International Journal of Drug Development and Research

Key words

Reversed phase HPLC, Paracetamol, Chlorppheniramine maleate, Phenylephrine hydrochloride, Simultaneous estimation

INTRODUCTION:

Combination of chlorpheniramine maleate (CPM), paracetamol (PCM) and phenylephrine hydrochloride (PE) is widely used for cough and cold treatment as an antihistamine, analgesic and decongestant [1]. CPM [(RS)-3-(4-chlorophenyl)-3- (2-pyridyl) propylmethylamine hydrogen maleate] is a histamine H1 antagonist used in allergic reactions, hay fever, rhinitis, urticaria, and asthma. PCM [(N- (4-hydroxyphenyl) acetamide)] is a compound with analgesic and antipyretic properties [2, 3]. It is much safer than aspirin in terms of gastric irritation, ulceration and bleeding. PE [(3-{(1R)-1-hydroxy-2- methylaminoethyl} phenol hydrochloride)] is α- adrenergic agonist used as a mydriatic, nasal decongestant, and cardio tonic agent [4]. Structures of all three drugs depicted in Figure 1. Pharmaceutical formulations for the relief common cold symptoms usually contain a high proportion of acetaminophen and small amounts of phenylephrine hydrochloride and chlorpheniramine maleate this create a problem during simultaneous estimation of drugs.

Literature survey reveled that there have been numerous publications describing different HPLC methods for quantification of these three compounds both individually and in combination with other drugs but few HPLC methods have been used for simultaneous determination of CPM, PCM and PE in cold formulations. The method of Senyuva and Ozden [5] permits the rapid determination of the three actives in combined pharmaceutical dosage forms using a Bondapak CN column nevertheless acetaminophen is not separated from the solvent front, with the corresponding quantification problems, Kanumula et al,[6] that uses wavelength programming and pseudoephedrine hydrochloride as internal standard and the method developed by Krieger [7] for the separation of acetaminophen in analgesic preparations containing chlorpheniramine maleate, phenylephrine hydrochloride, and other active components by HPLC. Olmo et al, developed a method using two cyano column for the separation of these three drugs with impurities, but with high retention time [8].

Hence, aim of present study is to develop a simpler, accurate and selective validated isocratic HPLC method with simple C-8 column for the simultaneous estimation of the three actives in tablet pharmaceutical formulations.

EXPERIMENT:

Chemical and Reagents:

Working reference standard of chlorpheniramine Maleate, Paracetamol and Phenylephrine hydrochloride were kindly gifted by Sun Pharma, Mumbai, India. HPLC grade Acetonitrile, Methanol, Water, Orthophosphoric acid, Di-sodium hydrogen ortho-phosphate was purchased from Rankem, Ranbaxy Fine Chemical Limited, New Delhi, India. Mobile Phase and solution for the injection were filtered through 0.45μm membrane before use.

Chromatographic Conditions:

The HPLC (Perkin Elmer) instrument was equipped with a model series 200 pump, vacuum degasser (PerkinElmer series 200), Rheodyne injector with a 20μl loop and UV-Visible detector (PerkinElmer series 200). Separation and quantitation was achieved on a reversed phase Princeton C8 analytical column (250 x 4.6mm, 5μm particle size) using mobile phase 0.01M Na2HPO4 buffer: acetonitrile with Total Chrom Navigator S 200 Perkin Elmer software. The pH of mobile phase was adjusted at 3 with 50% orthophosphoric acid. The analysis was performed at flow rate of 1 mLmin-1. The system was equilibrated with the mobile phase before injection. The column effluent was monitored on UV detector set at 230nm.

Preparation of Stock and Standard Solutions:

A stock solution of CPM, PCM and PE (1 mg mL-1) were prepared by dissolving 100 mg CPM, PCM and PE in 100ml mobile phase (70:30, 0.01M Na2HPO4 buffer: acetonitrile) Appropriate volumes of the stock solution were transferred to 10mL volumetric flasks and the solutions were diluted with mobile phase to furnish final concentrations of all the three drugs in the range 5–60μg mL-1.

Preparation of Pharmaceutical Formulations for Assay:

Twenty tablets were weighed, their mean weight determined and finely powdered. An equivalent weight to 500 mg Paracetamol, 10mg Phenylephrine hydrochloride and 4mg Chlorpheniramine Maleate weighed and transferred into a 10 ml volumetric flask and diluted to 10mL with mobile phase to get 5000 μgmL-1 PCM, 100 μgmL-1 PE and 400 μgmL-1 CPM stock solution. The resulting solution was centrifuged at 3000 rpm for 5 minute. Further dilution was performed with mobile phase to reach the calibration range for each compound.

Commercial Pharmaceutical Preparation:

Maxtra P (500mg paracetamol, 10mg phenylephrine hydrochloride and 4mg chlorpheniramine maleate) a tablet from Zuventus Healthcare Ltd., India were assayed.

