Preparation of Selective Sensors for Cyproheptadine Hydrochloride based on Molecularly Imprinted Polymer used N , N-Diethylaminoethyl Methacrylate as Functional Monomer

The research involved the preparation of four selective sensors for cyproheptadine hydrochloride using molecularly imprinted polymers (MIPs) method. The process of polymerization was used for preparation of MIP using cyproheptadine hydrochloride as the template with N, N-(diethylaminoethyl methacrylate) (NDMAT) as monomer, N, O-bismethacryloyl ethanol amide (NBMEA) as cross linker, and 2, 2azobisisobutyronitrile (AIBN) as initiator. Preparation of membrane as sensors for electrode construction based on different plasticizers, di-isodecyl adipate (DIA), diisobutylmaleate (DIBM), 2-nitrophenyldodecylether (NPDE) and 3-trimethyltrimellitata (TMTM). The selective sensors were tested and their susceptibility to the estimation of the cyproheptadine hydrochloride, the selectivity coefficients of inorganic ions, sugars, amino acids, and drugs were also studied. The experimental results showed that the best electrode was based on DIA and TMTM as plasticizers, displaying a linear range from1×10-1-3×10-4M and 1×10-1-2×10-4M with a Nernstian slope of 58.4 mV/decade and 55.8 mV/decade, correlation coefficient of 0.9998 and 0.9993. The detection limit was 3.3×10-5 M and 2.4×10-5, the lifetime was around 30 and 48 days respectively. The proposed electrodes were applied successfully to the determination of cyproheptagine in a pharmaceutical preparation.


INTRODUCTION
Molecular imprinting technique has been used successfully and widely to prepare polymers offering high affinity binding sites for a variety of molecules; including organic, inorganic, and even biological molecule or Ions [1].These materials are useful for various applications; such as biomaterials, sensor technologies, molecular and ionic separation, and catalysis.MIPs are recognition materials that have been used in sensors which possess attributes of both of the above class [2].MIPs are synthetic polymers that can be prepared inexpensively and readily often from commercially available starting materials.The recognition properties of MIPs can also be tailored using a molecular templation process as shown in Scheme 1.

/ 11
Several papers were published for drug determination by MIP such as ibuprofen [4,5,6].In this work MIP of cyproheptadine was prepared based on N, N-(diethylaminoethyl methacrylate) monomer and different types of plasticizers.The best electrodes were used for determination of cyproheptadine in pharmaceutical samples.

Synthesis of Imprinted Polymer (CYPH-MIP)
The polymerization process was done in 50 mL thick-walled glass tube with screw cap by dissolving 3 mmol (g) CYPH with methanol (18 mL).A functional monomer N, N-(diethylaminoethyl methacrylate) (NDMAT) 9 mmol (g), cross-linker N, O-bismethacryloyl ethanolamide (NBMEA) 27 mmol and correspondingly initiator 2, 2azobisisobutyronitrile (AIBN) 0.32 mmol (0.0775g) were then added to the tube to be mixed with the solution.The mixture dipped in water bath with a shaker for a period of 30 minutes, and then nitrogen passes through the mixture to remove the oxygen from the mixture solution.After complete removing oxygen from the mixture, the solution put in 45°C water bath to permit starting the polymerization reaction which continued 24 hr.
After the polymer matrix has been formed the particles of CYPH should be removed from the matrix using soxhlet extraction with MeOH/ acetonitrile (90/10, v/v) until the CYPH particles removed completely.To remove residual acetonitrile it must be washed with pure methanol and dried at 55 •C.The resulting bulk rigid polymer was crushed and sieved which obtained particles have size 125 μm used for synthesis the sensing membrane of the electrodes.

Preparation and Measurements of Membrane Electrode
In order to prepare a membrane capable of sensing drug, calculated quantities were mixing; 0.34 g PVC, 0.72 g of the plasticizer (DIA, DIBM, NPDE, and TMTM) and 0.08 g of the MIP.After mixing 7-8 mL of tetrahydrofuran (THF) was added as solvent Follow good stirring in order to obtain homogenization solution.The mixture was poured in a petri dish of 10 cm2 in diameter.To evaporate the THF, a mixture was left at room temperature for 24 hours.A certain quantities of MIP were used to prepare flexible membranes.The sensor membranes must be soaking in 10 -1 M of CYPH for electrode conditioning.Electrode potential was measured after an equilibrium was achieved in a steady state.

Pharmaceutical Samples
Ten grains were taken and weighed thoroughly after they were well grinded by using the mortar and then taking the equivalent of 50 mg of the powder into 100 mL volumetric flask and diluted to 50 mL with the same solvent using magnetic stirrers for 15 min.The solution was filtered by whattmann filter paper, and transferred into a 100 mL volumetric flask to get concentration of 1.0 x 10 -3 M CYPH.

