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A simple, rapid and precise method was developed for the quantitative simultaneous determination of chlorpheniramine maleate (CPM), phenyleprine hydrochloride (PE), paracetamol (PCM) and caffeine in a combined pharmaceutical dosage form. The method is based on High Performance Liquid Chromatography (HPLC) on a reversed-phase column, Inertsil, ODS (C18), 4.0 mm Ã- 250 mm & 5 Î¼m, using a mobile phase of 0.05M dibasic phosphate buffer and acetonitrile (93: 07; v/v). The chromatographic conditions were - flow rate 1.5ml/min, column temperature 30Â°C and detector wavelength of 215 nm. All the drugs were well resolved on the stationary phase and the retention times were 2.74, 3.48, 9.5 and 26.32 minutes for CPM, PE, PCM and Caffeine respectively. The method was validated and shown to be linear for CPM, PE, PCM and Caffeine with correlation coefficients 0.999, 0.998, 0.999 and 0.999 respectively. The relative standard deviation for six replicate measurements in the tablets was found less than 2%. The method was validated for precision and accuracy. The proposed method was successfully applied to the pharmaceutical dosage forms containing the above mentioned drug combination without any interference by the excipients.
Chlorpheniramine (CPM) (Fig.1,a) [3-(p-chlorophenyl)-3-(2- pyridyl)-N, N-dimethylpropylamine] (C16H19ClN2, C4H4O4) is a powerful first-generation alkyl amine antihistamine, H1-receptor antagonist, widely used for symptomatic relief of common cold and allergic rhinitis, with weak sedative properties ( Paton and Webster, 1983).
Phenylephrine hydrochloride (PE) (Fig.1,b) [(R)-1-(3-hydroxyphenyl) - 2- methylaminoethanol hydrochloride] (C9H13NO2, HCl) use as decongestant and promotes nasal and sinus drainage. It is available as oral tablets, chewable tablets, oral disintegrating tablet, capsules and sachets formulations (Savic et al., 2008).
Paracetamol (PCM) (Fig.1, c) (acetaminophen) [4-hydroxyacetanilide] (C8H9NO2) is one of the most popular over-the-counter analgesic and antipyretic drugs. Acetaminophen is a non-opiate, non-salicylate, centrally and peripherally acting analgesic agent. It is also useful in the management of more severe pain, where it allows lower dosages of additional NSAIDs or opioid analgesics, thereby minimizing overall side-effects. It is also used widely as antipyretic agent. Combined paracetamol treatment may increase the effect and decrease the dose-dependent side-effects of NSAIDs. Addition of paracetamol to NSAID is well tolerated and effective in the treatment of osteoarthritis flare pain. The combination of NSAIDs with acetaminophen holds promise for greater analgesia than either drug alone, but without the increased side effects associated with opioids in ambulatory dental patients (Dongre et al., 2009).
Caffeine (Fig.1,d) [3, 7-dihydro-1, 3, 7- trimethyl- 1H- purine- 2, 6- dione] (C8H10N4O2) is ranked number one drug worldwide and is usually employed as Central Nervous System stimulant. The use of the mixture of paracetamol and caffeine as an analgesic and antipyretic is well established in pharmaceutical formulation (Erdal, 1999).
The High Performance Liquid Chromatography (HPLC) method has been highly used in the quality control of drugs because of its sensitivity, reproducibility and specificity. Although HPLC is the current method of choice for the analysis of multicomponent pharmaceutical formulations, it requires a separation treatment and several injections during analysis. In chromatographic analysis, the main problem of this method involves the optimization of experimental conditions such as selection of column type, temperature of column, variety and composition of mobile phase, selection of specific wavelength and cheap instrumentation. In spite of the fact that this method undoubtedly provides more sensitive determination than the spectrophotometric methods, Reversed Phase Chromatography (RPC) is the first choice for most regular samples (Erdal et al., 2006). RPC is typically more convenient and rugged than other forms of LC and is more likely to result in a satisfactory final separation. High-performance RPC columns are efficient, stable and reproducible. Detection is often easier in RPC especially for UV detectors. Finally, most workers have more experience with RPC than with other HPLC methods (Snyder et al., 1997).
