Food Chemistry 158 (2014) 421–428
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Analytical Methods
Preparation, evaluation and application of diazinon imprinted polymers as the sorbent in molecularly imprinted solid-phase extraction and liquid chromatography analysis in cucumber and aqueous samples Davood Davoodi a, Mohammad Hassanzadeh-Khayyat b, Mitra Asgharian Rezaei b, Seyed Ahmad Mohajeri b,⇑ a b
Student Research Committee (SRC), Mashhad University of Medical Sciences, Mashhad, Iran Pharmaceutical Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran
a r t i c l e
i n f o
Article history: Received 5 August 2013 Received in revised form 28 January 2014 Accepted 19 February 2014 Available online 6 March 2014 Keywords: Affinity Cucumber Diazinon Functional monomer Molecularly imprinted polymer
a b s t r a c t A series of diazinon imprinted polymers (MIPs) were prepared and evaluated in the binding study in comparison with a non-imprinted polymer (NIP). The optimised MIP was evaluated as a sorbent, for extraction and preconcentration of diazinon from aqueous media and cucumber tissue. The HPLC-UV method was calibrated, in the range of 0.025–10 mg/kg. The results indicated that the optimised MIP had an excellent affinity for diazinon. The molecularly imprinted solid-phase extraction (MISPE) procedure was optimised with a recovery of 77–98%, in aqueous solution, and a recovery of 82–110%, in cucumber. The intra-day variation and inter-day variation values were less than 8.26% and 9.7%, respectively. Our data showed that, the MIP enabled the extraction of trace amounts of diazinon successfully from aqueous solution and cucumber, demonstrating the potential of MISPE for rapid, sensitive and cost-effective sample analysis. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Molecular imprinting is a method for synthesising specific recognition sites in polymeric matrices (Hadizadeh, Zakerian, & Mohajeri, 2013). In this process the functional monomers are used to interact with a template molecule in a pre-polymerization solution and a cross-linker monomer is applied to fix the spatial arrangement during polymerisation. After washing and removing the template molecule, the imprinted cavities are able to recognize the template and its similar analogues (Hadizadeh, Zakerian, et al., 2013). Molecularly imprinted polymers (MIPs) are cheap, robust materials with a high mechanical and chemical stability, reusable with different applications (Schirmer & Meisel, 2008). The MIPs could be applied as the sorbent in solid-phase extraction (Hadizadeh, Hassanpour Moghadam, & Mohajeri, 2013; Sahebnasagh, Karimi, & Mohajeri, 2013), as the stationary phase in liquid
Abbreviations: AIBN, 2,20 -azo-bis-iso-butyronitrile; Bmax, maximum binding sites; EGDMA, ethylene glycol dimethacrylate; KD, dissociation constant; MAA, methacrylic acid; MISPE, molecularly imprinted solid-phase extraction; NIP, nonimprinted polymer; ACN, acetonitrile. ⇑ Corresponding author. Tel.: +98 511 7112611; fax: +98 511 7112470. E-mail address: [email protected] (S.A. Mohajeri). http://dx.doi.org/10.1016/j.foodchem.2014.02.144 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
cromatography (Sun, Qiao, & Liu, 2006), as the carrier in drug delivery systems (Malaekeh-Nikouei, Ghaeni, Motamedshariaty, & Mohajeri, 2012; Malaekeh-Nikouei, Vahabzadeh, & Mohajeri, 2013) and as the recognition agents in biosensors (Alizadeh, Zare, Ganjali, Norouzi, & Tavana, 2009). Molecularly imprinted solidphase extraction (MISPE) is a technique which is commonly used for sample preconcentration and clean-up before analysis (Moller, Nilsson, & Crescenzi, 2001; Schirmer & Meisel, 2008; Sun, Schussler, Sengl, Niessner, & Knopp, 2008). The MISPE is a convenient, high speed and selective method in comparison with alternative extraction methods. Furthermore, MISPE can coupled with analytical procedures (Schirmer & Meisel, 2008). Therefore, the MIPs are widely used in development of selective sorbents for extraction and determination of trace analytes. Organophosphate pesticides (OPs) are used widely in agriculture, public health and domestic fields for controlling insects (Razavi, Hosseinzadeh, Abnous, Sadat Motamedahariaty, & Imenshahidi, 2013). Diazinon (O,O-Diethyl O-[4-methyl-6-(propan-2-yl)pyrimidin-2-yl] phosphorothioate) is an anti-cholinesterase organophosphate pesticide, which is commonly used against insects in agriculture and also for controlling silverfish, cockroaches, and ants in residential and commercial buildings (Prince, Fan, Skoczenski, & Bushway, 2001). Diazinon functions as an acetylcholinesterase
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(AChE) inhibitor. The inhibition of this enzyme causes an abnormal accumulation of acetylcholine in the synaptic cleft at muscarinic, nicotinic, and central sites results in the toxic effects such as headache, dizziness, convulsions, delirium, and depression (Liang, Balcer, Solomon, Messe, & Galetta, 2003). Also, other toxic effects of diazinon on hepatocytes, spleen, thymus, blood cells, lymph nodes, and heart have been reported in human and animals (Razavi et al., 2013). Several analytical methods have been developed for detection and measurement of anti-AChE OPs (Aprea, Colosio, Mammone, Minoia, & Maroni, 2002). Most of them need time-consuming procedure, sophisticated and expensive equipments with skilled operators. The maximum residue limit (MRL) of diazinon in cucumber, set by WHO, is 0.5 mg/kg (0.5 ppm) (Kabir, Rahman, Ahmed, Prodhan, & Akon, 2008). Diazinon is usually determined by GC methods (GC–MS, GC-ECD, GC-FPD, etc.) in cucumber and other fruits (Bagheri, Es’haghi, Es-haghi, & Mesbahi, 2012; Garrido Frenich, Martínez Vidal, Fernández Moreno, & Romero-González, 2009; Srivastava, Trivedi, Srivastava, Lohani, & Srivastava, 2011; Zhao et al., 2011). The extraction and preconcentration procedures are usually performed in above mentioned methods before analysis to increase sensitivity. The quantification limit set in these studies is around 0.001–0.01 mg/kg (0.001–0.01 ppm); which is significantly less than the MRL of diazinon in fruits (Bagheri, Es’haghi, Es-haghi, & Mesbahi, 2012; Garrido Frenich et al., 2009; Srivastava et al., 2011; Zhao et al., 2011). Obviously, the sensitivity of HPLC-UV method, for analysis of OPs, is much lower than that of GC methods. But the lower cost and more availability of HPLC methods are the important advantageous. The quantification limit of diazinon assay by HPLC-UV in different fruit juice or agriculture products was 0.05–0.54 mg/kg (0.05–0.54 ppm) (Gebreegzi, Foster, & Khan, 2000; Seebunrueng, Santaladchaiyakit, Soisungnoen, & Srijaranai, 2011). Preconcentration procedures are also necessary in HPLC methods. Thus, new approaches such as solid-phase extraction (SPE), supercritical fluid extraction (SFE), accelerated solvent extraction (ASE), and solid phase microextraction (SPME) have been of particular interest (Heidari, Shahtaheri, Alimohammadi, & Rahimi-Froshani, 2009; Shahtaheri, Abdollahi, Golbabaei, RahimiFroushani, & Ghamari, 2008; Shahtaheri, Ibrahimi, Golbabaei, Hosseini, & Fouladi Dehghi, 2007; Shahtaheri, Mesdaghinia, & Stevenson, 2005). Recently, because of stability, low cost and ease of preparation, molecular imprinting has become an interesting research field to prepare an specific sorbent (MIP) for SPE of compounds in environmental and occupational samples (Haupt, 2003). In addition, the MIPs can be also used as a sorbent for the analogues of the main template or other structurally similar molecules (Wang et al., 2011). It is of great interest to develop a new MISPE that uses diazinon as a template for the extraction of this toxin from aqueous media and cucumber tissue. The aim of the present study was development of an optimised MIP for diazinon, evaluation of its binding properties and development of MISPE for sample preparation, clean-up and preconcentration of toxin from aqueous media and cucumber tissue. In this study, we coupled MISPE with HPLC-UV assay as a rapid, easy, affordable and accessible method for determination of diazinon in cucumber. We tried to increase the sensitivity of the assay to an acceptable level by MISPE. 2. Experimental 2.1. Chemicals and materials Diazinon, methacrylic acid (MAA), and ethylene glycol dimethacrylate (EGDMA) were obtained from Sigma–Aldrich (Milwaukee, USA). 2,2’-Azobis-iso-butyronitrile (AIBN) was purchased from Acros (Geel, Belgium). All solvents used [acetonitrile (ACN), chloroform, methanol, acetone, acetic acid and dichloromethane] were of HPLC grade.
