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J. Sep. Sci. 2014, 37, 587–594
Ting Yang Chao Ma Huaixia Chen Yajie Zhang Xueping Dang Jianlin Huang Ministry of Education Key Laboratory for the Synthesis and Application of Organic Functional Molecules, Hubei Collaborative Innovation Center for Advanced Organic Chemical Materials, College of Chemistry and Chemical Engineering, Hubei University, Wuhan, China Received November 17, 2013 Revised December 12, 2013 Accepted December 13, 2013
Research Article
A molecularly imprinted organic–inorganic hybrid monolithic column for the selective extraction and HPLC determination of isoprocarb residues in rice An IPC-imprinted (IPC is isoprocarb) poly(methacrylic acid)/SiO2 hybrid monolithic column was prepared and applied for the recognition of the template. The hybrid monolithic column was synthesized in a micropipette tip using methyltrimethoxysilane as the inorganic precursor, 3-(methacryloxy)propyltrimethoxysilane as the coupling agent, and ethylene glycol dimethacrylate as the cross-linker. The synthesis conditions, including the porogenic solvent, coupling agent, volume ratio of the inorganic alcoholysate and organic part, were optimized. The prepared monolithic column was characterized by SEM and FTIR spectroscopy. A simple, rapid, and sensitive method for the determination of IPC in rice using the imprinted monolithic column microextraction combined with HPLC was developed. Several parameters affecting the sample pretreatment were investigated, including the eluent, washing solution, and loading sample volume. The linearity of the calibration curve was observed in the range of 9.0–1000 g/kg for IPC in rice with the correlation coefficient (r2 ) of 0.9983. The LOD was 3.0 g/kg (S/N = 3). The assay gave recovery values ranging from 91 to 107%. The proposed method has been successfully applied for the selective extraction and sensitive determination of IPC in rice and a satisfactory result was obtained. Keywords: Hybrid monolithic columns / Isoprocarb / Molecularly imprinted polymers / Rice DOI 10.1002/jssc.201301227
1 Introduction Isoprocarb (2-(1-methylethyl)phenyl methylcarbamate, IPC), one of the most important carbamate insecticides, has been used worldwide in agricultural and cotton spraying since 1970 [1]. IPC residue can be found in food, providing a potential risk to humans because it affects the nervous system by disrupting an enzyme that regulates acetylcholine, a neurotransmitter. To ensure food safety, international organizations have established maximum residue levels (MRLs) of 0.02–5 mg/kg for carbamates including IPC in crops [2]. The
Correspondence: Professor Huaixia Chen, Ministry of Education Key Laboratory for the Synthesis and Application of Organic Functional Molecules, Hubei Collaborative Innovation Center for Advanced Organic Chemical Materials, Hubei University, Wuhan 430062, China E-mail: [email protected] Fax: +86-27-88663043
Abbreviations: EF, enrichment factor; ER, extraction recovery; EGDMA, ethylene glycol dimethacrylate; HMIM, organic–inorganic hybrid MIPs monolithic column; HMIMME, HMIM microextraction; HNIM, hybrid nonimprinted polymer monolithic column; IF, imprinting factor; IPC, isoprocarb; KH570, 3-(methacryloxy)propyltrimethoxysilane; MAA, methacrylic acid; MIPs, molecularly imprinted polymers; MTMS, methyltrimethoxysilane; SPME, solid-phase microextraction C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chinese government has set a limit of 0.2 mg/kg for IPC in rice (GB2763–2005). An ideal method for the determination of IPC residue should have high sensitivity and selectivity and should be applicable to complex matrices. Some analytical methods have been developed for carbamates including IPC, such as GC [3, 4], ELISA [5, 6], MEKC [7, 8], biosensors [9], HPLC [10–12], and HPLC–MS [13–15]. But the thermal instability of IPC does not permit the direct determination by GC unless it is derived into thermally stable derivatives. LC–MS methods are the most powerful for pesticide residue analysis but the instrumentation is quite costly, and the laboratory-based method is also timeconsuming. For this reason, HPLC with different detectors has become the most commonly used technique for the determination of IPC residue. To enhance the sensitivity and selectivity of the HPLC method, different pretreatment methods, including liquid–liquid extraction (LLE) [16], SPE [17,18], microwave-assisted extraction (MAE) [19], solid-phase microextraction (SPME) [20, 21], and liquid-phase microextraction (LPME) [22,23], have been used for the preconcentration and cleanup of the IPC residue from different matrices prior to instrumental analysis. Although many of these techniques are suitable and effective, they are relatively time-consuming, require large volumes of organic solvents, and, especially, lack selectivity. Colour Online: See the article online to view Figs. 1 and 3 in colour. www.jss-journal.com
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Molecular imprinting technology is a powerful tool for the synthesis of materials with molecular recognition ability. Organic molecularly imprinted polymers (MIPs) have excellent pH stability and the easy availability of various monomers. However, these materials may shrink or swell when exposed to different mobile phases, and the various degrees of swelling in different solvents may considerably change the morphology of the polymer network [24, 25]. On the other hand, the inorganic MIPs will inevitably present cracking and shrinkage, although these materials can offer excellent mechanical strength and good solvent resistance [26]. However, organic– inorganic hybrid MIPs have been found to be highly advantageous as they exhibit flexibility, low density, and long shelf-life with excellent optical and mechanical properties [27–32]. Monolithic columns with the ideal porous structure that can accelerate the rate of mass transfer have become increasingly important as stationary phases with good separation efficiency. Nowadays, organic and inorganic MIP monolithic columns were introduced into the SPE and SPME procedure [33, 34], respectively. And the organic–inorganic hybrid MIPs monolithic columns (HMIMs) were used as the separation columns for CEC31 and HPLC [35]. To the best of our knowledge, organic–inorganic HMIMs have not been synthesized and applied for the SPME or SPE pretreatment of complex samples. In this article, we chose IPC as the template, methacrylic acid (MAA) as the organic functional monomer, methyltrimethoxysilane (MTMS) as the inorganic precursor, 3-(methacryloxy)propyltrimethoxysilane (KH570) as the coupling agent, and ethylene glycol dimethacrylate (EGDMA) as the cross-linker. KH570 was used to form covalent bonds between the organic and inorganic phases. A novel IPCimprinted poly(methacrylic acid)/SiO2 hybrid monolithic column was synthesized in a micropipette tip for the first time. The monolithic column was characterized and applied to the selective SPME for the efficient separation and fast enrichment of IPC residue from rice samples.