Literature survey shows that several HPLC methods have been reported for paraceatmol and its combinations in pharmaceuticals or in biological fluids. Most of them are used for the determination of binary combinations like PCM-CPM (Celma et al., 2000; Gasco-Lopez et al., 1997), or ternary combinations like PCM-caffeine-codeine (Murat, 2001), PCM-caffeine-propylphenazone (Avramova, 1989), PCM-caffeine-chlorpheniramine (Qil et al., 2002) and PCM-aspirin-caffeine (Hashem, 2010). Several HPLC methods have been reported for estimation of CPM and phenylephrine hydrochloride combinations in pharmaceuticals (Erka and Kartal, 1998; Marin et al., 2002; Heydari, 2008; Cieri, 2006; Amer et al., 2008; Hamide and Tuncel, 2002; Palabiyik and Onur, 2007 and Marin and Barbas, 2004). Few HPLC methods have been reported for the estimation of caffeine in combination like Paracetamol, Caffeine and Dipyrone (Altun M. L, 2002), Paracetamol, Caffeine and Acetylsalicylic acid (Dalibor et al., 2004).
Several Spectrophotometric methods have also been reported for the estimation of CPM and PE combinations (Erk, 2000; Savic et al., 2008; Erka and Kartal, 1998 and Kazemipour and Ansari, 2005). Capillary Electrophoresis method have been reported for the estimation of PCM, CPM and PE combination (Marin and Barbas, 2004), LC-MS method for the determination of paracetamol, caffeine, pseudoephedrine, chlorpheniramine and cloperastine in human plasma (Hao et al., 2010) and H-point standard addition method for the estimation of paracetamol and caffeine (Tavallali and Sheikhaei, 2009) have been reported.
However, there is no RP-HPLC method reported for the estimation of quaternary mixture of PCM, CPM, PE and caffeine. Since these drugs in combination is widely used in the infections of respiratory system, it is important to develop and validate analytical methods for their determination in dosage form. Thus, an attempt has been made to develop and validate HPLC method for the estimation of paraceatmol, chlorpheniramine maleate, phenylephrine hydrochloride and caffeine from the tablet formulation.
Equipment and chromatographic conditions
A high-performance liquid chromatographic system consisted of a degasser DGU-20 A5, pump LC-20 AT, oven CTO-10 ASVP, SPD M-20A photo diode array detector and a SIL-M 20AC prominence auto sampler with data processor LC solution (Shimadzu, Japan). All pH measurements were performed on a m pH System 362 (Systronics, India). Chromatographic separation was carried isocratically at room temperature with an Inertsil ODS C18, (250mm Ã- 4mm i.d., 5Î¼m) column from G. L. sciences Inc., Japan. Mobile phase was prepared by dissolving 6.8 gm of potassium dihydrogen phosphate in 1000 ml of double-distilled water. The buffer solution was sonicated for 10 minutes and finally the volume was making up to 1000 ml with the same. The pH of the dibasic phosphate buffer was adjusted to 4.0 with ortho-phosphoric acid. A mixture of 0.05M dibasic phosphate buffer and acetonitrile in the ratio of 93: 07 was prepared. Finally the mobile phase was filtered through a 0.45 Î¼m membrane filter and degassed for 10 minutes. The injection volumes for samples and standards were 20 Î¼l and eluted at a flow rate of 1.5 ml/min at 30Â°C. The eluents were monitored at 215 nm.
Materials and reagents
CPM, PE, PCM and Caffeine were kindly supplied by Plethico Pharmaceuticals Ltd. (India). Acetonitrile (HPLC grade) from Rankem, India. Methanol (HPLC grade) and Potassium dihydrogen phosphate (A. R. grade) was from Merck Mumbai, India and used as obtained. Water was deionised and double distilled. Pharmaceutical dosage form containing CPM, PE, PCM and Caffeine was obtained commercially. Each tablet was labelled to contain 4 mg CPM, 5 mg PE, 500 mg PCM and 30 mg Caffeine.
Preparation of standard solutions
A working standard solution containing CPM 4 Î¼g/ml, PE 5 Î¼g/ml, PCM 500 Î¼g/ml and Caffeine 30 Î¼g/ml was prepared by dissolving CPM, PE, PCM and Caffeine reference standard in diluents (Acetonitrile : Buffer, 50:50v/v). The mixture was sonicated for 15 minutes or until the reference standard dissolved completely.
