Anal Bioanal Chem DOI 10.1007/s00216-017-0671-5

RESEARCH PAPER

A highly selective electrochemical sensor based on molecularly imprinted polypyrrole-modified gold electrode for the determination of glyphosate in cucumber and tap water Chao Zhang 1 & Yongxin She 1 & Tengfei Li 2 & Fengnian Zhao 1 & Maojun Jin 1 & Yirong Guo 3 & Lufei Zheng 1 & Shanshan Wang 1 & Fen Jin 1 & Hua Shao 1 & Haijin Liu 4 & Jing Wang 1

Received: 13 July 2017 / Revised: 14 September 2017 / Accepted: 21 September 2017 # Springer-Verlag GmbH Germany 2017

Abstract An electrochemical sensor based on molecularly imprinted polypyrrole (MIPPy) was developed for selective and sensitive detection of the herbicide glyphosate (Gly) in cucumber and tap water samples. The sensor was prepared via synthesis of molecularly imprinted polymers on a gold electrode in the presence of Gly as the template molecule and pyrrole as the functional monomer by cyclic voltammetry (CV). The sensor preparation conditions including the ratio of template to functional monomers, number of CV cycles in the electropolymerization process, the method of template removal, incubation time, and pH were optimized. Under the optimal experimental conditions, the DPV peak currents of hexacyanoferrate/hexacyanoferrite changed linearly with Gly concentration in the range from 5 to 800 ng mL−1, with a detection limit of 0.27 ng mL−1 (S/N = 3). The sensor was used to detect the concentration of Gly in cucumber and tap Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00216-017-0671-5) contains supplementary material, which is available to authorized users. * Yongxin She [email protected] * Jing Wang [email protected] 1

Key Laboratory of Agro-product Quality and Food Safety, Ministry of Agriculture, Institute of Quality Standards & Testing Technology for Agro-Products, Chinese Academy of Agricultural Sciences, Beijing 100081, China

2

Department of Food Science, College of Agriculture, Hebei University of Engineering, Handan, Hebei 056021, China

3

College of Agriculture and Biology Technology, Zhejiang University, Zhejiang, Hangzhou 31000, China

4

Tibet Testing Center of Quality and Safety for Agricultural and Animal Husbandry Products, Lhasa, Tibet 850000, China

water samples, with recoveries ranging from 72.70 to 98.96%. The proposed sensor showed excellent selectivity, good stability and reversibility, and could detect the Gly in real samples rapidly and sensitively.

Keywords Electrochemical sensor . Molecularly imprinted polypyrrole . Glyphosate . Cucumber . Tap water

Introduction Glyphosate [Gly; N-(phosphonomethyl) glycine] (Scheme 1A), a non-selective post-emergence herbicide applied in agricultural, industrial, and domestic settings [1], can inhibit enolpyruvyl shikimate phosphate synthase (EPSPS), which is a crucial enzyme in the shikimate pathway [2, 3]. Due to its high efficiency, low toxicity and lability, Gly is widely used in orchards, mulberry fields, tea plantations, rubber and other fields. However, the transfer of Gly to the soil surface can cause the possibility of residue accumulation in water and food. Besides, the constant use of Gly may pose a risk on crop security, the ecological environment, and human health. Although Gly has ever been considered to be a Btoxicologically harmless^ element, the International Agency for Research on Cancer (Lyon, France) recently announced that Gly is likely carcinogenic to humans [4]. Furthermore, accumulation of Gly could have an effect on the central nervous system, causing respiratory, myocardial, and neuromuscular malfunctions, and it could eventually lead to death [5, 6]. Consequently, many governments have proposed limiting the maximum residue levels (MRLs) of Gly in water and agricultural products. The United States

Zhang C. et al.

Scheme 1 A Structure of the neutral form of glyphosate. B Structure of the neutral form of pyrrole

Environmental Protection Agency has stated that the MRL of Gly in drinking water is 700 μg L−1 [7]. Canada established the MRL of Gly in drinking water at 280 μg L−1 [8]. China put the maximum of Gly in water at 700 μg L−1 [9]. The European Union instituted the amount of any pesticide in drinking water at 0.1 μg L−1, regardless of its toxicological impact [10], and established the MRLs of Gly in cucumber, tomatoes, carrot, celery and leek at 100 μg L−1. Therefore, there is an urgent need to identify trace levels of Gly in agricultural commodities and drinking water quickly and accurately. Currently, the main detection methods of Gly include liquid chromatography (LC) [11–13], LC–tandem mass spectrometry (LC-MS/MS) [14, 15], gas chromatography (GC) [16], GC–tandem mass spectrometry [17], capillary electrophoresis [18], ion chromatography [19, 20], enzyme-linked immunoassays (ELISA) [21], fluorescence detection [22] and electrochemical luminescence methods [23]. Because of its high polarity, low solubility in organic solvents, and lack of suitable functional groups, most of the LC and GC methods reported for Gly involve derivatization [24]. Calcium and magnesium ions in samples interfered with the detection results for Gly in ion chromatography readily [25, 26]. To avoid the need for expensive instruments and sample derivatization, electrochemical sensors have been created for on-site, affordable and fast identification of Gly. Such electrochemical sensors have exhibited higher sensitivities than ELISA [27]. However, the reported electrochemical sensors lack sufficient selectivity for Gly in complicated matrices. Therefore, to achieve the trace detection of Gly in agricultural products and drinking water without any cross-reactivity and/or false positives, a simple, low cost, rapid, highly sensitive and highly specific detection method needs to be developed. Molecularly imprinted polymers (MIPs) have specific imprinting sites which can match with the target molecule in shape, size, and functional groups. This allows MIPs to specifically recognize target molecules in preference to other closely related compounds [28]. Various techniques to polymerize MIPs, such as soft lithography, molecular selfassembly with electropolymerization, and chemical grafting,