2 Materials and methods 2.1 Reagents and materials IPC (99.5%), carbary (99.5%), and cephalexin (99.5%) were purchased from Dikma Technologies (Beijing, China). EGDMA purchased from Acros (Morris Plains, NJ, USA) was extracted with 5% aqueous sodium hydroxide and water, then dried using anhydrous magnesium sulfate. MAA purchased from Fuchen Chemical Reagent Company (Tianjin, China) was distilled under vacuum prior to use. 2,2 Azobisisobutyronitrile was purchased from Shanghai No. 4 Chemical Reagent (Shanghai, China). KH570 and MTMS were purchased from Aladdin Reagent (Shanghai, China). Methanol and acetonitrile (HPLC grade) were obtained from Tedia (Fairfield, OH, USA). Ultrapure water was obtained on an Ultrapure Water System (Beijing, China).
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
J. Sep. Sci. 2014, 37, 587–594
2.2 Instrumentation and HPLC conditions An X-650 scanning electron microscope (Hitachi, Tokyo, Japan) was used to investigate the surface of the hybrid imprinted monolithic column. The IR spectra of EGDMA, MTMS, HMIM, and HNIM (hybrid nonimprinted polymer monolithic colum) were examined by a Fourier transform infrared spectrometer (Perkin Elmer, Waltham, MA, USA). The chromatographic analysis was carried out on a Dionex Summit U3000 HPLC system equipped with a manual injector and a Photodiode Array Detector (Dionex Technologies, Sunnyvale, CA, USA). A personal computer equipped with a Chromeleon Chem-Station program for LC was used to process chromatographic data. An amethyst C18 column (4.6 × 250 mm, 5 m) from Sepax Technologies (Newark, DE, USA) was connected with a guard column (cartridge 2.1 × 12.5 mm, 5 m, Agilent Technologies, PaloAlto, CA, USA) filled with the same packing material. The mobile phase was a mixture of methanol/water (65:35, v/v) and the flow rate was 1.0 mL/min. The column temperature was set at 30⬚C by a temperature controller (Nuohai Technologies, Hangzhou, China). The UV detector was set at a wavelength of 246 nm for the analytes. All injections were performed manually with a 20.0 L sample loop. An LSP01–1A longer pump (Baoding Longer Precision Pump, Baoding, China) was used for pumping. The 0.22 m membrane was obtained from Xingya Scavenging Material (Shanghai, China).
2.3 Preparation of IPC-imprinted hybrid monolithic column A sol–gel approach was used to prepare the IPC-imprinted organic–inorganic hybrid monolithic column. The prepolymer solution for MIP was prepared by dissolving 0.1 mmol of IPC in 1.1 mL of methanol. The solution was vortexed thoroughly. Then, 0.4 mmol MAA was added and kept at room temperature for 2 h using an ultrasonic method. A quantity of 0.4 mmol KH570 was added, blended, and kept at 40⬚C for 2 h in a water bath. At the same time, MTMS was dissolved in methanol and HNO3 (8:3:2, v/v/v) and stirred at 40⬚C for 1 h. The inorganic solution was added to the above organic prepolymer solution in the ratio of 3:1 (MTMS/KH570, n/n). The mixture was stirred at 40⬚C for 2 h. After that, 2.0 mmol EGDMA and 12.0 mg 2,2 -azobisisobutyronitrile were added. The mixture solution was degassed in an ultrasonic bath for 5 min to deoxygenize it. Next, 100 L of the mixture was transferred into a micropipette tip, which had been sealed at one end. Subsequently, the other end of the pipette tip was sealed with silicon rubber. After polymerization at 60⬚C for 24 h, the silicon rubber was removed. The resultant HMIM was washed with methanol to remove the template molecules. An HNIM was prepared following the same procedures without IPC in the synthesis.
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2.4 Preparation of aqueous standards and samples
3 Results and discussion
A total of 1.0 mg/mL IPC stock solution was prepared by dissolving 100 mg of IPC in 100 mL of methanol. The stock solutions were stored at 4⬚C and protected from light. Aqueous standards at various concentrations were daily prepared by diluting each stock solution with ultrapure water. The rice samples obtained from a local supermarket were ground. A total of 3.0 g of ground rice was accurately weighed and placed in a 10 mL centrifuge tube. Then, 5 mL of methanol was added into the tube, and mixed. Next, ultrasound-assisted extraction was carried out at room temperature for 20 min. Then, the tube was centrifuged at 4000 rpm for 10 min. The supernatant was filtered through a 0.22 m syringe filter and diluted to 50 mL with ultrapure water prior to extraction.
In order to evaluate the extraction efficiency of the HMIM microextraction (HMIMME) and obtain the optimized extraction conditions, enrichment factor (EF) and extraction recovery (ER) were used. And, the imprinting factor (IF) was used to evaluate the recognition abilities of the HMIMs. The definitions of EF, ER, and IF have been published [33].
3.1 Preparation of the imprinted monolith The synthesis conditions concluding the volume of porogenic solvent, mole number of KH570, ratio of organic and inorganic continents, and nature of inorganic precursor were optimized according to the morphology, flow rate, and IF value of the monolithic column. 3.1.1 Selection of porogenic solvent
2.5 Preparation of the extraction device The IPC-HMIM could be connected with syringes in different sizes simply without any other treatment. This extraction device was simple and convenient for extraction operation. A syringe infusion pump (Baoding Longer Precision Pump) was employed for the delivery of simple solution, washing solution, and desorption solvent.
2.6 Microextraction procedure The IPC-HMIM was washed with 2.0 mL of methanol and 0.5 mL of water at a flow rate of 0.05 mL/min, respectively. Then, an aliquot of 3.0 mL pretreated sample solution was loaded at a flow rate of 0.05 mL/min. Next, the monolithic column was washed with 200 L of chloroform and water, respectively. Then, the analyte was eluted with 150 L of a mixture of methanol/acetonitrile (6:4, v/v) at a flow rate of 0.05 mL/min. The eluent solution was removed using a 100 L HPLC microsyringe and injected into the HPLC system for analysis directly. All experiments were performed repeatedly and the mean values were used to plot curves and are given in the tables.
