Development of Validated HPTLC Method for Simultaneous Estimation of Fexofenadine Hydrochloride and Montelukast Sodium in Tablet Dosage form

Tamilselvi N¹*, Sruthi K¹, Arivukkarasu R¹, Vanathi P², Deepthi Visakh¹

¹Department of Pharmaceutical Analysis, K.M.C.H College of Pharmacy, Coimbatore -641048, Tamilnadu.

²Department of Pharmaceutical Chemistry, Karpagam College of Pharmacy, Coimbatore, Tamilnadu.

*Corresponding Author E-mail: tamildeiva@gmail.com

Received on 28.01.2016 Modified on 15.02.2016

Research J. Pharm. and Tech. 9(4): April, 2016; Page 469-473

DOI: 10.5958/0974-360X.2016.00087.1

ABSTRACT:

A simple, precise, speciﬁc and accurate high performance thin layer chromatographic method has been developed for the simultaneous estimation of Fexofenadine hydrochloride (FEXO) and Montelukast sodium (MONT) in pharmaceutical dosage form. The separation was carried out on Merck HPTLC aluminium plates of silica gel G60 F₂₅₄, (20 × 10 cm) with 250 µm thickness using hexane: ethyl acetate: propanol (2: 5: 3, v/v/v) as mobile phase. Densitometric measurement was carried out in the absorbance mode at 230 nm. The drugs were resolved satisfactorily with R values of 0.31 ± 0.01 and 0.57 ± 0.01 for FEXO and MONT, respectively. The linear regression analysis data for the calibration plots showed good linear relationship with r²=0.9996 and 0.9998for FEXO and MONT, respectively in the concentration range of 1200-6000 ng spot^-1 for FEXO and 100-500 ng spot^-1 for MONT. The method was validated for precision, robustness, specificity and accuracy. The limit of detection and quantitation were 393 and 1193 ng spot^-1, respectively for FEXO and 37 and 112 ng spot^-1, respectively for MONT. The proposed method can be successfully used to determine the drug content of marketed formulation.

KEYWORDS: Fexofenadine hydrochloride, Montelukast sodium, HPTLC, Method Development, Validation

INTRODUCTION:

Fexofenadine hydrochloride (FEXO) (Fig. 1) (0RS)-2-[4-[1-Hydroxy-4-[4-(hydroxy-diphenylmethyl)-1piperidyl] butyl] phenyl]-2-methyl-propanoic acid, is used to relieve the allergy symptoms of seasonal allergic rhinitis (hay fever), including runny nose; sneezing; and red, itchy, or watery eyes; or itching of the nose, throat, or roof of the mouth in adults^[1]. It is carboxylic acid metabolite of terfenadine, a non-sedating selective histamine H1 receptor antagonist. This drug contains an asymmetric carbon in its chemical structure and is administered clinically or is used as a P-glycoprotein probe as a racemic mixture of R- and S-enantiomers ^[2].

Montelukast sodium (MONT) (Fig. 2) is chemically (S, E)-2-(1-((1-(3-(2-(7-chloroquinolin-2yl) vinyl) phenyl)-3-(2-(2-hydroxypropan-2-yl) phenyl) propyl thio) methyl) cyclopropyl) acetic acid ^[3] is a Leukotriene receptor antagonist used in the treatment of chronic asthma and allergic rhinitis ^[4]. Literature survey reveals that Fexofenadine hydrochloride is estimated individually or in combination with other drugs by UV spectrophotometry ^[5,
6], RP-HPLC ^{[7, 8]}, HPTLC ^{[9, 10]}, in biological fluid by RP-HPLC ^[11], LC/MS ^[12], LC/MS/MS ^[13] and Stability indicating HPLC and TLC method ^[14]. Similarly for Montelukast sodium, UV spectrophotometry ^{[15, 16]}, spectrofluorimetry ^[17], RP-HPLC ^[18], HPTLC ^[19], plasma HPLC ^[20], LC/MS ^[21], and stability indicating HPLC methods ^{[22, 23]} have been reported. Above literature suggests that no method has been reported for simultaneous determination of FEXO and MONT by HPTLC. So, the present study is designed for the development and validation of simple, precise and accurate HPTLC method for the simultaneous determination of FEXO and MONT in tablet formulation. The proposed method is validated as per ICH guidelines ^[24].

Fig 1. Structure of Fexofenadine Hydrochloride

Fig 2. Structure of Montelukast Sodium

Materials and methods:

Chemicals and reagents:

Working standards of pharmaceutical grade FEXO (99.60 %, w/w) and MONT (100.0 %, w/w) were obtained as gift samples from Hetero Pharmaceuticals, Hyderabad, India. Fixed dose combination tablets (MONTAIR FX, Cipla Ltd.) containing 120 mg FEXO and 10 mg MONT were purchased from local pharmacy, Coimbatore, India. All chemicals and reagents of analytical grade were purchased from Merck Chemicals, Mumbai, India.