have been documented [29]. The electropolymerization techniques have some advantages, such as adherence of the polymeric films to the surface of electrodes with all shapes and sizes, and the capability to influence membrane thickness and deposition density by altering deposition conditions. Molecularly imprinted electrochemical sensors can combine the advantages of electrochemical detection and molecular imprinting technology, including dependability, speedy reaction, less expense, ease of operation, efficiency, high sensitivity, and nice selectivity. Currently, MIP-coated electrochemical sensors have been widely used to detect Parathion [30], hexazinone [31], trichlorfon [32], metolcarb [33], and other kinds of analytes [34]. In recent years, some studies based on the use of MIPs with Gly have been reported. Most of these studies investigated the application of MIPs in the extraction and preconcentration of Gly using MIPs as sorbents. Although electrodes modified with MIPs have been effectively applied in the selective and sensitive identification of numerous pesticides [35], there were just three studies relative to the identification of Gly published [36–38]. Each of the studies mentioned above has its advantages and disadvantages. Generally, solidified imprinted sensing films could be electropolymerized using functional monomer and molecular template. And the elution methods of molecular template mainly depend on the feature of functional monomer. Therefore, seeking appropriate functional monomers which can effectively remove the molecular template is one of the strategies to improve the performance of molecularly imprinted electrochemical sensor. Pyrrole (Py; Scheme 1B) is well known to be partially cross linked, and it also is one of the electro-monomers for the development of molecularly imprinting electrochemical sensors, so it is not necessary to use a crosslinker for polymerization of MIP [39]. Polypyrrole (Ppy) can be easily electropolymerized on numerous substrate materials even at a neutral pH, and also possesses nice stability, appropriate redox properties and high conductivity [38]. When a specific potential is applied, the positive charge of the backbone of PPy is taken away and oxygen-containing groups, including

A highly selective electrochemical sensor based on molecularly imprinted polypyrrole-modified gold...

carbonyl and carboxyl groups were formed, which could control the uptake/ejection of template molecules [40, 41]. As shown in these studies, polypyrrole molecularly imprinted polymeric membrane with good stability and porous structure could be obtained via the doping and overoxidation of Py, and the template molecules could be eluted from membrane rapidly and easily. This study described the preparation of a new electrochemical sensor for Gly using an electrosynthesized molecularly imprinted polypyrrole (MIPPy) on an Au electrode. The factors in sensor fabrication that affect the imprinting process and sensing performance were critically studied by comparing the effects of various supporting electrolytes in the electropolymerization process and various Ppy overoxidation methods for template extraction. In addition, the prepared sensor was characterized by scanning electron microscopy (SEM), atomic force microscope (AFM), cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and Fourier transform infrared (FTIR) spectra. The results showed that the Gly molecules could be extracted rapidly, and the sensor showed good stability, reversibility, sensitivity and selectivity for the detection of Gly. The sensor has been successfully used to detect Gly in cucumber and tap water.

Materials and methods Reagents and materials Gly, aminomethylphosphonic acid (AMPA), and pyrrole (Py, 99%) were obtained from Aladdin Industrial Corporation (Shanghai, China). Chlorpyrifos and aldicarb were purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany). Potassium ferricyanide (K3[Fe(CN)6]) and other chemicals used in the experiments were purchased from Beijing Chemical Reagent Co. (Beijing, China) and of at least analytical grade. The buffers used in this study were phosphatebuffered saline (PBS; 0.01 mol/L) and Britton–Robinson buffer solution (BR; 0.04 mol/L). Doubly deionized water (DDW, 18.2 MΩ cm−1) was purified using a Milli-Q Reagent water system plus (Millipore, Billerica, MA). The EIS solution included 0.1 M of KCl, 5 mM of Fe (CN)63−, and 5 mM of Fe(CN)64− redox probes in 40 mM of BR at pH 7.0. The cucumber samples were bought from organic supermarket. The tap water samples were from the community water supply. Instrumentation and operating parameters Electrochemical experiments were done with an electrochemical workstation (CHI630E, Shanghai CH Instruments, China) and a typical three-electrode system that uses a bare or modified Au electrode as the working electrode, a saturated

calomel electrode as the reference electrode, and a platinum wire as the counter electrode. Each electrochemical operation was done at room temperature. SEM images were recorded with a scanning electron microscope (S-4800, Hitachi, Japan). AFM images were obtained with an Atomic Force Microscope (SPM-960, Shimadzu, Japan). Fourier transform infrared (FTIR) spectra were collected using attenuated total reflectance with an FTIR spectrometer (Spectrum 400, Perkin Elmer Instrument, USA). The amount of Gly in samples was confirmed by LC-MS/MS with an Agilent 1200 Series Rapid Resolution LC system (Agilent Technologies, Palo Alto, CA, USA) along with a mass spectrometer (API 5500, Applied Biosystems, AB Sciex, Foster City, CA, USA) that included a turbo ion spray interface. Preparation of electrodes Bare Au electrodes (3 mm in diameter) were polished to a mirror finish with 0.3 μm and 0.05 μm alumina slurry, and then successively cleaned in an ultrasonic bath with water and ethanol. Before surface modification, each bare Au electrode was scanned in the potential range from −0.2 to +0.6 V in 5.0 mmol L−1 K3[Fe(CN)6] solution containing 0.1 M KCl supporting electrolyte until a pair of well-defined redox peaks was observed. T he M I P P y e l ec t r od e w a s co n s t r u c t ed b y t h e electropolymerization of Gly and Py on the surface of an Au electrode using CV in the potential range between −1.0 and +1.0 V at a scan rate of 100 mV/s for five cycles. The supporting electrolyte contained 7.4 mM Gly, 37.0 mM Py, and 0.04 M BR (pH = 5.0). After electropolymerization, the embedded Gly molecules were extracted from the PPy membranes with an overoxidation method that included scanning by CV in the potential range of −1.3 to +1.2 V at a scan rate of 100 mV/s in 0.1 M NaOH solution over 20 cycles. A MIPPy electrode with cavities was obtained. A non-imprinted polypyrrole (NIPPy) electrode was also prepared under the same experimental conditions except that the Gly template was omitted from the pre-polymer solution. Analytical procedures A schematic representation of the experimental procedure to detect Gly using the MIPPy electrode was shown in Fig. 1. To quantitatively identify Gly, the MIPPy electrode was incubated in an aqueous solution of formic acid (pH 4.0) with the required concentration of Gly for 18 min after template removal. The MIPPy electrode was then cleaned with DDW to eliminate any non-specifical adsorbates. Electrochemical determinations were performed by differential pulse voltammetry (DPV) in 5.0 mmol/L of K3[Fe(CN)6] solution with 0.1 mol/L of KCl. DPVs were recorded in the potential range between −0.2 and +0.6 V at a scan rate of 50 mV/s. The value

Zhang C. et al.

