DEVELOPMENT AND VALIDATION OF UV SPECTROPHOTOMETRIC METHOD FOR SIMULTANEOUS ESTIMATION OF MONTELUKAST SODIUM AND LEVOCETRIZINE DIHYDROCHLORIDE IN BULK AND TABLET DOSAGE FORMULATION.

ABOUT AUTHORS:
Gohel Bhavika A*, Patel Bhavna A, Parmar Sharddha J, Mital Sondagar M, Jadav Alpa V
P G Department of Pharmaceutical Sciences, Sardar Patel University,
V. V. Nagar, Gujarat
*pharma.bhavika@gmail.com

ABSTRACT
A new, simple, rapid and novel Spectrophotometric method has been developed for simultaneous estimation of Montelukast sodium and Levocetrizine dihydrochloride from tablet formulation. The method employs formation and solving of simultaneous equation using 230nm and 283nm as two analytical wavelengths which are absorbance maxima of Levocetrizine dihydrochloride and Montelukast sodium, respectively. The linearity was obtained in the concentration range of 5-25 g/mL for both the drugs. Recovery studies range from 98.45-100.30% for Levocetrizine dihydrochloride and 98.80-101.84% for Montelukast sodium. The results of analysis have been validated statistically and by recovery studies according to ICH guidelines. The proposed methods can be successfully applied in routine work for the determination of Levocetrizine dihydrochloride and Montelukast sodium in combined dosage form.

REFERENCE ID: PHARMATUTOR-ART-1779

INTRODUCTION:
Levocetrizine 2-(2-{4-[(4-chlorophenyl)(phenyl)methyl]piperazin-1-yl}ethoxy) acetic acid [5] (fig.1) is Histamine receptor(H1) antagonist indicated for the treatment of allergic rhinitis and chronic idiopathic urticaria. It is official in IP and EP.^{2, 3, 5}

Figure 1: STRUCTURE OF LEVOCETRIZINE DIHYDROCHLORIDE

Montelukast 2-[1-({[ (1R)-1-{3- [(E)-2-(7-chloroquinolin-2-yl)ethenyl]phenyl}-3-[2-(2-hydroxypropan-2-yl)phenyl]propyl]sulfanyl}methyl)cyclopropyl]acetic acid ^[4] (fig. 2) is a leukotriene receptor antagonist (LTRA) used for the maintenance and treatment of asthma and to relieve symptoms of seasonal allergies. It is official in IP 2010. ^{2, 4}

Figure 2: STRUCTURE OF MONTELUKAST SODIUM

The review of literature revealed that several methods are available for the determination of montelukast and Levocetrizine individually or in combination with other drugs. But no method mentioned in the literature explained the simultaneous estimation of montelukast and Levocetrizine in bulk, dosage forms and in biological fluids. The present study describes a simple, rapid, accurate, reproducible method for simultaneous estimation of Montelukast and Levocetrizine. The proposed method was validated according to ICH guidelines.⁷

MATERIAL AND METHODS

INSTRUMENTATION
Shimadzu UV-1800, UV-Visible double beam Spectrophotometer with matching pair of 1 cm quartz cuvettes (Shimadzu Corporation, Kyoto, Japan). The absorption spectra of solutions were recorded in 1 cm quartz cell over the range of 200?400 nm. All weighing was done on analytical balance (Denver instrument, Germany). A sonicator (Electroquip Ultra sonicator, Texas) was used in the study. Calibrated glass wares were used throughout the work.

MATERIALS AND REAGENTS
Chemical used was methanol (A.R.Grade, Sisco Chem Pvt Ltd, Andheri, Mumbai). Marketed formulation containing Levocetrizine (5 mg) and Montelukast (10 mg) (Montecip-LC) was obtained from the local pharmacy.

