This is a post-refereed version of the article published in the Journal of Electroanalytical Chemistry (2018) (no volume or page assigned yet)

Electrochemical determination of 2-isopropoxyphenol in glassy carbon and molecularly imprinted poly-pyrrole electrodes.

Bakhtiyar Qadera,b, Mark Baronb , Issam Hussainc, Jose Gonzalez-Rodriguezb,*

a Sulaimani Medicolegal institute, Qanat street, Sulaimani, Kurdistan regional government, Iraq.

b School of Chemistry, University of Lincoln, Brayford Pool, Lincoln LN6 7TS, UK. jgonzalezrodriguez@lincoln.ac.uk. Fax Number: +441522201109. Telephone: +441522886878.

c School of Life Sciences, University of Lincoln, Brayford Pool, Lincoln LN6 7TS, UK.

* Corresponding author

ABSTRACT: A simple, rapid and sensitive electrochemical method using a molecularly imprinted polymer (MIP) based on the electropolymerisation of Pyrrol (Py) was developed for the determination of 2-isopropoxyphenol (IPP) in model and real samples. The electrochemical behavior of IPP was investigated by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) on bare glassy carbon (GC) electrodes in a Britton-Robinson buffer (pH, 2) solution. IPP exhibited a quasi-reversible behavior on a GC electrode. An anodic peak for IPP showed good linearity over a concentration range from 0.21-75 µM (r2=0.999) with a limit of detection (LOD) of 0.21 µM in DPV. For the theoretical design of the MIP, to screen suitable functional monomers and to optimize monomer-template mole ratio, a computational approach was followed using density functional (B3LYP) and Semi-Empirical Parameterized Model number 3 (PM3) models. Pyrrole monomers in the presence of IPP template were electrochemically polymerized using CV on the working electrode. The sensor exhibited an oxidation peak at 0.737 V and an excellent linearity (r2=0.9969) toward increasing concentration of the template over the range 0.09-45 µM with a LOD of 0.09µM. Intra- and inter-day assay precisions, expressed as %RSD, were overall less than 8.67 % for both methods. The result of the selectivity experiment showed that the imprinted sensor has a good response and selectivity towards IPP. The developed sensors were successfully applied for the determination of IPP in real samples recovered from in vitro metabolism of Propoxur (PPX).

Keywords: 2-isopropoxyphenol, molecularly imprinted polymer, computational chemistry, cyclic voltammetry, differential pulse voltammetry.

1. Introduction

Propoxur (2-isopropoxyphenyl N-methyl Carbamate, PPX) is known as Baygon. PPX is one of the carbamates that are more frequently used these days for eradication of different species of pests, which have a harmful effect on human being and animals such as flies and mosquitoes (Selva and Paixão, 2016; Sun and Lee, 2003). World health organization (WHO) classified PPX as a moderately hazardous pesticide compound and daily intake of 0.02mg/kg was considered harmless to humans (Hardt and Angerer, 1999). Various routes of exposure to PPX, such as water resources, vegetable, fruit and food consumption, absorption through skin (Zafiropoulos et al., 2014) have been reported. PPX, like most of other carbamates, interferes with acetylcholine esterase present in postsynaptic nerve ending of the peripheral and central nervous system and leads to overstimulation of nerve impulses causing a wide range neurological sign and symptoms (Cochran, 1997; Selva and Paixão, 2016).

Previous in vitro study observed that CYP enzymes, in the presence of NADPH, are mainly metabolizing PPX into 2-isopropoxyphenol (IPP), 2-hydroxyphenyl N-methylcarbamate, and W-hydroxymethyl PPX (Sanchez-Arroy et al., 2001). Moreover, it was reported that the major metabolite of PPX in mammals is IPP in a rapid metabolic route via depropylation to O-hydroxyl phenyl N-carbamate and then hydrolysis to IPP (Cochran, 1997; Selva and Paixão, 2016; Suma et al., 2005). Furthermore, in aquatic media (distilled water, drinking water, rain water, river and sea water) two transformation products were detected from the degradation of PPX being IPP and N-methylformamide. PPX degradation was dramatically increased in the presence of irradiation and increasing media pH indicating that hydrolysis of PPX is light and pH dependant (Sun and Lee, 2003).