Gly

Au

Py

(polymerize)

(recognize)

(elute)

(electrical signal)

Fig. 1 Schematic representation of the experimental procedure to detect glyphosate

of the peak currents in DPVs was acted as the response signal of the sensor. Each measurement was done at room temperature and a calibration curve was prepared via plotting the change of peak current against Gly concentration. Analysis of real samples Before detection, the cucumber samples were extracted by acetic acid-water solution, and water samples were passed through a 0.22-μm membrane filter. No Gly was found in any of the samples by LC-MS/MS. The samples spiked with Gly standard solution were pretreatment by above mentioned methods. And the Gly concentration was determined by DPV using the MIPPy electrode. Recoveries were obtained by comparing the concentrations calculated using the calibration curve with the spiked Gly concentration. Spiked samples were also analyzed by LC-MS/MS [14].

Results and discussion Electropolymerization of mippy films on electrodes MIPPy electrodes were fabricated on cleaned Au electrodes through electropolymerization of Py with Gly as a template by CV for five cycles in BR. The initial scan revealed an irreversible oxidation peak of Py at about 1.0 V. The CVs in Fig. S1 (see Electronic Supplementary Material, ESM) show that in the second scan, the current decreased markedly in the potential range between − 0.3 and 1.0 V. Then, the peak current decreased slowly with additional cycling, finally approaching zero after five cycles. This phenomenon indicated the formation of PPy films on the electrode surface, which would hamper the further access of electroactive Py to the electrode surface. Hence, if the template was not electroactive, molecular diffusion towards the electrode was disrupted after the initial scan by the formation of a non-conductive polymeric layer, which prevented the template forming part of the layer and thereby decreases the peak current [42, 43].

The template diffused towards the electrode surface and was entrapped in the polymer matrix during polymerization of Py, which was necessary to obtain a voltammetric sensor based on a MIP. In this work, we designed a strategy involving template Gly entrapped within MIPPy and then removed from MIPPy with a pH-controlled solution. Figure 2 depicts a schematic representation of Gly imprinting and removal of Gly in the MIPPy electrode. The Gly might present to be anion at pH 5.0 [44]. The positively charged PPy membrane was obtained during the oxidation of Py, and the positively charged membrane could dope the anionic Gly with the electrostatic interactions. The oxygencontaining groups were introduced after the overoxidation of PPy membran at pH 11.0 of in the 0.1 M NaOH solution. The introduction of oxygen-containing groups in the PPy backbone repelled template Gly during the overoxidation, and cavities complementary to the molecular shape of a dopant anion were formed [45, 46]. Characterization of the surface morphology SEM was used to investigate the surface morphology of the polymeric films formed in the absence and presence of template Gly. As shown in Fig. 3, the MIP films before template removal (a) were compact and smooth. After template removal (b, c), the MIP films had a different morphology. To further character the surface morphologies of different films clearly, the surface morphologies were examined via AFM. Three dimensional (3D) images, surface-height, arithmetic mean roughness (Ra) and root mean square roughness (Rq) of bare electrode (a), NIP film (b), MIP film (c) and MIP film after removal of template (d, e) were shown in Fig. S2 (see ESM). The surface-height, Ra and Rq values for MIP film were lower than that for NIP film. This maybe resulted from the embedding of Gly, which made the membrane more dense and smooth. The surface-height, Ra and Rq values for the MIP film after removal of template were far higher than that for MIP film, which indicated that the film possesses larger surface area and rougher surface. In addition, the surface morphology of MIP film after removal of template in Fig. S2e (see ESM) was similar to that of SEM. Characterization of the imprinted sensor using FTIR spectroscopy To illustrate the imprinting and release of Gly in the polymeric electrodes, the molecular structures of Gly, MIPPy with bound Gly, MIPPy after removal of Gly, and MIPPy and NIPPy after reabsorption of Gly were characterized by FTIR spectroscopy. Fig. S3a (see ESM) shows the FTIR spectrum of Gly in the solid state. The bands at 1150, 1220, and 1267 cm−1; 1201 and 1421 cm−1; 1481 and 1556 cm−1; 979, 1241, and 1334 cm−1; and 1029 cm−1 correspond to

A highly selective electrochemical sensor based on molecularly imprinted polypyrrole-modified gold... Fig. 2 Schematic representation of imprinting and removal of Gly in the MIPPy electrode

the phosphate, carboxylic, amine, and CH2 groups, and the skeletal vibration of CCNC, respectively [47]. The

Fig. 3 SEM images of (a) MIPPy electrode surface (10 μm), (b) MIPPy electrode after template removal (5 μm), and (c) MIPPy electrode after template removal (2 μm)

spectrum of the NIPPy electrode (Fig. S3b) contains a band at 1698 cm−1 that is assigned to the typical C = C stretching of the PPy ring. The bands around 1285 cm−1 may correspond to the C–N stretching vibration of the ring and C–H bond in-plane deformation of PPy units. The broad peak at 3065 cm−1 corresponds to N–H stretching of PPy, while that at 980 cm−1 is assigned to the C–H out-of-plane vibration, indicating polymerization of Ppy [48]. Comparing the FTIR spectra of NIPPy and MIPPy films before overoxidation, it is meaningful to observe the shift of the peaks at 3242 and 1701 cm−1 in the MIPPy spectrum compared to those of NIPPy; the peaks correlate to the N-H and the C = C of the Py ring, and their shift may be due to the interaction between PPy and Gly. Additional proof of Gly entrapment inside imprinted cavities is the presence of characteristic peaks of the Gly template in the region between 2283 and 3242 cm−1, which appears only in the MIPPy spectrum. These peaks cannot be attributed to any vibrational mode of the polymer (as shown by the spectrum of NIPPy) and are not present in the spectrum of MIPPy following overoxidation. Moreover, after overoxidation, the spectrum of the MIPPy films including the expected modifications, and no peak shift is observed in the MIPPy spectrum in this case, confirming removal of template Gly. There are some admittedly slight differences in the FTIR spectra of NIPPy and MIPPy, but the spectra are very similar when the template removal of MIPPy (ESM Fig. S3b and S3c). Upon reabsorption of Gly by the MIPPy which was removed of the template molecule, two bands at 2283 and 998 cm−1, representing the stretching vibration mode of N-P and N-C groups, respectively, were observed. In the presence of Gly, the intensity of the peak at 2970 cm−1 increased in the spectrum of the MIPPy film, and the position of the band at 1639.22 cm−1, which is assigned to the C = C stretching vibration of the PPy backbone, shifted. The above changes of the FTIR spectrum of the MIPPy film were attributed to the interaction of PPy with Gly. Thus, FTIR spectroscopy confirmed that a

Zhang C. et al.