The choice of porogens is critical in the syntheses of the HMIMs. The porogen and its volume influence the pore diameter, pore size distribution, pore ratio, and mechanical strength of the monolithic column, which will influence on the permeability of the monolithic column in the next microextraction process. In general, the porogen must be able to dissolve the template, monomer, cross-linker, initiator, and inorganic precursor, and then make these compounds become homogeneous. Furthermore, the porogens can produce a larger surface area and adsorption capacity for the separation medium in the synthesis process. Toluene, methanol, and acetonitrile were chosen as porogens. The experimental results indicated that the HMIMs in the presence of toluene or acetonitrile were unevenly distributed and not tight. Methanol as the porogenic solvent could display better morphological characters and extraction efficiency for IPC. So, methanol was selected as the appropriate porogenic solvent. The volume of porogen was optimized while different volumes of 0.9, 1.0, 1.1, and 1.2 mL were used in the synthesis. The experimental results indicated that the monolithic column with 1.1 mL of methanol as the porogens could display a suitable flow rate and lower backpressure in the microextraction process. 3.1.2 Selection of the coupling agent KH570
2.7 Determination of the selectivity of the monolith To investigate the selectivity of the monolithic column, 1.0 mL of mixed solution of IPC, carbary, and cephalexin, whose concentrations were 59.5 ng/mL, was loaded on HMIM and HNIM at a flow rate of 0.05 mL/min, respectively. The monolithic columns were washed with 200 L of chloroform and water, respectively. Then, the analyte was eluted with 150 L of methanol at a flow rate of 0.05 mL/min. The eluent was analyzed by HPLC. C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
In the synthesis of hybrid materials, the coupling agent acts as a bridge that connects the inorganic precursor with organic cross-linker simultaneously. Therefore, the hybrid materials can overcome the disadvantages of single organic or inorganic materials. KH570 was selected as the coupling agent. The volume of KH570 was optimized in the study. Different volume ratios of KH570 to inorganic part 1:2, 1:3, 1:4, and 1:5 were investigated, respectively. The results showed that the monolithic column had better morphological characters and modest backpressure when the volume ratio of 1:3 was chosen. www.jss-journal.com
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3.1.3 Selection of the volume ratio of the inorganic alcoholysate and organic part
Table 1. The EF values of IPC in the HMIMs and HNIMs and the respective IF values
In this study, the volume ratios of the inorganic alcoholysate to organic part 1:1, 1:3, 1:5, and 1:6 were investigated, respectively. The results showed that the HMIMs were too soft and easy to shrink when the volume ratios of 1:1 and 1:3 were chosen. The monolithic column was easy to swell, which resulted in a high backpressure in the microextraction process when the volume ratio 1:6 was selected. To avoid the shrinkage and high backpressure of the hybrid polymer, the volume ratio 1:5 was chosen.
Compound
3.2 Characterization and specificity evaluation of the monolith The SEM images of the monoliths were obtained by using an X-650 scanning electron microscope and were used to observe the morphology of the HMIMs. It is readily observed that the monolithic column was uniform and regular, and there were many macropores and flow-through channels inlaid in the network skeleton of the monolithic column, which provided flow paths through the column. Due to the size and density of the macropore network, the monolithic column had a high external porosity and, consequently, a large permeability and low column hydraulic resistance. The pores allowed the mobile phase to flow through with low backpressure. Fourier transform infrared (FTIR) spectra in potassium bromide were recorded by a Spectrum 100 FTIR spectrophotometer. The results showed the IR spectra of EGDMA, MTMS, IPC HMIM, and HNIM. Comparing with the IR spectra of EGDMA, the C=O stretching vibration peak of 1728 cm−1 and the C–H stretching vibration peak of 2960 cm−1 appeared in the spectra of HMIMs. But the C=C stretching vibration peak of 1638 cm−1 became very weak. These results indicated that the cross-linker EGDMA had polymerized. Comparing with the IR spectrum of MTMS, the Si–O–Si stretching vibrations around 1105 cm−1 and Si–O vibrations around 796 cm−1 appeared in the IR spectrum of HMIM. The spectra of HNIM and HMIM showed similar locations and appearances of the major bands. These results showed that the polymers have been successfully synthesized. To evaluate the selectivity of the HMIM, carbary with a similar structure to IPC as the analog and cephalexin as nonanalog were tested. It can be seen from the Table 1 that the HMIM had a higher absorbability for IPC than HNIM, where the IF was 4.41. At the same time, the HMIM possessed higher extraction efficiency for carbary than HNIM. This was due to the similar structure of carbary and IPC. The results also indicated that the retention of cephalexin in HMIM was similar to that in HNIM. These results demonstrated the good selectivity of the synthesized HMIM for IPC. To prepare the HMIM, IPC was dissolved in methanol, and MAA was used as the functional monomer to produce hydrogen-bonding interactions with the template. Then, a stable donor–receptor complex between the template and C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
IPC Carbary Cephalexin
EF
IF
HMIMs
HNIMs
5.62 4.12 1.34
1.29 1.23 1.21
4.41 3.35 1.11
functional monomer was formed in the prepolymerization process. The existence of the complex resulted in the formation of well-defined specific binding sites in the polymers. The silane coupling agent KH570 acted as a bridge that connected the monomer with MTMS at the same time. Subsequently, the HMIM was prepared through the polymerization of EGDMA. After removal of the template molecule, the specific imprinting sites were maintained. The synthesis protocol of the HMIM and the recognition mechanism are indicated in Fig. 1. The flow-through pore size distribution was determined by mercury porosimetry, and the result is shown in Fig. 2. It can be seen that HMIM possessed larger flow-through pores (a narrow peak centered at 4.0 m), which could lead to higher permeability and favorable mass transfer during extraction. The specific surface areas and pore volumes from nitrogen adsorption–desorption experiments were 25.1 m2 /g and 0.062 cm3 /g for HMIM, and 21.8 m2 /g and 0.069 cm3 /g for HNIM, respectively. The similar surface areas and pore volumes of HMIM and HNIM showed that selectivity was due to the imprinted recognition properties of HMIM.