2.2. Synthesis of polymers Diazinon as the template and MAA (2.5 mmol) as the functional monomer, were dissolved in chloroform (5 mL) and kept at 4 °C for 1 h. The tube was placed in a shaker at 100 rpm for 1 h at room temperature. EGDMA (12.5 mmol) and AIBN (10 mg) were then added as the cross-linker and initiator, respectively. Different amounts of diazinon (0.833, 0.500, 0.416, 0.312 and 0.250 mmol) were applied to obtain diazinon/MAA molar ratios of 1/3, 1/5, 1/6, 1/8 and 1/10 in preparation of MIP1, MIP2, MIP3, MIP4 and MIP5, respectively. After sonication for 5 min the mixture was sparged, in an ice bath, with oxygen-free nitrogen for 5 min and heated at 60 °C for 24 h to complete polymerisation. The resultant rigid polymer monoliths were powdered and sieved through 200 and 400 mesh stainless steel sieves (particle size between 38 to 75 lm). After washing the polymer particles with a methanol– ACN (7:3, v/v) mixture (4 25 mL) and finally with methanol (5 25 mL), the mixtre was centrifuged at 3000 rpm for 10 min and the supernatant was analysed by HPLC. The washing step was continued until no diazinon or other chemicals were detected in supernatant. Non-imprinted polymer (NIP) was prepared under the same condition described above but in the absence of diazinon. 2.3. Batch adsorption procedure The binding properties of polymers were examined by batch adsorption experiments. Dry polymer (10 mg) was added to 1.5 mL methanol–water (pH = 10) (75:25, v/v) with diazinon and was shaken for 24 h at room temperature. After centrifugation of the solution (3000 rpm for 10 min) the concentration of diazinon in supernatant was analysed by HPLC. The amount of bound diazinon was measured from the initial and final concentrations in solution after equilibrium. Each experiment was repeated four times and mean ± SEM (standard error of mean) was reported. The imprinting factor (IF) was obtained from Eq. (1) (Hadizadeh, Zakerian, et al., 2013):
K MIP IF ¼ K NIP
ð1Þ
where K value was the partition coefficient for MIP or NIP and calculated in (Hadizadeh, Zakerian, et al., 2013):
Bound Diazinon=g polymer K¼ ½Free
ð2Þ
where [Free], was the final concentration of free unbound diazinon in the solution and bound diazinon was the amount of diazinon bound to per gram dry polymer after equilibrium.A Scatchard plot was drawn (Eq. 3) when the diazinon concentration was varied in the solution (Hadizadeh, Zakerian, et al., 2013):
Bound Bound Bmax þ ¼ ½Free KD KD
ð3Þ
The [Bound] value was the amount of diazinon bound to polymer after equilibrium; [Free] was the final diazinon concentration at equilibrium; Bmax was the maximum binding sites and KD represented the dissociation constant. The values of KD and Bmax could be obtained from the slope and intercept of the line drawn in a plot of Bound/Free vs. Bound. 2.4. Optimisation of MISPE protocol 150 mg of polymer (MIP5 or NIP), was suspended and packed into an empty polypropylene SPE cartridge. The column was regenerated with 3 mL methanol-acetic acid (90:10 v/v), 10 mL dichloromethane and 5 mL ACN and then conditioned with 5 mL methanol
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and 6 mL deionized water. An aqueous solution of diazinon (10 ppm) was prepared and 0.5 mL of this solution was loaded onto the column. Washing solvent was then transmitted through the column. Different solvent compositions [ACN, water, methanol– water (pH = 10.6, NaOH 0.025 M)] (Table 1) were studied to find a solvent with maximum selectivity for diazinon in washing step. Finally, 2 mL ACN was applied in elution step to complete the extraction of diazinon. The loading, washing and eluting fractions were analysed by HPLC.
2.5. Extraction of diazinon from cucumber and aqueous samples A polypropylene SPE cartridge was packed with 150 mg polymer (MIP5 or NIP) and then regenerated [with 3 mL methanolacetic acid (90:10, v/v), 10 mL dichloromethane and 5 mL ACN] and conditioned with 5 mL methanol and 6 mL deionized water. Cucumber was purchased and collected from different markets of the city (Mashhad, Iran), washed completely with deionized water and chopped into very small pieces. Then homogenised with manual homogenizer (Heidolph laborata 4003 digital) (3 15 min) and sonicated (Branson 5510) for 10 min to have perfect homogenised mixture. Then 5 g mixture was weighed accurately, mixed with 5 mL methanol–water (pH = 10.6, NaOH 0.025 M) and shaken for 1 h at room temperature. Then the mixture was centrifuged (6000 rpm for 10 min at -5 °C) with refrigerator-centrifuge (Hettich Zentrifugen universal 320R). 7 mL supernatant solution was loaded onto the MISPE column. Washing solvent was then passed through the column as described in method 9 [1 mL deionized water, 1 mL deionized water-ACN (70:30, v/v), 5 mL methanol–water (pH = 10.6, NaOH 0.025 M)ACN (40:40:20, v/v/v)]. Finally, 2 mL ACN was applied to perform complete extraction and elution of diazinon. For aqueous samples; 7 mL diazinon solution was loaded onto the column. The washing and elution step were the same as described above for cucumber samples. The final eluted fraction (2 mL ACN) was then dried under oxygen-free nitrogen stream and then resolved in 0.2 mL methanol before injection into HPLC. The concentration of diazinon was determined by HPLC.
2.6. Standard and calibration solutions A 10 mg diazinon (8.94 lL) was dissolved in 10 mL methanol to prepare stock solution (1000 ppm). The standard solutions used for spiking calibration samples were prepared from the stock solution by diluting with methanol. 100 lL of standard solutions was added to 900 mg homogenised cucumber tissue or deionized water to obtain calibration standards (0.025, 0.05, 0.1, 0.5, 1, 5 and 10 ppm) in aqueous and cucumber samples.
2.7. HPLC analysis Chromatographic determination of diazinon was carried out by a Younglin Acme 9000 system (South Korea), consisting of SP930D solvent delivery module, SDV50A solvent mixing vacuum degasser, column oven CTS30, UV730 dual wavelength UV/VIS detector set to 245 nm and ODSA C18 (4.6 150 mm, 5 lm) column. The data analysis was performed by Autochro- 3000 software. The injection volume was 20 lL, the flow rate was 0.8 ml/min and the column temperature was fixed at 30 °C. An isocratic method was used, and mobile-phase composition was ACN–methanol–water (80:10:10, v/v/v). 3. Results and discussion 3.1. Synthesis of MIPs Molecular recognition of the template molecule by imprinted polymers is based on the intermolecular interactions between the template molecule and functional groups in the polymer (Sun et al., 2008). Choice of functional monomer is one of the most important factors in preparation of MIPs (Alizadeh et al., 2009; Sun et al., 2008). Our previous studies indicated that the selection of a proper functional monomer leads to synthesise an MIP with strong intermolecular interactions between template molecule and MIP (Hadizadeh, Hassanpour Moghadam, et al., 2013; Hadizadeh, Zakerian, et al., 2013). Due to the presence of nitrogen, oxygen and sulphur sites in the chemical structure of diazinon and carboxylic acid group in MAA, the strong electrostatic and hydrogen bond interactions could be formed in organic and aqueous media. According to these facts and our previous experiments, MAA was selected as a functional monomer in molecular imprinting process. Solvent plays an important role in formation of monomer-template complex (Sahebnasagh et al., 2013). The polarity of the solvent influences the extent of the non-covalent pre-polymer complex before and during polymerisation. Aprotic solvents with low polarity e.g. chloroform are usually applied as the media to increase complex formation and facilitate non-covalent interactions (monomer-template) such as hydrogen bonding in imprinting process (Mohajeri, Karimi, Aghamohammadian, & Khansari, 2011). On the other hand, more polar solvents especially protic ones tend to dissociate the non-covalent weak interactions in the pre-polymer complex (Sahebnasagh et al., 2013). In the present work, the chloroform was applied as an organic solvent to optimise the molecular imprinting process. Another factor is the optimised template/ monomer molar ratio. Our previous works showed that the optimised ratio is usually obtained empirically (Hadizadeh, Zakerian, et al., 2013). In this work, MIP5 with diazinon/MAA molar ratio of 1/10 showed the most specificity in rebinding test and was selected as the optimised MIP. According to our data, spatial
Table 1 Composition and volume of the solvents used in washing and elution procedure. Methods
Washing procedure STEP1
1 2 3 4 5 6 7 8 9
3 mL 2 mL 1 mL 1 mL 1 mL 1 mL 1 mL 1 mL 1 mL
deionized deionized deionized deionized deionized deionized deionized deionized deionized
Elution procedure STEP2
water water water water water water water water water
1 mL 1 mL 1 mL 1 mL 1 mL 1 mL 1 mL 1 mL 1 mL
ACN (water/ACN, (water/ACN, (water/ACN, (water/ACN, (water/ACN, (water/ACN, (water/ACN, (water/ACN,
STEP3 5/5, 7/3, 7/3, 7/3, 7/3, 7/3, 7/3, 7/3,
v/v) v/v) v/v) v/v) v/v) v/v) v/v) v/v)
1 mL methanol 2mL (methanol/water,5/5, v/v) 1 mL (methanol/water,5/5, v/v) 1.5 mL (methanol/water,7/3, v/v) 2 mL (methanol/acetic acid,9/1, v/v) 2 mL (methanol/water/acetic acid,5/4/1, v/v/v) 5 mL (methanol/water/ACN,5/4/1, v/v/v) 2 mL (methanol/water/ACN,5/3/2, v/v/v) 5 mL (methanol/water/ACN,4/4/2, v/v/v)
2 mL 2 mL 2 mL 2 mL 2 mL 2 mL 2 mL 2 mL 2 mL
ACN ACN ACN ACN ACN ACN ACN ACN ACN
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specific than other MIPs. Thus, MIP5 was selected for development of solid-phase extraction of diazinon from cucumber matrix.
3.2. Rebinding test and Scatchard analysis
Fig. 1. Binding of Diazinon to 10 mg of the MIPs and NIP polymers in 1.5 mL methanol–water (pH = 10) (75:25, v/v), (n = 4). Each point represents mean ± SEM; diazinon concentration in was 20 lg L1.
