SYNTHESIS OF NEW MOLECULARLY IMPRINTED SOLID -PHASE UESD STYRENE AND ALLYL CHLORIDE BASE FUNCTIONAL MONOMER FOR DETERMINATION OF COCAINE BY GC-MASS AND ITS CLINICAL APPLICATIONS

This study was aimed to synthesize a new molecularly imprinted polymer using a functional monomer of styrene(STY) and allyl chloride(ALC) and N, N-methylene bis-acrylamide (MBAA) as a cross-linker for the selective extraction of cocaine from urine samples. Cocaine is used as a template to create a monolithic solid-phase micro extraction (SPME) fiber. All of these analytical procedures were utilized to extract, preconcentrate, and selectively determine Cocaine and its derivatives (SPME) with gas chromatography and mass spectrometry (GC/MS). Samples taken from a suspected cocaine addict who was given to the medico-legal directorate as a donation (Baghdad, Iraq). The analytes were monitored using UV-Vis , (GC MS), FTIR and Scanning electron microscopy (SEM). The RSD percent for two patients' repeated studies for three measurements range from (1.587-4.545) percent cocaine 20-100 ppm .


‫العراقية‬ ‫الزراعية‬ ‫العلوم‬ ‫مجلة‬
, it is no longer considered a Belite drug due to the notoriety achieved in recent years. Consumption of Cocaine is Numerous health conditions, such as respiratory diseases, neurological deficiency, as well as social challenges and death, are associated with (1, 2). The effects of start was appear within seconds for minutes of use and last anywhere from five to ninety minutes. (1) limited number cocaine has approved medicinal purposes during nose surgery, such the numbing and bleeding decreasing. Cocaine is addictive because of its effect on the brain's reward mechanism. (7) Short period, there is a substantial danger of dependency developing. (8) The risk of stroke increases, infarction myocardial, problems lung, blood infections, and sudden cardiac death in people who smoke it. (9).Polymers have also been manufactured in the same way, and their selectivity has been demonstrated to them when used as highprecision films (10).Since this method is characterized by high selection and economics in consuming biological samples. This method was used in the estimation of other narcotic substances in forensic medicine laboratories such as amphetamine (11)(12)(13).

Mips synthesis:
One mmol template (Cocaine) was diluted in 5 ml porogen (methanol), and 2.5 mmol (STY) ,3 mmol(ALC) was added. After agitating the mixture with an ultrasonic for 10 minutes, 35mmol of cross linker (MBAA) and 0.34 mg of the initiator (benzoyl peroxide) were added, and the solution mixture was bubbled with N 2 gas for 15 minutes before being placed in a water bath at 55°C overnight. Cocaine-MIPs were generated when the polymerization process was completed. In a drying oven, the synthesized MIPs were left for 1 hour at 30 degrees. After that, the combinations were crushed and ground using a mortar and a 125 mesh filter to obtain 125m particles. Use as extraction needles before extracting from the sampler. The prepared (MIP) material was poured into the plastic syringe (column). The urine or standard solution was poured from the column's higher end, with the solution moving at 70 rpm under vacuum.

Procedure sampling
Prepare a stock solution of cocaine at pH 8 with a concentration of (20, 40, 60, 80, 100 ppm) and a flow rate of 70 rpm through Colum. To remove matrix interference, the column was washed twice with 2 mL distilled water and then removed from the MIPs. Sampling device Five ml plastic syringe was utilized, the syringe was filled with different weights of MIPs that had been previously ground and sifted (0.1, 0.3, gm) (0.70 microns).

