and Simultaneous determination of paracetamol, dextromethorphan, chlorpheniramine, pseudoephedrine and major impurities of paracetamol and pseudoephedrine by using capillary electrophoresis

A capillary electrophoresis method was developed for the first time and optimized for the determination of paracetamol, pseudoephedrine, dextromethorphan, chlorpheniramine, 4-aminophenol and ephedrine in tablet formulation. Optimum electrophoretic conditions were achieved by using a background electrolyte of 75 mmol L -1 sodium borate buffer at pH 8.0, a capillary temperature of 30°C, a separation voltage of 30 kV and a pressure injection of the sample at 50 mbar for 10 s. Calibration graphs showed a good linearity with a coefficient of determination (R 2 ) of at least 0.999 for all compounds. Intraday and interday precision (expressed as relative standard deviation (RSD) %) were lower than 1.39% for capillary electrophoresis method. The developed method was demonstrated to be simple and rapid for the determination of paracetamol, pseudoephedrine, dextromethorphan, chlorpheniramine, 4-aminophenol and ephedrine in tablet formulation providing recoveries in the range between 99.62 and 100.57% for all analytes.


INTRODUCTION
Paracetamol (PAR) (Figure 1a) is an acetanilide derivative. It is known as chemically 4-hydroxy acetanilide having analgesic, antipyretic and anti-inflammatory action (Devi et al., 2013). Dextromethorphan (DEX) (Figure1b) (3-methoxy-17-methylmorphinan) is a dextrorotatory optical isomer of levomethorphan, a typical morphine like opioid. DEX is an effective cough suppressant with negligible side effects at antitussive dose range. However, DEX produces a very complex pharmacological profile at very high doses (Tran et al., 2016) Chlorpheniramine (CHL) (Figure 1c), is a powerful first-generation alkyl amine antihistamine that antagonises the H1-receptors. Its chemical name is 3-(p-chlorophenyl)-3-(2-pyridyl)-N,Ndimethyl propylamine. It is widely used for symptomatic relief of the common cold and allergic rhinitis with weak sedative properties (Jain, Sharma, 2015). Pseudoephedrine (PSE) (Figure 1d) is (1S,2S)-2-(methylamino)-1 phenylpropan-1-ol hydrochloride. It is a stereoisomer of ephedrine and has similar action. PSE is orally used for the symptomatic relief of nasal congestion. Also, it is commonly used in combination with other ingredients in pharmaceutical dosage forms intended for the relief of cough and cold symptoms (Abdelwahab, Abdelaleem, 2017).
Nowadays, miniaturized separation techniques such as CE, capillary electrochromatography (CEC) and micro/ capillary/nano liquid chromatography (micro LC/CLC/ nano-LC) have become greatly popular in pharmaceutical analysis. These separation methods are increasingly utilized in all processes of drug discovery as well as quality control of pharmaceutical preparations (Aturki et al., 2014).
The aim of this work is to develop a rapid and simple CE method for the simultaneous determination of PAR, 4-AP, PSE, EPH, DEX and CHL. Furthermore, the another aim of the study is to prove the accuracy of the method by applying the developed method to the pharmaceutical dosage form.

Preparation of solutions
The powdered sodium borate was weighed at 5.42 g and placed in a 100.0 mL flask and dissolved. Stock solution (150 mmol L -1 ) was stored at 4°C in the refrigerator. 75 mmol L -1 borate buffer solution was prepared from the stock sodium borate buffer solution and adjusted to pH 8.0. The background electrolyte (BGE) as a working buffer solution was prepared by adding 15% acetonitrile to 75 mmol L -1 borate buffer solution (pH 8.0).
The pure PAR in powder form was weighed to 10.0 mg precisely and placed in a 10.0 mL flask. This stock solution was waited for about 5 minutes at 40°C in an ultrasonic bath. Standard PAR solutions were prepared from this stock solution by diluting with BGE solution. Standard solutions of 4-AP, PSE, EPH, DEX and CHL were prepared by the same method. The stock solution of 4-AP was freshly prepared prior to each experiments because of its rapid deterioration and discoloration.
Twenty tablets of Gripamol ® containing PAR, PSE, DEX and CHL were precisely weighed and powdered in a mortar. The tablet powder, which is equal to the weight of a tablet, was weighed precisely and taken to a 100 mL flask. 6.0 mg of EPH and 6.0 mg of 4-AP were added to this flask. Finally, the mixture was completed to 100.0 mL. 10.0 mL of this solution was centrifuged at 5000 rpm for 10 minutes. 2.5 mL of the clear solution was taken by automatic pipette and this solution was completed with 10.0 mL of BGE solution; A solution containing 812.5 μg mL -1 PAR, 5 μg mL -1 CHL, 75 μg mL -1 pPSE, 37.5 μg mL -1 DEX and 15 μg mL -1 EPH and 15 μg mL -1 4-AP was obtained.