The conventional batch adsorption method involves the incubation of ligand solution with polymer at different concentrations and measurement of ligand bound to polymer after a fixed time (Dineiro et al., 2006; Mohajeri et al., 2011; Pap & Horvai, 2004). The data showed that all MIPs had higher affinity for diazinon than NIP polymer. According to these results MIP5 had superior binding properties than other MIPs (Fig. 1).The IF values of MIP1, MIP2, MIP3, MIP4 and MIP5 were 8.53, 8.97, 10.11, 12.08 and 37.86, respectively. The data indicated that the IF of MIP5 is significantly higher than that of other MIPs. Therefore, this polymer was selected as the optimised MIP for Scatchard analysis and extraction procedure. Also diazinon binding to MIP5 was more than NIP at all concentrations (Fig. 2). From the Scatchard plot and equation, two kinds of binding sites were discerned in MIP5. One kind represented low affinity binding sites (Y ¼ 0:0013Xþ0:1254; R2 ¼ 0:8424) with dissociation constant (KD) and maximum binding capacity (Bmax) values of 1000 lM and 125 lmol/ g MIP, respectively; while the data for high affinity binding sites (Y ¼ 0:0818X þ 0:7192; R2 ¼ 0:9795) were 12.34 lM (KD) and 8.87 lmol/g MIP (Bmax), respectively. The KD value of MIP-template interactions in other studies ranged from low lM to M (histamineMIP KD = 11.11 lM(Sahebnasagh et al., 2013), theophylline-MIP KD = 1.5 M (Sun et al., 2006)). The small KD in this work (12.34 lM) was low enough to be selected as a sorbent in solidphase extraction of diazinon from biological fluids.
3.3. Optimisation of the MISPE procedure
Fig. 2. Adsorption isotherm of MIP5 and NIP using batch adsorption test (n = 4). Each point represents mean ± SEM. Experiment conditions: 10 mg of polymer was incubated in 1.5 mL methanol–water (pH = 10) (75:25, v/v), (n = 4), with different concentrations of diazinon for 24 h at room temperature.
arrangement of MAA around the template and non-covalent interactions between MAA and diazinon in pre-polymerization solution is significantly more effective in diazinon/MAA ratio of 1/10. Therefore, the imprinted cavities and binding sites in MIP5 work more
Washing procedure is the most important step in solid-phase extraction method. We tested different solvents to obtain maximum selectivity and recovery of diazinon (Table 1). The highest recovery and selectivity was obtained in method 9 (100% recovery for MIP and 0% for NIP). Thus, the composition of [1 mL deionized water, 1 mL deionized water–ACN (70:30, v/v), 5 mL methanol– water (pH = 10.6, NaOH 0.025 M)-ACN (40:40:20, v/v/v)] was selected as the optimised solvents in washing steps (Table 2). In our previous works we have used a solvent or a mixture of solvents to optimize the washing step in solid-phase extraction process (Sahebnasagh et al., 2013). Also, ACN was strong enough to completely elute diazinon from MIP column; thus 2 mL ACN was selected as eluting solvent.
Table 2 The percentage recovery of diazinon on MISPE and NISPE procedures in washing and elution steps. Methods
Washing procedure STEP1
1 2 3 4 5 6 7 8 9
STEP2
Elution procedure STEP3
Total
MISPE (%)
NISPE (%)
MISPE (%)
NISPE (%)
MISPE (%)
NISPE (%)
MISPE (%)
NISPE (%)
MISPE (%)
1.91 ± 0.09a 0.86 ± 0.15 0.00 0.00 0.00 0.00 0.00 0.00 0.00
2.28 ± 0.35 1.06 ± 0.20 0.00 0.00 0.00 0.00 0.00 0.00 0.00
72.53 ± 3.0 8.06 ± 0.20 0.00 0.00 0.00 0.00 0.00 0.00 0.00
81.24 ± 2.7 8.36 ± 0.25 0.00 0.00 0.00 0.00 0.00 0.00 0.00
18.56 ± 0.60 43.70 ± 2.85 32 ± 2.64 41.10 ± 1.62 20.70 ± 1.58 44.71 ± 3.99 8.42 ± 0.20 1.92 ± 0.07 0.00
12.11 ± 1.42 48.36 ± 1.26 36.20 ± 1.98 61.37 ± 3.25 24.53 ± 1.47 53.01 ± 2.20 96.3 ± 1.01 98.37 ± 0.55 100.03 ± 0.07
93.01 ± 3.70 52.63 ± 3.21 32 ± 2.64 41.10 ± 1.62 20.70 ± 1.58 44.71 ± 3.99 8.42 ± 0.20 1.92 ± 0.07 0.00
95.63 ± 4.48 57.8 ± 1.72 36.20 ± 1.98 61.37 ± 3.25 24.53 ± 1.47 53.01 ± 2.20 96.3 ± 1.01 98.37 ± 0.55 100.03 ± 0.07
6.99 ± 3.62 47.36 ± 2.81 68 ± 2.64 58.89±1.62 79.29 ± 1.58 55.29 ± 3.99 91.57 ± 0.2 98.07 ± 0.07 100.00
a All experiments were carried out in triplicate and the results are expressed as mean ± standard deviation. MISPE: molecularly imprinted solid-phase extraction. NISPE: non-imprinted polymer solid-phase extraction.
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Fig. 3. Chromatograms of a standard solution of diazinon (1 lgmL1) before (a) and after solid-phase extraction using MIP5 (b) or NIP (c) polymers as the sorbent. Washing condition for MISPE: 1 mL deionized water, 1 mL deionized water–ACN (70:30, v/v), 5 mL methanol–water (pH = 10.6, NaOH 0.025 M)-ACN(40:40:20, v/v/v), elution condition: 2 mL ACN.
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Fig. 4. Chromatograms of a cucumber tissue spiked with diazinon (1 lg mL1) after solid-phase extraction with MIP5 (a) or NIP (b) as the stationary phase. Washing condition for MISPE: 1 mL deionized water, 1 mL deionized water–ACN (70:30, v/v), 5 mL methanol–water (pH = 10.6, NaOH 0.025 M)–ACN(40:40:20, v/v/v), elution condition: 2 mL ACN.
3.4. Extraction of diazinon from aqueous samples and cucumber Aqueous and cucumber samples were loaded onto the MIP5 column and MISPE procedure was carried out as described before. Aqueous samples: Due to the low sensitivity of HPLC-UV, the minimum concentration determined in standard solutions was 0.5 ppm. Thus, the standard curve was also plotted in the range of 0.5–10 ppm (Y ¼ 22:26X þ 1:2545; R2 ¼ 0:9996). The standard curve was obtained from HPLC-UV analysis of standard solutions prepared in aqueous media without any extraction or preconcentration process. But, calibration curve was plotted after molecularly imprinted solid-phase extraction and HPLC-UV analysis of diazinon in aqueous samples. The calibration curve of HPLC data in the range of 0.025–10 ppm was established (Y ¼ 484:08X þ 40:21; R2 ¼ 0:9983). The recovery of diazinon in MISPE was 77–98%. The limit
of detection (LOD) and limit of quantification (LOQ) of this assay were calculated 0.0037 ppm and 0.025 ppm, respectively. The lowest detectable concentration of diazinon was determined as LOD; while the lowest quantified concentration was selected as LOQ in this assay. Intra-day precision values for diazinon concentrations of 0.025 and 10 ppm, were 3.93 and 1.29%; while inter-day variation values of these concentrations were 4.19 and 2.23%, respectively. The precision values were calculated as relative standard deviation of the aqueous samples (n = 4) in one day (Intra-day precision) or four different days (inter-day variation). These data obviously indicated that MISPE could effectively increase the sensitivity (20 times) of HPLC analysis of diazinon in aqueous samples. Also the precision values demonstrated the repeatability of the assay. Fig. 3 illustrated the chromatograms of a standard solution of diazinon (Fig. 3a), an aqueous sample with the same concentration after MISPE and HPLC analysis (Fig. 3b) and the same aqueous
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sample after doing the solid-phase extraction process using NIP polymer as the sorbent and HPLC analysis (Fig. 3c). This figure indicated the efficacy of MISPE method in sample preconcentration and preparation before HPLC analysis of diazinon; whereas NIP, as the sorbent in SPE, could not extract the diazinon with the same washing and eluting protocol applied for MIP. Cucumber samples: The initial experiments (before diazinon spiking) showed that there was a small amount of diazinon in cucumber samples and we could not find a sample without toxin. Thus, the additional standard method was applied in calibration procedure. It meant that diazinon was spiked into the cucumber tissue which contained a fixed amount (X ppm) of this toxin. The calibration curve of HPLC data in the range of 0.025–10 ppm was established (Y ¼ 343:37X þ 86:42; R2 0:998). According to the equation when Y = 0, then X = 0.251 ppm (equivalent to 0.251 mg/kg) which represented the fixed amount of diazinon in cucumber tissue. The recovery of diazinon extraction was between 82% and 110%. The LOD and LOQ of the assay were 0.0037 ppm and 0.025 ppm, respectively. Intra-day precision values for diazinon concentrations of 0.025 and 10 ppm, were 1.14% and 8.26%; while interday variation values of these concentrations were 3.53% and 9.7%, respectively. According to the results a 15-fold increase in sensitivity was observed in assay after using MISPE before HPLC. The LOQ of this assay (0.025 ppm equivalent to 0.025 mg/kg) was significantly less than MRL of diazinon in cucumber (0.5 ppm equivalent to 0.5 mg/kg). The LOQ in this work is less than the previous values reported (0.05–0.54 ppm) for HPLC-UV developed assays in fruits or agricultural products. Thus, the calibrated method can be applied for analysis in cucumber. These data indicated the sensitivity and repeatability of the calibrated MISPE method for HPLC analysis of diazinon in cucumber. Fig. 4 showed the chromatograms of a cucumber sample after MISPE and HPLC analysis (Fig. 4a) and the same sample after doing the solid-phase extraction process using NIP polymer as the sorbent and HPLC analysis (Fig. 4b). Fig. 4a (compared to Fig. 3a), demonstrated the ability of MISPE method in sample preconcentration before HPLC analysis; while no peak was detected in the chromatogram of the same cucumber sample in Fig. 4b which used NIP as the sorbent in SPE. As mentioned before, the sensitivity of some analytical methods e.g. GC is higher than HPLC-UV in determination of diazinon. The LOQ value in GC procedures ranged from 0.001 to 0.01 ppm (Bagheri, Es’haghi, Es-haghi, & Mesbahi, 2012; Garrido Frenich et al., 2009; Srivastava et al., 2011; Zhao et al., 2011). But, these methods are sometimes time-consuming; need sophisticated and expensive equipments with skilled operators. Obviously, HPLC is easier, more accessible and affordable and cheaper than above mentioned procedures. We used MISPE method to increase the sensitivity to the suitable level. Thus, our developed method could be easily applied with an acceptable sensitivity, accuracy and repeatability for analysis of diazinon in cucumber. Also, the MIP synthesised in our study could be evaluated for preconcentration in other analytical methods such as GC. In our previous works we have also showed that the molecularly imprinted polymers could be effectively applied as the sorbent in solid-phase extraction of other chemicals such as histamine from biological samples (Sahebnasagh et al., 2013). In the next step, the calibrated assay was applied for determination of diazinon in real cucumber samples. 10 samples from different parts of the city (Mashhad) were gathered and tested using MISPE preconcentration procedure. According to our results, the diazinon concentration in cucumber was between 0.264 and 0.295 ppm (equivalent to 0.264–0.295 mg/kg). The amounts of diazinon were less than the MRL (0.5 ppm) (Kabir et al., 2008) that is set by WHO. This study demonstrated the role of molecular imprinting in producing specific cavities for diazinon and its ability