MIPPy film with the ability to recognize Gly was successfully constructed. Electrochemical characterization of different imprinted sensors To confirm that Gly molecules were embedded in the imprinted PPy membranes, CV and EIS measurements of different imprinted electrodes were conducted in an electrolyte containing 5 mmol/L K3Fe(CN)6 and 0.5 mol/L KCl. As shown in Fig. 4A, the current response of K3Fe(CN)6 at a bare Au electrode (curve a) was larger than that at the MIPPy electrode (curve d). The peak current of the MIPPy electrode decreased to nearly zero, which showed that a non-conductive polymer film was present on the electrode surface, and the film hindered electron transfer between the K3Fe(CN)6 redox

probe and electrode. After removal of the Gly template, the peak current of the MIPPy sensor increased (curve b), which suggested that cavities were formed in the MIPPy membrane after removal of the template Gly. Following reabsorption of Gly by the imprinted electrode, the peak current of the sensor decreased (curve c). These results showed that Gly could not only readily leave the cavities of the MIPPy electrode, but also be reabsorbed in these cavities. EIS is implemented to examine the electron transfer kinetics of the various electrodes; every EIS plot has two areas: a semicircle and a linear region. The semicircle is related to the electron transfer resistance (Ret), which is connected with the dielectric and insulating features at the electrode/electrolyte connection. The linear regions corresponds to a diffusion process. Figure 4B (a, b, c, and d) shows the EIS data obtained for a bare Au electrode, MIPPy electrode after template removal, NIPPy electrode, and MIPPy electrode with bound Gly, respectively. The bare electrode displayed a larger response and smaller radius than the other electrodes, indicating that the bare electrode possessed the smallest Ret. This was because there was no conductive polymer membrane influencing Ret of the Au electrode. The MIPPy and NIPPy electrodes were covered with the passivated polymer membrane, so their semicircle radii and Ret were larger than those of the bare electrode. The MIPPy electrode produced a semicircle with a larger radius than that of the NIPPy electrode because Gly was not electroactive and thus hindered electron transfer. The MIPPy electrode contained numerous cavities after the removal of template Gly, which could provide channels for electron transfer. Therefore, Ret of the MIPPy electrode after template removal was smaller than that of the MIPPy electrode with bound Gly.

Adsorption characteristics of the mippy electrodes To understand the adsorption characteristics of the MIPPy electrodes, the adsorption isotherms of MIPPy and NIPPy electrodes in the presence of Gly were obtained (Fig. 5). The isotherms are typical of the adsorption of imprinted polymers. Equation (1) was implemented to investigate the Langmuir adsorption kinetic process of the electrodes [49]: ΔI p ¼ ΔI pm :

Fig. 4 (A) Cyclic voltammograms of differently modified electrodes in the K3[Fe(CN)6] standard solution. (a) the bare gold electrode, (b) MIPPymodified gold electrode after elution, (c) MIPPy electrode with bound Gly and (d) MIPPy-modified gold electrode. (B) Electrochemical impedance spectroscopy of differently modified electrodes in the K3[Fe(CN)6] standard solution. (a) bare gold electrode, (b) MIP-modified gold electrode after elution, (c) NIP-modified gold electrode, (d) MIP-modified gold electrode. Inset graph of B is the enlargement of a and b

c kþc

ð1Þ

Because adsorbing a certain quantity of Gly would change the current of the MIP sensor, here we define that ΔIp (μA) is proportional to the amount of Gly that adsorbed on the MIP films, which is used to evaluate the properties of a MIP sensor; c (ng mL−1) is the equilibrium solution concentration of Gly; ΔIpm(μA) is the current change response of saturation capacity; and k (ng mL−1) is the dissociative constant. Figure 5

A highly selective electrochemical sensor based on molecularly imprinted polypyrrole-modified gold...

Ratio of template molecules to functional monomers

Fig. 5 Adsorption isotherms of (a) MIPPy and (b) NIPPy electrodes in the presence of glyphosate

shows ΔIpm of the redox probe after the MIP sensor had adsorbed a certain concentration of Gly. As shown in Fig. 5, with increasing of Gly concentration, the content of Gly absorbed by the MIPPy electrodes increased obviously and then reached a saturation value. In contrast, the response of the NIPPy electrode did not change obviously with Gly concentration. In addition, k of the MIPPy sensor was larger than that of the NIPPy as summarized in Table 1. These results showed that the MIPPy films contained selective binding sites for Gly, and the obtained MIPPy sensor was effectively imprinted with the template molecules.

Electropolymerization cycles The number of electropolymerization cycles applied to prepare the MIPPy electrode affected the sensor’s sensitivity and linearity. Electrodes were prepared with different numbers of CV cycles to identify the favorable number of CV cycles in the process of electropolymerization. A thin PPy membrane of few imprinted sites would be prepared with too few CV cycles. Although a thicker sensing film would be prepared with excessive number of cycles, it was difficult to remove templates [30]. In this work, the highest current difference for Gly at the MIPPy electrode was obtained using five electropolymerization cycles.

Table 1 Kinetic parameters of the recognition process of Gly for the different electrodes Curve

ΔIpm(μA)

R2

k(s)

A(MIP) B(NIP)

