Presentation on theme: "Capillary Electrophoresis as a Means of Quality Control for Cough Syrup Containing Ephedrine Tumbur Hutabarat Faculty of Biopharmaceutical Sciences (Pharmacognosy),"— Presentation transcript:
Capillary Electrophoresis as a Means of Quality Control for Cough Syrup Containing Ephedrine Tumbur Hutabarat Faculty of Biopharmaceutical Sciences (Pharmacognosy), University of Leiden, The Netherlands
Introduction Due to natural abundance, extracts from Ephedra sinica, Ephedra vulgaris, Ephedra herba and Ephedera equisetina are still being used in medicinal formulation for cough mixture as a source of ephedrine, although chemically it can now be synthesized successfully 1. Besides, its common reputation for diaphoretic purposes in Chinese herbal medicine, 2 now ephedrine get its way to modern pharmaceuticals for exciting nervous system, the systole of blood vessel and the lysis of spasm of bronchial smooth muscle 3. Ephedrine hydrochloride is reported to reduce viscosity of tenacious sputum and is used as an expectorant 4. Nevertheless, cough syrup usually contain a number of active ingredients to cater for therapeutic needs of cough 5. These ingredients along with ephedrine need to be qualitatively and quantitatively identified in pharmaceutical production prior to and after production. Active ingredients including ephedrine of cough syrup have been determined by methods of HPLC 5,6, differential derivative spectroscopy 4, amperometry 7, PVC membrane electrode 8 and atomic emission spectrometry 9.
Above-mentioned methods usually involve long preparative procedures, expensive organic solvents, and require complex instrumentation and difficult data analysis. Capillary electrophoresis, however, seems promising in minimizing the previously mentioned problems. Separation technique of capillary electrophoresis is based on the differences in the mobility exhibited by different molecules in an electrical field 10, even though solute size and pH do have an influence on the mobility 11. Sample of cough mixture will be weighed (about 0.5 gram) and be dissolved in 50 ml of methanol. Then the mixed solution will be ultra-sonicated for about 20 minutes and then vortex mixed. An aliquot of (60µL) of the tablet extract will be added to 100µL of the internal standard and then diluted to 1 ml with distilled water. Stock solution of known amount of cough mixture ingredients will be also prepared similarly to produce a standard curve. The samples will be then hydro-dynamically injected(5 s) at the anodic end of the system which will be runned for 9 minutes. Method
In figure 1., it can be seen that basic set up of CE consist of a high voltage power supply, two buffer reservoirs, a capillary column, and a detector. Apparatus and Reagents Nevertheless, for this proposal modern instrument of an Applied Biosystem CE units (ABI, Model 270 A, San Jose, CA) equipped with a Hewlett Packard integrator (Model HP3395, Palo alto, CA) will be used. The capillary column will be polyimide coated fused silica capillary, 72 cm x 50 µm id with applied voltage of 20kV and detection will be at 220 nm at temperature of 20 ± 1 0 C. High Voltage Power Supply Detector I Plexiglass box Capillary Solvent Reservoirs Washing solvents before and after injection for 20 minutes: NaOH (0.1M) and water, while running solvent used is Phosphate Buffer (0.05 M) adjusted to pH 8.0. Internal Standard used is methyl-p-hydroxy benzoate (1 mg/ml). Figure 1. Basic Set Up of CE
Discussions Figure 2. Expected electropelogram of Cough Mixture containing Ephedrin Figure 3. Electropelogram of Mixture of ephedrine and pseudoephedrin. Peaks: 1 = methyltriphenylphosphonium iodide (0.060 mg/ml; 2 = pseudoephedrine (0.080 mg/ml; 3 =ephedrine (0.080 mg/ml). Obtained by using Buffer containing 0.01M Valine and ajusted with ammonia to pH of 10. Figure 2 shows that all the essential ingredients of the cough mixture can be separated within 9 minutes of the run and hence they can be nicely quantified by comparing their peak ratio. Through this system, actually at higher pH the migration time of the ingredients also increases. The separation Ephedrine by using CE is superior than RP-LC in that it has shorter run time and no peak broadening. 6 The retention time of Ephedrine in Figure 3 is much shorter than in Figure 2. It is obtained by adjusting the pH of the solvent with ammonia which also reported to enhance the absorption signal. 3 By using the proposed system, pH below 7.0 peak overlapping may occur and above pH of above 9, peak broadening might be expected. 11
V eo + - Each particle packing bears its own electrical double layer x Figure 4. Diagramatic representation of electroosmotic flow in uncoated silica capilaries; x = the analyte, V eo = electroosmotic velocity. Phenomenon at an electrified column Cations are attracted towards the cathode and their speed is augmented by the electromagnetic flow. Anions, although electrophoretically attracted toward the anode are swept towards the cathode with the bulk flow of the electrophoretic column. Under this conditions, cations with the highest charge/mass ratio migrate first, followed by cations with reduced ratio. All the unresolved neutral components are then migrated as their charge/mass ratio is zero. Finally, the anions migrate. Anions with lower charge/mass ration migrate earlier than those with greater charge/mass ratio. 12 Li et al. noted that at low pH there were positive charges on the ephedrine molecule and a markable positive mobility on fused silica gel was observed with the help solvent system made of Tris-NaOH-H 3 PO 4 -Acetonitrile. 3 Moreover, a noticeable increase or decrease of plat number is influenced by the manipulation of acetonitrile due to its effect on adsorption and diffusion of analyte and on dissociation of silanol in the inner wall of the capillary.
Quantification by Capillary Electrophoresis The ratio between peak area of ephedrine from the cough mixture and the peak area of known standard (ephedrine from standard curve) can be used to calculate the concentration of the ephedrine in the mixture. Nevertheless, the linear equation generated from the standard curve will be sufficient enough to quantify the concentration of analytes in the capillary electrophoresis; y = a + bx. In this way, handling the data analysis of capillary electrophoresis will be relatively similar to other analytical methods such as HPLC and GC. The use of barium ion as the leading ion and amino acids such as -alanine 13, alanine, valine, isoleucine and leucine as the counter ions. 2 are beneficial in separation of ephedrine from other ingredients. Moreover, in the sample preparation, the mixture is dissolved in methanol which is a modifier to improve the separation of ephedrine as well. 3 In this proposed system, part of polyimide coating of the capilarry column will be burned as the point of detection. Important factors in detection through capillary electrophoresis are light source, aperture size and background light, optical path length and signal amplification. 12
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