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in sample preconcentration and clean-up before HPLC analysis in cucumber and aqueous samples. Also, the results indicated the efficacy, sensitivity and repeatability of HPLC-UV assay using MISPE procedure for determination of diazinon in real samples. Therefore, the MIP synthesised in this study can be evaluated for extraction and analysis of diazinon in other agricultural products or biological fluids. 4. Conclusion The results obtained in this work confirmed the effect of molecular imprinting technique in preparation of polymer with high affinity for diazinon. The binding properties could be modified by varying the diazinon/MAA molar ratios. The optimised MIP was applied for molecularly imprinted solid-phase extraction (MISPE) of diazinon from aqueous samples and cucumber tissue in the range of 0.025 to 10 mg/kg. The calibrated MISPE method had a recovery of 77–98%, in aqueous solution, and a recovery of 82–110%, in cucumber. The results indicated and acceptable sensitivity, repeatability and accuracy for assay. The intra-day variation and interday variation values were less than 8.26% and 9.7%, respectively. The LOD and LOQ of the assay were 0.0037 and 0.025 ppm, respectively. Finally, the calibrated method was applied for determination of diazinon in real samples in Mashhad. The amounts of diazinon in all samples were less than its MRL level. These results demonstrated that diazinon-specific SPE column characterised by high recoveries and sample preparation factors could solve several problems related to determination of diazinon by HPLC-UV. Acknowledgements We gratefully acknowledge the Vice Chancellor of Research, Mashhad University of Medical Sciences for financial support through grant number 89939. The results described in this paper were part of a PharmD student (Davood Davoodi) thesis. References Alizadeh, T., Zare, M., Ganjali, M. R., Norouzi, P., & Tavana, B. (2009). A new molecularly imprinted polymer (MIP)-based electrochemical sensor for monitoring 2,4,6-trinitrotoluene (TNT) in natural waters and soil samples. Biosensors & Bioelectronics, 25(5), 1166–1172. Aprea, C., Colosio, C., Mammone, T., Minoia, C., & Maroni, M. (2002). Biological monitoring of pesticide exposure: a review of analytical methods. Journal of Chromatography B, 769(2), 191–219. Bagheri, H., Es’haghi, A., Es-haghi, A., & Mesbahi, N. (2012). A high-throughput approach for the determination of pesticide residues in cucumber samples using solid-phase microextraction on 96-well plate. Analytica Chimica Acta, 740, 36–42. Dineiro, Y., Menendez, M. I., Blanco-Lopez, M. C., Lobo-Castanon, M. J., MirandaOrdieres, A. J., & Tunon-Blanco, P. (2006). Computational predictions and experimental affinity distributions for a homovanillic acid molecularly imprinted polymer. Biosensors & Bioelectronics, 22(3), 364–371. Garrido Frenich, A., Martínez Vidal, J. L., Fernández Moreno, J. L., & RomeroGonzález, R. (2009). Compensation for matrix effects in gas chromatography– tandem mass spectrometry using a single point standard addition. Journal of Chromatography A, 1216(23), 4798–4808. Gebreegzi, Y. T., Foster, G. D., & Khan, S. U. (2000). simultaneous determination of carbaryl, malathion, fenitrothion, and diazinon residues in sesame seeds (Sesamum indicum L.). Journal of agricultural and food chemistry, 48(11), 5165–5168. Hadizadeh, F., Hassanpour Moghadam, M., & Mohajeri, S. A. (2013). Application of molecularly imprinted hydrogel for the preparation of lactose-free milk. Journal of the Science of Food and Agriculture, 93(2), 304–309. Hadizadeh, F., Zakerian, A., & Mohajeri, S. A. (2013). Non-covalently lactose imprinted polymers and recognition of saccharides in aqueous solutions. Journal of the Iranian Chemical Society, 10(2), 207–212. Haupt, K. (2003). Imprinted polymers—tailor-made mimics of antibodies and receptors. Chemical Communications, 21(2), 171–178. Heidari, H., Shahtaheri, S., Alimohammadi, M., & Rahimi-Froshani, A. (2009). Trace analysis of xylene in occupational exposures monitoring. Iranian Journal of Public Health, 38(1), 89–99. Kabir, K., Rahman, M., Ahmed, M., Prodhan, M., & Akon, M. (2008). Determination of residue of diazinon and carbosulfan in brinjal and quinalphos in yard long bean
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under supervised field trial. Bangladesh Journal of Agricultural Research, 33(3), 503–513. Liang, T., Balcer, L., Solomon, D., Messe, S., & Galetta, S. (2003). Supranuclear gaze palsy and opsoclonus after Diazinon poisoning. Journal of Neurology, Neurosurgery & Psychiatry, 74(5), 677–679. Malaekeh-Nikouei, B., Ghaeni, F. A., Motamedshariaty, V. S., & Mohajeri, S. A. (2012). Controlled release of prednisolone acetate from molecularly imprinted hydrogel contact lenses. Journal of Applied Polymer Science, 126(1), 387–394. Malaekeh-Nikouei, B., Vahabzadeh, S. A., & Mohajeri, S. A. (2013). Preparation of a molecularly imprinted soft contact lens as a new ocular drug delivery system for dorzolamide. Current Drug Delivery, 10(3), 279–285. Mohajeri, S. A., Karimi, G., Aghamohammadian, J., & Khansari, M. R. (2011). Clozapine recognition via molecularly imprinted polymers; bulk polymerization versus precipitation method. Journal of Applied Polymer Science, 121(6), 3590–3595. Moller, K., Nilsson, U., & Crescenzi, C. (2001). Synthesis and evaluation of molecularly imprinted polymers for extracting hydrolysis products of organophosphate flame retardants. Journal of Chromatography A, 938(1–2), 121–130. Pap, T., & Horvai, G. (2004). Characterization of the selectivity of a phenytoin imprinted polymer. Journal of Chromatography A, 1034(1–2), 99–107. Prince, A. E., Fan, T. S., Skoczenski, B. A., & Bushway, R. J. (2001). Development of an immunoaffinity-based solid-phase extraction for diazinon. Analytica Chimica Acta, 444(1), 37–49. Razavi, M., Hosseinzadeh, H., Abnous, K., Sadat Motamedahariaty, V., & Imenshahidi, M. (2013). Crocin restores hypotensive effect of subchronic administration of diazinon in rats. Iranian Journal of Basic Medical Sciences, 16(1), 64–72. Sahebnasagh, A., Karimi, G., & Mohajeri, S. A. (2014). Preparation and evaluation of histamine imprinted polymer as a selective sorbent in molecularly imprinted solid-phase extraction coupled with high performance liquid chromatography analysis in canned fish. Food Analytical Methods, 7(1), 1–8. Schirmer, C., & Meisel, H. (2008). Molecularly imprinted polymers for the selective solid-phase extraction of chloramphenicol. Analytical and Bioanalytical Chemistry, 392(1–2), 223–229. Seebunrueng, K., Santaladchaiyakit, Y., Soisungnoen, P., & Srijaranai, S. (2011). Catanionic surfactant ambient cloud point extraction and high-performance
liquid chromatography for simultaneous analysis of organophosphorus pesticide residues in water and fruit juice samples. Analytical and Bioanalytical Chemistry, 401(5), 1707–1716. Shahtaheri, S., Abdollahi, M., Golbabaei, F., Rahimi-Froushani, A., & Ghamari, F. (2008). Monitoring of mandelic acid as a biomarker of environmental and occupational exposures to styrene. Iranian Journal of Environmental Research, 2(2), 169–176. Shahtaheri, S. J., Ibrahimi, L., Golbabaei, F., Hosseini, M., & Fouladi Dehghi, B. (2007). Solid phase extraction for 1-hydroxypyrene as a biomarker of occupational exposure to PAHs prior to high performance liquid chromatography. Iranian Journal of Chemistry & Chemical Engineering, 26(4), 75–81. Shahtaheri, S. J., Mesdaghinia, A., & Stevenson, D. (2005). Evaluation of factors influencing recovery of herbicide 2, 4-D from drinking water. Iranian Journal of Chemistry and Chemical Engineering, 24(1), 33–40. Srivastava, A. K., Trivedi, P., Srivastava, M., Lohani, M., & Srivastava, L. P. (2011). Monitoring of pesticide residues in market basket samples of vegetable from Lucknow City, India: QuEChERS method. Environmental Monitoring and Assessment, 176(1–4), 465–472. Sun, H. W., Qiao, F. X., & Liu, G. Y. (2006). Characteristic of theophylline imprinted monolithic column and its application for determination of xanthine derivatives caffeine and theophylline in green tea. Journal of Chromatography A, 1134(1–2), 194–200. Sun, Z., Schussler, W., Sengl, M., Niessner, R., & Knopp, D. (2008). Selective trace analysis of diclofenac in surface and wastewater samples using solid-phase extraction with a new molecularly imprinted polymer. Analytica Chimica Acta, 620(1–2), 73–81. Wang, S., Li, Y., Wu, X., Ding, M., Yuan, L., Wang, R., Wen, T., Zhang, J., Chen, L., & Zhou, X. (2011). Construction of uniformly sized pseudo template imprinted polymers coupled with HPLC–UV for the selective extraction and determination of trace estrogens in chicken tissue samples. Journal of Hazardous Materials, 186(2), 1513–1519. Zhao, W.-J., Sun, X.-K., Deng, X.-N., Huang, L., Yang, M.-M., & Zhou, Z.-M. (2011). Cloud point extraction coupled with ultrasonic-assisted back-extraction for the determination of organophosphorus pesticides in concentrated fruit juice by gas chromatography with flame photometric detection. Food Chemistry, 127(2), 683–688.