PREPARATION OF STANDARD STOCK SOLUTIONS
Levocetrizine dihydrochloride and Montelukast sodium (10 mg each) were separately weighed and transferred to a 10 ml volumetric flask and drugs were dissolved in methanol to get a solution of 1000 μg/ml. 1 ml of stock solution was diluted up to 10 ml using methanol to give 100 μg/ml of both drug separately. After proper dilutions, 10 μg/ml MT and CT were scanned in the UV-region i.e. 400 to 200 nm.

CALIBRATION CURVE
A calibration curve was plotted over a concentration range of 5-25 μg/mL for CT and MT individually. Accurately measured standard stock solution of CT (0.5, 1, 1.5, 2 & 2.5 mL) and were transferred to a separate series of 10 mL of volumetric flasks and diluted to the mark with Methanol. The same procedure was followed for MT. The absorbance of each solution was measured at the wavelengths of 230nm and 283nm. Calibration curves were constructed for CT and MT by plotting concentration versus absorbance at both wavelengths. Each reading was average of five determinations.

SELECTION OF ANALYTICAL WAVELENGTH
The stock solutions of CT and MT were separately diluted in Methanol to get a concentration of 10 μg/ml of CT and 10 μg/ml of MT and scanned in the wavelength range of 200-400nm. The absorbance of various dilutions of CT and MT were measured at 230nm and 283nm and calibration curves were plotted. The absorptivities (A1%, 1cm) of each drug at both the wavelengths were also determined. The absorbance and absorptivity values at the particular wavelengths were calculated and substituted in the following equation, to obtain the concentration.

SIMULTANEOUS EQUATION METHOD¹
The wavelength 230nm and 283nm (λmax of CT and MT respectively) was selected for the formation of the simultaneous equations. The absorptivity of MT at 230nm and 283nm was found to be 399.47 and 340.11, respectively. The absorptivity of CT at 230nm was found to be 343.71; while at 283nm it was negligible.

The absorbance and the absorptivity values at the particular wavelength were calculated and substituted in the following equation, to obtain the concentration.
CMT= (A1ax2 – A2ax1) / (ax2ay1 – ax1ay2).
CCT = (A2ay1 – A1ay2) / (ax2ay1 – ax1ay2).

Where,
CCT = Concentration of Levocetrizine dihydrochloride,
CMT = Concentration of Montelukast sodium,
A1 = Absorbance of sample at 230nm,
A2 = Absorbance of sample at 283nm,
ax1 = Absorptivity of Levocetrizine dihydrochloride at 230nm and
ax2 = Absorptivity of Levocetrizine dihydrochloride at 283 nm,
ay1 = Absorptivity of Montelukast sodium at 230nm and
ay2 = Absorptivity of Montelukast sodium at 283nm.

PREPARATION OF SAMPLE SOLUTION AND FORMULATION ANALYSIS
Twenty tablets of brand Montecip-LC containing 5 mg of Levocetrizine dihydrochloride and 10 mg of Montelukast sodium were weighed, average weight determined and finely powdered with the help of mortar and pestle. Appropriate quantity of powder from each tablet equivalent to 10 mg of Montelukast sodium was accurately weighed and transferred to 100 ml volumetric flask and volume was made up to 100 ml with methanol (100 μg/mL) then sonicated for 15 minutes. Necessary dilutions of filtrate were made with methanol to get final concentration 10 μg/ml of Montelukast sodium and 5 μg/ml of Levocetrizine dihydrochloride. Absorbances of this solution were measured at 230nm (λmax of Levocetrizine dihydrochloride) and 283nm (λmax of Montelukast sodium). Values were substituted in the formulae to obtain concentration. Results are shown in the Table II.

METHOD VALIDATION
Validation of the developed method was done according to the ICH Guideline.

LINEARITY
For each drug, appropriate dilutions of standard stock solutions were assayed as per the developed methods. The Beer-Lambert’s concentration range is 5-25 μg/mL for both Levocetrizine dihydrochloride and Montelukast sodium. The calibration curves (fig. 4 and 5) were constructed by plotting drug concentration versus the absorbance values of spectrum at 230nm for CT and 283nm for MT. Statistical data for calibration curves is depicted in Table I.