Parent compound or its metabolites in biological samples are mainly responsible for the pesticide toxicity (Hardt and Angerer, 1999; Schmidt et al., 2013; Forde et al., 2015). A study conducted on Caribbean countries revealed that IPP was observed in seven out of 10 countries with a detection frequency around 30 % among the carbamate metabolites searched (Forde et al., 2015). A linear relationship between PPX oral ingestion and total IPP amount excreted in urine samples has also been proved. Therefore, IPP can be used as a suitable parameter and biomarker for biological monitoring of PPX exposure toxicity (Berman et al., 2016; Hardt and Angerer, 1999).

Many analytical techniques have been mentioned in the literature for measuring and monitoring PPX and IPP in different types environmental and biological samples including spectrophotometry (Kamanavalli and Ninnekar, 2000), High Performance Liquid Chromatography (HPLC) (Khodadoust et al., 2014; ; Koc et al., 2008; Suma et al., 2005), Gas Chromatography-Mass Spectrometry (GC-MS) (Bravo et al., 2005; Corrion et al., 2005; Hardt and Angerer, 1999; Schmidt et al., 2013), Chemiluminescence (CL) (Perez-Ruiz et al., 2007). These methods are labour -intensive, require a great deal of time, high cost and require substantial trained staff. To overcome the shortcomings of the previous methods, an alternative tool should be inexpensive, able to be applied in-field analysis, simple to operate and highly sensitive and selective. Electrochemical techniques have all these properties and can produce methods of choice for the determination of pesticides in various samples. As the presence of pesticide compounds in the environment is a matter of concern for public health, the development of reliable and robust analytical method is imperative for evaluation of human exposure and health risks (Corrion et al., 2005; Kumar et al., 2005).

Electrochemical methods using a DPV technique were developed for measuring PPX using glassy carbon and boron-doped diamond electrodes in various water samples (Selva and Paixão, 2016). Additionally, voltammetric behaviour of PPX was also studied following its alkaline hydrolysis (Ni et al., 2005). To the best of our knowledge no electrochemical method has been reported for the analysis of its major metabolite, IPP. In this work, we developed a voltammetric method for determination of IPP using GC electrode and to achieve greater sensitivity and selectivity, the working electrode was modified with an imprinted polymer (MIP). Modification with MIPs was selected as these can be easily prepared; they are cheap and stable in wide range of chemical and physical conditions (Piletsky and Turner, 2002; Zhao et al., 2013). The basic principle of molecular imprinting is a selective recognition of target molecule in a network of polymeric matrix via its binding sites mimic biological receptors (Alizadeh et al., 2013; Luo et al., 2013). Nowadays computational studies are increasingly used for designing MIP. Various quantum chemical methods such as density function theory (DFT) can be adopted to select the best functional monomer for MIP design and are based on the energy of the monomer-template interaction (Nezhadlia and Mojarraba, 2014; Pardeshi et al., 2013; Tadi and Monghare; 2013). In this study, a DFT-B3LYP with 6-31G, computational approach was applied for the selection of best interacting monomer with IPP and the optimization of the matching monomer-template ratio was done by Semi-Empirical using PM3.

2. Materials and methods

2.1. Chemical and reagents

2-Isopropoxyphenol (IPP), chlorferron (CFN), disulfoton-sulfoxide(DSX), fenamiphos (FNP), strychnine (STN), sodium chloride, glacial acetic acid and pyrrole were obtained from Sigma (Sigma-Aldrich, UK). Phosphoric acid, Hydrochloric Acid and Potassium Hydroxide were all purchased from fisher scientific (Fisher Scientific, UK); Britton-Robinson buffer solution was made up with phosphoric acid, Glacial Acetic acid and Sodium Chloride; the pH value was adjusted with NaOH and HCl. Fumed silica (particle size0.007 μm) and aluminium oxide (particle size 0.05 μm) used for polishing the glassy carbon electrode were both bought from Sigma-Aldrich (Sigma-Aldrich, UK). Acetonitrile (HPLC grade) used in sonication of the GC electrode was purchased from fisher (Fisher Scientific, UK); Water was purified using an ELGA purification system to a specific resistance 18 MΩ and used to prepare all solutions. Artificial human plasma was purchased from sigma (Sigma Aldrich, UK).