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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Analytical Methods
Preparation, evaluation and application of diazinon imprinted polymers as the sorbent in molecularly imprinted solid-phase extraction and liquid chromatography analysis in cucumber and aqueous samples Davood Davoodi a, Mohammad Hassanzadeh-Khayyat b, Mitra Asgharian Rezaei b, Seyed Ahmad Mohajeri b,⇑ a b
Student Research Committee (SRC), Mashhad University of Medical Sciences, Mashhad, Iran Pharmaceutical Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran
a r t i c l e
i n f o
Article history: Received 5 August 2013 Received in revised form 28 January 2014 Accepted 19 February 2014 Available online 6 March 2014 Keywords: Affinity Cucumber Diazinon Functional monomer Molecularly imprinted polymer
a b s t r a c t A series of diazinon imprinted polymers (MIPs) were prepared and evaluated in the binding study in comparison with a non-imprinted polymer (NIP). The optimised MIP was evaluated as a sorbent, for extraction and preconcentration of diazinon from aqueous media and cucumber tissue. The HPLC-UV method was calibrated, in the range of 0.025–10 mg/kg. The results indicated that the optimised MIP had an excellent affinity for diazinon. The molecularly imprinted solid-phase extraction (MISPE) procedure was optimised with a recovery of 77–98%, in aqueous solution, and a recovery of 82–110%, in cucumber. The intra-day variation and inter-day variation values were less than 8.26% and 9.7%, respectively. Our data showed that, the MIP enabled the extraction of trace amounts of diazinon successfully from aqueous solution and cucumber, demonstrating the potential of MISPE for rapid, sensitive and cost-effective sample analysis. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Molecular imprinting is a method for synthesising specific recognition sites in polymeric matrices (Hadizadeh, Zakerian, & Mohajeri, 2013). In this process the functional monomers are used to interact with a template molecule in a pre-polymerization solution and a cross-linker monomer is applied to fix the spatial arrangement during polymerisation. After washing and removing the template molecule, the imprinted cavities are able to recognize the template and its similar analogues (Hadizadeh, Zakerian, et al., 2013). Molecularly imprinted polymers (MIPs) are cheap, robust materials with a high mechanical and chemical stability, reusable with different applications (Schirmer & Meisel, 2008). The MIPs could be applied as the sorbent in solid-phase extraction (Hadizadeh, Hassanpour Moghadam, & Mohajeri, 2013; Sahebnasagh, Karimi, & Mohajeri, 2013), as the stationary phase in liquid
Abbreviations: AIBN, 2,20 -azo-bis-iso-butyronitrile; Bmax, maximum binding sites; EGDMA, ethylene glycol dimethacrylate; KD, dissociation constant; MAA, methacrylic acid; MISPE, molecularly imprinted solid-phase extraction; NIP, nonimprinted polymer; ACN, acetonitrile. ⇑ Corresponding author. Tel.: +98 511 7112611; fax: +98 511 7112470. E-mail address: [email protected] (S.A. Mohajeri). http://dx.doi.org/10.1016/j.foodchem.2014.02.144 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
cromatography (Sun, Qiao, & Liu, 2006), as the carrier in drug delivery systems (Malaekeh-Nikouei, Ghaeni, Motamedshariaty, & Mohajeri, 2012; Malaekeh-Nikouei, Vahabzadeh, & Mohajeri, 2013) and as the recognition agents in biosensors (Alizadeh, Zare, Ganjali, Norouzi, & Tavana, 2009). Molecularly imprinted solidphase extraction (MISPE) is a technique which is commonly used for sample preconcentration and clean-up before analysis (Moller, Nilsson, & Crescenzi, 2001; Schirmer & Meisel, 2008; Sun, Schussler, Sengl, Niessner, & Knopp, 2008). The MISPE is a convenient, high speed and selective method in comparison with alternative extraction methods. Furthermore, MISPE can coupled with analytical procedures (Schirmer & Meisel, 2008). Therefore, the MIPs are widely used in development of selective sorbents for extraction and determination of trace analytes. Organophosphate pesticides (OPs) are used widely in agriculture, public health and domestic fields for controlling insects (Razavi, Hosseinzadeh, Abnous, Sadat Motamedahariaty, & Imenshahidi, 2013). Diazinon (O,O-Diethyl O-[4-methyl-6-(propan-2-yl)pyrimidin-2-yl] phosphorothioate) is an anti-cholinesterase organophosphate pesticide, which is commonly used against insects in agriculture and also for controlling silverfish, cockroaches, and ants in residential and commercial buildings (Prince, Fan, Skoczenski, & Bushway, 2001). Diazinon functions as an acetylcholinesterase
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(AChE) inhibitor. The inhibition of this enzyme causes an abnormal accumulation of acetylcholine in the synaptic cleft at muscarinic, nicotinic, and central sites results in the toxic effects such as headache, dizziness, convulsions, delirium, and depression (Liang, Balcer, Solomon, Messe, & Galetta, 2003). Also, other toxic effects of diazinon on hepatocytes, spleen, thymus, blood cells, lymph nodes, and heart have been reported in human and animals (Razavi et al., 2013). Several analytical methods have been developed for detection and measurement of anti-AChE OPs (Aprea, Colosio, Mammone, Minoia, & Maroni, 2002). Most of them need time-consuming procedure, sophisticated and expensive equipments with skilled operators. The maximum residue limit (MRL) of diazinon in cucumber, set by WHO, is 0.5 mg/kg (0.5 ppm) (Kabir, Rahman, Ahmed, Prodhan, & Akon, 2008). Diazinon is usually determined by GC methods (GC–MS, GC-ECD, GC-FPD, etc.) in cucumber and other fruits (Bagheri, Es’haghi, Es-haghi, & Mesbahi, 2012; Garrido Frenich, Martínez Vidal, Fernández Moreno, & Romero-González, 2009; Srivastava, Trivedi, Srivastava, Lohani, & Srivastava, 2011; Zhao et al., 2011). The extraction and preconcentration procedures are usually performed in above mentioned methods before analysis to increase sensitivity. The quantification limit set in these studies is around 0.001–0.01 mg/kg (0.001–0.01 ppm); which is significantly less than the MRL of diazinon in fruits (Bagheri, Es’haghi, Es-haghi, & Mesbahi, 2012; Garrido Frenich et al., 2009; Srivastava et al., 2011; Zhao et al., 2011). Obviously, the sensitivity of HPLC-UV method, for analysis of OPs, is much lower than that of GC methods. But the lower cost and more availability of HPLC methods are the important advantageous. The quantification limit of diazinon assay by HPLC-UV in different fruit juice or agriculture products was 0.05–0.54 mg/kg (0.05–0.54 ppm) (Gebreegzi, Foster, & Khan, 2000; Seebunrueng, Santaladchaiyakit, Soisungnoen, & Srijaranai, 2011). Preconcentration procedures are also necessary in HPLC methods. Thus, new approaches such as solid-phase extraction (SPE), supercritical fluid extraction (SFE), accelerated solvent extraction (ASE), and solid phase microextraction (SPME) have been of particular interest (Heidari, Shahtaheri, Alimohammadi, & Rahimi-Froshani, 2009; Shahtaheri, Abdollahi, Golbabaei, RahimiFroushani, & Ghamari, 2008; Shahtaheri, Ibrahimi, Golbabaei, Hosseini, & Fouladi Dehghi, 2007; Shahtaheri, Mesdaghinia, & Stevenson, 2005). Recently, because of stability, low cost and ease of preparation, molecular imprinting has become an interesting research field to prepare an specific sorbent (MIP) for SPE of compounds in environmental and occupational samples (Haupt, 2003). In addition, the MIPs can be also used as a sorbent for the analogues of the main template or other structurally similar molecules (Wang et al., 2011). It is of great interest to develop a new MISPE that uses diazinon as a template for the extraction of this toxin from aqueous media and cucumber tissue. The aim of the present study was development of an optimised MIP for diazinon, evaluation of its binding properties and development of MISPE for sample preparation, clean-up and preconcentration of toxin from aqueous media and cucumber tissue. In this study, we coupled MISPE with HPLC-UV assay as a rapid, easy, affordable and accessible method for determination of diazinon in cucumber. We tried to increase the sensitivity of the assay to an acceptable level by MISPE. 2. Experimental 2.1. Chemicals and materials Diazinon, methacrylic acid (MAA), and ethylene glycol dimethacrylate (EGDMA) were obtained from Sigma–Aldrich (Milwaukee, USA). 2,2’-Azobis-iso-butyronitrile (AIBN) was purchased from Acros (Geel, Belgium). All solvents used [acetonitrile (ACN), chloroform, methanol, acetone, acetic acid and dichloromethane] were of HPLC grade.