32.00 1.34

0.98 0.23

88 30

The ratio of template molecules to functional monomers in the polymerization process affected the affinity and recognition performance of the resulting imprinted membrane. Addition of an appropriate template concentration would produce a sensor with optimum sensing properties and ensure a high binding capacity for Gly. In our investigations, a series of MIPPy sensors with template-to-monomer ratios of 1:3, 1:5, and 1:8 were respectively fabricated. And then they were used to determine the same concentration of Gly. Figure 6A illustrates the current responses of the sensors produced with different ratios of template to monomer. The largest change of current response was obtained for the MIPPy sensor fabricated with a template/ monomer ratio of 1:5. When the ratio was lower (1:3) or higher (1:8) than 1:5, the response of the sensors to Gly were smaller than that of the sensor with the ratio of 1:5. These findings showed that the proposed MIPPy sensor possessed more effective recognition sites for Gly when the optimal molar ratio of template to monomer molecules was used. Effect of pH on the absorption capacity of the MIPPy electrode The strongest current response was observed when the MIPPy electrode was immersed in the buffer containing 0.2% formic acid. Thus, formic acid was chosen as the optimum absorption system for the detection of Gly by the MIPPy sensor. The pH of the detection system could affect the ionic state of Gly, which influences the absorption of Gly by MIPPy. In this work, the effect of solution pH (from 3.0 to 7.0) on the absorption of Gly by the MIPPy electrode was studied by the current response in DPV. As shown in Fig. 6B, the current response of the MIPPy sensor to Gly increased from pH 3.0 to 4.0, and then decreased from pH 4.0 to 7.0. Thus, pH 4.0 was chosen as the optimum pH in the Gly detection process by the MIPPy sensor. This pH could facilitate the interaction between Gly and the imprinted film. Incubation time The incubation time is an important factor affecting the sensitivity of molecular imprinted sensors. Therefore, after removal of the template molecules from the MIPPy electrode, the sensor was incubated in formic acid (pH 4.0) containing Gly (50 ng mL−1). The peak current change over time was recorded using DPV. Figure 6C displays the response of the sensor over time. The peak current increased rapidly from 10 to 18 min, indicating the rapid, effective recognition ability of the MIPPy electrode for the target Gly molecules. When the incubation time reached 18 min, the peak current leveled off gradually. Therefore, the optimum adsorption time for the MIPPy electrode was 18 min.

Zhang C. et al.

template was not able to be fully extracted. To take out the template within PPy, we used an overoxidation process, which involved scanning the potential range between −1.3 and +1.2 V for 20 cycles in 0.1 mol/L of NaOH solution. After this process, all Gly molecules were extracted from the MIPPy film. As shown in Fig. S4 (see ESM), the oxidation potential of Py was from 0.8 to 1.0 V. With increasing number of scan cycles, the absolute current at −0.3 and 0.8 V increased gradually, indicating that PPy had been peroxidated. Because of the interaction between the affinity reagent OH− and the active sites of PPy, oxygen-containing moieties such as carboxylate and carbonyl groups were introduced onto the Py unit. These oxygen-containing groups repelled anions in the film [50], causing the release of the Gly template molecules, and formation of electron transfer channels in the polymer film.

Fig. 6 Effect of template-monomer molar ratio (A), pH of the solvent (B), and incubation time (C) on responses of the electrochemical MIPPy sensors

Template removal conditions To realize specific absorption, good sensitivity, and reproducibility, the template molecules should be removed as thoroughly as possible. The universal protocol for template removal was to employ organic reagents or buffer solution as an eluent. However, this method took a long time and the

Fig. 7 (A) Differential pulse voltammetry of a MIPPy-modified gold electrode in electrolytes containing 5.0 mmol/L K3[Fe(CN)6], 0.1 mol/L KCl, and Gly concentrations of (a) 5, (b) 10, (c) 50, (d) 100, (e) 200, (f) 300, (g) 500, and (h) 800 ng/mL. (B) The calibration curve

A highly selective electrochemical sensor based on molecularly imprinted polypyrrole-modified gold... Table 2

Comparison of the performance of glyphosate sensors

Method

Linear range (ng/mL)

LOD (ng/mL)

References

Molecularly imprinted polypyrrole (MIPPy)-electrochemical sensor

5–800

0.27

Our work

Double-template imprinted polymer nanofilm-modified pencil graphite electrode

3.98–176.23 10−6–1 400–1200

0.35 0.8 × 10−6 92

36 37 38

1.69–845 500–35,000

1.69 46

51 52

0–10,816

98.02

8

MIP-MOF electrochemical sensor MIP/PB/Au/TiO2 electrochemical sensor Porous copper nanowires electrode Chemiluminescence-molecular imprinting sensor Surface plasmon resonance biosensor

Calibration curve and detection limit

Selectivity of the imprinted sensor

The analytical performance of the MIPPy sensor was investigated by measuring its response when incubated with various concentrations of Gly under the optimized conditions. The peak current of the MIPPy electrode decreased with the increase of Gly concentration. There was a linear relationship between Gly concentration and the response of the MIPPy electrode from 5 to 800 ng mL−1, as shown in Fig. 7. The linear regression equation for this relationship was I = − 12.85537lgC + 75.9205 (R2 = 0.99816). The limit of detection (LOD) for Gly by the MIPPy electrode was calculated using the equation LOD = 3 Sb/m, where Sb is the standard deviation of the blank reaction and m is the slope of the calibration plot. The calculated LOD was 0.27 ng mL−1 of Gly, which suggested the sensor was quite sensitive. The properties of the MIPPy sensor were compared with those of other published Gly sensors and the results were shown in Table 2 [8, 36–38, 51, 52]. The satisfactory LOD for the present MIPPy sensor compared with those of reported ones showed that the performance of the MIPPy sensor was reasonable.

Selective recognition of the template is an important feature of sensors based on MIPs [27]. To evaluate the selectivity of the MIPPy electrochemical sensor, AMPA, which is a metabolite of Gly, chlorpyrifos, and aldicarb were selected as interfering substances. Figure 8 shows the peak current changes of MIPPy and NIPPy electrodes immersed in solutions containing 100 ng mL−1 of Gly, AMPA, chlorpyrifos, and aldicarb respectively for 18 min by DPV measurements, which were carried out as previously described. The MIPPy sensor displayed a stronger affinity for Gly compared with the other substances at the same concentration, suggesting that the sensor may selectively identify Gly in the company of additional compounds. Further, the imprinting effect was examined by contrasting the selectivities of the MIPPy and NIPPy electrodes. The NIPPy electrode exhibited a smaller response to Gly, which was almost the same as that to other compounds, compared with that of the MIPPy electrode. Consequently, the high selectivity of the MIPPy sensor was attributed to the cavities in MIPPy that enhanced the shape and functional groups of Gly created within the polymerization process.