3.3 Optimization and application of the monolith In order to optimize the HMIMME conditions, several extraction parameters were investigated, including the type and volume of eluent, washing solution, and sample volume. Sample solutions were spiked with IPC at 59.5 ng/mL to perform the experiments. 3.3.1 Optimization of extraction conditions The selection of an appropriate eluent solvent is of high importance for the HMIMME process. Considering the solubility of IPC and consistency to the mobile phase used in HPLC, methanol and acetonitrile were selected. Different proportions of methanol with acetonitrile as the eluent were tested. The experimental results showed that the mixture of methanol with acetonitrile (6:4, v/v) as eluent offered the higher recovery. ER and EF of IPC were studied while different eluent volumes of 80–200 L of methanol/acetonitrile (6:4, v/v) were used. The results showed that ER increased and EF decreased while increasing the eluent volume from 80 to 200 L. One hundred and www.jss-journal.com
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Figure 1. Schematic representation of the preparation and extraction procedures of the HMIMs.
fifty microliter methanol/acetonitrile (6:4, v/v) was enough to provide a quantitative elution of the analyte from the sorbent. Water, cyclohexane, chloroform, and ethanol were selected as the washing solution. The experimental results showed that the recoveries were very low with cyclohexane C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
and ethanol as the washing solutions. Water and chloroform were suitable as the washing solution. To remove oil- and water-soluble impurities and get rid of the effect of chloroform on HPLC separation column, 200 L of chloroform and 200 L of water were selected to wash the monolithic column in order. www.jss-journal.com
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Figure 2. Pore size distribution profile of HMIM by mercury intrusion porosimetry.
The effect of sample volume was optimized by loading sample solutions 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, and 4.0 mL at a constant flow rate, respectively. The results showed that EF of IPC increased with the increasing of sample volume from 1.0 to 4.0 mL. However, ER began to decrease when the sample volume increased. To achieve sufficient sensitivity within a short time, 3.0 mL of sample solution was selected in the following test. 3.3.2 Analytical approach The obtained HMIMME was coupled with HPLC and UV detection to establish a method for the determination of IPC in rice samples under the optimized extraction conditions. Good linearity was achieved in the range of 9.0–1000 g/kg for IPC in blank rice samples with correlation coefficient (r2 ) of 0.9983. The LOD (S/N = 3) and LOQ (S/N = 10) were 3.0 and 9.0 g/kg for rice, respectively. The LOD of the analyte in rice was lower than the Chinese safe maximum (0.2 mg/kg). 3.3.3 Sample analysis Three kinds of rice samples (from Hanzhong, northeast China, and Fuxian, respectively) were selected for the application of the developed HMIMME–HPLC method. The results showed that these rice samples were free of IPC residue. The chromatograms of spiked rice samples before and after treatment by HNIM and HMIM are shown in Fig. 3. The results indicated that after pretreatment by HMIMME, the majority of interfering substances in the rice samples were eliminated. Furthermore, the sensitivity of the analyte in the rice was greatly enhanced with the HMIMME–HPLC analysis. The HNIM possessed extraction capability much lower than that of HMIMs for IPC. The results indicated that the specific recognition of the HMIM to IPC existed in the polymer. To validate the established method, blank rice samples were spiked with IPC at three levels of 83.3, 500, and 833 g/kg. The recoveries were 91–107% with the RSD from 3.1 to 8.9%, as shown in Table 2. These results indicated that C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 3. HPLC chromatograms of blank rice sample (1), 50 g/kg spiked rice sample (2), 50 g/kg spiked rice extracted with HNIMME (3), and 50 g/kg spiked rice extracted with HMIMME (4).
the established method was suitable for the extraction and determination of trace amounts of IPC in complex samples. 3.3.4 Comparison of the IPC-imprinted monolith extraction with other methods The efficiency of the presented HMIMME–HPLC–UV method for the determination of IPC in rice was compared with other reported methods such as LLE–GC–MS (LLE is liquid–liquid extraction); matrix solid-phase dispersion (MSPD) with gel permeation chromatography (GPC) and GC coupled with MS; dispersive SPE (DSPE) with GC coupled to MS, liquid-liquid microextraction (LLME)–HPGC–UV; and reversed electrode polarity stacking mode (REPSM) MEKC with photodiode array detection (PDA) from the aspects of the linear range, LOD, and RSD. As listed in Table 3, the LOD of the HMIMME–HPLC for IPC was obviously lower than those of other reported methods, even lower than those of the tandem GC–MS methods. These results revealed that the HMIMME–HPLC is a sensitive, rapid, low-cost, and reproducible technique that can be used for selective extraction and sensitive determination of IPC in complex samples.
4 Concluding remarks In summary, we have described the preparation and application of a novel IPC-imprinted poly(methacrylic acid)/SiO2 hybrid monolithic column by using a mild sol–gel process. The monolithic column was synthesized in a micropipette tip. So, the micromonolithic column could be connected with syringes in different sizes simply without any other treatment to perform microextraction process. The derived HMIMs showed high selectivity and enrichment ability for IPC. After optimization of the extraction conditions, the HMIMME coupled with HPLC–UV was used for the selective extraction and determination of IPC in rice samples successfully. www.jss-journal.com
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Table 2. Linear range, LODs, precision, and recoveries of the HMIMME–HPLC method for the determination of IPC in rice samples
Sample
Rice from Hanzhong Rice from northeast China Rice from Fuxian
Spiked (g/kg)
83.3 500 833 83.3 500 833 83.3 500 833
Recovery (%; n = 5)
95 91 103 97 94 105 92 97 107
RSD (%; n = 5) Intraday
Interday
7.7 3.5 3.1 7.4 4.3 5.7 5.8 4.3 3.7
8.9 4.3 5.2 7.8 5.1 6.0 7.5 5.2 5.4
Linear equation
LOD (g/kg)
LOQ (g/kg)
Y = 3.702 × 10−2 X − 1.279 × 10−2 (r2 = 0.9983)
3.0
9.0
Table 3. Comparison of the HMIMME method with other sample preparation techniques for the determination of IPC in real samples
Extraction method
Analytical method
Linear range (mg/kg)
LLE MSPD DSPE LLME REPSM HMIMs microextraction
GC–MS GPC–GC–MS GC–MS HPLC–UV MEKC–PDA HPLC
0.05–0.5 0.05–0.4 0.04–8.00 (mg/L) 0.010–5.0 0.009–1
RSD (%)
LOD (mg/kg)
Sample
Ref.
