DEVELOPMENT AND VALIDATION OF A HPLC METHOD FOR THE SIMULTANEOUS DETERMINATION OF PSEUDOEPHEDRINE, DEXTROMETHORPHAN, AND TRIPROLIDINE IN THEIR COMBINED COUGH AND COLD SYRUPS

6011 | P a g e F e b r u a r y 2 0 1 7 w w w . c i r w o r l d . c o m DEVELOPMENT AND VALIDATION OF A HPLC METHOD FOR THE SIMULTANEOUS DETERMINATION OF PSEUDOEPHEDRINE, DEXTROMETHORPHAN, AND TRIPROLIDINE IN THEIR COMBINED COUGH AND COLD SYRUPS Imad Osman Abu Reid a* and Elrasheed Ahmed Gadkariem a a Department of Pharmaceutical Chemistry, Al Ribat National University, Khartoum, Sudan * iabureid@hotmail.com ABSTRACT


INTRODUCTION
Cough and cold medications usually contain a complex mixture of nitrogenous compounds as active ingredients. These are usually present in varying and very different proportions, have diverse properties inherent to their formulation and desired action, and often possess some similar physical and chemical properties, which turns their separation difficult. Moreover, in the case of HPLC analysis, these basic drugs strongly interact with the reversed-phase stationary materials, causing peak asymmetry and lowering separation efficiency [1]. Due to these characteristics, quality control of preparations against the cough and cold always offers an interesting analytical challenge [2].
Pseudoephedrine (PS), triprolidine (TR), and dextromethorphan (DX) are nitrogenous compounds ( Figure 1) widely used as active ingredients in combined cold medicine formulations due to their decongestant (PS), antihistaminic (TR) and cough suppressant (DX) activities [3]. Their commercial associations exhibit important mass differences among the analytes (PS:TR up to 24:1, w/w) , in addition being liquids common formulations of these products require addition of preservatives such as methylparaben (MP), prorylparaben (PP), or sodium benzoate (SB). All these aspects increase the analytical challenge. The use of strong cation exchanger (SCX) materials, such as propylsulfonic acid, which are highly acidic (pKa ˂1) and effectively ionized at all pH values, makes the development of separations for basic compounds relatively straightforward, besides its other advantage related to its specificity for the analysis of basic compounds when present in mixture with acidic/neutral compounds as the latter are either not retained or poorly retained [4][5][6].
Literature survey revealed the interest in the determination of PS, TR, and DX in pharmaceutical formulations either alone or in combination with other active principles . The simultaneous analysis of the three components has been performed by a few methods only, including UV spectrophotometry [30][31][32] and HPLC [33].
The objective of the present work was to develop and validate a high-performance chromatographic methodology for the simultaneous determination of PS, DX, and TR in their combined syrup formulations, using cation-exchange stationary phase. Experimental design techniques as a rational, cost-effective and convenient tool to speed up the process were employed for development of the proposed method [34].
The novelty of the proposed method stems from its ability within a reasonable analysis time, to separate such a complex mixture without interference from the co-formulated additives and without prior sample pre-treatment. The cost effectiveness of the method in term of using inexpensive reagents and chemicals is another benefit added.
Experimental design, data analysis and desirability function calculations were performed by using Design-Expert ® trial version7.0.0. (Stat-Ease Inc., Minneapolis). Statistical analyses were carried using Microsoft Excel 2013 software (Microsoft, USA).

Reagents and Materials
Analytical grade reagents: ammonium acetate (E. Merck, Germany), glacial acetic acid (Scharlau Chemie, Spain), HPLCgrade methanol (Scharlau Chemie, Spain) and double-distilled water were employed for the preparation of the samples and for chromatographic analysis.

Optimization Standard
One ml from a stock solution containing 1.3 mg/mL of triprolidine HCl and 1.0 mg/mL of prorylparaben (PP) in methanol was transferred into a 20 ml volumetric flask containing accurately weighed about 35 mg pseudoephedrine HCl and 20 mg dextromethorphan HBr. To this mixture 10 mL of 50% v/v aqueous methanol diluent were added , content of the flask was sonicated for 5 minutes, cooled and made to mark using the same diluent.

Stock standard solution
Stock standard solution was prepared by initially dissolving accurately weighed about 180 mg pseudoephedrine HCl, 10 mg triprolidine HCl and 60 mg dextromethorphan HBr in 20 mL volumetric flask using 10 mL of 50% v/v aqueous methanol diluent. The content of the flask was then sonicated for 5 minutes, cooled and adjusted to mark using the same diluent.
Other standard solutions were prepared by proper dilution of aliquots from the stock standard solution with 50% v/v aqueous methanol.

Sample preparation
The sample density was determined and then sample weight accurately equivalent to 5 mL was taken in 20 mL volumetric flask, 10 mL of 50% aqueous methanol diluent were added and sample was then sonicated for 5 minutes, cooled and made to mark using same diluent. The sample was filtered through 0.45 µm nylon filter and 20 µl were injected into the HPLC system.

Chromatographic Procedure
Chromatographic separations were carried out on a Partisil 10 SCX column (10 µm particle size, 250 mm×4.6 mm I.D.).During the optimization experiments the mobile phases were prepared to contain different proportions of methanolwater-ammonium acetate, the pH of the mobile phases was adjusted with 10% glacial acetic acid. The detection wavelength was set to 265 nm to obtain maximum sensitivity for quantitation of triprolidine since it is present in small amount in the product; pseudoephedrine and dextromethorphan were also monitored at this wavelength. 20µl were injection volumes were used throughout the experimental work.