Reproducibility, stability and repeatability of the imprinted sensor

Fig. 8 Current change responses of glyphosate, AMPA, chlorpyrifos, aldicarb for MIPPy and NIPPy-modified gold electrodes. Pesticide concentration 100 ng mL−1

To investigate the reproducibility of MIPPy electrode formation, five molecularly imprinted electrodes were prepared under the same experimental conditions described above. The peak current values of each sensor after incubation in 50 ng mL−1 Gly solution were tested for five times. The template was removed from the sensor with 0.1 mol L−1 NaOH solution after each measurement. The relative standard deviation was 3.1% for five successive assays, which revealed that the imprinted sensor had good reproducibility and regeneration ability. The stability of the proposed sensor was also examined by measuring its DPV response to a standard solution of Gly (50 ng mL−1). The sensor response was decreased to 80.89% of the initial response after 3 weeks of being stored in DDW at

Zhang C. et al. Table 3 Determination results for glyphosate in real samples (n = 3)

Samples

Added (ng/mL)

Found (ng/mL)

Recovery (%, n = 3)

RSD (%, n = 3)

Tap water

10.00 20.00

9.88 15.72

98.80 78.60

4.48 3.83

50.00

49.48

98.96

2.80

10.00 20.00

7.27 16.05

72.70 80.25

2.52 1.07

50.00

42.58

85.15

2.44

Cucumber

room temperature. This confirmed that the sensor had good long-term stability. Determination of Gly in real samples To evaluate the suitability of the developed sensor for practical applications, the sensor was used to detect Gly in cucumber and tap water. In this case, the initial detection by LC-MS/MS showed that there was no Gly in the original samples. Both cucumber and tap water samples were spiked with three different concentrations of Gly standard solution (10, 20 and 50 ng mL−1) and then tested by DPV measurements using the imprinted sensor under the optimized conditions. To prove the accuracy of the developed electrochemical imprinted sensor for determination of Gly in real samples, the concentration of Gly spiked in samples was also measured by LC-MS/MS according to a method based on that of Guo et al. [53]. The analytical results were presented in Table 3. The recoveries of Gly from the real samples varied from 72.7 to 98.96% for the MIPPy electrode, and the RSD% were within the range of 1.07–4.48% for Gly in real samples. The three concentrations of Gly in real samples detected by LC-MS/MS were approximately the same as those detected by the developed electrochemical imprinted sensor. There was no substantial difference among the results acquired with the MIPPy sensor and LC-MS/MS technique based on t test evaluation with Microsoft Excel software (p < 0.05). These results demonstrated that the prepared electrochemical imprinted sensor possesses high reliability and high accuracy for the detection of Gly in real samples.

Conclusion In this study, a simple and effective electrochemical molecular imprinted sensor for Gly was prepared with molecularly imprinted overoxidized PPy. A detailed evaluation of the parameters that impact the imprinting effect of PPy, and performance of senor was carried out focusing on the electrolyte pH adopted to MIP electropolymerization and the overoxidation conditions used for template removal from MIPPy. The MIPPy–template interaction and template entrapment in imprinted cavities were confirmed by FTIR evaluation.

Under the optimized conditions, the sensor not only showed good sensitivity, selectivity, and recognition ability for Gly, but also displayed superb stability. The senor could be prepared rapidly because the template could be removed via the electrochemical overoxidation of Ppy. The sensor presented a broader linear range and lower LOD for Gly than others reported previously and possessed rapid binding kinetics to Gly. The sensor has been successfully used to detect Gly in cucumber and tap water, indicating that it will be suitable for practical applications. Acknowledgments This work was supported by the National Natural Science Foundation of China (Contact No. 31471654), the National Key Technology R&D Program for the 12th five-year plan (2014BAD13B0505), the Agricultural Public Welfare Project and Innovative Research Team in Chinese academy of agricultural sciences (201203094), and the Key Laboratory of Agrifood Safety and Quality, Ministry of Agriculture of China (2016-KF-10). Compliance with ethical standards Conflict of interest The authors declare that they have no conflict of interest.

References 1.

Duke SO, Powles SB. Glyphosate: a once-in-a-century herbicide. Pest Manag Sci. 2008;64(4):319–25. https://doi.org/10.1002/ps. 1518. 2. Fauvelle V, Nhu-Trang TT, Feret T, Madarassou K, Randon J, Mazzella N. Evaluation of titanium dioxide as a binding phase for the passive sampling of glyphosate and aminomethyl phosphonic acid in an aquatic environment. Anal Chem. 2015;87(12):6004–9. https://doi.org/10.1021/acs.analchem.5b00194. 3. Singh B, Singh K. Microbial degradation of herbicides. Crit Rev Microbiol. 2016;42(2):245–61. https://doi.org/10.3109/1040841x. 2014.929564. 4. Guyton KZ, Loomis D, Grosse Y, El Ghissassi F, Benbrahim-Tallaa L, Guha N, et al. Carcinogenicity of tetrachlorvinphos, parathion, malathion, diazinon, and glyphosate. Lancet Oncol. 2015;16(5): 490–1. https://doi.org/10.1016/s1470-2045(15)70134-8. 5. Gress S, Lemoine S, Seralini GE, Puddu PE. Glyphosate-based herbicides potently affect cardiovascular system in mammals: review of the literature. Cardiovasc Toxicol. 2015;15(2):117–26. https://doi.org/10.1007/s12012-014-9282-y. 6. Kier LD. Review of genotoxicity biomonitoring studies of glyphosate-based formulations. Crit Rev Toxicol. 2015;45(3): 209–18. https://doi.org/10.3109/10408444.2015.1010194.

A highly selective electrochemical sensor based on molecularly imprinted polypyrrole-modified gold... 7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