2.2. Instruments and Apparatus

Voltammetric experiments were performed using a Metrohm 757 VA Computrace (Metrohm Ltd., UK), data processing was performed using Metrohm version 1.0 Ct757 software (Metrohm Ltd., UK) and run in a personal computer (Compaq® DeskPro, Windows® 95). A conventional three electrode system consisting of a glassy carbon (GC) electrode, as the working electrode, a Ag/AgCl electrode, as reference electrode, and platinum as an auxiliary electrode were used for all the experiments (Metrohm Ltd., UK).

Prior to running all experiments the GC electrode was polished to a mirror-like surface successively with activated aluminium oxide and 0.007 μm silica slurry. The electrode was thoroughly washed with water and then treated with acetonitrile in an ultrasonic bath for about 5 minutes. Electrochemical experiments were carried out in a 50-mL voltammetric cell at room temperature after an initial purging of the solution under nitrogen gas for 300 seconds. A digital pH meter (Hanna instrument microprocessor pH 210 meter) was used when preparing buffer solutions. An ultrasound bath (Kerry, UK) was used for electrode sonication. A digital pH meter (Hanna instrument microprocessor pH 210 m1ter) was used when preparing buffer solutions. An ultrasound bath (Kerry, UK) was used for electrode sonication.

One hundred micro molar individual standard stock solutions of IPP was prepared in acetonitrile and stored at -20 ˚C in the dark bottle. All working solutions were freshly prepared from standard stock solution and kept in plastic bottle at -4 ˚C fridge. All measurements were done using CV in a potential window 0.4 to 1 V with sweep rate of 0.1 V/s and DPV scan was run from 0.5 to 1 V with a scan rate of 0.0248 V/s.

2.3. Modification of a GC electrode with a poly-pyrrole imprinted polymer

The electro-polymerization was performed in an electrolyte solution which contains 4 mM phenol, 1 mM DSN, and 100 mM BR buffer solution (pH, 2). The copolymerization of the Py and IPP were done by cyclic voltammetry in a potential range of -0.6 V to +1 V (vs Ag/AgCl) with a scan rate of 0.1 V/s for 5 scan cycles, after initial purging of the mixture under nitrogen gas for 300 seconds. The IPP molecules were removed from the polymeric film by immersing the MIP electrode into a stirred mixture of acetic acid and acetonitrile at a ratio of 1:5 (v/v). Finally, the molecularly imprinted GC electrode was then dried under nitrogen gas. The non-imprinted polymer (NIP) was also prepared by following the same electro-polymerization and template removal steps but without the presence of the template molecule, IPP, in the electrolyte mixture.

2.4. Computational approach

All calculations were carried out on a computer with 8 GB memory and an Intel ® core ™ i5-6200u CPU @2.30 GHz. Quantum calculations were carried out using Spartan 14, V1.1.4 software. The electronic binding energies were calculated through Density Functional Theory (DFT) and the geometry optimization was performed at the B3LYP/6-31G level. Finally, the molar concentration ratio between template and monomer was studied using Semi-Empirical model (PM3), developed, in order to select the most appropriate ratio. Using Spartan software, the chemical structure of the template (IPP), monomers and all template-monomer complexes were drawn (each chemical structure representing one mole in the polymerisation solution) and calculations for the interaction between the different molecules and complexes created were performed in order to assess their stability and interaction energies.