Instrumentation and chromatographic conditions:

A Camag HPTLC system comprising of Camag Linomat V automatic sample applicator, Hamilton syringe, Camag TLC Scanner 3, Camag Win CATS software, Camag twin-trough chamber and ultrasonicator was used during the study. The HPTLC plates were prewashed with methanol and activated at 110⁰ 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 on silica gel precoated HPTLC aluminum plate 60 F, (20 × 10 cm) with 250 µm thickness using a Camag Linomat V applicator. Linear ascending development was carried out in 20 cm × 10 cm twin trough glass chamber saturated with the mobile phase. The mobile phase was consisted of hexane: ethyl acetate: propanol (2:5:3, v/v/v). The optimized chamber saturation time with mobile phase was 20 min using saturation pads at room temperature (25⁰C ± 2). The length of chromatogramrun was 70 mm. Densitometric scanning was performed using a Camag TLC scanner III in the reﬂectance-absorbance mode and operated by win CATS software. The slit dimension was kept at 6mm × 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 spectrumbetween 200 and 400 nm. All determinations were performed at ambient temperature with a detection wavelength of 230 nm. Concentrations of the compound chromatographed weredetermined from the intensity of the diffused light. Evaluation was by peak areas with linearregression.

Standard solutions and calibration graphs

Mixed stock standard solution containing 12 mg mL^-1 of FEXO and 1 mg mL^-1of MONT was prepared in methanol by dissolving 300 mg of FEXO and 25 mg of MONT in 25 ml methanol. Mixed stock standard solution was further diluted with methanol to obtain working standard solutions in a concentration range of 1200-6000 ng/spot for FEXO and 100-500 ng/spot for MONT. Each concentration was applied six times on the HPTLC plate. The plate was then developed using the mobile phase.. Standard chromatogram of FEXO and MONT was shown in fig 5.The peak areas of Fexofenadine Hydrochloride and Montelukast Sodium were recorded and calibration graph was plotted against concentration of standards.

Sample preparation:

For analysis of tablet dosage form, twenty tablets, each containing 120 mg fexofenadine and 10 mg montelukast, were weighed and their average weight was calculated. The tablets were finely powdered and powder equivalent to 120mg of FEXO was accurately weighed and transferred into 10 ml of volumetric flask containing 5 mL of methanol, Sonicated for 30 min and make up to the mark with methanol to give 12000 µg/ml (ng/spot) of FEXO and1000 µg/ml (ng/spot) of MONT. The solution was centrifuged for 15 min at 600 rpm, filtered through Whattman No 41 filter paper and the residue was washed with methanol. The volume of the filtrate was adjusted to 10 ml with the same solvent. This above solution was further diluted with methanol to get the concentrations of 2400 and 4800 µg/ml (ng/spot) for FEXO and 200 and 400 µg/ml (ng/spot) for MONT. The formulation was assayed by spotting 1 µl of the solution on to the plate followed by development and scanning. The concentrations of the drugs were calculated from peak area obtained using standard calibration graph.

Fig 5.Chromatogram of Fexofenadine Hydrochloride and Montelukast Sodium

Method validation:

The optimized HPTLC method was validated with respect to the following Parameters as per the ICH guidelines (ICH, Q2 R₁ Validation of analytical procedure, 2005)^[21].

Linearity:

The linearity of analytical method is its ability to elicit test results that are directly proportional to the concentration of analyte in sample within a given range. The range of analytical method is the interval between the upper and lower levels of analyte that have been demonstrated to be determined within a suitable level of precision, accuracy and linearity. A series of standard drug solution were applied to a pre- washed TLC plate. The plate was developed, dried and subjected to densitometric measurements in absorbance mode at wavelength 230 nm using Camag TLC Scanner 3. A calibration plot was constructed by plotting peak area against standard concentration respectively (Table 1).The linear regression data showed good linear relationship over a concentration range of 1200 to 6000 ng/spot for Fexofenadine, 100 to 500 ng /spot for Montelukast. The standard chromatograms are shown in (Fig.3 and 4).

Precision:

Intraday precision:

Intraday precision was found out by carrying out the analysis of the standard drug at three different concentration 1200, 2400, 3600 ng/spot for FEXO and 100, 200, 300 ng/spot for MONT in the linearity range of drugs for six times on the same day. Each concentrations were applied in duplicate and percentage RSD was calculated (Table 2).