0.7 ≤21
J. Sep. Sci. 2014, 37, 587–594
Ting Yang Chao Ma Huaixia Chen Yajie Zhang Xueping Dang Jianlin Huang Ministry of Education Key Laboratory for the Synthesis and Application of Organic Functional Molecules, Hubei Collaborative Innovation Center for Advanced Organic Chemical Materials, College of Chemistry and Chemical Engineering, Hubei University, Wuhan, China Received November 17, 2013 Revised December 12, 2013 Accepted December 13, 2013
Research Article
A molecularly imprinted organic–inorganic hybrid monolithic column for the selective extraction and HPLC determination of isoprocarb residues in rice An IPC-imprinted (IPC is isoprocarb) poly(methacrylic acid)/SiO2 hybrid monolithic column was prepared and applied for the recognition of the template. The hybrid monolithic column was synthesized in a micropipette tip using methyltrimethoxysilane as the inorganic precursor, 3-(methacryloxy)propyltrimethoxysilane as the coupling agent, and ethylene glycol dimethacrylate as the cross-linker. The synthesis conditions, including the porogenic solvent, coupling agent, volume ratio of the inorganic alcoholysate and organic part, were optimized. The prepared monolithic column was characterized by SEM and FTIR spectroscopy. A simple, rapid, and sensitive method for the determination of IPC in rice using the imprinted monolithic column microextraction combined with HPLC was developed. Several parameters affecting the sample pretreatment were investigated, including the eluent, washing solution, and loading sample volume. The linearity of the calibration curve was observed in the range of 9.0–1000 g/kg for IPC in rice with the correlation coefficient (r2 ) of 0.9983. The LOD was 3.0 g/kg (S/N = 3). The assay gave recovery values ranging from 91 to 107%. The proposed method has been successfully applied for the selective extraction and sensitive determination of IPC in rice and a satisfactory result was obtained. Keywords: Hybrid monolithic columns / Isoprocarb / Molecularly imprinted polymers / Rice DOI 10.1002/jssc.201301227
1 Introduction Isoprocarb (2-(1-methylethyl)phenyl methylcarbamate, IPC), one of the most important carbamate insecticides, has been used worldwide in agricultural and cotton spraying since 1970 [1]. IPC residue can be found in food, providing a potential risk to humans because it affects the nervous system by disrupting an enzyme that regulates acetylcholine, a neurotransmitter. To ensure food safety, international organizations have established maximum residue levels (MRLs) of 0.02–5 mg/kg for carbamates including IPC in crops [2]. The
Correspondence: Professor Huaixia Chen, Ministry of Education Key Laboratory for the Synthesis and Application of Organic Functional Molecules, Hubei Collaborative Innovation Center for Advanced Organic Chemical Materials, Hubei University, Wuhan 430062, China E-mail: [email protected] Fax: +86-27-88663043
Abbreviations: EF, enrichment factor; ER, extraction recovery; EGDMA, ethylene glycol dimethacrylate; HMIM, organic–inorganic hybrid MIPs monolithic column; HMIMME, HMIM microextraction; HNIM, hybrid nonimprinted polymer monolithic column; IF, imprinting factor; IPC, isoprocarb; KH570, 3-(methacryloxy)propyltrimethoxysilane; MAA, methacrylic acid; MIPs, molecularly imprinted polymers; MTMS, methyltrimethoxysilane; SPME, solid-phase microextraction C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chinese government has set a limit of 0.2 mg/kg for IPC in rice (GB2763–2005). An ideal method for the determination of IPC residue should have high sensitivity and selectivity and should be applicable to complex matrices. Some analytical methods have been developed for carbamates including IPC, such as GC [3, 4], ELISA [5, 6], MEKC [7, 8], biosensors [9], HPLC [10–12], and HPLC–MS [13–15]. But the thermal instability of IPC does not permit the direct determination by GC unless it is derived into thermally stable derivatives. LC–MS methods are the most powerful for pesticide residue analysis but the instrumentation is quite costly, and the laboratory-based method is also timeconsuming. For this reason, HPLC with different detectors has become the most commonly used technique for the determination of IPC residue. To enhance the sensitivity and selectivity of the HPLC method, different pretreatment methods, including liquid–liquid extraction (LLE) [16], SPE [17,18], microwave-assisted extraction (MAE) [19], solid-phase microextraction (SPME) [20, 21], and liquid-phase microextraction (LPME) [22,23], have been used for the preconcentration and cleanup of the IPC residue from different matrices prior to instrumental analysis. Although many of these techniques are suitable and effective, they are relatively time-consuming, require large volumes of organic solvents, and, especially, lack selectivity. Colour Online: See the article online to view Figs. 1 and 3 in colour. www.jss-journal.com
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Molecular imprinting technology is a powerful tool for the synthesis of materials with molecular recognition ability. Organic molecularly imprinted polymers (MIPs) have excellent pH stability and the easy availability of various monomers. However, these materials may shrink or swell when exposed to different mobile phases, and the various degrees of swelling in different solvents may considerably change the morphology of the polymer network [24, 25]. On the other hand, the inorganic MIPs will inevitably present cracking and shrinkage, although these materials can offer excellent mechanical strength and good solvent resistance [26]. However, organic– inorganic hybrid MIPs have been found to be highly advantageous as they exhibit flexibility, low density, and long shelf-life with excellent optical and mechanical properties [27–32]. Monolithic columns with the ideal porous structure that can accelerate the rate of mass transfer have become increasingly important as stationary phases with good separation efficiency. Nowadays, organic and inorganic MIP monolithic columns were introduced into the SPE and SPME procedure [33, 34], respectively. And the organic–inorganic hybrid MIPs monolithic columns (HMIMs) were used as the separation columns for CEC31 and HPLC [35]. To the best of our knowledge, organic–inorganic HMIMs have not been synthesized and applied for the SPME or SPE pretreatment of complex samples. In this article, we chose IPC as the template, methacrylic acid (MAA) as the organic functional monomer, methyltrimethoxysilane (MTMS) as the inorganic precursor, 3-(methacryloxy)propyltrimethoxysilane (KH570) as the coupling agent, and ethylene glycol dimethacrylate (EGDMA) as the cross-linker. KH570 was used to form covalent bonds between the organic and inorganic phases. A novel IPCimprinted poly(methacrylic acid)/SiO2 hybrid monolithic column was synthesized in a micropipette tip for the first time. The monolithic column was characterized and applied to the selective SPME for the efficient separation and fast enrichment of IPC residue from rice samples.
