VALIDATION OF HPLC METHOD FOR SIMULTANEOUS DETERMINATION OF PSEUDOEPHEDRINE HCl, GUAIFENESIN, CHLORPHENIRAMINE MALEATE AND DEXTROMETHORPHAN HBr

Objectives: Pseudoephedrine HCl, Guaifenesin, Chlorpheniramine Maleate and Dextromethorphan HBr combination is a common combination cough syrup. Many validated methods are available for the determination of each compound alone and in combination with other drugs. The local pharmaceutical industry used to analyze such combination in individual assessment which is efforts and time consuming. The objective of this study is to validate a method for simultaneous determinations of the four compounds in one single injection. Methods: HPLC method had been develop using detector at 210 nm, column C18 4.6 mm × 250 mm, 3µm and mobile phase of Potassium dihydrogen orthophosphate, acetonitrile, orthophosphoric acid, triethanolamine and water. The column oven temperature is 40 0 C, flow rate 0.8 ml/min and 60 minutes run time. The method had been validated according to the ICH guidelines with respect to method specificity, linearity and range, precision, accuracy and robustness. Limit of detection, quantitation limit and solution stability had been assessed. Results: The average retention times the 4 compounds are 5.5, 12.63, 15.85, 50.44 minutes. The RSD% is less than 1%, the theoretical plates is more than 2000, the tailing factor is not more than 2 and the resolution between the peaks was found to be above 20. The Method showed an appropriate linearity having correlation coefficient r 2 0.9996 – 0.9998. The RSD% of results for two analysts in two different apparatus in two days was less than 2. The test solution is stable for 48 hours. Conclusion: The method is simple and fulfilled all acceptable criteria for all validation parameters. The method is qualified enough to be used for routine analysis of products containing the four components.


INTRODUCTION
Validation of an analytical procedure is the process by which it is established, by laboratory studies, that the performance characteristics of the procedure meet the requirements for the intended analytical application 1 . As per the ICH guidelines, the validation process of the method includes the specificity, linearity and range, precision, accuracy, solution stability, assay of pharmaceutical product and robustness 2 .

Compounds structural formula:
Pseudoephedrine is a systemic decongestant, Quiafenesin is used as an expectorant and to liquefy the bronchial secretion, chlorpheniramine is used for symptomatic relief of allergy, and dextromethorphan is a cough suppressant 3, 4 . The USP HPLC method for its individual assay uses water/ methanol/glacial acetic acid as mobile phase, 4.6 mm×250 mm column packed with L1 10µm, 276 nm detector and 2ml/min rate flow. The retention time is 7 min 1 . The USP method for assay of solution three or more of Acetaminophen, Chlorpheniramine Maleate, Dextromethorphan HBr and Pseudoephedrine HCL uses menthol/ water, monobasic potassium phosphate, triethylamine, sodium lauryl sulphate and phosphoric acid as mobile phase. 2m/min flow rate 1 . Many studies to assay Guaifenesin alone and in combination of other drugs had been done using Spectrophotometric methods HPLC methods and volumetric methods 5- 15 . The separation and determination of psudoephedrine, dextromethorphan, diphenhydramine and chlorpheniramine in cold medicines had been done using Non-aqueous Capillary electrophoresis 16 . A HPLC method for simultaneous determination of the four compound plus pyrilamine and pheniramine had been performes using Kromasil C18 column, mobile phase of methanol and dihydrogen phosphate at pH 3 and wavelength 220 nm, run time of 13 minutes had been achieved 17 . The objective is to validate a method for quantitative determination of Pseudoephedrine HCL, Guaifenesin, Chlorpheniramine Maleate and Dextromethorphan HBr simultaneously in one single HPLC injection.  8%. Dilute to volume by water and adjust the pH to 3 with orthophosphoric acid or Sodium hydroxide. Preparation of diluent: use the mobile phase as a diluent. Preparation of the Standard: 100 mg Guaifenesin, 30 mg Pseudoephedrine HCL, 10 mg Dextromethorphan and 2 mg Chlorpheniramine maleate working standards into 100 ml volumetric flask, add 60 diluent, shake and sonicate for 5 minutes, cool and make up to volume with diluent. Mix well, transfer to 10 ml to 50 ml volumetric flask make up to volume with the diluent, mix and filter using 0.45 µL nylon syringe filter. Preparation of the Sample: Transfer 2 ml of the sample of specific gravity 1.2779 g/cm 3 = 2.5558 grams to 100 m volumetric flask, add 60 ml diluent, shake well for 10 minutes, make up to volume with diluent, filter using0.45 µL nylon syringe filter 16, 17 . Procedure Equilibrate the column with mobile phase for sufficient time until stable baseline is obtained. Separately inject equal volumes 20µL of the standard preparation and the assay preparation into the chromatographic system, record the chromatogram and measure the areas of the major peaks. Inject the blank once, the standard solution for 6 replicates and the sample preparation in triplicates. The tailing factor for each peak should not be more than 2 and the RSD should not be more than 2.

Figure 3: The peak purity without interference of Placebo and excipients.
Calculate the quantity in percentage by the formula: Where, D is the density in mg/ml, W u is the weight in mg of the sample taken, R u and R s are the peak areas responses from the assay preparation and the standard preparation respectively, P is the potency of tested API in % and L is the labeled quantity. Steps

RESULTS AND DISCUSSION Precision
The Table 1 presents the average of 6 injection of the standard. The RSD% for the retention times and he peaks areas of all substances is less than 1%, the theoretical plates is more than 2000, the tailing factors are not more than 2 and the resolution between the peaks is more than 2. Thus complying, the precision acceptance criteria.

Specificity
Using placebo suspension in the same weight and way of the sample test, following the same procedure, no interference from the placebo was observed at the retention time of the drugs peaks ( Figure 3). Peak purity demonstrates that the observed chromatographic peak is attributed to a single component that the excipients were not interfering with the component peaks at the specific retention time. The acceptance criteria for the peak purity are to be attributed to 90 -100% purity.

Range
The data obtained from the accuracy studies may be used to assess the range of the method. 50% to 150% of the target concentration is utilized.    The tailing factor for compounds should not be more than 2.0. 3. The RSD% of the peaks areas of the replicates of either the standard solution or the compounds should not be more than 2.0%. 4. The resolution between the peaks of the compounds should be ≥ 2.0. The method fulfilled the acceptance criteria as the number of the theoretical plates in all variables is more than 2000, the RSD% of the retention time and peaks area are less than 2.0%, the tailing factor for all peaks of the different variables are less than 2.0 and the resolution between the peaks is more than 2.0. Thus, the method satisfied the requirements for robustness on changing the column temperature, on changing the detective wavelength and on changing the flow rate.

Solution Stability
The test had been carried out by initial testing then after preservation of the test solution for 6 hours, 12 hours, 18 hours, 24 hours and 48 hours. The RSD% for the peaks areas of all compounds is less than 2%, therefore, the standard preparation is stable for 48 hours at room temperature.

CONCLUSION
The analytical method used for determination of Pseudoephedrine HCL, Guaifenesin, Chlorpheniramine Maleate and Dextromethorphan HBr in syrup as fourin-one was found to be consistent and precise and in conformance with the acceptable criteria of validation parameters of specificity, system suitability, linearity and range, precision, accuracy, reproducibility and robustness. The method is fully validated and can be used in routine testing for simultaneous determination of such combination products.