Optimization of method
Optimum conditions were investigated by changing the parameters of BGE solution (type of solution, pH and concentration of BGE solution, and effect of organic solvent) and other parameters (capillary diameter, injection time, applied voltage, temperature and detector wavelength) for the simultaneous analysis of PAR, 4-AP, PSE, EPH, DEX and CHL.
Validation studies CE method was developed and validated according to the International Council on Harmonisation (ICH Guideline (Q2A) (R1) Validation of Analytical Procedures: Text and Methodology, 2005) guidelines with respect to specificity, linearity, accuracy, precision, etc. The limit of detection (LOD) value was found according to the concentration in which the S / N (signal / noise) ratio was 3. The limit of quantification (LOQ) value was found by measuring the value in which the S / N ratio was 10. Repeatability studies were carried out in two different parameters. Intra-day precision studies were performed on the same day by using three parallel solutions. Also, inter-day precision studies were performed on different days by using three parallel solutions. For this purpose, the mixture containing the standard compounds at the same concentration was analyzed seven times with the developed method and the RSD (relative standard deviation) values were calculated. Recovery studies were performed to determine the accuracy of the developed method. The accuracy of the method was determined by calculating the mean% recoveries of seven determination of the six compounds at 100 mg tablet -1 . For this aim, standard solutions were added to the known amounts of mixture solution contaning tablet ingredients and impurities. The recovery values were calculated using the calibration lines prepared with standard solutions from the electropherograms obtained from the injection of these solutions.

Selection of the BGE solution and components
In the CE analysis, the separation, sensitivity, migration time and peak shape of the analytes are closely related to BGE solution. Therefore, it is significant to choose a proper BGE solution. Aqueous buffers are mostly used as BGE in CE. In the literatüre (Capella-Peiro et al., 2006;Dong et al., 2005)., phosphate, acetate and borate buffers were found to be suitable for the separation of selected analytes. Phosphate, acetate and borate buffers were prepared at different pH values to determine optimum working buffer solution and sodium borate (pH 8.0) buffer was determined as BGE solution. The strongest acidic pKa values are 9.46 and 10.4 for PAR and 4-AP, respectively. Therefore, they are in neutral form at studied pH. Similarly, the strongest basic pKa values are 9. 52, 9.52, 9.85 and 9.47 for EPH, PSE, DEX, and CHL, respectively. These compounds are in cationic form at studied pH. In CE studies, the change in migration time depends on the cationic, anionic or neutral form of compounds. PAR and 4-AP were in neutral form in this study, therefore, they were detected at a later time than other compounds. Moreover, EPH, PSE, DEX, and CHL were detected at a earlier time than PAR and 4-AP because of cationic forms of EPH, PSE, DEX, and CHL. When the migration times (Table SI) of the compounds were examined, it can be said that pH 8.0 is suitable for analysis.
Simultaneous determination of paracetamol, dextromethorphan, chlorpheniramine, pseudoephedrine and major impurities of paracetamol and pseudoephedrine by using capillary electrophoresis BGE concentrations affect not only the resolution of compounds but also the sensitivity and migration time in the CE (Tang, Xu, Xu, 2019). Therefore, BGE concentration plays an significant role in the analysis. Different sodium borate concentrations on the separation of six compounds have been examined for a better separation. As it is shown in Figure 2, when the concentration of sodium borate was 75.0 mmol L -1 , each compounds can be efficiently separated with a well sensitivity. When the concentration of sodium borate was 25.0 mmol L -1 and 50.0 mmol L -1 some of the compounds were not well separated because of the large velocity of electroosmotic flow (EOF). When the concentration of sodium borate was increased to 100.0 mmol L -1 , the peak shapes became poor. The reason of this may be high electrical conductivity and Joule heat. As a result, the 75.0 mmol L -1 concentration of sodium borate was selected as the optimum concentration for further experiments.
The addition of an organic solvent as a modifier to an aqueous buffer solution offers a number of advantages for CE analysis. The main goal of using organic solvents as an additive to the buffer electrolyte in CE is to modulate separation, selectivity, sensitivity and resolution through direct effects on the physicochemical properties of the buffer electrolyte, the capillary wall zeta potential and the analytes themselves (Yang, Boysen, Hearn, 2004). Besides, increasing the solubility of some analytes, enhancing the stability of In this study, percentage of ACN was changed from 0 to 30% (v/v). As it is shown in Figure S1, when the percentage of ACN is 15%, each compounds can be well separated with a good sensitivity. PAR, 4-AP and PSE have relatively lower logP values than other compounds. When percentage of ACN was increased, shape of these peaks improved.
Capillary temperature was investigated over the range of 15-30°C. Increasing the separation temperature increased both resolution and selectivity. Also, peak shapes were improved. It was seen that 30°C is suitable for separation ( Figure S3). Therefore, 30°C was considered as optimum temperature.