2 Materials and methods 2.1 Reagents and materials IPC (99.5%), carbary (99.5%), and cephalexin (99.5%) were purchased from Dikma Technologies (Beijing, China). EGDMA purchased from Acros (Morris Plains, NJ, USA) was extracted with 5% aqueous sodium hydroxide and water, then dried using anhydrous magnesium sulfate. MAA purchased from Fuchen Chemical Reagent Company (Tianjin, China) was distilled under vacuum prior to use. 2,2 Azobisisobutyronitrile was purchased from Shanghai No. 4 Chemical Reagent (Shanghai, China). KH570 and MTMS were purchased from Aladdin Reagent (Shanghai, China). Methanol and acetonitrile (HPLC grade) were obtained from Tedia (Fairfield, OH, USA). Ultrapure water was obtained on an Ultrapure Water System (Beijing, China).
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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2.2 Instrumentation and HPLC conditions An X-650 scanning electron microscope (Hitachi, Tokyo, Japan) was used to investigate the surface of the hybrid imprinted monolithic column. The IR spectra of EGDMA, MTMS, HMIM, and HNIM (hybrid nonimprinted polymer monolithic colum) were examined by a Fourier transform infrared spectrometer (Perkin Elmer, Waltham, MA, USA). The chromatographic analysis was carried out on a Dionex Summit U3000 HPLC system equipped with a manual injector and a Photodiode Array Detector (Dionex Technologies, Sunnyvale, CA, USA). A personal computer equipped with a Chromeleon Chem-Station program for LC was used to process chromatographic data. An amethyst C18 column (4.6 × 250 mm, 5 m) from Sepax Technologies (Newark, DE, USA) was connected with a guard column (cartridge 2.1 × 12.5 mm, 5 m, Agilent Technologies, PaloAlto, CA, USA) filled with the same packing material. The mobile phase was a mixture of methanol/water (65:35, v/v) and the flow rate was 1.0 mL/min. The column temperature was set at 30⬚C by a temperature controller (Nuohai Technologies, Hangzhou, China). The UV detector was set at a wavelength of 246 nm for the analytes. All injections were performed manually with a 20.0 L sample loop. An LSP01–1A longer pump (Baoding Longer Precision Pump, Baoding, China) was used for pumping. The 0.22 m membrane was obtained from Xingya Scavenging Material (Shanghai, China).
2.3 Preparation of IPC-imprinted hybrid monolithic column A sol–gel approach was used to prepare the IPC-imprinted organic–inorganic hybrid monolithic column. The prepolymer solution for MIP was prepared by dissolving 0.1 mmol of IPC in 1.1 mL of methanol. The solution was vortexed thoroughly. Then, 0.4 mmol MAA was added and kept at room temperature for 2 h using an ultrasonic method. A quantity of 0.4 mmol KH570 was added, blended, and kept at 40⬚C for 2 h in a water bath. At the same time, MTMS was dissolved in methanol and HNO3 (8:3:2, v/v/v) and stirred at 40⬚C for 1 h. The inorganic solution was added to the above organic prepolymer solution in the ratio of 3:1 (MTMS/KH570, n/n). The mixture was stirred at 40⬚C for 2 h. After that, 2.0 mmol EGDMA and 12.0 mg 2,2 -azobisisobutyronitrile were added. The mixture solution was degassed in an ultrasonic bath for 5 min to deoxygenize it. Next, 100 L of the mixture was transferred into a micropipette tip, which had been sealed at one end. Subsequently, the other end of the pipette tip was sealed with silicon rubber. After polymerization at 60⬚C for 24 h, the silicon rubber was removed. The resultant HMIM was washed with methanol to remove the template molecules. An HNIM was prepared following the same procedures without IPC in the synthesis.
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2.4 Preparation of aqueous standards and samples
3 Results and discussion
A total of 1.0 mg/mL IPC stock solution was prepared by dissolving 100 mg of IPC in 100 mL of methanol. The stock solutions were stored at 4⬚C and protected from light. Aqueous standards at various concentrations were daily prepared by diluting each stock solution with ultrapure water. The rice samples obtained from a local supermarket were ground. A total of 3.0 g of ground rice was accurately weighed and placed in a 10 mL centrifuge tube. Then, 5 mL of methanol was added into the tube, and mixed. Next, ultrasound-assisted extraction was carried out at room temperature for 20 min. Then, the tube was centrifuged at 4000 rpm for 10 min. The supernatant was filtered through a 0.22 m syringe filter and diluted to 50 mL with ultrapure water prior to extraction.
In order to evaluate the extraction efficiency of the HMIM microextraction (HMIMME) and obtain the optimized extraction conditions, enrichment factor (EF) and extraction recovery (ER) were used. And, the imprinting factor (IF) was used to evaluate the recognition abilities of the HMIMs. The definitions of EF, ER, and IF have been published [33].
3.1 Preparation of the imprinted monolith The synthesis conditions concluding the volume of porogenic solvent, mole number of KH570, ratio of organic and inorganic continents, and nature of inorganic precursor were optimized according to the morphology, flow rate, and IF value of the monolithic column. 3.1.1 Selection of porogenic solvent
2.5 Preparation of the extraction device The IPC-HMIM could be connected with syringes in different sizes simply without any other treatment. This extraction device was simple and convenient for extraction operation. A syringe infusion pump (Baoding Longer Precision Pump) was employed for the delivery of simple solution, washing solution, and desorption solvent.
2.6 Microextraction procedure The IPC-HMIM was washed with 2.0 mL of methanol and 0.5 mL of water at a flow rate of 0.05 mL/min, respectively. Then, an aliquot of 3.0 mL pretreated sample solution was loaded at a flow rate of 0.05 mL/min. Next, the monolithic column was washed with 200 L of chloroform and water, respectively. Then, the analyte was eluted with 150 L of a mixture of methanol/acetonitrile (6:4, v/v) at a flow rate of 0.05 mL/min. The eluent solution was removed using a 100 L HPLC microsyringe and injected into the HPLC system for analysis directly. All experiments were performed repeatedly and the mean values were used to plot curves and are given in the tables.
