, Mohammed Awadelkareem Hamad2VALIDATION OF HPLC METHOD FOR SIMULTANEOUS DETERMINATION OF PSEUDOEPHEDRINE HCl, GUAIFENESIN, CHLORPHENIRAMINE MALEATE AND DEXTROMETHORPHAN HBr
Abdrhman Mahmoud Gamil1, Mohammed Awadelkareem Hamad2
1Department of Pharmaceutics, Al-Neelain University, Sudan.
2Department of Chemistry, Blue Nile Research Centre, Sudan.
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 400C, 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 r2 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.
Key words: Chemical method validation, chlorpheniramine, chromatographic system validation, dextromethorphan, guaifenesin, pseudoephedrine.
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 application1. 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 robustness2.
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 suppressant3,4. 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 min1. 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. Column 4.6 mm×150 mm, L11, 214 nm detector and 2m/min flow rate1. Many studies to assay Guaifenesin alone and in combination of other drugs had been done using Spectrophotometric methods HPLC methods and volumetric methods5-15. The separation and determination of psudoephedrine, dextromethorphan, diphenhydramine and chlorpheniramine in cold medicines had been done using Non-aqueous Capillary electrophoresis16. 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 achieved17. 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.
Figure 1: The structural formulas of the compounds.
Figure 2: Chromatogram for system suitability.
MATERIALS AND METHODS
Purified water, Blue Nile Research Centre, Sudan. Potassium Dihydrogen Orthophophate and Acetonitrile HPLC grade, Sharlau Chemie, Spain. Triethanolamine 99.8% AR, Chem lab NV; Belgium. Orthophosphoric acid 88% Luba Chemie. Chlorphenir-amine Maleate, Guaifenesin, Dextromethorphan hydro-bromide and Pseudoephedrine working standards and test samples. High Performance Liquid Chromatography, Prominece – LC 2030, Shimadzu, Japan.Software Lab solution, Shimadzu, Japan.Column; insert Sustain C18; 4.6 mm× 250 mm; 3 µm. Electronic Balance AY 220, Schimadzu. pH meter Mi 150; Hanna instruments, Romania. Rocking Shaker SK-330-pro, USA.Sonicator 621.05.003 IsolabograreGmpH instruments, Germany.
Chromatographic System
Column: insert Sustain C18; 4.6 mm× 250 mm; 3 µm.
Flow rate: 0.8 ml/min.
Wavelength 210 nm.
Detector: PDA.
Oven temperature: 40?C.
Injection volume: 20 µL.
Run time: 60 min.
Preparation of 0.2 M Potassium dihydrogen orthophosphate: dissolve 27.218 gram in 700 ml water and complete to 1000 ml.
Preparation of mobile phase: to 550 ml of 0.2 M Potassium dihydrogen Orthophosphate in a 1 litre
volumetric flask add 200 ml of Acetoniltrile, 30 ml of 10% Orthophosphoric acid and 1 ml Triethanolamine
99.8%. 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/cm3= 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 filter16,17.
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.
Table 1: Results of the method Precision.
Figure 3: The peak purity without interference of Placebo and excipients.
Steps on Method Validation18,19
1. Develop a validation protocol or operating procedure for the validation.
2. Define the application, purpose, and scope of the method.
3. Define the performance parameters and acceptance criteria.
4. Define validation experiments.
5. Verify relevant performance characteristics of equipment.
6. Qualify materials (e.g., standards and reagents).
7. Perform pre-validation experiments.
8. Adjust method parameters or/and acceptance criteria if necessary.
9. Perform full internal (and external) validation experiments.
10. Develop SOPs for executing the method in the routine.
11. Define criteria for revalidation.
12. Define type and frequency of system suitability tests and/or analytical quality control
(AQC) checks for the routine.
13. Document validation experiments and results in the validation report.
Table 2: Levels of concentration of Standard µg/ml.
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.
Table 3: Peak area and RSD% for linearity.
Table 4: Linearity Results
Table 5: Results for Accuracy.
Table 7: Results of robustness on change in column temperature.
Table 8: Resolution of peaks at different Temperature
Table 9: Results of Change in the Wavelength.
Table 10: Resolution of peaks at different Wavelengths.
The Peak for Pseudoephedrine is detected at 5.154 min, for Guaifenesin 12.615 min, for Chlorpheniram-ine15.83 min and for Dextromethorphan 50.362 min giving rise to peak purity 99.16%, 92.2%, 94.95% and 96.28% as shown in figures 4,5,6,7 respectively.
The acceptance criteria for the correlation Coefficient r2should be ≥ 0.999 for the range of concentration 75 – 125% of the target concentration. Thus, the method comply the requirement for linearity.
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.
Figure 8: Linearity Chromatograms.
Table 11: Results of robustness on change of flow rate.
Table 12: Resolution of peaks in changing the rate flow.
Table 13: The average and RSD% of peak areas for solution stability.
Accuracy
According to the ICH guide lines Q2 the accuracy is assessed using three replicates of each of the concentrations 50%, 100% and 150% were analyzed for theoretical values, RSD and percent recovery. Since the acceptance criteria is that the measured recovery should be 95%- 105%, so the method comply the requirement for accuracy16,17.
Precision
Repeatability
10 replicates of the sample were used and the mean, stand deviation and relative standard deviation were obtained. The FDA and ICH stated that the RSD should be ± 1% for the drug substance and ± 2% for the drug product. Thus, the method fulfilled the repeatability criterion.
Intermediate Precision
Intermediate precision within laboratory variations had been demonstrated by two analysts, using two HPLC systems on different days and evaluating the relative percent purity data across the two HPLC systems at three concentration levels; 50%, 100% and 150%. The following results were obtained: S1A and S1B is the RSD% of concentration 50% for analysts A and B. S2A and S2B is the RDS% of concentration 100% for analysts A and B. S3A and S3B is the RSD% of concentration 150% for analysts A and B. Two diffident systems at two different days technique were used. S2a + S2b are 0.52, 0.27, 0.09, and 0.17 for the four compounds respectively. S3a +S3b are 0.97, 1.0, 0.34, and 0.21 for the four compounds respectively.
Since the acceptance criterion for intermediate precision is that the results obtained by two analysts using two instruments at different days should have statistical RSD≤2%, thus the method comply the acceptable criteria20.
Robustness
Effect of change in column temperature
Acceptance Criteria for Robustness
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 four-in-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.
ACKNOWLEDGEMENT
The team and manager of Blue Nile Research Centre were greatly acknowledged for their great support and encouragement.
AUTHOR’S CONTRIBUTION
The manuscript was carried out, written, and approved in collaboration with all authors.
CONFLICT OF INTEREST
No conflict of interest associated with this work.
REFERENCES