Wan YQ, Ma YQ, Mao XJ. Simultaneous determination of organotin compounds in white wine by gas chromatography-mass spectrometry. Anal Lett. 2012;45(13):1799–809. Ding X, Yang KL. Development of an oligopeptide functionalized surface plasmon resonance biosensor for online detection of glyphosate. Anal Chem. 2013;85(12):5727–33. https://doi.org/10.1021/ ac400273g. Smitiene V, Semasko I, Vickackaite V. Speciation of methyltins by dispersive liquid-liquid microextraction and gas chromatography with mass spectrometry. J Sep Sci. 2014;37(15):1989–95. https:// doi.org/10.1002/jssc.201400074. Richter J, Fettig I, Philipp R, Jakubowski N. Tributyltin—critical pollutant in whole water samples—development of traceable measurement methods for monitoring under the European Water Framework Directive (WFD) 2000/60/EC. Environ Sci Pollut Res. 2015;22(13):9589–94. https://doi.org/10.1007/s11356-0154614-4. Druart C, Delhomme O, de Vaufleury A, Ntcho E, Millet M. Optimization of extraction procedure and chromatographic separation of glyphosate, glufosinate and aminomethylphosphonic acid in soil. Anal Bioanal Chem. 2011;399(4):1725–32. https://doi.org/10. 1007/s00216-010-4468-z. Raina-Fulton R. A review of methods for the analysis of orphan and difficult pesticides: glyphosate, glufosinate, quaternary ammonium and phenoxy acid herbicides, and dithiocarbamate and phthalimide fungicides. J AOAC Int. 2014;97(4):965–77. https://doi.org/10. 5740/jaoacint.SGERaina-Fulton. Arkan T, Molnar-Perl I. The role of derivatization techniques in the analysis of glyphosate and aminomethyl-phosphonic acid by chromatography. Microchem J. 2015;121:99–106. https://doi.org/10. 1016/j.microc.2015.02.007. Steinborn A, Alder L, Michalski B, Zomer P, Bendig P, Martinez SA, et al. Determination of glyphosate levels in breast milk samples from Germany by LC-MS/MS and GC-MS/MS. J Agric Food Chem. 2016;64(6):1414–21. https://doi.org/10.1021/acs.jafc. 5b05852. Christoph Gehring MR. Determination of inter-leaf translocated free glyphosate in Arabidopsis thaliana using liquid chromatography tandem mass spectrometry (LCMS/MS) after derivatization with fluorenylmethyloxycarbonyl chloride (FMOC-Cl). Anal Bioanal Chem. 2014;s2(01). doi:https://doi.org/10.4172/21559872.s2-007. Catrinck TCPG, Aguiar MCS, Dias A, Silvério FO, Fidêncio PH, de Pinho GP. Study of the reaction derivatization glyphosate and aminomethylphosphonic acid (AMPA) with N, O-bis(trimethylsilyl)trifluoroacetamide. Am J Anal Chem. 2013;04(11):647–52. https://doi.org/10.4236/ajac.2013.411077. Kudzin ZH, Gralak DK, Drabowicz J, Łuczak J. Novel approach for the simultaneous analysis of glyphosate and its metabolites. J Chromatogr A. 2002;947(1):129–41. Lanaro R, Costa JL, Cazenave SO, Zanolli-Filho LA, Tavares MF, Chasin AA. Determination of herbicides paraquat, glyphosate, and aminomethylphosphonic acid in marijuana samples by capillary electrophoresis. J Forensic Sci. 2015;60(Suppl 1):S241–7. https:// doi.org/10.1111/1556-4029.12628. Dimitrakopoulos IK, Thomaidis NS, Megoulas NC, Koupparis MA. Effect of suppressor current intensity on the determination of glyphosate and aminomethylphosphonic acid by suppressed conductivity ion chromatography. J Chromatogr A. 2010;1217(22): 3619–27. https://doi.org/10.1016/j.chroma.2010.03.048. Popp M, Hann S, Mentler A, Fuerhacker M, Stingeder G, Koellensperger G. Determination of glyphosate and AMPA in surface and waste water using high-performance ion chromatography coupled to inductively coupled plasma dynamic reaction cell mass spectrometry (HPIC-ICP-DRC-MS). Anal Bioanal Chem. 2008;391(2):695–9. https://doi.org/10.1007/s00216-008-2037-5.

21.

Selvi AA, Sreenivasa MA, Manonmani HK. Enzyme-linked immunoassay for the detection of glyphosate in food samples using avian antibodies. Food Agric Immunol. 2011;22(3):217–28. https://doi. org/10.1080/09540105.2011.553799. 22. Wang D, Lin B, Cao Y, Guo M, Yu Y. A highly selective and sensitive fluorescence detection method of glyphosate based on an immune reaction strategy of carbon dot labeled antibody and antigen magnetic beads. J Agric Food Chem. 2016;64(30):6042– 50. https://doi.org/10.1021/acs.jafc.6b01088. 23. Cai QH, Chen XP, Qiu B, Lin ZY. Electrochemiluminescent detection method for glyphosate in soybean on carbon fiber-ionic liquid paste electrode. Chin J Chem. 2011;29(3):581–6. 24. Khrolenko MV, Wieczorek PP. Determination of glyphosate and its metabolite aminomethylphosphonic acid in fruit juices using supported-liquid membrane preconcentration method with highperformance liquid chromatography and UV detection after derivatization with p-toluenesulphonyl chloride. J Chromatogr A. 2005;1093(1–2):111–7. https://doi.org/10.1016/j.chroma.2005.07. 062. 25. da Mata K, Corazza MZ, de Oliveira FM, de Toffoli AL, Teixeira Tarley CR, Moreira AB. Synthesis and characterization of crosslinked molecularly imprinted polyacrylamide for the extraction/ preconcentration of glyphosate and aminomethylphosphonic acid from water samples. React Funct Polym. 2014;83:76–83. https:// doi.org/10.1016/j.reactfunctpolym.2014.07.004. 26. Khenifi A, Derriche Z, Forano C, Prevot V, Mousty C, Scavetta E, et al. Glyphosate and glufosinate detection at electrogenerated NiAl-LDH thin films. Anal Chim Acta. 2009;654(2):97–102. https://doi.org/10.1016/j.aca.2009.09.023. 27. She YX, Cao WQ, Shi XM, Lv XL, Liu JJ, Wang RY, et al. Classspecific molecularly imprinted polymers for the selective extraction and determination of sulfonylurea herbicides in maize samples by high-performance liquid chromatography-tandem mass spectrometry. J Chromatogr B. 2010;878(23):2047–53. 28. Han Y, Yang C, Zhou Y, Han D, Yan H. Ionic liquid-hybrid molecularly imprinted material-filter solid-phase extraction coupled with H PL C f o r d e t e r m i n a t i o n o f 6- b e n z y l a d e ni n e a n d 4 chlorophenoxyacetic acid in bean sprouts. J Agric Food Chem. 2017;65(8):1750–7. https://doi.org/10.1021/acs.jafc.6b03922. 29. Weiss R, Freudenschuss M, Krska R, Mizaikoff B. Improving methods of analysis for mycotoxins: molecularly imprinted polymers for deoxynivalenol and zearalenone. Food Addit Contam. 2003;20(4):386–95. https://doi.org/10.1080/ 0265203031000065827. 30. Yang Q, Sun Q, Zhou T, Shi G, Jin L. Determination of parathion in vegetables by electrochemical sensor based on molecularly imprinted polyethyleneimine/silica gel films. J Agric Food Chem. 2009;57(15):6558–63. https://doi.org/10.1021/jf901286e. 31. Toro MJU, Marestoni LD, Sotomayor MDPT. A new biomimetic sensor based on molecularly imprinted polymers for highly sensitive and selective determination of hexazinone herbicide. Sensor Actuators B Chem. 2015;208:299–306. https://doi.org/10.1016/j. snb.2014.11.036. 32. Gao W, Wan F, Ni W, Wang S, Zhang M, Yu J. Electrochemical sensor for detection of trichlorfon based on molecularly imprinted sol–gel films modified glassy carbon electrode. J Inorg Organomet Polym. 2011;22(1):37–41. https://doi.org/10.1007/s10904-0119593-4. 33. Pan MF, Fang GZ, Liu B, Qian K, Wang S. Novel amperometric sensor using metolcarb-imprinted film as the recognition element on a gold electrode and its application. Anal Chim Acta. 2011;690(2):175–81. https://doi.org/10.1016/j.aca.2011.02.034. 34. Li J, Zhang Z, Xu S, Chen L, Zhou N, Xiong H, et al. Label-free colorimetric detection of trace cholesterol based on molecularly imprinted photonic hydrogels. J Mater Chem. 2011;21(48):19267. https://doi.org/10.1039/c1jm14230e.

