Simultaneous Estimation of Ambroxol Hydrochloride and Cetirizine Hydrochloride in Pharmaceutical Tablet Dosage Form by Simultaneous Equation Spectrophotometric Method : A Quality Control Tool for Dissolution Studies

Ambroxol Hydrochloride and Cetirizine Hydrochloride are used for the treatment of bronchitis, cough, and allergy. A simple, economical, accurate, and precise method for simultaneous estimation of Ambroxol Hydrochloride and Cetirizine Hydrochloride in tablet dosage formhas been developed. Simultaneous equationmethod based onmeasurement of absorbance at twowavelengths, that is, 244 nm and 230 nm, λmax of Ambroxol Hydrochloride and Cetirizine Hydrochloride in pH 6.8 phosphate buffer. Both of these drugs obeyed Beer-Lambert’s law in the concentration range of 2–18μg/mL for Ambroxol Hydrochloride and 2–20 μg/mL for Cetirizine Hydrochloride. The high values of correlation coefficient (R) indicated good linearity of calibration curve for both the drugs. The accuracy and precision of method were determined and the method was validated statistically. Result of percentage recovery study confirms the accuracy of proposedmethod. As per the ICHguidelines, themethod validation parameters checked were linearity, accuracy, precision, and assay of drug formulation. Based on the results obtained, it can be concluded that the proposed simultaneous equation spectrophotometric method for simultaneous estimation of Ambroxol Hydrochloride and Cetirizine Hydrochloride is rapid, economical, accurate, precise, and reproducible. Hence, the proposed method can be employed for quantitative estimation of Ambroxol Hydrochloride and Cetirizine Hydrochloride from their tablet dosage form.


Introduction
Ambroxol Hydrochloride [AMB HCl] (Figure 1) officially in Indian Pharmacopoeia (IP) is chemically trans-4-[(2-amino-3, 5-dibromobenzyl) amino]-cyclohexanol Hydrochloride.It is a white or yellowish crystalline powder.It is sparingly soluble in water, soluble in methanol, and practically insoluble in methylene chloride [1,2].Ambroxol Hydrochloride is a potent mucolytic and mucokinetic, capable of inducing bronchial secretion.It depolymerises mucopolysaccharides directly and by liberating lysosomal enzymes network of fibres in tenacious sputum is broken.It is particularly useful if mucus plugs are present.Ambroxol Hydrochloride (AMB) is semisynthetic derivative of vasicine obtained from Indian shrub Adhatoda vasica.It is a metabolic product of bromhexine, used in a variety of respiratory disorders including chronic bronchitis and also used in the treatment of cough [3].
Cetirizine Hydrochloride [CET HCl] (Figure 2) is chemically [2-[4-[(4-chlorophenyl) phenylmethyl]-1piperazinyl]ethoxy]acetic acid and the active metabolite of the piperazine H 1 -receptor antagonist hydroxyzine.It is white or almost white powder, freely soluble in water, practically insoluble in acetone and in methylene chloride [4,5].It is a nonsedative second generation antihistamine drug used in the treatment of seasonal allergic rhinitis, perennial allergic rhinitis, and chronic urticaria and also used as adjuvant in seasonal asthma.Cetirizine inhibits the release of histamine and of cytotoxic mediators from platelets, as well as eosinophil chemotaxis during the secondary phase of allergic response [3].The combination of these two drugs is not official in any pharmacopoeia; hence, no official method is available for the simultaneous estimation of Ambroxol Hydrochloride and Cetirizine Hydrochloride in their combined dosage form.From the literature survey, it was found that many chromatographic methods [HPTLC and RP-HPLC] have been reported for estimation of Ambroxol Hydrochloride and Cetirizine Hydrochloride in their combined dosage form and no spectrophotometric method for simultaneous estimation of Ambroxol Hydrochloride and Cetirizine Hydrochloride has been reported so far [6][7][8][9][10][11][12][13].Hence an attempt has been made to develop new simultaneous equation spectrophotometric method which is simple, rapid, reproducible, and economical method for simultaneous estimation of Ambroxol Hydrochloride and Cetirizine Hydrochloride in tablet dosage form.

Apparatus.
A double beam UV visible spectrophotometer (UV-1800 Shimadzu, Japan) was used.Absorption and overlain spectra of both test and standard solutions were recorded over the wavelength range of 200-400 nm using 1 cm quartz cell at fast scanned speed and fixed slit width of 1.0 nm.All weighing of ingredients was done on digital weighing balance (DV 215 CD Ohaus, USA) and bath sonicator (PCI Analytical Pvt., Ltd.) was also used in study.

