Method Development and Validation for the Simultaneous Determination of Fexofenadine Hydrochloride and Montelukast Sodium in Drug Formulation Using Normal Phase High-Performance Thin-Layer Chromatography

A simple, precise, speciﬁc, and accurate high-performance thin-layer chromatographic method has been developed for the simultaneous determination of fexofenadine hydrochloride (FEX) and montelukast sodium (MTKT) in pharmaceutical dosage form. The separation was carried out on Merck HPTLC aluminum plates of silica gel G60 F 254 , (20 × 10cm) with 250 μ m thickness using toluene: ethyl acetate: methanol: ammonia (30%) (0.5: 7: 2: 0.5, v/v/v/v) as mobile phase. HPTLC separation of the two drugs followed by densitometric measurement was carried out in the absorbance mode at 220 nm. The drugs were resolved satisfactorily with R f values of 0 . 21 ± 0 . 01 and 0 . 59 ± 0 . 01 for FEX and MTKT, respectively. The linear regression analysis data for the calibration plots showed good linear relationship with r 2 = 0 . 9996 and 0.9998 for FEX and MTKT, respectively, in the concentration range of 2400–10800 ngspot − 1 for FEX and 200–900 ng spot − 1 for MTKT. The method was validated for precision, robustness, speciﬁcity, and accuracy. The limits of detection and quantitation were 100 and 300 ngspot − 1 , respectively, for FEX and 50 and 100 ngspot − 1 , respectively, for MTKT. The proposed developed HPTLC method can be applied for identiﬁcation and quantitative determination of FEX and MTKT in bulk drug and drug formulation.

Similarly for montelukast sodium, UV spectrophotometry [23,24], spectrofluorometry [25], RP-HPLC [26,27], HPTLC [26,28], in biological fluid by HPLC [29][30][31][32], LC/MS [33,34], and stability indicating HPLC methods [35,36] have been reported. According to literature research no method has been reported for simultaneous determination of FEX and MTKT by HPTLC and HPLC. HPTLC method is cost effective, rapid, and less time consuming. In HPTLC many samples are simultaneously used and solvent requirement is low. The development and validation of simple, precise, and accurate HPTLC method for the simultaneous determination of FEX and MTKT in tablet formulation is described in the present study. The proposed method is validated as per ICH guidelines [37].

Instrumentation and Chromatographic
Conditions. The HPTLC plates were prewashed with methanol and activated at 110 • C for 5 min prior to chromatography. The samples were spotted in the form of bands 6 mm width with a Camag 100 microlitre sample syringe (Hamilton, Bonaduz, Switzerland) on silica-gel-precoated HPTLC aluminum plate 60 F 254 , ((20 × 10 cm) with 250 μm thickness; E. Merck, Darmstadt, Germany, supplied by Anchrom Technologies, Mumbai) using a Camag Linomat V applicator (Switzerland). A constant application rate of 0.1 μLs −1 was used and the space between two bands was 6 mm. Linear ascending development was carried out in 20 cm × 10 cm twin trough glass chamber (Camag, Muttenz, Switzerland) saturated with the mobile phase. The mobile phase was consisted of toluene: ethyl acetate: methanol: ammonia (30%) (0.5: 7: 2: 0.5, v/v/v/v) and 20 mL was used per chromatography run. The optimized chamber saturation time with mobile phase was 30 min using saturation pads at room temperature (25 • C ± 2). The length of chromatogram run was 80 mm and run time was 20 min. Densitometric scanning was performed using a Camag TLC scanner III in the reflectance-absorbance mode and operated by winCATS software (V1.1.4, Camag). The slit dimension was kept at 5 mm × 0.45 mm and the scanning speed was 10 mm s −1 . The source of radiation used was a deuterium lamp emitting a continuous UV spectrum between 200 and 400 nm. All determinations were performed   at ambient temperature with a detection wavelength of 220 nm. Concentrations of the compound chromatographed were determined from the intensity of the diffused light. Evaluation was by peak areas with linear regression.