Zhang C. et al. 35.

Yan X, Deng J, Xu J, Li H, Wang L, Chen D, et al. A novel electrochemical sensor for isocarbophos based on a glassy carbon electrode modified with electropolymerized molecularly imprinted terpolymer. Sensors Actuator B Chem. 2012;171-172(8):1087–94. 36. Prasad BB, Jauhari D, Tiwari MP. Doubly imprinted polymer nanofilm-modified electrochemical sensor for ultra-trace simultaneous analysis of glyphosate and glufosinate. Biosens Bioelectron. 2014;59:81–8. https://doi.org/10.1016/j.bios.2014.03.019. 37. Do MH, Florea A, Farre C, Bonhomme A, Bessueille F, Vocanson F, et al. Molecularly imprinted polymer-based electrochemical sensor for the sensitive detection of glyphosate herbicide. Int J Environ An Ch. 2015;95(15):1489–501. https://doi.org/10.1080/03067319. 2015.1114109. 38. Xu J, Zhang Y, Wu K, Zhang L, Ge S, Yu J. A molecularly imprinted polypyrrole for ultrasensitive voltammetric determination of glyphosate. Microchim Acta. 2017; https://doi.org/10. 1007/s00604-017-2200-9. 39. Chen L, Wang X, Lu W, Wu X, Li J. Molecular imprinting: perspectives and applications. Chem Soc Rev. 2016;45(8):2137–211. https://doi.org/10.1039/c6cs00061d. 40. Lin M, Hu X, Ma Z, Chen L. Functionalized polypyrrole nanotube arrays as electrochemical biosensor for the determination of copper ions. Anal Chim Acta. 2012;746:63–9. https://doi.org/10.1016/j. aca.2012.08.017. 41. Şahin M, Şahin Y, Özcan A. Ion chromatography-potentiometric detection of inorganic anions and cations using polypyrrole and overoxidized polypyrrole electrode. Sensors Actuators B Chem. 2008;133(1):5–14. https://doi.org/10.1016/j.snb.2008.01.004. 42. Nezhadali A, Rouki Z, Nezhadali M. Electrochemical preparation of a molecularly imprinted polypyrrole modified pencil graphite electrode for the determination of phenothiazine in model and real biological samples. Talanta. 2015;144:456–65. 43. Vasilescu A, Marty JL. Electrochemical aptasensors for the assessment of food quality and safety. Trac Trend Anal Chem. 2016;79: 60–70. 44. Koskinen WC, Marek LEJ, Hall KE. Analysis of glyphosate and aminomethylphosphonic acid in water, plant materials and soil. Pest Manag Sci. 2016;72(3):423–32. https://doi.org/10.1002/ps.4172.

45.

Gomez-Caballero A, Diaz-Diaz G, Bengoetxea O, Quintela A, Unceta N, Goicolea MA, et al. Water compatible stir-bar devices imprinted with underivatised glyphosate for selective sample cleanup. J Chromatogr A. 2016;1451:23–32. 46. Shiigi H, Yakabe H, Kishimoto M, Kijima D, Zhang Y, Sree U, et al. Molecularly imprinted overoxidized polypyrrole colloids: promising materials for molecular recognition. Microchim Acta. 2003;143(2–3):155–62. 47. Santana HD, Toni LRM, Benetoli LODB, Zaia CTBV, Rosa M, Zaia DAM. Effect in glyphosate adsorption on clays and soils heated and characterization by FT–IR spectroscopy. Geoderma. 2006;136(3–4):738–50. 48. Tan F, Cong L, Li X, Zhao Q, Zhao H, Quan X, et al. An electrochemical sensor based on molecularly imprinted polypyrrole/ graphene quantum dots composite for detection of bisphenol A in water samples. Sensors Actuators B Chem. 2016;233:599–606. 49. Chen YP, Liu B, Lian HT, Sun XY. Preparation and application of urea electrochemical sensor based on chitosan molecularly imprinted films. Electroanalysis. 2011;23(6):1454–61. 50. Özcan L, Şahin Y. Determination of paracetamol based on electropolymerized-molecularly imprinted polypyrrole modified pencil graphite electrode. Sensors Actuators B Chem. 2007;127(2):362–9. 51. Poorahong S, Thammakhet C, Thavarungkul P, Kanatharana P. One-step preparation of porous copper nanowires electrode for highly sensitive and stable amperometric detection of glyphosate. Chem Pap. 2015;69(3). doi:https://doi.org/10.1515/chempap-20150038. 52. Zhao P, Yan M, Zhang C, Peng R, Ma D, Yu J. Determination of glyphosate in foodstuff by one novel chemiluminescencemolecular imprinting sensor. Spectrochim Acta A Mol Biomol Spectrosc. 2011;78(5):1482–6. https://doi.org/10.1016/j.saa.2011. 01.037. 53. Guo H, Riter LS, Wujcik CE, Armstrong DW. Direct and sensitive determination of glyphosate and aminomethylphosphonic acid in environmental water samples by high performance liquid chromatography coupled to electrospray tandem mass spectrometry. J Chromatogr A. 2016;1443:93–100.