2.5. In vitro incubation of PPX

Pig Liver microsomes prepared and stored from a previous study (Alshamaileh, 2017) were utilized for the metabolic studies of PPX. Stored Pig Liver microsomes (PLM) at -80 ˚C were thawed and diluted to 10 mg/L. An incubation mixture consisted of PPX (final concentration of 0.250 mM), PLM (0.5 mg/mL microsomal protein), MgCl2 (10 mM), and NADPH system (40 µL) in a total volume of 0.4 mL potassium phosphate buffer (0.1 M, pH 7.4) following previously published study with minor modifications (Usmani et al., 2004). NADPH system was daily prepared by adding (10 mM Glucose 6-Phosphate, 1 mM NADP and 2 U/mL) into 0.1 M potassium phosphate buffer (pH 7.4). Before adding NADPH, the mixture was vortex and incubated at 37 ˚C for 10 min in a shaking water bath. The incubation mixture begins to react by the addition of NADPH for 1 hour. The process was terminated by adding 100 μL ice cold methanol to incubated system. Samples were centrifuged for 10 minutes at 13,000 x G. The supernatant was filtered through 0.45 μm filters and evaporated under blow of nitrogen gas and then reconstituted with 1 mL of BRB (pH, 2) before measuring the concentration of PPX using the developed electrochemical sensor.

3. Results and Discussion

3.1. 2-Isopropoxyphenol voltammetry in glassy carbon electrodes

Voltammetric behaviour of IPP was evaluated by CV at GC electrode in 100 mM BR buffer solution. The CV of IPP (Fig.1) shows one oxidation peak at 0.752 V and two reduction peaks at first scan cycle.

Figure 1. Cyclic voltammograms of 1mM IPP in 0.1 M BR buffer solution pH 2 (a) first scan cycle; (b) second scan cycle) and (c) blank sample. Voltammetric measurement; potential window 0 to 1.2 V, Initial potential 0 V, scan rate 100 mV/s.

The first reduction peak is well-defined and located at +0.484 V while the second reduction peak is slightly faint and located at +0.275 V. On the second scan cycle, a second anodic peak at 0.573 V was observed in addition to previous peaks. This peak appears to correspond to the main reduction peak. This general behavior is similar to that obtained with phenol, (Enache, Oliveira-Brett, 2011), with the quinone-hydroquinone pairs roughly defined and similar to that indicated in figure 1 for IPP. It should be noted that in the oxidation of IPP the predominant product is the para-isomer of quinone.

The first anodic peak was well-defined, more intense than other peaks and linearly proportional to increasing of concentration IPP. Therefore, this peak was used for quantitative analysis of the IPP.

The influence of the pH on the IPP oxidation peak was examined with DPV using the Briton-Robinson buffer in a pH range 3.0-9.0. The peak for the analyte shifted with increasing pH indicating that protons are involved in the overall electrode reactions (Zheng et al., 2005). The peak current intensity was increased with increasing acidity of the buffer solution. The highest current intensity response for the IPP peak was observed at pH 3, as shown in Fig. 2A. Representation of Ep vs pH is linear with a slope of -53 mV/pH (Fig. 2B).

The effect of scan rates on the current response for the oxidation peak of IPP was also investigated within the range 10–1000 mV/s. Representations log Ip vs logv and EP vs logv are linear with slopes of +0.4766 and +52.0 mV/dec, respectively (Fig. 2C y 2D). This peak current is controlled by mass transport (Gowda and Nandibewoor, 2014; Guziejewski et al., 2012) and the peak potential shifted to more positive potentials when the scan rate was increased indicating that the overall electron transfers are quasi-reversible (Asad Ullah et al., 2015; Zheng et al., 2005). Also, this result and the slope obtained in the study with the pH are compatible with a bielectronic electrooxidation and the transfer of two protons in the global reaction. This observation is suggesting that the reaction process at electrode is diffusion controlled.

Figure 2: A) Differential Pulse voltammogram of 0.1 mM IPP at different pH value in 0.1 M BR buffer on bare GC electrode; B) influence of pH on Potential peak of IPP; C) the value of logarithm of peak current versus vs logarithm of scan rates ranging from 10-1000 mV/s for IPP anodic peak; D) Linear dependence of the peak potential of IPP with the inverse logarithm of scan rate ranging from 10-1000 mV/s.