Preparation of sample solutions
Twenty tablets, each containing 4 mg CPM, 5 mg PE, 500 mg PCM and 30 mg caffeine were accurately weighed and finely powdered. A quantity of powder equivalent to 4 mg of CPM, 5 mg of PE, 500 mg of PCM and 30 mg of Caffeine was accurately weighed and transferred into a 200 ml calibrated volumetric flask. About 50 ml of diluent was added and ultrasonicated for 15 minutes; the volume was then adjusted to the mark. The resulting solution was filtered, using 0.45 Âµm filter to ensure the absence of particulate matter. 5 ml of the filtered solution was diluted to 25 ml in the volumetric flask with the diluent for analysis.
The method was validated for the parameters like system suitability, specificity, range and linearity, accuracy, precision and robustness according to International Conference on Harmonization (ICH) guidelines for validation of analytical procedures (ICH - Guidelines Q2B, 1996).
The system suitability was assessed by five replicate analyses of the drugs at a concentration of 50 Î¼g/ml. System suitability of the method was evaluated by analyzing the repeatability, peaks symmetry (symmetry factor), theoretical plates of the column, resolution between the peaks, tailing factor and relative retention time. The specificity of the chromatographic method was determined to ensure separation of CPM, PE, PCM and Caffeine. Specificity was also determined in the presence of excipients used in formulation, CPM, PE, PCM and Caffeine was spiked (at a concentration of 4 Î¼g/ml CPM, 5 Î¼g/ml PE, 500 Î¼g/ml PCM and 30 Î¼g/ml Caffeine) in placebo and chromatogram was observed and compared with that of reference standard. The purity of the peak was checked using a PDA detector.
The linearity of the analytical method is its ability to elicit test results that are directly, or by a well-defined mathematical transformation, proportional to the concentration of analyte in samples within a given range. The linearity is important to demonstrate that the response of the measurement of detector system is linear over the range of interest of the method. This was determined by means of calibration graph using increasing amounts of a standard solution (80, 90, 100, 110 and 120%) of all four drugs. These standards were tested six times in agreement to the International Conference on Harmonization (ICH). A calibration curve was constructed and the proposed method was evaluated by its correlation coefficient and intercept value, calculated in the corresponding statistical study (ANOVA) (p < 0.05). Characteristic parameters for regression equation (y = mx+c) of the HPLC method obtained by least squares treatment of the results was used to confirm the good linearity of the method developed. The correlation co-efficient between concentration and peak area found must not be less than 0.995.
The accuracy of this method was the closeness of the test results obtained by that method to the true value and established across its range. Accuracy was determined by means of recovery experiments, by spiked addition of active drug to placebo formulations. It was shown that the recoveries were independent of the concentration of the active drug over a reasonable concentration range normally 80 to 120 % of the nominal concentration. The accuracy of the assay was measured by analyzing samples of CPM, PE, PCM and caffeine, by spiking known amount of drugs in the placebo, at three concentration levels (80%, 100% and 120%).
According to the ICH recommendations, precision must be considered at two levels, repeatability and intermediate precision. Repeatability refers to the use of the analytical procedure within a laboratory over a short period of time using the same analyst with the same equipment. On that account, a six-sample replicates were consecutively tested in the same equipment at a concentration of 100% of all four analytes of the regular analytical working value. The intermediate precision expresses the within laboratory variations, was assessed by using different equipments, analysts and days. The relative standard deviation (% RSD) was determined in order to assess the precision of the assay and it was not be more than 2.0 %.
Robustness studies were performed on method precision sample concentration 4 Î¼g/ml CPM, 5 Î¼g/ml PE, 500 Î¼g/ml PCM and 30 Î¼g/ml Caffeine by making slight variations in flow rate, amount of acetonitrile, and detection wavelength changes one at a time.
RESULTS AND DISCUSSION
The use of HPLC methods for the quantitation of drug has become a routine consideration in the quality control of drugs and drug products. The goal of this study was to develop a rapid, easy, accurate, precise and reliable HPLC method for the analysis of CPM, PE, PCM and caffeine from tablets or other formulations using the most commonly employed C18 column with PDA detector.