2.2. Synthesis of polymers Diazinon as the template and MAA (2.5 mmol) as the functional monomer, were dissolved in chloroform (5 mL) and kept at 4 °C for 1 h. The tube was placed in a shaker at 100 rpm for 1 h at room temperature. EGDMA (12.5 mmol) and AIBN (10 mg) were then added as the cross-linker and initiator, respectively. Different amounts of diazinon (0.833, 0.500, 0.416, 0.312 and 0.250 mmol) were applied to obtain diazinon/MAA molar ratios of 1/3, 1/5, 1/6, 1/8 and 1/10 in preparation of MIP1, MIP2, MIP3, MIP4 and MIP5, respectively. After sonication for 5 min the mixture was sparged, in an ice bath, with oxygen-free nitrogen for 5 min and heated at 60 °C for 24 h to complete polymerisation. The resultant rigid polymer monoliths were powdered and sieved through 200 and 400 mesh stainless steel sieves (particle size between 38 to 75 lm). After washing the polymer particles with a methanol– ACN (7:3, v/v) mixture (4 25 mL) and finally with methanol (5 25 mL), the mixtre was centrifuged at 3000 rpm for 10 min and the supernatant was analysed by HPLC. The washing step was continued until no diazinon or other chemicals were detected in supernatant. Non-imprinted polymer (NIP) was prepared under the same condition described above but in the absence of diazinon. 2.3. Batch adsorption procedure The binding properties of polymers were examined by batch adsorption experiments. Dry polymer (10 mg) was added to 1.5 mL methanol–water (pH = 10) (75:25, v/v) with diazinon and was shaken for 24 h at room temperature. After centrifugation of the solution (3000 rpm for 10 min) the concentration of diazinon in supernatant was analysed by HPLC. The amount of bound diazinon was measured from the initial and final concentrations in solution after equilibrium. Each experiment was repeated four times and mean ± SEM (standard error of mean) was reported. The imprinting factor (IF) was obtained from Eq. (1) (Hadizadeh, Zakerian, et al., 2013):
K MIP IF ¼ K NIP
ð1Þ
where K value was the partition coefficient for MIP or NIP and calculated in (Hadizadeh, Zakerian, et al., 2013):
Bound Diazinon=g polymer K¼ ½Free
ð2Þ
where [Free], was the final concentration of free unbound diazinon in the solution and bound diazinon was the amount of diazinon bound to per gram dry polymer after equilibrium.A Scatchard plot was drawn (Eq. 3) when the diazinon concentration was varied in the solution (Hadizadeh, Zakerian, et al., 2013):
Bound Bound Bmax þ ¼ ½Free KD KD
ð3Þ
The [Bound] value was the amount of diazinon bound to polymer after equilibrium; [Free] was the final diazinon concentration at equilibrium; Bmax was the maximum binding sites and KD represented the dissociation constant. The values of KD and Bmax could be obtained from the slope and intercept of the line drawn in a plot of Bound/Free vs. Bound. 2.4. Optimisation of MISPE protocol 150 mg of polymer (MIP5 or NIP), was suspended and packed into an empty polypropylene SPE cartridge. The column was regenerated with 3 mL methanol-acetic acid (90:10 v/v), 10 mL dichloromethane and 5 mL ACN and then conditioned with 5 mL methanol
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and 6 mL deionized water. An aqueous solution of diazinon (10 ppm) was prepared and 0.5 mL of this solution was loaded onto the column. Washing solvent was then transmitted through the column. Different solvent compositions [ACN, water, methanol– water (pH = 10.6, NaOH 0.025 M)] (Table 1) were studied to find a solvent with maximum selectivity for diazinon in washing step. Finally, 2 mL ACN was applied in elution step to complete the extraction of diazinon. The loading, washing and eluting fractions were analysed by HPLC.
2.5. Extraction of diazinon from cucumber and aqueous samples A polypropylene SPE cartridge was packed with 150 mg polymer (MIP5 or NIP) and then regenerated [with 3 mL methanolacetic acid (90:10, v/v), 10 mL dichloromethane and 5 mL ACN] and conditioned with 5 mL methanol and 6 mL deionized water. Cucumber was purchased and collected from different markets of the city (Mashhad, Iran), washed completely with deionized water and chopped into very small pieces. Then homogenised with manual homogenizer (Heidolph laborata 4003 digital) (3 15 min) and sonicated (Branson 5510) for 10 min to have perfect homogenised mixture. Then 5 g mixture was weighed accurately, mixed with 5 mL methanol–water (pH = 10.6, NaOH 0.025 M) and shaken for 1 h at room temperature. Then the mixture was centrifuged (6000 rpm for 10 min at -5 °C) with refrigerator-centrifuge (Hettich Zentrifugen universal 320R). 7 mL supernatant solution was loaded onto the MISPE column. Washing solvent was then passed through the column as described in method 9 [1 mL deionized water, 1 mL deionized water-ACN (70:30, v/v), 5 mL methanol–water (pH = 10.6, NaOH 0.025 M)ACN (40:40:20, v/v/v)]. Finally, 2 mL ACN was applied to perform complete extraction and elution of diazinon. For aqueous samples; 7 mL diazinon solution was loaded onto the column. The washing and elution step were the same as described above for cucumber samples. The final eluted fraction (2 mL ACN) was then dried under oxygen-free nitrogen stream and then resolved in 0.2 mL methanol before injection into HPLC. The concentration of diazinon was determined by HPLC.
2.6. Standard and calibration solutions A 10 mg diazinon (8.94 lL) was dissolved in 10 mL methanol to prepare stock solution (1000 ppm). The standard solutions used for spiking calibration samples were prepared from the stock solution by diluting with methanol. 100 lL of standard solutions was added to 900 mg homogenised cucumber tissue or deionized water to obtain calibration standards (0.025, 0.05, 0.1, 0.5, 1, 5 and 10 ppm) in aqueous and cucumber samples.