Effect of capillary column and temperature
Another important parameter in CE studies is the capillary column used. During the studies, the effect of capillary diameter was investigated and two capillaries of 50 μm and 75 μm diameter were used for this purpose. It is observed that sensitivity, peak symmetry and resolution are better with 75 μm diameter capillary as shown in Figure S2.

Effect of sample pH, selection of wavelength and injection time
pH can affect the surface charge of analytes, pH of the sample can have a great effect on the migration time. In this study, the effect of sample's pH values were examined in the range of 3.0 to 11.0. As shown in Figure  S4, the peak shapes and resolution is not suitable at acidic or basic pH values. Therefore, pH 7.0 was chosen as the optimal sample's pH (in terms of sensitivity) and was used in further analysis.
Optimization of CE method conditions as well as the selection of optimum detection wavelength was based on peak area which offered the advantage of having a higher reproducibility than would be obtained using peak area or peak height.
Absorption value of analytes was scanned in the range of 190-260 nm. Then, 3D absorption spectrum (absorptionmigration time-wavelength) was recorded in Figure S5. It was found that all analytes absorbed in the range of 192-200 nm and maximum absorption was choosen at 192 nm.
The effect of injection time was investigated by applying a pressure of 50 mbar for 5-20 s. However, increasing the injection time within this range had no noticeable effect on migration time. Despite the fact that well resolution between the peaks was observed at a 20-s of injection time, higher injection times were not applied due deterioration of peak symmetry. Therefore, 10 s injection time was choosen as optimum.
The calibration curves show a linear correlation of the analytical signal for all compounds. The LOD was calculated according to the IUPAC criteria as 3.0 times se/b1 and the LOQ was determined as 10 times se/b1 where se is the square root of the residual variance of the standard curve and b1 is the analytical sensitivity. The LOD values achieved by the proposed methodology is sufficient for the determination of compounds (Muñoz et al., 2019). The precision of the method, which includes the intra and inter day

Analysis of pharmaceutical dosage forms
In order to evaluate the applicability, recovery and possible matrix effect of the proposed CE method, the commercial tablet (Gripamol ® ) with reported amount of 325 mg PAR, 30 mg PSE, 2 mg CHL and 15 mg DEX was examined for assay analysis. Obtained electropherograms were presented in Figure 3. The quantifications (Table III) for each analyte were calculated from the calibration curves.  Table II. Simultaneous determination of paracetamol, dextromethorphan, chlorpheniramine, pseudoephedrine and major impurities of paracetamol and pseudoephedrine by using capillary electrophoresis  In order to determine the accuracy of the developed method, recovery studies were conducted. For this purpose, known amounts of standard PAR, 4-AP, PSE, CHL, EPH and DEX solutions were added to the previously determined mixture of tablet and impurity solutions. The recovery values were calculated by using the calibration lines prepared with standard solutions from the electropherograms obtained from the injection of these solutions. The results of the recovery study are shown in Table III. As it can be seen, the recovery values obtained for each compounds are close to 100%, indicating the accuracy of the developed method.
A combination of PAR, PSE, DEX and CHL are used in pharmaceutical dosage forms for reducing symptoms which are associated with the common cold. There are many studies on determination of these compounds. From the reported methods, they are detected by HPLC, LC-MS/MS, CE or CZE. In this study, a sample preparation procedure such as liquid-liquid extraction and solid phase extraction purification is not required. This article compares the established method with the reported methods, as shown in Table IV. From Table IV, it can be seen that the sensitivity of the proposed method is high and the recoveries is satisfactory. Proposed method can meet the needs of routine analysis. The developed method offers a wider working range especially for PAR. LOQ results show that the developed method is relatively sensitive compared to other methods (Palabiyik, Onur, 2010). 100.14 Simultaneous determination of paracetamol, dextromethorphan, chlorpheniramine, pseudoephedrine and major impurities of paracetamol and pseudoephedrine by using capillary electrophoresis

CONCLUSION
The simultaneous analysis of active substances and excipients in the pharmaceutical preparation is important in routine laboratory experiments. In this study, CE method was optimized and validated for the simultaneous determination of PAR, PSE, DEX, CHL, 4-AP and EPH in pharmaceutical dosage forms. Experimental conditions were optimized by employing a step by step approach. Moreover, this method was fully validated according to the ICH guidelines under optimized conditions. Proposed method was presented numerous advantages, such as use of minimum amounts of organic solvents, rapidity, low cost, simplicity, ease of operation and high selectivity. Good recoveries, high reproducibility and interference-free electropherograms were also achieved. High percentage of recovery results showed that the proposed method was free from interferences of commonly used excipients and additives in the formulations. Finally, new CE method was developed for the first time for analysis of selected compounds. The proposed method is suitable for quality control laboratories where economy and time are essential.