DMIPs Chemical composition of the synthesized DMIPs and NIPs were characterized with FT-IR (Fig. 2A). Absorption peaks of 998 cm−1 and 966 cm−1 assigned to glycosidic bonds were found both in RAF (curve a) and DMIPs (curve b), and NIPs synthesized using the same procedure but without the template molecule RAF did not show these two absorption peaks (curve c). After washing with HAc-MeOH, the intensity of 998 cm−1 and 966 cm−1 in DMIPs was disappeared (curve d), indicating the removal of template molecule RAF. Also, absorptions of 1732 cm−1 and 1259 cm−1 resulted from carboxyl groups in the functional monomer MAA existed in all the three polymers (curve b, c and d). The SEM image of DMIPs and NIPs were shown in Fig. 2B. DMIPs were uniform spherical morphology with about 1.0 μm in diameter, while NIPs exhibited irregular bulk shapes. Taken together, these results demonstrated that DMIPs could be successfully prepared using RAF as a template molecule.
2.6. Sample pretreatment Environmental water samples were obtained from rivers or reservoir located in the city of Yantai. The samples were filtered through a 0.22 μm membrane and loaded through DMIPs cartridge as described above. 3. Results and discussion 3.1. Synthesis of DMIPs for AAs Appropriate dummy template molecules could offer an effective way to circumvent the template leakage problem associated with the preparation and application of traditional MIPs. In this study, RAF was selected as a dummy template to synthesize MIPs for enriching AAs. The chemical structures of RAF and AAs are shown in Fig. 1. The spatial locations of three or four six-membered ring structure in AAs are similar with RAF compound. Also, the abundant OH groups existing in RAF could provide hydrogen bonding interaction with COOH groups in MAA. During the polymerization process of DMIPs, the molar ratio of template, functional monomer and crosslinker is the main factor influencing the morphology and final adsorption capacity of polymers.
3.3. Binding study of DMIPs and scatchard analysis Equilibrium absorption and scatchard analysis experiments were used to evaluate the binding properties of the synthesized RAF-DMIPs. Firstly, DMIPs particles (10 mg) were mixed with 2 mL of 20 μg/ mL KAM water solution at different periods of shaking time (0.5–4 h) to
Fig. 1. The structure of raffinose and representative molecular structures of AAs. 3
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Table 2 Adsorption capacity of dummy molecularly imprinted polymers with different molar ratios. Analyte
STP KAM APM GNT TOM PRM
Template molecule：Functional monomer：Cross-linking agent 1:6:10
1:10:10
1:14:10
1:6:20
1:10:20
1:14:20
1:6:50
1:10:50
1:14:50
50 130 172 176 156 176
80 176 182 190 174 182
110 190 196 198 190 196
80 140 180 184 176 184
84 150 182 192 178 188
80 164 186 194 182 188
70 120 164 174 146 174
70 130 172 180 160 182
76 144 178 188 170 186
Note: the unit of adsorbing data was μg g−1.
Fig. 2. (A):FT-IR spectra of RAF (a), DMIPs (b), NIPs (c) and DMIPs with template removing (d); (B) Scanning electron micrographs of DMIPs (a) and NIPs (b).
Fig. 3. (A) Adsorption isotherms of RAF-DMIPs and NIPs for KAM; (B) Scatchard plot analysis of RAF-DMIPs and NIPs. 4
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Fig. 4. Effect of different elution solvents (A), washing solvents (B) and sample loading volumes (C) on recoveries (%) of STP, KAM, APM, GNT, TOM, PRM.
obtain the saturation adsorption time by dynamic absorption experiment (Fig. S2). A saturation adsorption time of 2 h was finally set. Subsequently, the DMIPs particles and NIPs (10.0 mg) were mixed with kanamycin (2.0 mL) from 10 μg/mL to 2000 μg/mL for 2 h, respectively. Fig. 3A showed the adsorption isotherms of KAM on DMIPs and NIPs adsorbents. In spite of adsorption capacity of DMIPs and NIPs both improving with the increasing concentrations of KAM, the adsorption amount of DMIPs (128 mg/g) displayed an obviously higher capacity than that of NIPs (80 mg/g) when the initial concentration was 800 mg/ L. This may be due to the predetermined specific binding sites selectively binding to kanamycin molecules, while NIPs having no selective sites. Moreover, the subsequent Scatchard analysis of the polymers was evaluated. The Scatchard equation is:
Q/Ce = (( Qmax
on MAA and AAs. In this experiment, 5 mL water samples spiked with 5 μg/mL of each AAs was loaded into the pre-conditioned cartridge (3 mL of methanol, 3 mL ultra-pure water). Then, the cartridges were followed by eluting with FA-H2O (0.5%, 1%, 2% v/v), FA-ACN (10:90, 20:80, 30:70), respectively. The recoveries improving with the increasing concentration of FA in CAN and reached a maximum while 3 mL 1% FA-H2O was used (Fig. 4A). 1% and 2% FA-H2O witnessed no significant changes. Therefore, 3 mL 1% FA-H2O was selected as the elution solution. 3.4.2. Washing solvents During the sample loading process, the retention of AAs was mainly attributed to the specific imprinting interaction between analytes and the cavity of DMIPs. Also, there was no-specific interaction due to the inter-molecular interactions. In order to achieve the optimized imprinting selectivity and remove non-target molecules, the washing solution including 3 mL of H2O, ACN-H2O (20:80, 50:50, 80:20 v/v), and ACN were used for washing, respectively. As shown in Fig. 4B, the recoveries of STP, KAM, APM, GNT, TOM and PRM were all close to 100% when both H2O and ACN were performed. Considering the consumption of organic reagents, 3 mL of H2O was finally selected as the washing solution.