Scanning Electron Microscopy (SEM)
To know the shapes and sizes of MIP particles a scanning electron microscopy (SEM) was used.The morphology of MIP and NIP membranes for cyproheptadine hydrochloride before and after washing is showed by electron microscope in Figure 2.

Measurements and Analysis
To ascertain the formation of the template, a spectral identification must be performed, Fourier-transform infrared spectroscopy (FTIR) spectra of CYPH-MIP before template removal and after template removal were recorded in the range of 400-4000 cm -1 by the KBr disk method as shown in Figures 3, 4 and 5.  From the FTIR spectrum of CYPH-MIP(NDMAT) before template removal showed five variation absorption bands at 3010,2923 cm -1 (C-H) aromatic,1299 cm -1 (C-N) stretching,747 and 711 cm -1 for out of plane-mono-sub when comparing with the FTIR spectrum of CYPH-MIP (NDMAT) after template removal show disappearance the four bands which proved that CYPH was removed from template, shown in Table 1.
Different plasticizers di-isodecyl adipate (DIA), di-isobutylmaleate (DIBM), 2-nitrophenyldodecylether (NPDE) and 3-trimethyltrimellitata (TMTM) used for preparation four electrodes of different compositions based upon the different viscosities.The results of electrode specification were obtained from the calibration curves are shown in Figure 6 and the characteristics listed in Table 2.The results from Table 2 show best characteristics appeared for the sensor with plasticizers DIA and DIBM which have a near-Nernstian with slopes of 58.4 and 55.8 mV/decade and a detection limit of 3.3x10 -5 and 2.4x10 -5 M, respectively.

Effect of pH
These electrodes with different concentrations 1.0 × 10 -2 , 1.0×10 -3 and 1.0 × 10 -4 M CYPH solutions.In this work used (1.0 M HCl and 0.1 M NaOH) for control to the acidic and basic medium by adding small volumes and the potential measured.From the figure Note that the range of the pH of the different concentrations is allowed 3.1-9.7 this is an indication of the work of electrodes in a wide range of medium.

Response Time and Reversibility of the Electrode
Electrode response time is an important feature that must be studied in order to reach the time required for ion exchange, and then the potential is measured taking into consideration the difference in concentration.The CYPH concentrations range 1.0 × 10 -5 to 1.0 × 10 -4 M with about 10 -30 seconds as shown in Figure 8.In this work, it was observed that the response time decreases in the case of high concentrations and increases in the case of low concentrations because access to the equilibrium state in high concentration shorter than the low solutions this proves the response time dependent upon the concentration of CYPH.

Interfering Ions Affection
Two different methods were applied to determine the selectivity coefficient, separate solution method (SSM), which based on Nickolsky-Eisenman equation and matched potential method (MPM) when the involved ions have unequal charges.From the results in Table 3, show the preparation electrodes were selective to CHPY comparing with interfering ions used as inorganic ions, sugars, amino acids, and drugs.The calculated values of selectivity coefficient appeared no interfering obtained between and CYPH and interfering ions.
To remove the effect of interference using the method of standard additions is the best method.The method includes an addition of five times different volumes of standard CYPH to the synthetic solution and the measurements were repeated several times, these methods appear low relative error (%RE) when used to determine the concentration of CYPH.
Classic analysis to determination CYPH was done by used potentiometric titration Technique by used a 10 -4 M N-bromo succinamide (NBS) as a titrant.A typical titration plot was shown in Figure 10.
From the results in Tables 2 and 4 shows the synthesis electrodes I and II were proved to be useful in the potentiometric determination of CYPH in pharmaceutical preparations using different analysis methods.

CONCLUSION
The preparation of sensors for electrodes based on MIP-cyproheptadine using N,N-(diethylaminoethyl methacrylate) as monomer with different plasticizers gave excellent results for determination of the drugs in pharmaceutical samples.Good results were obtained showed high sensitivity, reasonable selectivity, fast static response, long-term stability and low detection limit.

Scheme 1 .
Scheme 1. Schematic representation of the principle of molecular imprinting technology

Figure 2 .
SEM photograph of the surface of MIP, a) after washing b) before washing

Figure
Figure2ashowed a conglomerate consistent of particles on the surface about 5 μm may indicate that the polymer bond is consistent.Figure2bshowed clear holes about 10 μm after removed the particles from template using soxhlet extraction.Micro analysis shows very small particles and spherically shaped polymeric particles with small sizes around (2.50-4.16)µm.

Figure 8 . 3 .
Figure 8. Response time for step changes in concentration of cyproheptadine hydrochloride

Table 2 .
The characteristics of CYPH-MIP ISE selective electrode using (NDMAT) as a functional monomers and different plasticizers

Table 4 .
Determination of cyproheptadine hydrochloride ion standard solutions by different potentiometric technique * Each measurement was repeated three times