Standard Solutions and Calibration Graphs.
Mixed stock standard solution containing 12 mg mL −1 of FEX and 1 mg mL −1 of MTKT was prepared in methanol by dissolving 300 mg of FEX and 25 mg of MTKT in 25 mL methanol. Mixed stock standard solution was further diluted with methanol to obtain working standard solutions in a concentration range of 2400-10800 ng spot −1 for FEX and 200-900 ng spot −1 for MTKT. Each concentration was applied six times on the HPTLC plate. The plate was then developed using the previously described mobile phase. The peak areas were plotted against the corresponding concentrations to obtain the calibration graphs. Linear calibration curves were generated using least-squares linear-regression analysis.

Sample Preparation.
To determine the content of FEX and MTKT simultaneously in pharmaceutical dosage form MONTAIR FX (label claim: 120 mg FEX and 10 mg MTKT per tablet, B. no. ACF1010, Cipla Ltd.), twenty tablets were weighed and finely powdered. An accurate weight of the powder equivalent to 120 mg of FEX and 10 mg of MTKT was weighed. This was then transferred into a 100 mL volumetric flask containing 50 mL methanol, sonicated for 30 min, and made up to the mark with methanol. This solution was filtered through a 0.45 μm nylon syringe filter. The previous concentration achieved was 1200 ng μL −1 of FEX and 100 ng μL −1 of MTKT. 6 μL volume was spotted for six times to achieve a final concentration of 7200 ng spot −1 for FEX and 600 ng spot −1 for MTKT. The plate was developed in the previously described chromatographic conditions. The peak area of the spots was measured at 220 nm for FEX and MTKT,  precision of the method was checked by performing same procedure on different days under the same experimental conditions. The repeatability of sample application and measurement of peak area were expressed in terms of relative standard deviation (% RSD) and standard error (SE). An amount of the sample powder equivalent to the label claim of FEX and MTKT was accurately weighed and assayed. System repeatability was determined by six replicate applications and measurement of sample solution at a concentration of 7200 ng spot −1 for FEX and 600 ng spot −1 for MTKT and the peak areas for real sample were expressed in terms of relative standard deviation (% RSD). , v/v/v/v)) were tried and chromatograms were run. The amount of mobile phase was varied over the range of ±5%. The time from spotting to chromatography and from chromatography to scanning was varied by +10 min. The robustness of the method was determined at three different concentration levels of 2400, 7200, and 10800 ng spot −1 for FEX and 200, 600, and 900 ng spot −1 for MTKT.

Limit of Detection and Limit of Quantitation.
The detection limit of an individual analytical procedure is the lowest amount of analyte in a sample that can be detected but not necessarily quantitated as an exact value. The quantitation limit of an individual analytical procedure is the lowest amount of analyte in a sample that can be quantitatively determined with suitable precision and accuracy. In order to estimate the limit of detection (LOD) and limit of quantitation (LOQ), the signal-to-noise ratio (S/N) of 3 and 10 was determined for six replicate determinations.

Specificity.
Specificity of the method was determined by means of complete separation of pure drugs in the presence of other excipients normally present in the formulation. Peak purity of FEX and MTKT was assessed by comparing their respective spectra at peak start (S), peak apex (M), and peak end (E) position of the spots.
2.6.5. Accuracy. Accuracy of the proposed method was carried out by applying the method to pharmaceutical dosage form (FEX and MTKT combination tablets) to which known amounts of FEX and MTKT standard powder corresponding to 80, 100, and 120% of label claim had been added (standard addition method). The absolute recovery was calculated by comparing the peak areas obtained from standard solution of FEX and MTKT with the peak areas of samples of different concentration. Six determinations at each level of concentration were performed and the results obtained were compared with expected results.