Interday precision:

Inter day precision was found out by carrying out the analysis of the standard drugs at three different concentrations 1200, 2400, 3600 ng/ spot for FEXO and 100, 200, 300 ng/spot for MONT in the linearity range of drugs for six times and the percentage RSD was calculated (Table 2)

Table 1.Linearity range of Fexofenadine HCL and Montelukast Sodium

Concentration FEXO (ng/spot )	Peak area *	Concentration MONT (ng / spot)	Peak area *
1200	4166	100	2046.21
2400	7942.55	200	3998.64
3600	11793.29	300	5699.95
4800	15386.97	400	7490.03
6000	18918.04	500	9280.11

Fig 3.Calibration curve of Fexofenadine Hydrochloride

Fig 4. Calibration curve of Montelukast Sodium

Table 2. Intra-day and inter-day precision of the developed method

Concentration (ng/spot)	Intraday			Interday
Concentration (ng/spot)	Peak area	SD	%RSD*	Peak area	SD	%RSD*
FEXO
1200	7160	64.83	0.90	7341	66.46	1.11
2400	9637	61.15	0.63	9542	59.61	0.73
3600	12591	75.49	0.59	12424	73.48	0.81
MONT
100	2486	33.38	1.34	2295	20.88	0.91
200	4112	36.26	0.88	4312	53.03	1.23
300	5628	75.39	0.95	5812	83.69	1.44

Table 4. Accuracy (Recovery studies)

Drug	Label Claim (mg/tab)	Spike Level (%)	Amount of drug added (ng/spot)	Amount of drug recovered (ng/spot)	Percentage Recovery	%RSD*
FEXO	120	50	600	595.92	99.32	0.98
		100	1200	1206.1	100.5	1.02
		150	1800	1781.1	98.95	0.75
MONT	10	50	50	50.09	100.19	1.16
		100	100	99.7	99.7	1.31
		150	150	149.6	99.54	0.92

Sensitivity:

The sensitivity of the method was estimated in terms of the Limit of Quantification and Limit of Detection. LOD and LOQ were determined by applying decreasing amount of the drug in triplicate on the plate. The lowest concentration at which the peak is detected is called ‘Limit of Detection’ and the lowest concentration at which the peak is quantified is called ‘Limit of Quantification’.

Limit of detection (LOD):

The L.O.D. was estimated from the set of 5 calibration curves.

LOD = 3.3 × (S.D./Slope)

Where,

S.D. = Standard deviation of the Y- intercepts of the 5 calibration curves.

Slope = Mean slope of the 5 calibration curves.

Limit of quantification (LOQ):

The L.O.Q. was estimated from the set of 5 calibration curves.

LOQ = 10 × (S.D./Slope)

Where,

S.D. = Standard deviation of the Y- intercepts of the 5 calibration curves.

Slope = the mean slope of the 5 calibration curves.

LOQ and LOD of MONT and FEXO is described in table (5).

Table 5. LOD and LOQ of Fexofenadine Hydrochloride and Montelukast Sodium

Parameter	FEXO (ng/spot)	MONT (ng/spot)
LOD	393	37
LOQ	1193	112

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. The specificity of the method was ascertained by peak purity profiling studies. Peak purity of MONT and FEXO was assessed by comparing their respective spectrum at peak start (S), peak apex (M) and peak end (E) position of the spots. The peak purity was determined on win CATS software using statistical equation.

Stability of the plate:

To test the stability of the drugs on the TLC plates, the freshly prepared solutions of the analyte were applied to the plates and developed and scanned at different intervals. No decomposition of the drug was observed during chromatogram development. No significant decrease in peak area was found for a stock solution after storage at room temperature for 4 hours. These observations suggest that the drug is stable under the typical processing and storage conditions of the analytical procedure (Table 6).

Table.6. Stability of plate

Volume applied

Time in hours

Peak area

FEXO

MONT

1µl

7341

7234

7159

7013

6895

6712

2546

2397

2104

1954

1865

1734

RESULTS AND DISCUSSION:

Under experimental conditions described, calibration curve, assay of tablets, recovery studies, precision studies, LODs & LOQs were performed. Using appropriate dilutions of standard stock solution, the two solutions were scanned separately. A critical evaluation of proposed method was performed by statistical analysis of data where slope, intercept, correlation coefficient were studied. Beer’s law is obeyed in the concentration range 100-500 ng/spot for MONT and 1200-6000 ng/spot for FEXO and correlation coefficient of 0.9988 and 0.9995for FEXO and MONT respectively. The proposed method was also evaluated by the assay of commercially available tablets containing FEXO and MONT (n = 6). The % assay was found to be 99.74 % for FEXO and 99.3 % for MONT. The accuracy and reproducibility is evident from the data as results are close to 100 % and standard deviation is low.

CONCLUSION:

The developed HPTLC technique is precise, speciﬁc, and accurate method for analysis of FEXO and MONT 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 a rapid and cost-effective for routine analysis of FEXO and MONT in tablet or capsule formulation

ACKNOWLEDGEMENT:

Authors thank Hetero Pharmaceuticals, Hyderabad, India for providing gift sample of standards FEXO and MONT and K.M.C.H College of Pharmacy for providing facilities for carrying out this study.

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