The choice of porogens is critical in the syntheses of the HMIMs. The porogen and its volume influence the pore diameter, pore size distribution, pore ratio, and mechanical strength of the monolithic column, which will influence on the permeability of the monolithic column in the next microextraction process. In general, the porogen must be able to dissolve the template, monomer, cross-linker, initiator, and inorganic precursor, and then make these compounds become homogeneous. Furthermore, the porogens can produce a larger surface area and adsorption capacity for the separation medium in the synthesis process. Toluene, methanol, and acetonitrile were chosen as porogens. The experimental results indicated that the HMIMs in the presence of toluene or acetonitrile were unevenly distributed and not tight. Methanol as the porogenic solvent could display better morphological characters and extraction efficiency for IPC. So, methanol was selected as the appropriate porogenic solvent. The volume of porogen was optimized while different volumes of 0.9, 1.0, 1.1, and 1.2 mL were used in the synthesis. The experimental results indicated that the monolithic column with 1.1 mL of methanol as the porogens could display a suitable flow rate and lower backpressure in the microextraction process. 3.1.2 Selection of the coupling agent KH570
2.7 Determination of the selectivity of the monolith To investigate the selectivity of the monolithic column, 1.0 mL of mixed solution of IPC, carbary, and cephalexin, whose concentrations were 59.5 ng/mL, was loaded on HMIM and HNIM at a flow rate of 0.05 mL/min, respectively. The monolithic columns were washed with 200 L of chloroform and water, respectively. Then, the analyte was eluted with 150 L of methanol at a flow rate of 0.05 mL/min. The eluent was analyzed by HPLC. C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
In the synthesis of hybrid materials, the coupling agent acts as a bridge that connects the inorganic precursor with organic cross-linker simultaneously. Therefore, the hybrid materials can overcome the disadvantages of single organic or inorganic materials. KH570 was selected as the coupling agent. The volume of KH570 was optimized in the study. Different volume ratios of KH570 to inorganic part 1:2, 1:3, 1:4, and 1:5 were investigated, respectively. The results showed that the monolithic column had better morphological characters and modest backpressure when the volume ratio of 1:3 was chosen. www.jss-journal.com
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3.1.3 Selection of the volume ratio of the inorganic alcoholysate and organic part
Table 1. The EF values of IPC in the HMIMs and HNIMs and the respective IF values
In this study, the volume ratios of the inorganic alcoholysate to organic part 1:1, 1:3, 1:5, and 1:6 were investigated, respectively. The results showed that the HMIMs were too soft and easy to shrink when the volume ratios of 1:1 and 1:3 were chosen. The monolithic column was easy to swell, which resulted in a high backpressure in the microextraction process when the volume ratio 1:6 was selected. To avoid the shrinkage and high backpressure of the hybrid polymer, the volume ratio 1:5 was chosen.
Compound
3.2 Characterization and specificity evaluation of the monolith The SEM images of the monoliths were obtained by using an X-650 scanning electron microscope and were used to observe the morphology of the HMIMs. It is readily observed that the monolithic column was uniform and regular, and there were many macropores and flow-through channels inlaid in the network skeleton of the monolithic column, which provided flow paths through the column. Due to the size and density of the macropore network, the monolithic column had a high external porosity and, consequently, a large permeability and low column hydraulic resistance. The pores allowed the mobile phase to flow through with low backpressure. Fourier transform infrared (FTIR) spectra in potassium bromide were recorded by a Spectrum 100 FTIR spectrophotometer. The results showed the IR spectra of EGDMA, MTMS, IPC HMIM, and HNIM. Comparing with the IR spectra of EGDMA, the C=O stretching vibration peak of 1728 cm−1 and the C–H stretching vibration peak of 2960 cm−1 appeared in the spectra of HMIMs. But the C=C stretching vibration peak of 1638 cm−1 became very weak. These results indicated that the cross-linker EGDMA had polymerized. Comparing with the IR spectrum of MTMS, the Si–O–Si stretching vibrations around 1105 cm−1 and Si–O vibrations around 796 cm−1 appeared in the IR spectrum of HMIM. The spectra of HNIM and HMIM showed similar locations and appearances of the major bands. These results showed that the polymers have been successfully synthesized. To evaluate the selectivity of the HMIM, carbary with a similar structure to IPC as the analog and cephalexin as nonanalog were tested. It can be seen from the Table 1 that the HMIM had a higher absorbability for IPC than HNIM, where the IF was 4.41. At the same time, the HMIM possessed higher extraction efficiency for carbary than HNIM. This was due to the similar structure of carbary and IPC. The results also indicated that the retention of cephalexin in HMIM was similar to that in HNIM. These results demonstrated the good selectivity of the synthesized HMIM for IPC. To prepare the HMIM, IPC was dissolved in methanol, and MAA was used as the functional monomer to produce hydrogen-bonding interactions with the template. Then, a stable donor–receptor complex between the template and C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
IPC Carbary Cephalexin
EF
IF
HMIMs
HNIMs
5.62 4.12 1.34
1.29 1.23 1.21
4.41 3.35 1.11
functional monomer was formed in the prepolymerization process. The existence of the complex resulted in the formation of well-defined specific binding sites in the polymers. The silane coupling agent KH570 acted as a bridge that connected the monomer with MTMS at the same time. Subsequently, the HMIM was prepared through the polymerization of EGDMA. After removal of the template molecule, the specific imprinting sites were maintained. The synthesis protocol of the HMIM and the recognition mechanism are indicated in Fig. 1. The flow-through pore size distribution was determined by mercury porosimetry, and the result is shown in Fig. 2. It can be seen that HMIM possessed larger flow-through pores (a narrow peak centered at 4.0 m), which could lead to higher permeability and favorable mass transfer during extraction. The specific surface areas and pore volumes from nitrogen adsorption–desorption experiments were 25.1 m2 /g and 0.062 cm3 /g for HMIM, and 21.8 m2 /g and 0.069 cm3 /g for HNIM, respectively. The similar surface areas and pore volumes of HMIM and HNIM showed that selectivity was due to the imprinted recognition properties of HMIM.