Selection of Analytical
Wavelength. UV spectrum of FEX and MTKT showed maximum absorbance at 220 nm and 344 nm, respectively ( Figure 3). Further, in situ HPTLC spectral overlain of FEX and MTKT was taken and 220 nm was selected as scanning wavelength (Figure 4).

Optimization of Mobile Phase.
Optimization of mobile phase was done with a view to separate FEX and MTKT drugs. Since initially tested mobile phase, which was composed of toluene, ethyl acetate, methanol, and ammonia (30%) (2.5: 7: 2.5: 1, v/v/v/v) showing good peak shape but incomplete separation, was observed as the R f of FEX was 0.40 and R f of MTKT was 0.47 [26], Several other combinations of the same mobile phase components were tested. Such combinations included toluene, ethyl acetate, methanol, and ammonia (30%) ((2.5: 7: 3: 1), (2: 7: 2.5: 1), and (2: 7: 2.5: 0.5)). However, since these mobile phases did not lead to the aimed result so, mobile phase was changed to ethyl acetate, methanol, and ammonia (30%) (7: 1.5: 0.5 v/v/v) which was also the reported earlier [22]. The result showed good separation with R f of 0.20 and 0.60 for FEX and MTKT, respectively, but MTKT peak showed significant tailing. Toluene (0.1, 0.3, 0.5 mL) was then added to mobile phase and in the subsequent run it was found that toluene was responsible for improving the peak shape of MTKT.  (Table 1). Residual analysis was performed to ascertain linearity ( Figure 5). Table 2 show that proposed method provides acceptable intraday and interday variation of FEX and MTKT with respect to working standard.

Precision. The % RSD values depicted in
The repeatability of real sample application and measurement of peak areas were expressed in terms of % RSD and were found to be 0.61 and 0.26 for FEX and MTKT, respectively.

3.5.
Robustness. The standard deviation of the peak areas was calculated for each parameter and the % RSD was found to be less than 2%. The low values of the % RSD, as shown in Table 3, indicated the robustness of the method.

Limit of Detection and Limit of Quantitation.
The signal/noise ratios 3 : 1 and 10 : 1 were considered as LOD and LOQ, respectively. The LOD and LOQ were found to be 100, 300 ng spot −1 and 50, 100 ng spot −1 for FEX and MTKT, respectively.

3.7.
Specificity. The specificity was noticed by the complete separation of FEX and MTKT peaks. The peak purity of was assessed by comparing their respective spectra at the peak start, apex, and peak-end positions of the spot, that is, r(S, M) = 0.9999 and r(M, E) = 0.9999 for FEX and r(S, M) = 0.9997 and r(M, E) = 0.9998 for MTKT. Table 4 satisfactory recovery percentage in the limit of 98-102% with small relative standard deviations (% RSD) is obtained at various added concentrations. The results indicate that the method is highly accurate for simultaneous determination of FEX and MTKT.

Accuracy. As shown from the data in
3.9. Analysis of a Marketed Formulation. Using the proposed chromatographic method, assay of FEX and MTKT in their tablets (MONTAIR FX, label claim: 120 mg FEX and 10 mg MTKT per tablet, B. no. ACF1010, Cipla Ltd.) was carried out. The peaks at R f 0.21 for FEX and 0.59 for MTKT were observed in the densitogram of the drug samples extracted from tablets. There was no interference from the excipients commonly present in the tablets ( Figure 6). Satisfactory results were obtained for both drugs in a good agreement with the label claim. The drug content was found to be 99.82% ± 0.98 (%RSD of 0.98) and 100.01% ± 1.06 (% RSD of 1.07) for FEX and MTKT, respectively.

Conclusion
The developed HPTLC technique is precise, specific, robust, and accurate method for analysis of FEX and MTKT in pharmaceutical preparations. The procedure can be readily used for selective analysis of drugs and repeatable results are obtained without interference from auxiliary substances. The method can be used for analysis of a few formulations on a single plate and is rapid and cost-effective for routine analysis of FEX and MTKT in tablet or capsule formulation.