Reagents and Materials. Ambroxol Hydrochloride and
Cetirizine Hydrochloride were supplied as gift sample by Trojan Pharma, Baddi, India.All other chemicals and reagents used were of analytical grade.

Selection of Common
Solvent.Phosphate buffer of pH 6.8 was selected as common solvent for developing spectral characteristics of drug.The selection was made after assessing the solubility of the drugs in different solvents.

Preparation of Standard Stock Solution.
Standard stock solutions of both Ambroxol Hydrochloride and Cetirizine Hydrochloride were prepared by dissolving 10 mg of AMB HCl and 10 mg of CET HCl separately in 10 mL of pH 6.8 phosphate buffer solution and sonicated for 15 minutes in bath sonicator and filtered through Whatman filter paper in order to get dilution of 1 mg/1 mL, that is, 1000 g/mL.

Determination of Absorption (𝜆) Maximas
. By appropriate dilution of standard stock solutions of AMB HCl and CET HCl with pH 6.8 phosphate buffer, solution containing 10 g/mL of AMB HCl and 10 g/mL of CET HCl was scanned separately and then in mixture form in the range of 200-400 nm.

Calibration Plots for Ambroxol Hydrochloride and
Cetirizine Hydrochloride.The calibration plots were constructed for Ambroxol Hydrochloride and for Cetirizine Hydrochloride using pH 6.8 phosphate buffer solutions at their respective absorption maximas in order to find the linearity range of drugs at their respective absorption maximas..The absorbances were measured at the selected wavelengths and absorptivities for both drugs were determined at both wavelengths.The concentrations of drugs in sample solution were determined by using following formula:

Development of Simultaneous Equation
where  A and  C are the concentration of Ambroxol Hydrochlorideand Cetirizine Hydrochloride, respectively,  1 and  2 are absorbance at 244 nm and 230 nm, respectively,  1 and  2 are absorptivities of Ambroxol Hydrochlorideat 244 nm and 230 nm, respectively and  1 and  2 are absorptivities of Cetirizine Hydrochloride at 244 nm and 230 nm, respectively.

Validation of Proposed Method.
The optimized UV spectrophotometric method was completely validated according to the procedure described in ICH guidelines.The performance parameters evaluated for the method were linearity, precision, accuracy, limits of detection and quantitation, and assay of drug.2.8.1.Linearity.The linearity of measurement was evaluated by analyzing different concentrations of the standard solution of Ambroxol Hydrochlorideand Cetirizine Hydrochloride.

Precision (Repeatability).
The precision of the instrument was checked by repeated scanning and measurement of absorbance of solutions ( = 6) for Ambroxol hydrochlorideand cetirizine hydrochloride (10 g/mL for both drugs) without changing the parameter of the proposed spectrophotometry method.The intraday and interday precision studies (intermediate precision) were carried out by estimating the corresponding responses 3 times on the same day and on 3 different days for three different concentrations of AMB HCl (8, 12, and 16 g/mL) and CET HCl (6, 10, and 14 g/mL), and the results are reported in terms of relative standard deviation.

Limit of Detection
and Limit of Quantitation.The limit of detection (LOD) and limit of quantitation (LOQ) were separately determined based on standard deviation of the intercept and the slope of the calibration curve by using ( 3) and ( 4), respectively: where,  is standard deviation of -intercept and  is slope of calibration curve.

Recovery (Accuracy) Studies.
In order to check the accuracy, reproducibility, and precision of the proposed method, recovery study was carried out by taking standard mixture solution of both AMB and CET and absorbances were determined at 244 nm and 230 nm, respectively.

Assay of Drug.
Ten tablets (200 mg) were powdered in a mortar pestle and the blend equivalent to 7.5 mg of Ambroxol Hydrochloride and 5 mg of ctirizine Hydrochloride was weighed and dissolved in 100 mL of pH 6.8 phosphate buffer solutions.The solution was sonicated for 15 minutes, filtered through Whatman filter paper, and suitably diluted with pH 6.8 phosphate buffer and the drug content was analyzed from simultaneous equation method by using double beam UV spectrophotometer at 244 nm and 230 nm, respectively.

Determination of Absorption Maximas.
Wavelengths of absorption maximas were determined for both drugs.AMB HCl showed absorption maximas one at 244 nm and another one at 308 nm, respectively.244 nm was selected as  max of Ambroxol Hydrochloride.CET HCl showed maximum absorbance at 230 nm with reference to British Pharmacopoeia and Indian Pharmacopoeia.
From the overlain spectra of Ambroxol Hydrochlorideand Cetirizine Hydrochloride (Figure 3), two wavelengths 244 nm and 230 nm and  max of Ambroxol Hydrochlorideand Cetirizine Hydrochloride were selected for further spectroscopic studies.Therefore, for simultaneous equation method, wavelengths selected for analysis were 244 nm for AMB HCl and 230 nm for CET HCl.