2.7. HPLC analysis Chromatographic determination of diazinon was carried out by a Younglin Acme 9000 system (South Korea), consisting of SP930D solvent delivery module, SDV50A solvent mixing vacuum degasser, column oven CTS30, UV730 dual wavelength UV/VIS detector set to 245 nm and ODSA C18 (4.6 150 mm, 5 lm) column. The data analysis was performed by Autochro- 3000 software. The injection volume was 20 lL, the flow rate was 0.8 ml/min and the column temperature was fixed at 30 °C. An isocratic method was used, and mobile-phase composition was ACN–methanol–water (80:10:10, v/v/v). 3. Results and discussion 3.1. Synthesis of MIPs Molecular recognition of the template molecule by imprinted polymers is based on the intermolecular interactions between the template molecule and functional groups in the polymer (Sun et al., 2008). Choice of functional monomer is one of the most important factors in preparation of MIPs (Alizadeh et al., 2009; Sun et al., 2008). Our previous studies indicated that the selection of a proper functional monomer leads to synthesise an MIP with strong intermolecular interactions between template molecule and MIP (Hadizadeh, Hassanpour Moghadam, et al., 2013; Hadizadeh, Zakerian, et al., 2013). Due to the presence of nitrogen, oxygen and sulphur sites in the chemical structure of diazinon and carboxylic acid group in MAA, the strong electrostatic and hydrogen bond interactions could be formed in organic and aqueous media. According to these facts and our previous experiments, MAA was selected as a functional monomer in molecular imprinting process. Solvent plays an important role in formation of monomer-template complex (Sahebnasagh et al., 2013). The polarity of the solvent influences the extent of the non-covalent pre-polymer complex before and during polymerisation. Aprotic solvents with low polarity e.g. chloroform are usually applied as the media to increase complex formation and facilitate non-covalent interactions (monomer-template) such as hydrogen bonding in imprinting process (Mohajeri, Karimi, Aghamohammadian, & Khansari, 2011). On the other hand, more polar solvents especially protic ones tend to dissociate the non-covalent weak interactions in the pre-polymer complex (Sahebnasagh et al., 2013). In the present work, the chloroform was applied as an organic solvent to optimise the molecular imprinting process. Another factor is the optimised template/ monomer molar ratio. Our previous works showed that the optimised ratio is usually obtained empirically (Hadizadeh, Zakerian, et al., 2013). In this work, MIP5 with diazinon/MAA molar ratio of 1/10 showed the most specificity in rebinding test and was selected as the optimised MIP. According to our data, spatial
Table 1 Composition and volume of the solvents used in washing and elution procedure. Methods
Washing procedure STEP1
1 2 3 4 5 6 7 8 9
3 mL 2 mL 1 mL 1 mL 1 mL 1 mL 1 mL 1 mL 1 mL
deionized deionized deionized deionized deionized deionized deionized deionized deionized
Elution procedure STEP2
water water water water water water water water water
1 mL 1 mL 1 mL 1 mL 1 mL 1 mL 1 mL 1 mL 1 mL
ACN (water/ACN, (water/ACN, (water/ACN, (water/ACN, (water/ACN, (water/ACN, (water/ACN, (water/ACN,
STEP3 5/5, 7/3, 7/3, 7/3, 7/3, 7/3, 7/3, 7/3,
v/v) v/v) v/v) v/v) v/v) v/v) v/v) v/v)
1 mL methanol 2mL (methanol/water,5/5, v/v) 1 mL (methanol/water,5/5, v/v) 1.5 mL (methanol/water,7/3, v/v) 2 mL (methanol/acetic acid,9/1, v/v) 2 mL (methanol/water/acetic acid,5/4/1, v/v/v) 5 mL (methanol/water/ACN,5/4/1, v/v/v) 2 mL (methanol/water/ACN,5/3/2, v/v/v) 5 mL (methanol/water/ACN,4/4/2, v/v/v)
2 mL 2 mL 2 mL 2 mL 2 mL 2 mL 2 mL 2 mL 2 mL
ACN ACN ACN ACN ACN ACN ACN ACN ACN
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specific than other MIPs. Thus, MIP5 was selected for development of solid-phase extraction of diazinon from cucumber matrix.
3.2. Rebinding test and Scatchard analysis
Fig. 1. Binding of Diazinon to 10 mg of the MIPs and NIP polymers in 1.5 mL methanol–water (pH = 10) (75:25, v/v), (n = 4). Each point represents mean ± SEM; diazinon concentration in was 20 lg L1.
The conventional batch adsorption method involves the incubation of ligand solution with polymer at different concentrations and measurement of ligand bound to polymer after a fixed time (Dineiro et al., 2006; Mohajeri et al., 2011; Pap & Horvai, 2004). The data showed that all MIPs had higher affinity for diazinon than NIP polymer. According to these results MIP5 had superior binding properties than other MIPs (Fig. 1).The IF values of MIP1, MIP2, MIP3, MIP4 and MIP5 were 8.53, 8.97, 10.11, 12.08 and 37.86, respectively. The data indicated that the IF of MIP5 is significantly higher than that of other MIPs. Therefore, this polymer was selected as the optimised MIP for Scatchard analysis and extraction procedure. Also diazinon binding to MIP5 was more than NIP at all concentrations (Fig. 2). From the Scatchard plot and equation, two kinds of binding sites were discerned in MIP5. One kind represented low affinity binding sites (Y ¼ 0:0013Xþ0:1254; R2 ¼ 0:8424) with dissociation constant (KD) and maximum binding capacity (Bmax) values of 1000 lM and 125 lmol/ g MIP, respectively; while the data for high affinity binding sites (Y ¼ 0:0818X þ 0:7192; R2 ¼ 0:9795) were 12.34 lM (KD) and 8.87 lmol/g MIP (Bmax), respectively. The KD value of MIP-template interactions in other studies ranged from low lM to M (histamineMIP KD = 11.11 lM(Sahebnasagh et al., 2013), theophylline-MIP KD = 1.5 M (Sun et al., 2006)). The small KD in this work (12.34 lM) was low enough to be selected as a sorbent in solidphase extraction of diazinon from biological fluids.
3.3. Optimisation of the MISPE procedure
Fig. 2. Adsorption isotherm of MIP5 and NIP using batch adsorption test (n = 4). Each point represents mean ± SEM. Experiment conditions: 10 mg of polymer was incubated in 1.5 mL methanol–water (pH = 10) (75:25, v/v), (n = 4), with different concentrations of diazinon for 24 h at room temperature.
arrangement of MAA around the template and non-covalent interactions between MAA and diazinon in pre-polymerization solution is significantly more effective in diazinon/MAA ratio of 1/10. Therefore, the imprinted cavities and binding sites in MIP5 work more
Washing procedure is the most important step in solid-phase extraction method. We tested different solvents to obtain maximum selectivity and recovery of diazinon (Table 1). The highest recovery and selectivity was obtained in method 9 (100% recovery for MIP and 0% for NIP). Thus, the composition of [1 mL deionized water, 1 mL deionized water–ACN (70:30, v/v), 5 mL methanol– water (pH = 10.6, NaOH 0.025 M)-ACN (40:40:20, v/v/v)] was selected as the optimised solvents in washing steps (Table 2). In our previous works we have used a solvent or a mixture of solvents to optimize the washing step in solid-phase extraction process (Sahebnasagh et al., 2013). Also, ACN was strong enough to completely elute diazinon from MIP column; thus 2 mL ACN was selected as eluting solvent.
Table 2 The percentage recovery of diazinon on MISPE and NISPE procedures in washing and elution steps. Methods
Washing procedure STEP1
1 2 3 4 5 6 7 8 9
STEP2
Elution procedure STEP3
Total
MISPE (%)
NISPE (%)
MISPE (%)
NISPE (%)
MISPE (%)
NISPE (%)
MISPE (%)
NISPE (%)
MISPE (%)
1.91 ± 0.09a 0.86 ± 0.15 0.00 0.00 0.00 0.00 0.00 0.00 0.00
2.28 ± 0.35 1.06 ± 0.20 0.00 0.00 0.00 0.00 0.00 0.00 0.00
72.53 ± 3.0 8.06 ± 0.20 0.00 0.00 0.00 0.00 0.00 0.00 0.00
81.24 ± 2.7 8.36 ± 0.25 0.00 0.00 0.00 0.00 0.00 0.00 0.00
18.56 ± 0.60 43.70 ± 2.85 32 ± 2.64 41.10 ± 1.62 20.70 ± 1.58 44.71 ± 3.99 8.42 ± 0.20 1.92 ± 0.07 0.00
12.11 ± 1.42 48.36 ± 1.26 36.20 ± 1.98 61.37 ± 3.25 24.53 ± 1.47 53.01 ± 2.20 96.3 ± 1.01 98.37 ± 0.55 100.03 ± 0.07
93.01 ± 3.70 52.63 ± 3.21 32 ± 2.64 41.10 ± 1.62 20.70 ± 1.58 44.71 ± 3.99 8.42 ± 0.20 1.92 ± 0.07 0.00
95.63 ± 4.48 57.8 ± 1.72 36.20 ± 1.98 61.37 ± 3.25 24.53 ± 1.47 53.01 ± 2.20 96.3 ± 1.01 98.37 ± 0.55 100.03 ± 0.07
6.99 ± 3.62 47.36 ± 2.81 68 ± 2.64 58.89±1.62 79.29 ± 1.58 55.29 ± 3.99 91.57 ± 0.2 98.07 ± 0.07 100.00
a All experiments were carried out in triplicate and the results are expressed as mean ± standard deviation. MISPE: molecularly imprinted solid-phase extraction. NISPE: non-imprinted polymer solid-phase extraction.
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Fig. 3. Chromatograms of a standard solution of diazinon (1 lgmL1) before (a) and after solid-phase extraction using MIP5 (b) or NIP (c) polymers as the sorbent. Washing condition for MISPE: 1 mL deionized water, 1 mL deionized water–ACN (70:30, v/v), 5 mL methanol–water (pH = 10.6, NaOH 0.025 M)-ACN(40:40:20, v/v/v), elution condition: 2 mL ACN.
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Fig. 4. Chromatograms of a cucumber tissue spiked with diazinon (1 lg mL1) after solid-phase extraction with MIP5 (a) or NIP (b) as the stationary phase. Washing condition for MISPE: 1 mL deionized water, 1 mL deionized water–ACN (70:30, v/v), 5 mL methanol–water (pH = 10.6, NaOH 0.025 M)–ACN(40:40:20, v/v/v), elution condition: 2 mL ACN.