ACCURACY
Accuracy was confirmed by recovery study as per ICH guidelines Q2R1 at three different concentration levels 80%, 100%, 120% by replicate analysis (n = 3). Known amounts of standard solutions of MT and CT were added at 80%, 100% and 120% level to sample solutions of MT and CT (10 μg/ml for MT and 5 μg/ml for CT ) and then percentage of drug content was calculated. From the recovery study it is clear that the method is accurate for quantitative estimation of Levocetrizine dihydrochloride and Montelukast sodium in combined dosage form as the statistical parameters are within the acceptance range. The results are shown in Table III.

PRECISION
The reproducibility of the proposed method was determined by performing the assay for the same day at three different time (intraday precision) and on three different days (inter day precision). Precision studies were performed by preparing nine determinations covering the specified range for the procedure (3 x 3 replicates for each concentration). Low % RSD shows that the method has good precision. The results of intraday and inter day precision were expressed in % RSD was tabulated in Table III.

LIMIT OF DETECTION
It is the lowest amount of analyte in a sample that can be detected but not necessarily quantitated under the stated experimental conditions. Limit of detection can be calculated using following equation as per ICH guidelines. 7
LOD = 3.3 × SD/S

Where,
SD = Standard deviation of the response and
S = Slope of the corresponding calibration curve.

LIMIT OF QUANTIFICATION
It is the lowest concentration of analyte in a sample that can be determined with the acceptable precision and accuracy under stated experimental conditions.
Limit of quantification can be calculated using following equation as per ICH guidelines. 7
LOQ = 10 × SD/S

Where,
SD = Standard deviation of the response and

S = Slope of the corresponding calibration curve.

RESULTS

Table I: SPECTRAL AND LINEARITY CHARACTERISTIC DATA

Parameter	Levocetrizine dihydrochloride	Montelukast sodium
λ_max	230nm	283nm
Beer’s Law Limit (μg/mL)	5-25 μg/mL	5-25 μg/mL
Correlation coefficient (r²)	0.9984	0.9997
Regression Equation	y = 0.0327x + 0.018	y = 0.0335x + 0.0055
Slope	0.0327	0.0335
Intercept	0.018	0.0055
LOD (μg/ml)	2.368	2.266
LOQ (μg/ml)	5.471	6.869

Table II: RESULT OF ANALYSIS OF TABLET FORMULATION

Brand

Drug Name

Lable Claim

in mg

% Lable Claim

Found

RSD

Montecip-LC

98.09%

0.509

0.518

99.06%

0.205

0.206

Table III:RESULT OF RECOVERY STUDY AND PRECISION

Parameter	Drug	Levocetrizine dihydrochloride	Montelukast sodium
Accuracy	80%	98.45%	98.80%
	100%	100.30%	99.89%
	120%	99.72%	101.84%
Precision	Inter-day(RSD)	0.7-1.55	0.6-0.9
Precision	Intra-day(RSD)	0.8-1.2	0.9-1.4

Figure 3: OVERLAY SPECTRA OF MONTELUKAST SODIUM (10 μg/ml)AND LEVOCETRIZINEDIHYDROCHLORIDE (10 μg/ml )

Figure 4: CALIBRATION CURVE OF LEVOCETRIZINEDIHYDROCHLORIDE

Figure 5: CALIBRATION CURVE OF MONTELUKAST SODIUM

DISCUSSION
The overlain spectra of the CT and MT in methanol were recorded and λmax values of both drugs were noted. The drugs were found to obey beers law at the selected wavelength. Simultaneous equation was developed and amount of each drug was calculated in marketed formulations. The recovery of each drug was found to be satisfactory and within the limits. The validation of proposed methods showed that SD and %RSD values are within the limits. Hence, the method can be routinely adapted for the quality control of the drugs and is easier and statistically validated.

ACKWOLEDGEMENTS
The authors are highly thankful to P. G. Department of Pharmaceutical Sciences, Sardar Patel University, Vallabh Vidyanagar-388120, Gujarat, India for providing all the facilities to carry out the work.

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