Q)) /KD
where Q was the amount of KAM adsorbed to polymers, Ce was the free KAM concentration at equilibrium, KD was the equilibrium dissociation constant and Qmax was the maximum number of binding sites. From Fig. 3B, two different straight lines were obtained, inferring that the binding sites in DMIPs were heterogeneous. The linear regression equation for the left part and right part was Q/Ce = −91.043x +1954.6 (R2 = 0.89) and Q/Ce = −2.9141x+779.68 (R2 = 0.88), respectively. The calculated Qmax were 23.46 mg/g and 246.96 mg/g, furtherly indicating the good adsorbing capacity towards KAM.
3.4.3. Sample loading pH and volume The pH value of samples is an important factor influencing the recognition capacity of DMIPs and the existing forms of analytes. In our study, sample loading pH was optimized by percolating water samples with different pH values from 3.0 to 11.0 adjusted by aqueous ammonia and FA (Fig. 4C). The recoveries of AAs was higher than 85.0% in the pH 5.0, 7.0 and decreased substantially with too acid or too alkaline environment due to possible ionization of AAs and interfering hydrogen bond between MAA and AAs. Considering the easy operation and maximum recovery, sample loading pH was set at 7. In order to obtain the optimum extraction and enriching efficiency, different loading volumes of spiked water sample were also evaluated. 5, 10, 20, 30, 50, 70, 90, 100 mL samples spiked with 5 μg/mL of each
3.4. Optimization of the DMISPE procedure 30 mg of RAF-DMIPs was packed into 3 mL SPE cartridges by dry method. In order to obtain the maximum recovery, the adsorption and elution conditions including elution/washing solutions, sample loading volumes were optimized. 3.4.1. Elution condition The optimum composition of elution solvent could desorb the analytes from the adsorbent to the utmost extent. The mainly interaction between the synthesized DMIPs and analytes was hydrogen bond based 5
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AAs and even after 15 times recycling use of the cartridge, the recoveries of AAs were more than 92% (Fig. 6). Thus, the DMIPs could provide excellent recycling capacity and guarantee the accuracy of six trace AAs due to the predetermined recognition cavities, chemical and mechanical stability. 3.7. Method validation Fig. S3 presented the extracted ion chromatograms of AAs solutions. A series of aqueous solutions containing each of STP, KAM from 10 to 200 ng/mL, and APM, GNT, TOM and PRM from 50 to 1000 ng/mL were prepared for calibration curve. Analysis was performed with five replicates for each level. Linearity, correlation coefficient (r2), limit of detections (LODs) and relative standard deviations (RSDs) were carefully studied (Table 3). The results showed all the analytes exhibited good linearity with r2 larger than 0.9921. The LODs (S/N = 3) were from 0.006 ng/mL to 0.6 ng/mL. A repeatability study (the spiked samples of 10 ng/mL for each of the AAs) was also carried out by performing five parallel experiments, and RSDs ranged from 5.3% to 9.3%. Those parameters indicated that the developed method had high sensitivity and good repeatability.
Fig. 5. The recoveries of DMIPs for extraction of different AAs.
3.8. Analysis of environmental water samples The established SPE cartridge based on DMIPs adsorbents was evaluated for its practical application for extracting AAs from environmental water samples (tap water, lake water and irrigation water samples). The results were listed in Tables 4 and 2 ng/mL of STP was detected in lake water samples. The typical residual ion chromatogram of STP was showed in Fig. S4. Different types of environmental water samples were spiked at three different concentration levels (2, 4 and 20 ng/mL for STP and KAM, and 10, 20 and 100 ng/mL for TOM, GNT, APM and PRM, respectively). The recoveries fell into 70.8%–108.3% and RSDs were from 2.6% to 11.4%.
Fig. 6. The recoveries of AAs with different recycling times.
AAs was loaded into DMIPs cartridge, respectively. The recoveries of six AAs remained almost unchanged (higher than 95%) when at 5, 10, 20, 30, 50 mL samples (Fig. 4D). In spite of the slightly decreasing recoveries with subsequent volume, considering sample loading time consuming, a volume of 50 mL was conducted in the real sample process.
3.9. Comparison with other methods The developed DMISPE-HILIC-HPLC-MS/MS method was compared with previously represented literatures for determining AAs in different matrices (Table S1). In spite of less number of detected AAs in our developed method, the other parameters (LOQs, recovery, simplicity, amount of sample) were better than traditional adsorbents (C18, exchange resin) [7,9,29], and the traditional method usually consisted of tedious process with two consecutive pretreatment method (e.g. C18-weak cation exchange) [9,29]. It was noteworthy that this method showed several advantages of less packed adsorbent, larger loading sample, low cost, and high sensitivity in comparison with other MISPE method, especially superior to the reported synthetic MIPs (AAs as template) [23].