RESULTS AND DISCUSSION:

Method Development and Optimization:

Chromatographic conditions were optimized with pH, mobile phase, wavelength and flow rate for the separation CPM, PCM and PE on a C8 column. During the optimization of the method pH was tested from 6.7 to 2.75 (adjusted by addition of orthophosphoric acid) and two organic solvents (methanol and acetonitrile) were tested. Different percentage of acetonitrile (20 to 70%) was investigated which showed variation in retention of solutes with mobile phase composition follows the usual trends and showed faster elution of all studied compounds. But there was a problem of resolution between compounds. Therefore, a percentage of 30% was selected as an optimum as it offers a compromise between resolution and analysis time. A low pH was used to promote rapid elution and better peak shape of CPM and thus reduce total analysis time. The concomitant effects of optimum eluent composition, pH of the mobile phase and flow rate for the determination of CPM, CM and PE by HPLC were studied.

The conditions finally adopted were a C8 column (250 x 4.6mm, 5μm particle size), mobile phase 0.01M Na2HPO4 buffer: acetonitrile (70: 30) with pH 3, flow rate 1mLmin-1, 20 μl injection volume at ambient temperature. A typical chromatogram of standard drugs and marketed formulation are shown in figure 2 and figure 3 respectively.

Linearity:

The calibration curves were established with seven different concentrations in the range 5-60μg for all the three drugs i.e. CPM, PCM and PE. Each solution was injected three times and a regression equation was established by plotting the peak mean area to concentration of drugs. The best fit for the calibration curve could be achieved by linear regression equation and regression coefficient values (r2) of CPM, PCM and PE was found to be 0.9998, 0.9996 and 0.9996 respectively indicating a high degree of linearity for all drugs.

Specificity:

Specificity is the ability of the method to measure the analyte response in the presence of its potential impurities and degradation products [9]. The specificity studies revealed the absence of any other excipients interference, since none of the peaks appeared at the same retention time, as shown in figure 2. The interaction study of all drugs in standard solution was also carried out by comparing peak of each drug, individually Vs peaks of drug mixture.

Limit of Detection and Quantitation:

The limit of detection (LOD) was the lowest concentration of the analyte in a sample that could be detected under the stated experimental condition and limit of quantitation (LOQ), the lowest concentration of the active ingredients in a sample that could be determined with acceptable precision and accuracy. According to International Conference on Harmonization (ICH) recommendation, the approach based on the standard deviation (SD) of the response and slope (m) was used for determining the detection and quantitation limits [10]. LOD can be calculated according to formula: LOD = 3.3 (SD/m) and LOQ can be calculated according to formula: LOQ = 10 (SD/m). LOD values were found to be 0.36μgmL-1 for CPM, 0.36μgmL-1 for PCM and 0.28μgmL-1 for PE. LOQ values were found to be 1.1μgmL-1 for CPM, 1.1μgmL-1 for PCM and 0.86μgmL-1 for PE. Different statistical data are given in table 1.

Precision:

The precision of analytical procedure expresses degree of the agreement among individuals test when the procedure is applied repeatedly to multiple sampling of a homogeneous sample. Precision are considered at two levels: repeatability (intraday) and intermediate precision (inter-day). Intraday precision was determined by replicates analysis of the sample on one day. Inter-day precision was determined on three successive days and the corresponding results were expressed as the relative standard deviation (RSD) [11]. The RSD values for intra and inter-day precision were varied from 0.06% to 1.45% and from 0.05% to 1.75% respectively. The data of repeatability and intermediate precision are presented in table 2.

Accuracy and Recovery:

The accuracy of an analytical procedure expresses the closeness of agreement between the value, which is accepted either as a conventional true value or an accepted reference value and value found. It was determined by applying the analytical method to synthetic mixture of excipients to which known quantities of the drugs, corresponding to 80%, 100% and 120% of the label claim had been added [12]. Each concentration was analyzed three times and recovery and RSD were calculated. According to the obtained results a good accuracy was observed from this method. The excipients in pharmaceutical formulation do not interfere in the analysis of these compounds in their pharmaceutical formulation. The data of recovery studies are compiled in table 3.

Assay of Pharmaceutical Dosage Form:

The proposed method was successfully used to determine CPM, PCM and PE in their dosage forms. The amount of each compound was calculated using calibration curve method. Satisfactory results as listed in table 4 were obtained for each compound in good agreement with label claims. The results obtained indicate that the content of drug corresponds to the drug label, which confirms good accuracy of the proposed method. Indeed, the assay percentage with respect to label claims were ranged 99.64 to 100.58% which showed that estimation of dosage form was accurate.

System Suitability:

System suitability parameters must be checked to ensure that the system is working correctly during the analysis [13]. System suitability data for the method were determined by analysis of standard working solutions. The resolution, capacity factor, tailing factor, theoretical plates, retention volume and asymmetric factor were determined and listed in table 5. From the table it is apparent that column efficiency is adequate, peaks well resolved from each other and from the void volume.

CONCLUSION:

An isocratic HPLC method has been developed for the determination of chlorpheniramine maleate, paracetamol and phenylephrine hydrochloride in tablet dosage form, in less than 8 minute. The method is simpler and more accurate than the developed methods. Results from validation experiments showed that the method is reliable and accurate and can therefore be used for routine analysis of the tablets for both quality control and assessment of stability.

Tables at a glance

Table 1 Table 2 Table 3 Table 4 Table 5

Figures at a glance

Figure 1 Figure 2 Figure 3

References

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