A Novel and Validated RP-HPLC Method for the Simultaneous Determination of Fexofenadine Hydrochloride and Montelukast Sodium in Bulk Drug and Pharmaceutical Dosage Forms

Figure 1: Structure of [a] Fexofenadine hydrochloride  [b] Montelukast Sodium

There for the present research work aims to develop a simple, sensitive, accurate and reproducible method for simultaneous estimation of Montelukast Sodium and Fexofenadine hydrochloride in combined dosage form by RP-HPLC method.

2. MATERIALS AND METHODS

2.1. Chemicals and reagents

Active pharmaceutical ingredient of Montelukast Sodium and Fexofenadine hydrochloride was obtained as a gift sample from Anglo french Pharmaceutical Pvt. Ltd and Micro labs  Pvt. Ltd, India.

2.2. Instrumentation and chromatographic conditions

The chromatographic analysis was performed using an Agilent 1120 Compact LC HPLC system equipped with a gradient pump, Rheodyne injector, UV–Visible variable-wavelength detector, and standard flow cell. Chromatographic separation was achieved on an X-Bridge C18 column (250 mm × 4.5 mm, 5 µm) maintained under ambient conditions. The mobile phase was optimized after evaluating different solvent combinations and ratios and consisted of trifluoroacetic acid buffer (pH 2.1) and methanol in a ratio of 70:30 (v/v). The system was operated at a flow rate of 1.0 mL/min with an injection volume of 20 µL and UV detection at 215 nm. The total chromatographic run time was 10 min. Under the optimized conditions, well-resolved peaks were obtained with retention times of 3.863 min for fexofenadine hydrochloride and 7.333 min for montelukast sodium. Analytical weighing was performed using a Shimadzu AUX 220 balance, while an Equitron sonicator was used for sample and solvent preparation. A Super Fit vacuum pump and Tarsons filtration assembly with 0.45 µm nylon membrane filters (Merck Millipore) were used for filtration. A Shimadzu UV-1800 double-beam UV–Visible spectrophotometer was used for wavelength selection and spectral analysis. Chromatographic data acquisition, evaluation, and storage were performed using EZChrome Elite software.

2.3 Preparation of mobile phase:

The buffer solution was prepared by dissolving 1g of 0.1% of Trifluoroacetic acid buffer in 1000 ml HPLC grade water (20 mM). The pH of the resulting solution was adjusted to 2.1 by using HPLC grade Triethylamine. HPLC experiments were carried out using a binary pump, pump A containing Methanol and pump B containing Trifluoroacetic acid buffer.

2.4 Standard solutions preparation  of Fexofenadine Hydrochloride and Montelukast Sodium

The standard stock solution of Fexofenadine Hydrochloride and Montelukast Sodium was prepared by dissolving 50 mg of the drug into 50 ml of the volumetric flask in Methanol (HPLC grade), volume was made up to the mark with the same solvent. This gave the concentration of 1000 µg/ml for both Fexofenadine Hydrochloride and Montelukast Sodium (Stock-1). Further dilution of 10 ml in 100 ml volumetric flask makes up the volume with Methanol gives 100 µg ml-1(stock-2). From stock solution-2, 6 dilutions were prepared between 120 µg/ml for Fexofenadine Hydrochloride and 60 µg/ml .Montelukast Sodium from the above dilutions of working concentration made up by using Methanol in the ratio of 70:30 Trifluoroacetic acid buffer: Methanol.

Figure 2: Chromatogram for Fexofenadine Hydrochloride and Montelukast Sodium

1. RESULTS AND DISCUSSION

3.1 Validation of analytical method

1.Linearity and range:

By using the working standard, aliquots of 10-120µg/ml,of  Fexofenadine hydrochloride and montelukast sodium, were prepared with methanol. Six dilutions of each of the above-mentioned concentrations were prepared separately and from these six dilutions, 20µl of each concentration was injected into the HPLC system. Then their chromatogram was recorded. Peak areas were recorded for all the peaks and a standard calibration curve of peak area against concentration was plotted.

Figure 3: Linearity Chromatogram for Fexofenadine Hydrochloride and Montelukast Sodium 40-60µg/ml

Figure 4: Linearity graph of Fexofenadine Hydrochloride and Montelukast sodium

Table 1: Linearity data for Fexofenadine Hydrochloride and Montelukast Sodium

Specfication r2 = 1

2.Precision

The precision of the assay was determined in terms of intra-day and inter-day variation in the peak area for a set of drug solutions 50-50µg/ml, assayed six times on the same day and on different 2 days.

a                                                                         b

Figure 5: Intraday Chromatogram for Fexofenadine Hydrochloride and Montelukast Sodium a) Morning b) Afternoon

a                                                                        b

Figure 6: Intraday Chromatogram for Fexofenadine Hydrochloride and Montelukast Sodium a) Day 1& b) Day 2

1. Accuracy
2. The accuracy was carried out for both Fexofenadine Hydrochloride and Montelukast Sodiumdrugs at 80%, 100%, and 120% levels. % recovery was determined.

a                                                                     b

C

Figure 7: Chromatogram for accuracy of  a) 80%, b) 100% , c) 120%

4.Limit of detection and limit of quantification

LOD and LOQ were calculated according to ICH recommendations where the approach is based on the signal-to-noise ratio. Chromatogram signals obtained with known low concentrations of analytes were compared with the signals of the blank samples. A signal–to–noise ratio of 3:1 and 10:1 was considered for calculating LOD and LOQ respectively.

Table 2: LOD and LOQ for simultaneous estimation of Fexofenadine Hydrochloride and Montelukast Sodium

5.System Suitability

The resolution, number of theoretical plates, Capacity Factor, S/N (6 Sigma) and peak asymmetry were calculated for the standard solutions.

Table 3: Optimum conditions in RP-HPLC method for simultaneous estimation of  Fexofenadine Hydrochloride and Montelukast Sodium