3.5. Specific recognition As we could see from Fig. 1, the molecule structure of SPC was similar with RAF, but much different from other AAs. In order to evaluate the specific recognition ability of DMIPs, the water samples spiked at 5 μg/mL of seven AAs were treated by DMIPs-based cartridges. The recovery of structural analogue-SPC was compared with other six AAs in Fig. 5 and the recovery (51%) of SPC was obviously lower than STP、 KAM、APM、GNT、TOM and PRM (94.7%, 98.0%, 99.0%, 98.5%, 229 95.0%, 99.0%), indicating that DMIPs possessed excellent class-specific recognition capacity towards six targeted AAs.
4. Conclusion
3.6. Recycling times of DMIPs
In this study, the selective dummy molecularly imprinted polymers for AAs was successfully synthesized using raffinose as dummy template. The obtained DMIPs exhibited uniform sizes and high selective adsorption capacity to six AAs, suitable for SPE adsorbents, avoiding
Furthermore, the recycling capacity of DMIPs cartridge were investigated by loading 50 mL water samples spiked with 5 μg/mL of each Table 3 The parameters of the developed method for the six AAs. Neonicotinoid
Linear range (ng/mL)
Regression equation
r2
LODs (ng/mL)
LOQs (ng/mL)
RSD (%) (n = 5)
STP KAM APM GNT TOM PRM
10–200 10–200 50–1000 50–1000 50–1000 50–1000
y y y y y y
0.9969 0.9921 0.9958 0.9971 0.9954 0.9970
0.006 0.25 0.5 0.6 0.6 0.6
0.02 0.8 1.5 1.8 1.8 1.8
7.6 5.3 6.2 9.3 7.2 6.8
= = = = = =
−11200 + 1890x −3080 + 366x −3640 + 89.1x −10100 + 276x −77900 + 241x −810 + 23.7x
6
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Table 4 The recoveries of the six AAs from tap water, lake water and irrigation water samples. Analyte
Spiked (ng/mL)
Tap water (ng/mL) Found (ng/mL)
STP
KAM
APM
GNT
TOM
PRM
0 2 4 20 0 2 4 20 0 10 20 100 0 10 20 100 0 10 20 100 0 10 20 100
nd 1.83 3.93 20.3 nd 1.94 3.8 20.7 nd 10.54 20.72 96.5 nd 9.34 21.2 94.5 nd 9.45 19.86 101.9 nd 9.31 21.24 88.6
Lake water (ng/mL) R (%)
RSD (%)
91.5 98.2 101.3
8.3 7.6 4.9
96.9 95.2 103.5
4.2 5.7 9.7
105.4 103.6 96.5
6.3 8.2 5.6
93.4 106.3 94.5
5.7 11.4 8.1
94.5 99.3 101.9
4.7 6.2 7.1
93.1 106.2 88.6
8.2 7.6 3.7
Found (ng/mL) 2.0 1.89 3.75 19.04 nd 1.95 4.08 19.9 nd 9.52 19.74 99.3 nd 10.25 19.46 99.2 nd 10.35 19.48 105.4 nd 10.83 20.44 98.6
Irrigation water (ng/mL) R (%)
RSD (%)
94.4 93.6 95.2
4.3 3.6 8.2
97.6 102.1 99.4
8.6 4.2 7.5
95.2 98.7 99.3
5.2 4.7 4.8
102.5 97.3 99.2
8.3 4.1 5.6
103.5 97.4 105.4
5.4 3.9 8.1
108.3 102.2 98.6
7.2 8.3 4.7
Found (ng/mL) nd 1.52 2.84 14.4 nd 1.98 3.82 16.5 nd 10.15 19.66 87.9 nd 9.92 20.54 98.6 nd 9.95 19.88 99.8 nd 9.56 19.84 94.4
R (%)
RSD (%)
76.4 70.8 71.6
7.2 4.1 3.5
99.2 95.4 81.1
7.3 9.6 5.1
101.5 98.3 87.9
8.2 4.5 7.7
99.2 102.7 98.6
5.2 6.9 8.4
99.5 99.7 99.8
6.3 10.5 8.6
95.6 99.2 94.4
4.1 2.6 5.4
Note: nd means not found.
the defect of template leakage in the sample pretreatment. The developed method based on DMIPs cartridges coupled with HILIC-HPLC-MS/ MS showed good recoveries, reproducibility and high sensitivity for extraction of STP, KAM, APM, GNT, TOM and PRM from environmental water samples. The DMIPs adsorbents possess great potential application for detecting residual AAs in other matrix.
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Acknowledgements [10]
This work was supported by the Natural Science Foundation of Shandong Province, China (ZR2019BCE091) and the National Natural Science Foundation of China (Contact No. 21305121)
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Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.talanta.2019.120385.
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