Potentiometric Sensor Based on Molecularly Imprinted Polymer for Determination of Tramadol in a Biologic Fluid Model and Urine

In this study, a potentiometric sensor based on a molecularly imprinted polymer (MIP) was developed for determination of tramadol. At optimized condition the electrode exhibited a Nernestian response (30.5±1.0 mV/decade) in a concentration range of (1×10 -6 to 1×10 -2 M). The potential response of the electrode was constant in the pH range of 1.0-9.0. The electrode demonstrated a response time about 15s. Selectivity coefficient of the sensor with structural similarity to tramadol was evaluated by matched potential method (MPM). The electrode was examined successfully for determination of tramadol in biological fluids and urine with a good accuracy and precision


Introduction
Over the last few years, molecular imprinting technique has been received high attention in the field of polymer science and chemical analysis, owing to its predetermined selectivity for target molecular, high affinity and robustness [1,2]. Molecularly imprinted polymer (MIP) can be synthesized in the presence of functional monomers, template molecules and cross-linking agent by covalent, non-covalent and sacrificial spacer methods [3][4][5]. After removing of the template molecules from the polymerized material, binding sites of the MIP with molecular recognition properties are formed.
In this condition, cavities complementary in size and shape to the template for the substance rebinding process will be formed, MIPs can be used in several applications such as chromatographic separation, catalysis facilitation, and artificial reception drug delivery [3,[6][7][8].
Several MIP characteristics such as their stability, less cost ease of producing as compared to their biological counterparts including antibodies and enzymes, have made them to gain wide acceptance as new molecular recognition materials in chemical sensors [9,10].
There are various chemical sensors preparation using MIPs for molecular recognition followed by appropriate signal transduction including capacitance, conductometry and spectroscopy [11][12][13].
For chemical sensing, potentiometric sensors are generic and highly successful. They do not require the template molecules to diffuse through the electrode membranes for generation of membrane potentials have shown to be very promising, unlike sensors based other transduction techniques, so the MIP based potentiometric sensors are very promising and have gotten a rapid response time but still the reports of MIP based potentiometric sensor are rather rare [14][15][16][17][18][19].
Tramadol hydrochloride, is a synthetic analgesic with morphinelike effect causes its analgesic effect, it binding to receptors in the brain and inhibiting reuptake of norepinephrine and serotonin.
Tramadol like other narcotics is a painkiller and if it is abused, may have a therapeutic plasma concentration that is in the range of 100-300 mg/l. Tramadol is rapidly and almost completely absorbed after an administration, but its absolute bioavailability is only 65-70 % [20]. The current work, proposed an innovative

Materials and Methods
Tramadol hydrochloride from Daropakhsh Co. Thermo Nicolet FTIR Spectrometer. A MP225 Metter-Toledo pHmeter was used for the pH adjustments.

Synthesis of MIP and NIP
The MIP was prepared by mixing tramadol (0.25 mmol,) as the template, it was dissolved in (25 ml) chloroform, and MAA

Electrode Preparation
Membrane contained MIP or NIP (58 mg). NaTPB (1 mg), as additive, DOP (60 mg), as plasticizer and 3 mg PVC was dissolved in 2 ml of THF. A Pyrex tube with about 5 mm was dipped into the mixture so that a membrane thickness of 0.4 mm was formed.
The tube was then pulled out from the mixture and kept at room temperature for about 24 h. The tube was then filled with an internal filling solution (10 -3 M tramadol hydrochloride). The electrode was finally conditioned for 24 h.

Emf Measurements
The following sensor was assembled for the conduction of the

Optimization of The Membrane
The influence of membrane composition on the potential response was tested and the results shown in (Table 1). The ratio of the optimized membrane was kept constant in the optimization of membrane composition. It was found that the membrane with NaTPB and DOP provided a better response with a wider linear response range. Experiments showed that the response slop of the MIP based membrane could be improved to achieve theoretical value with the lipophilic borate. The selectivity and linearity of the membrane electrode also depends on the amount of the MIP which determined the number of the binding sites. Table 1 indicates that the membrane with 33 wt % of PVC, 60 wt % of DOP, 1.2 wt % of NATPB and 5.8 wt % of MIP showed the best performance.

Characterization of Imprinted Polymers
After template extraction and washing procedure, the FT-IR

Effect of Internal Solution
The effect of the internal solution concentration on the potential response of the sensor was investigated. The concentration of tramadol was changed between1.0×10 -4 and 1.0 × 10 -3 mol l -1 and the potential responses of the sensor were measured. It was found that the concentration variation of the internal solution showed a significant difference in the corresponding potential response.
Therefore, the 1×10 -3 mol l -1 internal solution which showed the best response to the target molecule was used for smooth functioning of the sensor (Figure 2).

Effect of pH
The pH dependence of the membrane sensor was tested with

Calibration Curve
The potential response curves of MIP and NIP based membrane shown in (Figure 4). The MIP membrane showed a Nernstian response of 30.5±1 mVper decade over the concentration range of 1.0×10 -6 to 1.0×10 -2 mol l -1 . However, the NIP-based and blank membranes could not be observed with NIP and blank.

Interference Studies
Interference studies was determined by matched potential

Detection of Tramadol in Biologic Fluid Model
In order to investigate the applicability of the new sensor to detect the drug in the biological fluids the drug solution was diluted with the biological fluid model sample. The tramadol content of the solution was detected by the proposed electrode. The result of recovery studies summarized in (Table 2).

Discussion
In this study, a new polymeric membrane ion-selective electrode based on MIP for tramadol has been described. The sensor can be used for measuring tramadol in urine and plasma samples. The analytical applicability of proposed sensor was checked by the determination and recovery of tramadol in urine and biologic fluid model and also titration method. Additives could decrease the membrane resistance, reduce anion interference and improve selectivity of the electrode [21]. It is notable that the experimental conditions such as the stirring or flow rate, the ionic concentration and composition of test solution, the concentration and composition of the solution to which the electrode was exposed before experiment measurement was performed, any previous usages or preconditioning of the electrode, and testing temperature have an effect on the experimental response time of sensor [22]. Interference studies method is recommended to overcome the difficulties associated with the method based on the Nicolsky-Eisenman equation [21,22]. The prepared electrode showed promising results which could be applied in improved versions to help in determination of tramadol in various solutions for acceptable analytical purposes.