Real sample
Urine samples of suspected cocaine were collected and forwarded to forensic medicine at the judge's order (Baghdad, Iraq). To remove any precipitated material, the centrifuge sample was spun at 5000 rpm for 10 minutes.
Cocaine was immediately impregnated in the urine supernatant, and the non-pointed and squid samples were extracted by column. Extraction procedure MIPs Cocaine solid phase extraction (SPE) column was used to extract cocaine from the urine. A 3 ml plastic syringe was previously filled with MIP to make this column (0.2 g). The supernatant from the centrifuged urine sample was poured into the space above the packing of the SPE column at a flow rate of 1 mL/min (75 rpm). The eluent was collected in a small beaker after 1ml of distilled water and 1ml of methanol/acetic acid (32:8, v/v) were added to the column. The eluent was dried for 10 minutes before adding 1 ml (1:100, v/v) acetic acid: methanol, collecting the eluent in the same beaker, and drying the residue in a water bath at 50°C. Later, the solution was cooled to room temperature, the solvent was evaporated to dryness under stream of nitrogen, and the residues and sample were ready to inject into the GC/MS.

RESULTS AND DISCUSSION
Mips cocaine synthesis Self-assembly (non-covalent) bulk polymerization was used to install two Cocaine Self-assembly (non-covalent) bulk polymerization was used to install two cocaine MIPs. Monomer styrene and allyl chloride were employed to synthesis the MIPs was essential in analyzing interactions with the template. FTIR analysis is an important chemical characterization process Tables 1,2.

Isotherm adsorption
For understanding the mechanism of adsorption template on the polymer surface. The isothermal equilibrium obtained were analyzed to show the isothermal Langmer model or Freundlich models [14]. This is achieved by plotting the binding capacity (Q) against the free concentration of the drug, Q is calculated according to the following equation:

Q = [(C i -C f ) V s ×1000] / M MIP
Isotherm adsorption experiments were carried out by adding 0.1g and 0.3g of cocaine MIP into 15 mL of cocaine concentration ranged from 10ppm to 40ppm. The solution was kept for several hours around 10 hours at room temperature and the solid phase was separated by centrifugation. The remain concentration of cocaine was measured by Uv-Visible spectrophotometer at wavelength equal to 273 nm and the data for adsorption isotherm were used for a Scatchard analysis. Capacity factor was calculated for two cocaine MIPs mass 0.1g and 0.3g using cocaine concentration range from 10 ppm to 40 ppm. Table (2) shows the values of Q, Q/ Cf of cocaine MIPs based on styrene and allyl chloride as monomers and N, N-methylene bis-acrylamide (MBAA) as a cross-linker. important because it determines the time required for extraction. It should be enough to prevent wasting time by controlling the total analysis time. The effect of sample loading flow rate in the 5-50 rpm range was studied to estimate the effect of contact time between MIP and sample solution on recovery as shown in Table (4 and 5).  From the following Table 3, the experiment needs a minimum amount of time in order to preserve time. Complete extraction was achieved at any peristaltic pump flow rate from 10 to 70 rpm. The following Figure (2) shows the flow rate per minute with time in minutes. The time decreased as the flow rate increased and we fixed the flow rate at 70 rpm where the time was 5 min and this time was used in the following experiments: removed in a homogeneous column by washing time from 60 s to 5 min, the matrix peaks were visibly suppressed. This can be demonstrated by extracting an empty urine sample with a 3-minute wash step. The application of the same washing step to the urine sample increased, and satisfactory results were obtained from cocaine: no reduction in this was found. A plastic syringe containing 0.1-0.3 g MIPs (styrene and allyl chloride) were taken while passing different concentration of cocaine into the urine samples. in the 20-100 ppm range successfully under optimal conditions. The results are shows in Table 6.

CONCLUSION
In this study, cocaine was extracted from urine samples using a MIP Cocaine Solid Phase Extraction (SPE) column. Which used styrene and allyl chloride as a monomers. The drug can be estimated based on small concentrations and multiple mixtures. The first step was the preparation of molecularly imprinted cocaine polymers, in which small proportions of the drug and at different times of drug metabolism could be concentrated and quantified. The second step was to obtain a concentration using solid-phase extraction, thus combining a molecularly printed polymer with solid-phase micro-extraction (SPME) to obtain a pre-concentration and a one-step estimation process for better accuracy, sensitivity and selectivity . UV-Vis used to identification the results of extraction parameters . MIP fibers have been successfully applied in the selective extraction of cocaine in urine samples with relative recovery ranging (95-102.5).