3.3 Optimization and application of the monolith In order to optimize the HMIMME conditions, several extraction parameters were investigated, including the type and volume of eluent, washing solution, and sample volume. Sample solutions were spiked with IPC at 59.5 ng/mL to perform the experiments. 3.3.1 Optimization of extraction conditions The selection of an appropriate eluent solvent is of high importance for the HMIMME process. Considering the solubility of IPC and consistency to the mobile phase used in HPLC, methanol and acetonitrile were selected. Different proportions of methanol with acetonitrile as the eluent were tested. The experimental results showed that the mixture of methanol with acetonitrile (6:4, v/v) as eluent offered the higher recovery. ER and EF of IPC were studied while different eluent volumes of 80–200 L of methanol/acetonitrile (6:4, v/v) were used. The results showed that ER increased and EF decreased while increasing the eluent volume from 80 to 200 L. One hundred and www.jss-journal.com
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Figure 1. Schematic representation of the preparation and extraction procedures of the HMIMs.
fifty microliter methanol/acetonitrile (6:4, v/v) was enough to provide a quantitative elution of the analyte from the sorbent. Water, cyclohexane, chloroform, and ethanol were selected as the washing solution. The experimental results showed that the recoveries were very low with cyclohexane C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
and ethanol as the washing solutions. Water and chloroform were suitable as the washing solution. To remove oil- and water-soluble impurities and get rid of the effect of chloroform on HPLC separation column, 200 L of chloroform and 200 L of water were selected to wash the monolithic column in order. www.jss-journal.com
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Figure 2. Pore size distribution profile of HMIM by mercury intrusion porosimetry.
The effect of sample volume was optimized by loading sample solutions 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, and 4.0 mL at a constant flow rate, respectively. The results showed that EF of IPC increased with the increasing of sample volume from 1.0 to 4.0 mL. However, ER began to decrease when the sample volume increased. To achieve sufficient sensitivity within a short time, 3.0 mL of sample solution was selected in the following test. 3.3.2 Analytical approach The obtained HMIMME was coupled with HPLC and UV detection to establish a method for the determination of IPC in rice samples under the optimized extraction conditions. Good linearity was achieved in the range of 9.0–1000 g/kg for IPC in blank rice samples with correlation coefficient (r2 ) of 0.9983. The LOD (S/N = 3) and LOQ (S/N = 10) were 3.0 and 9.0 g/kg for rice, respectively. The LOD of the analyte in rice was lower than the Chinese safe maximum (0.2 mg/kg). 3.3.3 Sample analysis Three kinds of rice samples (from Hanzhong, northeast China, and Fuxian, respectively) were selected for the application of the developed HMIMME–HPLC method. The results showed that these rice samples were free of IPC residue. The chromatograms of spiked rice samples before and after treatment by HNIM and HMIM are shown in Fig. 3. The results indicated that after pretreatment by HMIMME, the majority of interfering substances in the rice samples were eliminated. Furthermore, the sensitivity of the analyte in the rice was greatly enhanced with the HMIMME–HPLC analysis. The HNIM possessed extraction capability much lower than that of HMIMs for IPC. The results indicated that the specific recognition of the HMIM to IPC existed in the polymer. To validate the established method, blank rice samples were spiked with IPC at three levels of 83.3, 500, and 833 g/kg. The recoveries were 91–107% with the RSD from 3.1 to 8.9%, as shown in Table 2. These results indicated that C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 3. HPLC chromatograms of blank rice sample (1), 50 g/kg spiked rice sample (2), 50 g/kg spiked rice extracted with HNIMME (3), and 50 g/kg spiked rice extracted with HMIMME (4).
the established method was suitable for the extraction and determination of trace amounts of IPC in complex samples. 3.3.4 Comparison of the IPC-imprinted monolith extraction with other methods The efficiency of the presented HMIMME–HPLC–UV method for the determination of IPC in rice was compared with other reported methods such as LLE–GC–MS (LLE is liquid–liquid extraction); matrix solid-phase dispersion (MSPD) with gel permeation chromatography (GPC) and GC coupled with MS; dispersive SPE (DSPE) with GC coupled to MS, liquid-liquid microextraction (LLME)–HPGC–UV; and reversed electrode polarity stacking mode (REPSM) MEKC with photodiode array detection (PDA) from the aspects of the linear range, LOD, and RSD. As listed in Table 3, the LOD of the HMIMME–HPLC for IPC was obviously lower than those of other reported methods, even lower than those of the tandem GC–MS methods. These results revealed that the HMIMME–HPLC is a sensitive, rapid, low-cost, and reproducible technique that can be used for selective extraction and sensitive determination of IPC in complex samples.
4 Concluding remarks In summary, we have described the preparation and application of a novel IPC-imprinted poly(methacrylic acid)/SiO2 hybrid monolithic column by using a mild sol–gel process. The monolithic column was synthesized in a micropipette tip. So, the micromonolithic column could be connected with syringes in different sizes simply without any other treatment to perform microextraction process. The derived HMIMs showed high selectivity and enrichment ability for IPC. After optimization of the extraction conditions, the HMIMME coupled with HPLC–UV was used for the selective extraction and determination of IPC in rice samples successfully. www.jss-journal.com
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Table 2. Linear range, LODs, precision, and recoveries of the HMIMME–HPLC method for the determination of IPC in rice samples
Sample
Rice from Hanzhong Rice from northeast China Rice from Fuxian
Spiked (g/kg)
83.3 500 833 83.3 500 833 83.3 500 833
Recovery (%; n = 5)
95 91 103 97 94 105 92 97 107
RSD (%; n = 5) Intraday
Interday
7.7 3.5 3.1 7.4 4.3 5.7 5.8 4.3 3.7
8.9 4.3 5.2 7.8 5.1 6.0 7.5 5.2 5.4
Linear equation
LOD (g/kg)
LOQ (g/kg)
Y = 3.702 × 10−2 X − 1.279 × 10−2 (r2 = 0.9983)
3.0
9.0
Table 3. Comparison of the HMIMME method with other sample preparation techniques for the determination of IPC in real samples
Extraction method
Analytical method
Linear range (mg/kg)
LLE MSPD DSPE LLME REPSM HMIMs microextraction
GC–MS GPC–GC–MS GC–MS HPLC–UV MEKC–PDA HPLC
0.05–0.5 0.05–0.4 0.04–8.00 (mg/L) 0.010–5.0 0.009–1
RSD (%)
LOD (mg/kg)
Sample
Ref.
0.7 ≤21