3.4. Extraction of diazinon from aqueous samples and cucumber Aqueous and cucumber samples were loaded onto the MIP5 column and MISPE procedure was carried out as described before. Aqueous samples: Due to the low sensitivity of HPLC-UV, the minimum concentration determined in standard solutions was 0.5 ppm. Thus, the standard curve was also plotted in the range of 0.5–10 ppm (Y ¼ 22:26X þ 1:2545; R2 ¼ 0:9996). The standard curve was obtained from HPLC-UV analysis of standard solutions prepared in aqueous media without any extraction or preconcentration process. But, calibration curve was plotted after molecularly imprinted solid-phase extraction and HPLC-UV analysis of diazinon in aqueous samples. The calibration curve of HPLC data in the range of 0.025–10 ppm was established (Y ¼ 484:08X þ 40:21; R2 ¼ 0:9983). The recovery of diazinon in MISPE was 77–98%. The limit
of detection (LOD) and limit of quantification (LOQ) of this assay were calculated 0.0037 ppm and 0.025 ppm, respectively. The lowest detectable concentration of diazinon was determined as LOD; while the lowest quantified concentration was selected as LOQ in this assay. Intra-day precision values for diazinon concentrations of 0.025 and 10 ppm, were 3.93 and 1.29%; while inter-day variation values of these concentrations were 4.19 and 2.23%, respectively. The precision values were calculated as relative standard deviation of the aqueous samples (n = 4) in one day (Intra-day precision) or four different days (inter-day variation). These data obviously indicated that MISPE could effectively increase the sensitivity (20 times) of HPLC analysis of diazinon in aqueous samples. Also the precision values demonstrated the repeatability of the assay. Fig. 3 illustrated the chromatograms of a standard solution of diazinon (Fig. 3a), an aqueous sample with the same concentration after MISPE and HPLC analysis (Fig. 3b) and the same aqueous
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sample after doing the solid-phase extraction process using NIP polymer as the sorbent and HPLC analysis (Fig. 3c). This figure indicated the efficacy of MISPE method in sample preconcentration and preparation before HPLC analysis of diazinon; whereas NIP, as the sorbent in SPE, could not extract the diazinon with the same washing and eluting protocol applied for MIP. Cucumber samples: The initial experiments (before diazinon spiking) showed that there was a small amount of diazinon in cucumber samples and we could not find a sample without toxin. Thus, the additional standard method was applied in calibration procedure. It meant that diazinon was spiked into the cucumber tissue which contained a fixed amount (X ppm) of this toxin. The calibration curve of HPLC data in the range of 0.025–10 ppm was established (Y ¼ 343:37X þ 86:42; R2 0:998). According to the equation when Y = 0, then X = 0.251 ppm (equivalent to 0.251 mg/kg) which represented the fixed amount of diazinon in cucumber tissue. The recovery of diazinon extraction was between 82% and 110%. The LOD and LOQ of the assay were 0.0037 ppm and 0.025 ppm, respectively. Intra-day precision values for diazinon concentrations of 0.025 and 10 ppm, were 1.14% and 8.26%; while interday variation values of these concentrations were 3.53% and 9.7%, respectively. According to the results a 15-fold increase in sensitivity was observed in assay after using MISPE before HPLC. The LOQ of this assay (0.025 ppm equivalent to 0.025 mg/kg) was significantly less than MRL of diazinon in cucumber (0.5 ppm equivalent to 0.5 mg/kg). The LOQ in this work is less than the previous values reported (0.05–0.54 ppm) for HPLC-UV developed assays in fruits or agricultural products. Thus, the calibrated method can be applied for analysis in cucumber. These data indicated the sensitivity and repeatability of the calibrated MISPE method for HPLC analysis of diazinon in cucumber. Fig. 4 showed the chromatograms of a cucumber sample after MISPE and HPLC analysis (Fig. 4a) and the same sample after doing the solid-phase extraction process using NIP polymer as the sorbent and HPLC analysis (Fig. 4b). Fig. 4a (compared to Fig. 3a), demonstrated the ability of MISPE method in sample preconcentration before HPLC analysis; while no peak was detected in the chromatogram of the same cucumber sample in Fig. 4b which used NIP as the sorbent in SPE. As mentioned before, the sensitivity of some analytical methods e.g. GC is higher than HPLC-UV in determination of diazinon. The LOQ value in GC procedures ranged from 0.001 to 0.01 ppm (Bagheri, Es’haghi, Es-haghi, & Mesbahi, 2012; Garrido Frenich et al., 2009; Srivastava et al., 2011; Zhao et al., 2011). But, these methods are sometimes time-consuming; need sophisticated and expensive equipments with skilled operators. Obviously, HPLC is easier, more accessible and affordable and cheaper than above mentioned procedures. We used MISPE method to increase the sensitivity to the suitable level. Thus, our developed method could be easily applied with an acceptable sensitivity, accuracy and repeatability for analysis of diazinon in cucumber. Also, the MIP synthesised in our study could be evaluated for preconcentration in other analytical methods such as GC. In our previous works we have also showed that the molecularly imprinted polymers could be effectively applied as the sorbent in solid-phase extraction of other chemicals such as histamine from biological samples (Sahebnasagh et al., 2013). In the next step, the calibrated assay was applied for determination of diazinon in real cucumber samples. 10 samples from different parts of the city (Mashhad) were gathered and tested using MISPE preconcentration procedure. According to our results, the diazinon concentration in cucumber was between 0.264 and 0.295 ppm (equivalent to 0.264–0.295 mg/kg). The amounts of diazinon were less than the MRL (0.5 ppm) (Kabir et al., 2008) that is set by WHO. This study demonstrated the role of molecular imprinting in producing specific cavities for diazinon and its ability
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in sample preconcentration and clean-up before HPLC analysis in cucumber and aqueous samples. Also, the results indicated the efficacy, sensitivity and repeatability of HPLC-UV assay using MISPE procedure for determination of diazinon in real samples. Therefore, the MIP synthesised in this study can be evaluated for extraction and analysis of diazinon in other agricultural products or biological fluids. 4. Conclusion The results obtained in this work confirmed the effect of molecular imprinting technique in preparation of polymer with high affinity for diazinon. The binding properties could be modified by varying the diazinon/MAA molar ratios. The optimised MIP was applied for molecularly imprinted solid-phase extraction (MISPE) of diazinon from aqueous samples and cucumber tissue in the range of 0.025 to 10 mg/kg. The calibrated MISPE method had a recovery of 77–98%, in aqueous solution, and a recovery of 82–110%, in cucumber. The results indicated and acceptable sensitivity, repeatability and accuracy for assay. The intra-day variation and interday variation values were less than 8.26% and 9.7%, respectively. The LOD and LOQ of the assay were 0.0037 and 0.025 ppm, respectively. Finally, the calibrated method was applied for determination of diazinon in real samples in Mashhad. The amounts of diazinon in all samples were less than its MRL level. These results demonstrated that diazinon-specific SPE column characterised by high recoveries and sample preparation factors could solve several problems related to determination of diazinon by HPLC-UV. Acknowledgements We gratefully acknowledge the Vice Chancellor of Research, Mashhad University of Medical Sciences for financial support through grant number 89939. The results described in this paper were part of a PharmD student (Davood Davoodi) thesis. References Alizadeh, T., Zare, M., Ganjali, M. R., Norouzi, P., & Tavana, B. (2009). A new molecularly imprinted polymer (MIP)-based electrochemical sensor for monitoring 2,4,6-trinitrotoluene (TNT) in natural waters and soil samples. Biosensors & Bioelectronics, 25(5), 1166–1172. Aprea, C., Colosio, C., Mammone, T., Minoia, C., & Maroni, M. (2002). Biological monitoring of pesticide exposure: a review of analytical methods. Journal of Chromatography B, 769(2), 191–219. Bagheri, H., Es’haghi, A., Es-haghi, A., & Mesbahi, N. (2012). A high-throughput approach for the determination of pesticide residues in cucumber samples using solid-phase microextraction on 96-well plate. Analytica Chimica Acta, 740, 36–42. Dineiro, Y., Menendez, M. I., Blanco-Lopez, M. C., Lobo-Castanon, M. J., MirandaOrdieres, A. J., & Tunon-Blanco, P. (2006). Computational predictions and experimental affinity distributions for a homovanillic acid molecularly imprinted polymer. Biosensors & Bioelectronics, 22(3), 364–371. Garrido Frenich, A., Martínez Vidal, J. L., Fernández Moreno, J. L., & RomeroGonzález, R. (2009). Compensation for matrix effects in gas chromatography– tandem mass spectrometry using a single point standard addition. Journal of Chromatography A, 1216(23), 4798–4808. Gebreegzi, Y. T., Foster, G. D., & Khan, S. U. (2000). simultaneous determination of carbaryl, malathion, fenitrothion, and diazinon residues in sesame seeds (Sesamum indicum L.). Journal of agricultural and food chemistry, 48(11), 5165–5168. Hadizadeh, F., Hassanpour Moghadam, M., & Mohajeri, S. A. (2013). Application of molecularly imprinted hydrogel for the preparation of lactose-free milk. Journal of the Science of Food and Agriculture, 93(2), 304–309. Hadizadeh, F., Zakerian, A., & Mohajeri, S. A. (2013). Non-covalently lactose imprinted polymers and recognition of saccharides in aqueous solutions. Journal of the Iranian Chemical Society, 10(2), 207–212. Haupt, K. (2003). Imprinted polymers—tailor-made mimics of antibodies and receptors. Chemical Communications, 21(2), 171–178. Heidari, H., Shahtaheri, S., Alimohammadi, M., & Rahimi-Froshani, A. (2009). Trace analysis of xylene in occupational exposures monitoring. Iranian Journal of Public Health, 38(1), 89–99. Kabir, K., Rahman, M., Ahmed, M., Prodhan, M., & Akon, M. (2008). Determination of residue of diazinon and carbosulfan in brinjal and quinalphos in yard long bean
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