To assess the number of transferred electrons involved in the oxidation reaction of IPP, a plot of peak potential (Ep) versus the inverse logarithm of scan rate (ln ʋ) was done in sweep rate range 10-1000 mV/s that is seen in Fig.2D. The linear relationship was obtained as follow equation:

Ep = 0.0226 ln ʋ + 0.8307, R2=0.982 (2)

On other hand, According to Laviron’s equation (1974).

Ep (3)

Where α is the electron transfer coefficient, R is the gas constant, T the temperature, F is the Faraday׳s constant, K standard heterogeneous rate constant of the reaction, E formal redox potential, and n is the number of electrons. Accordingly, the slope of Ep vs. ln ʋ can be used for calculation of αn. Here the value of is equal to 0.0226 (Asad Ullah et al., 2015; Hong et al., 2013) and from calculation, αn is equal to 1.12.

On other side, based on Bard and Faulkner equation (2002), α can be calculated from this equation:

α= 47.7/Ep-Ep1/2 mV (4)

where Ep/2 is the potential where the current is at half the peak value. For this system, we got the value of α to be 0.59. Hence, the number of electrons (n) is shared in oxidation reaction of IPP is equal to 1.9 to 2. Thus, the proposed mechanism of oxidation for the hydrolysis product of PPX as shown in figure 3 is consistent with the IPP oxidation reaction shown in figure 1.

Figure 3. Proposed electrochemical oxidation- reduction mechanism for IPP at GC

electrode.

3.2. Calculation of the analytical parameters for IPP in GC electrodes

The anodic peak of IPP had a clear response when concentrations of the analyte in the supporting electrolyte solution were increased. The calibration curves showed good linear responses within the concentration range from 0.21 to 75 µM with correlation coefficients of r2=0.999 using DPV. The limit of detection (LOD) was calculated using 3S/P and the limit of quantification (LOQ) was calculated as 10 S/P, where S is the standard deviation of nine measurements of the lowest concentration and P is a slope of linear regression. LOD and LOQ were 0.21 µM and 0.69 µM respectively.

Thus, the sensitivity of the proposed method is nearly the same to those methods routinely used for detection of prenatal exposure to PPX using maternal and umbilical cord blood (Corrion et al., 2005) and even better than other methods utilized determination of PPX based on hydrolysis of PPX with sodium hydroxide (Zanella et al., 2002; Kumar et al., 2005; Ni et al., 2005). The recovered concentration of a 25 µM IPP solution analysed by DPV was found to be 24.55 µM (98.2 %). Intra-day precision (n=5) was found to be 2.87 % and inter-day precision (n=5) was found to be 8.67 % (as %RSD).

3.3. Computational approach for the selection of functional monomer in the fabrication of IPP-MIP.

The basic procedure in the production of MIPs is the formation of a complex between template and a suitable functional monomer. Therefore, the selection of an adequate functional monomer is a key factor for MIP design (Batlokwa, 2011). A Density Functional Theory (DFT) approach at B3LYP/6-311G(d) level was used to find the best conformational optimization to be electrochemically polymerised on a glassy carbon electrode for IPP and 7 functional monomers ([Phenol (Ph), Pyrrole (Py), Aniline (A), 2-aminophenol (OAP), o-aminobenzoic acid (ABA), 3,4 ethelenedioxythiophen (EDOT), 0-phenelendiamine (OPD)]. One chemical structure for each monomer was separately matched with one IPP molecule using DFT in a vacuum. Calculation energies of IPP, Monomers and IPP-Monomer complex were obtained and the binding energy, ΔE was calculated per below equation (Nezhadalia and Mojarraba, 2014):

ΔE = E(template–monomer) − E(template) − ∑E (monomer) (5)

In principle, the most suitable monomer for designing the MIP should have the highest binding energy in the monomer-template complex interaction [Gholivand et al., 2010; Nezhadalia and Mojarraba, 2014). The calculated energy for IPP-Py showed this combination had the highest binding value of -0.006891 kJ/molecule with IPP-OPD being the weakest (-0.004520 kJ/molecule). Therefore, Pyrrole was selected as the best functional monomer for designing an IPP-MIP/GC.