Methods development and optimization
This isocratic-mode method with PDA detection was developed for the determination of the active ingredients, CPM, PE, PCM and caffeine, at 100% level. Firstly, the reversed-phase column, Inertsil ODS C18, (250mm Ã- 4mm i.d., 5Î¼m) was tested. This column provides efficient and reproducible separation of the components while minimizing solvent usage. Consequently, it was selected for the method development. The system suitability studies were carried out as specified in ICH guidelines.
The mobile phase consisted of dibasic phosphate buffer solution and acetonitrile at various ratios (80:20, 85:15, 90:10 and 93:07 v/v) was tested as starting solvent. The variation at the mobile phase leads to considerable changes in the chromatographic parameters. However, the proportion buffer: acetonitrile at a ratio of 93:07 (v/v) yielded the best results.
In order to study the effect of excipients on quantification of CPM, PE, PCM and caffeine, a placebo was analysed. The results revealed that there is no interference of the excipients. Based on the highest UV absorbance for CPM, PE, PCM and caffeine, 215 nm was chosen for detection of this new HPLC method at which the best detector responses for all substances were obtained.
System suitability testing
Typical system suitability results are summarized in table 1. All the values for the system suitability parameters were within the limits. System suitability test are an integral part of chromatographic methods and are used to verify that the resolution and reproducibility of the system are adequate for the analysis to be performed.
Range and linearity
The range of an analytical method is the interval between the upper and lower analytical concentration of a sample where the method has shown to demonstrate acceptable accuracy, precision, and linearity. The linearity of the method was observed in the expected concentration range demonstrating its suitability for analysis. The calibration curve was constructed with five concentrations (fig. 2). The regression statistics are shown in table 2. The goodness-of-fit (r2) was found to be > 0.999 and value of intercept was less than 2% of the response of 100% concentration in all the cases indicating functional linear relationship between the concentration of analyte and area under the peak.
The accuracy was evaluated by the recovery of CPM, PE, PCM and caffeine at three different levels (80, 100 and 120%). The results of accuracy studies are shown in table 3. Recoveries of CPM, PE, PCM and caffeine were 99.38-101.48%, 100.29-101.68%, 100.65-101.29% and 98.74-100.25% respectively with coefficients of variation ranged from 0.10% to 0.29%, 0.34%-0.85%, 0.21%-0.24% and 0.09%-0.13% respectively which is evident that the method is accurate within the desired range.
Precision is a measure of the ability of the method to generate reproducible results. The precision of this method was determined by repeatability (intra-day) and intermediate precision (inter-day) and was expressed as % R.S.D. of a series of measurement. The experimental values obtained for the repeatability of CPM, PE, PCM and Caffeine in samples are presented in Table 4. The result obtained shows R.S.D. of < 2%, indicating good intra-day precision. Inter-day variability was also calculated from assays on 2 days, a mean R.S.D was < 2% (Table 5). All the data were within the acceptance criteria.
Specificity is the ability of a method to discriminate between the analyte of interest and other components that are present in the sample. For demonstrating the specificity of the method for drug formulation, the drug was spiked, wherein the excipients used in different formulation products did not interfere with the drug peak and thus the method was specific for CPM, PE, PCM and Caffeine (Fig.3). The method demonstrated good resolution between the peaks and was found to be free of interference.
Label claim recoveries from tablets
The proposed method was evaluated in the assay of commercially available tablets containing CPM (4 mg), PE (5 mg), PCM (500 mg) and Caffeine (30 mg). Six replicate determinations (n=6) were carried out on an accurately weighted amount of the pulverized tablets equivalent to 4 mg of CPM, 5 mg of PE, 500 mg of PCM and 30 mg of Caffeine. The label claim found is shown in table 6. Chromatogram of the sample is shown in fig. 4.
A reversed-phase High Performance Liquid chromatographic method was developed and it was validated for the simultaneous determination of Chlorpheniramine maleate, Phenylephrine Hydrochloride, Paracetamol and Caffeine and proved to be more convenient and effective for the quality control and identity of these drugs in pharmaceutical dosage forms. The method is novel, simple, precise, accurate, specific, selective and linear over the concentration range tested (80-120%) with a correlation coefficient better than 0.9991. The good percentage recovery in tablet dosage forms suggests that the excipients present in the dosage forms have no interference in the determination. The percent RSD was also less than 2% showing high degree of precision of the proposed method. So, the method is suitable for the determination of the drugs in tablets without interference from commonly used excipients, and could be used in a quality control laboratory for routine sample analysis.