Calibration Plots for Ambroxol Hydrochloride and Cetirizine Hydrochloride.
From the calibration plot of Ambroxol Hydrochloride and Cetirizine Hydrochloride at their respective absorption maximas, the linearity was observed in the concentration range of 2-18 g/mL for Ambroxol Hydrochlorideand 2-20 g/mL for Cetirizine Hydrochloride, which were validated by the least square method.Coefficient of correlation () was found to be 0.999 for both Ambroxol Hydrochloride and Cetirizine Hydrochloride as given in Table 3.The high value of correlation coefficient () also indicates good linearity of calibration curve for both drugs..The absorptivity values were found approximately the same for all the concentrations; hence both drugs obeyed Beer-Lambert's law in indicated concentration range as given in Tables 1 and 2.

Development of Simultaneous Equation
The following equation was designed for the simultaneous estimation of Ambroxol Hydrochloride and Cetirizine Hydrochloride in tablet dosage form: where  A and  C are the concentration of Ambroxol Hydrochlorideand Cetirizine Hydrochloride, respectively,  1 and  2 are absorbance values at 244 nm and 230 nm, respectively, 0.025 and 0.019 are absorptivities of Ambroxol Hydrochlorideat 244 nm and 230 nm, respectively and 0.0063 and 0.0338 are absorptivities of Cetirizine Hydrochloride at 244 nm and 230 nm, respectively.By solving (5) and ( 6) simultaneously, the concentration of Ambroxol Hydrochloride and Cetirizine Hydrochloride in tablet dosage form can be calculated.

Validation of Proposed Method.
The validation parameters were studied at all wavelengths for the proposed method.
The optical characteristics such as  max , Beer-Lambert's law  limit, Regression equation, slope, intercept, correlation coefficient, LOD, and LOQ were calculated and are summarized in Table 3.
As per the ICH guidelines, the method validation parameters checked were linearity, accuracy, precision, and assay of drug.Precision studies were carried out to study the intraday and interday variations of the responses.Instrumental precision study was carried out by repeatability study.The low RSD value and LOD and LOQ values indicate that the method is precise and sensitive.The recovery studies and assay of drug for Ambroxol Hydrochloride and Cetirizine Hydrochloride were given in Tables 4 and 5.
Based on the results obtained, it can be concluded that the proposed spectrophotometric method for simultaneous estimation of Ambroxol Hydrochloride and Cetirizine Hydrochloride is rapid, economical, accurate, precise, and reproducible.The utility of the developed method has been demonstrated by analysis of combined dose tablet formulation.Hence, the proposed method can be employed for quantitative estimation of Ambroxol Hydrochloride and Cetirizine Hydrochloride in combined tablet dosage form.Simultaneous equation method can be used to carry out dissolution study in tablet formulation of these drugs.

Conclusion
The developed spectrophotometric method, that is, simultaneous equation method is found to be simple, sensitive, accurate, and precise and can be used for routine analysis of Ambroxol Hydrochloride and Cetirizine Hydrochloride in combined table dosage form.The developed method was validated as per ICH guidelines.The results demonstrated that simultaneous equation method by spectrophotometer could be useful technique for estimation of Ambroxol Hydrochloride and Cetirizine Hydrochloride in tablet dosage form and cost of analysis is less as compared to RP-HPLC method.Hence simultaneous equation method can be conveniently used for routine quality control analysis of AMB HCl and CET HCl in its pharmaceutical tablet formulations.

Table 1 :
Absorbance and absorptivity values of Ambroxol Hydrochloride at  max 244 nm and 230 nm, respectively.Absorbance at 230 nm ± S.D Absorptivity at 244 nm Absorptivity at 230 nm * Absorbance at 244 nm ± S.D * * Each value is the average of three determinations.

Table 2 :
Absorbance and absorptivity values of Cetirizine Hydrochloride at  max 244 nm and 230 nm, respectively.
* Absorbance at 244 nm ± S.D * Absorbance at 230 nm ± S.D Absorptivity at 244 nm Absorptivity at * Each value is the average of three determinations.

Table 3 :
Optical characteristics of Ambroxol Hydrochloride and Cetirizine Hydrochloride.

Table 4 :
Recovery studies for Ambroxol Hydrochloride and Cetirizine Hydrochloride.

Table 5 :
Drug content uniformity of Ambroxol Hydrochloride and Cetirizine Hydrochloride.