3.4. Fabrication and optimization of the IPP imprinted sensor

The MIP film was prepared by electro polymerization on the surface of a bare glassy carbon electrode using CV in a potential range -0.6 to 1 V and scan rate 100 mV/s in BR buffer solution (pH, 2) (Mamo and Gonzalez-Rodriguez, 2014). For the synthesis of the MIP some factors involving concentrations of functional monomer and template, number of scanning cycles and washing time play an imperative role. Theoretically, the concentration of monomers should be higher than template concentration and an excess concentration may interfere with the formation of the imprinted binding sites and in turn affect the sensitivity of the synthesized MIP (Hrichi et al., 2014; Schweiger et al., 2015). Computationally, functional monomer-template concentration ratios were also optimized using Semi-empirical (PM3) calculations. The calculations showed a ratio 1:4 (IPP:Py) produced the highest binding energy and more stable configurations for IPP-MIP formation (Tadi and Monghare, 2013).

After the optimization of the molar ratio, the thickness of the MIPs during electro polymerisation was controlled by modifying the scan cycles. Generally, less scan cycle leads to formation of thinner polymer films and a fewer number of binding sites. The number of cycles also affects the stability and sensitivity of formed MIPs (Li et al., 2015; Schweiger et al., 2015). In this study, different scanning cycles, including 1cycle, 3 cycles, 5 cycles, 7 cycles, and 10 cycles, were studied. It was found, as seen in Fig 4A, that a 5-cycle polymerization produced the highest current response for the imprinted sensor. Extraction time for the template removal from the generated MIP film was another significant step in the preparation of a viable molecularly imprinted electrochemical sensor. An acetic acid: acetonitrile (1:5 v/v) solution was used to elute the template molecules from the polymer. As shown in fig. 4B, at incubation time zero no peak current can be detected because no recognition binding site formed yet. Then the peak current increased with increasing incubation time from 1 to 10 min and then levels off after 10 mins, suggesting that the template molecules were removed completely from the MIP (Tan et al., 2015). Therefore, a washing time of 10 mins was selected for the following measurements.

Figure 4. Optimization of factors influences MIP preparation on the response of the sensor to 15μM of IPP: (A) number of scan cycles and (B) incubation time.

3.5. Analytical parameters for MIP-IPP/GC sensor

The analytical performance of the IPP-MIP sensor was investigated by DPV in a BR buffer (pH,2). The peak currents were proportional to the increased concentration of IPP in the range of (0.09-45) µM with a correlation coefficient R2= 0.9969. Limit of detection (LOD) and the limit of quantification (LOQ) were calculated based on the IUPAC recommendation (Liu et al., 2016) and were were 0.09 µM and 0.3 µM, respectively, suggesting the developed sensor is more sensitive than the bare GC electrode.

The recovered concentration of a 25 µM IPP solution analysed by DPV was found to be 25.44 µM (101.7%). Intra-day precision (n=5) was found to be 2.30 % and Inter-day precision (n=5) was found to be 4.32 % (as %RSD). In general, the precisions of developed MIP are better than precision on bare GC indicating that developed MIP sensor is more reliable and precise.