Validation
The optimized method was validated in according to the ICH guideline [35], the method linearity in the relevant working ranges, precision, and accuracy were evaluated. System suitability parameters were also determined.

Optimization Design and Analysis
Before starting an optimization procedure, the curvature term was investigated using factorial design with center points investigates. ANOVA generated for 2 k factorial design showed that curvature is significant for all the responses (the resolution between each pair of adjacent analytes peaks ) since p-value was less than 0.05. This implies that quadratic should be considered to model the separation process [36]. In order to obtain second order predictive model Box-Behnken design was employed [37]. The optimum composition of the mobile phase was determined with series of mobile phases containing water: methanol mixtrues in the range of (45 -50% v/v) containing ammonium acetate (35 -45 mM/ L) with the pH being varied between 5-6.
Derringer's desirability function was used to optimize this process since the process involves optimization of three responses at a time [38]. The resolutions between adjacent peak pairs were the responses considered for the purpose of optimization. The use of response surface methodologies ( Figure 2) indicated that the optimum mobile phase composition was 50% v/v methanol-water mixture having ionic strength 35.07 mM/ L and pH 5.58. The elution order of the analytes from the column was PS , DX and TR respectively; this order of elution can be related to their acid dissociation constants (pka) since PS (pka = 9.8) , DX (pka = 8.3) and TR (pka = 6.5) [39]. PS being the storgest base among the three compounds was eluted first, followed by DX and TR respectively, under the optimized experimental conditions. This finding is consistent with the earlier observation [40], that if two bases are fully protonated then the stronger base is eluted before (has less affinity for the stationary phase) than the weaker one.
Based on this finding it is possible to predict the elution order of basic compounds in a mixture under cation exchange conditions through the knowdeldge of their pka value , moreover this property can be used to adjust selectivity by proper selection of the mobile phase pH and manipulating its ionic strength and aqueous/organic ratio .

Method Validation
The optimized method was validated in agreement with the ICH guideline [35]. Accordingly, method linearity in the relevant working ranges, precision and accuracy were evaluated. System suitability was also determined.

Range and Linearity
Method range and linearity were evaluated with six mixtures of standards at the following concentrations: 188.0 -1880.0 µg/ml PSE, 61.3 -613.0 µg/ml DX, and 8.60 -86.0 µg/ml of TR, covering up to 125% of the expected concentration of the analytes in the formulation. Samples were injected in triplicate and calibrations were obtained by plotting the standard drug concentrations versus peak areas of the individual drugs. The calibration curves were defined by the equations shown in Table 1, and the residuals were spread uniformly and at random around the regression lines, passing the normality distribution test (p<0.05). In addition, correlation coefficients were higher than 0.99 and the confidence intervals of the intercepts contained the zero, confirming method linearity.

Precision
Method precision was verified in its repeatability and intermediate precision aspects according to the ICH guideline Q2 (R1) [35]. Six replicate determinations of the samples containing 100%of their corresponding expected concentrations in the pharmaceutical product were injected and the relative standard deviation (RSD) of their recoveries was determined. The observed RSD levels (Table 1), which were below 2%, were considered satisfactory. For verification of the intermediate precision the process was repeated on another day using fresh reagents and samples, the samples were injected at random during two different days. This evidenced that the outcome of the determination was statistically similar regardless the day of the assay and the reagents preparation in the determinations. In addition, in all cases almost quantitative and consistent (RSD<2%) drug recoveries were recorded [41]. These results confirmed that the method is precise.

Accuracy
Method accuracy was demonstrated using standard addition method by evaluating analyte recoveries from a pre-assayed pharmaceutical formulation sample, containing 60% of the declared amounts of the drugs, which was fortified with known amounts of the three analytes, to reach concentration levels of 60% -120% of the expected drug concentrations in the pharmaceutical dosage form. The observed RSD levels (Table 1), which were below 2%, were considered satisfactory. This confirmed that the method enables the accurate determination of the analytes [41].

Limits of Detection LOD and Quantification LOQ
In order to assess that the validated concentration ranges of the analytes were above their LOQ values, the LOD and LOQ were determined employing the ICH method based on the calibration curve [35].

System Suitability Parameters
System suitability parameters must be checked to ensure that the system is working correctly during the analysis. The test was carried out by injecting five replicates of the optimization standard. The method performance data including column efficiencies (N), resolutions between adjacent peeks (Rs), and asymmetry factor (As) are listed in Table 2. All the parameters were in good agreements with the theoretically required ones [42].

CONCLUSION
A reliable and rapid liquid chromatography method for the simultaneous determination of PSE, DX, and PSE in syrup has been developed and validated. Experimental designs were employed for rational method optimization and demonstration of its suitability for the intended purpose. The use of strong cation exchange column enabled separation of the active principles within 10 min, despite their widely different properties; using very simple, relatively cheap and easy to prepare mobile phase. It was also possible to achieve their quantification in spite of the important abundance differences among the analytes (PSE: TR up to 24:1, w/w). In addition, the results indicate that the method is sensitive, linear, precise, and accurate, with regards to the mixture under investigation. Therefore, the method can be safely applied to the quality control of combined cough and cold medicines containing PSE, DX, and TR.