3.6. Selectivity Study

To verify the selectivity of the proposed IPP-MIP sensor, current responses were measured by DPV in the presence of four potential interferences, namely chlorferon (CFN), disulfoton-sulfone (DSO), fenamiphos (FNP), and strychnine (STN) used for the assessment of the IPP-MIP/GC sensor selectivity because these substances have the oxidation peaks close to that of IPP and CFN and DSO are metabolites of Coumaphos and Disulfoton pesticides. The selectivity experiments (Table 1) were carried out by detecting the current response of a 10 µM IPP solution using the imprinted sensor in the presence of up to 5-folds concentration of the interference species. All spiked interfering elements did not show significant interference to the IPP oxidation signal. However, with increasing concentrations of interfering species, particularly FNP and CFN, the current response slightly decreased indicating that more than 5-fold of CFN and FNP concentration may influence sensor selectivity slightly (10 µmol/L, the signals change below 16 %). These results confirm that the sensor has a high selectivity even in presence of up to 5-folds interference species due to the highly specific recognition between the binding sites and IPP molecules (Hrichi et al., 2014).

Table 1: Effect of interferences on the differential pulse voltammetric response for different IPP concentrations using the IPP-MIP/GC electrode.

Interferent molecules

Concentration (µM)a

Signal change

(%)b

RSD (%)

(N=3)

Chlorferon

10

30

50

-5.7

-8.6

-15.9

9.73

7.41

3.68

Disulfoton-sulfone

10

30

50

-1.3

-5.0

-8.6

2.09

1.88

2.26

Fenamiphos

10

30

50

-4.2

-10.1

-15.9

2.15

2.29

11.85

Strychnine

10

30

50

-1.3

-3.5

-7.2

3.77

4.28

5.1

a Spiked concentration to 10 µM IPP solution.

b Percent increase of analytical signal following the addition of interferent molecule.

3.7. Application of the electrochemical sensors in real samples

The developed electrochemical methods were used for determination of IPP in real samples prepared from an in vitro study of PPX metabolism. One thousands µL from the incubated samples of this study were evaporated and then reconstituted with 1 mL B-R buffer solution (pH=2). Alkaline hydrolysis of PPX was avoided to get rid of any interference effect of hydrolyzed PPX products on IPP recovered from in vitro metabolism. Hence all results were recorded specific for the oxidation peak of IPP. The sample specimens were measured by DPV at both the bare GC and IPP-MIP sensors and each sample measurements repeated three times. Table 2 includes the results from three different incubated samples. Moreover, the peak current response is much clearly observed at IPP-MIP electrode than GC electrode due to its high selectivity recognition of binding site of IPP molecules. Thus, using both bare and fabricating electrodes together can be used for quantitative and qualitative detection of IPP from in vitro metabolism study.

Table 2: Application of the developed electrochemical sensors for the determination of IPP in real samples.

Sample No

Electrode

Recovered concentration (µM)

RSD (%)

N=3

Sample 1

GC

2.70

16.65

IPP-MIP/GC

2.92

10.58

Sample 2

GC

5.00

19.14

IPP-MIP/GC

7.02

16.97

Sample 3

GC

9.06

8.48

IPP-MIP/GC

9.14

10.04

4. Conclusion

The main objective of this research was to produce a highly sensitive and selective electrochemical method for the determination of different IPP concentrations in different biological fluids. On a bare GC electrode using cyclic voltammetry and differential pulse voltammetry IPP was found to be electroactive and showed two oxidations and two reduction peaks in a quasi-reversible behavior. One oxidation peak of IPP was chosen for quatitative analysis. The peak showed a very good linearity, sensitivity and a reliable precision and recovery in agreement with ICH guidelines. Using computation aid and DFT models, pyrrole monomer was selected as the best monomer for designing a MIP-IPP sensor. The influence of different preparation conditions including the ratio of template/monomer, scan cycles and extraction time were optimized for the sensor. The developed sensor showed a very good response toward IPP molecules and was linearly proportional to increased concentrations of IPP with a low limit of detection that can be used for biomonitoring of its parent compound. The IPP-MIP sensor revealed a high selectivity toward IPP molecules in presence of potential interferences: chlorferon (CFN), disulfoton-sulfone (DSO), fenamiphos (FNP), and strychnine (STN). The applicability of the proposed sensor for the measuring of IPP in real samples was also performed with good recoveries and precision.

Acknowledgements

The authors gratefully acknowledge the financial support of this work by the Higher Committee for Educational Development, Iraq.

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Potential (V)

Currrent (µA)