SIMULTANEOUS ESTIMATION OF LEVOCETRIZINE DIHYDROCHLORIDE AND AMBROXOL HYDROCHLORIDE IN SYRUP DOSAGE FORM BY USING UV SPECTROPHOTOMETRIC METHOD

• S. S. Pekamwar, Madhav Shelke

ABSTRACT: A simple, economical, rapid, accurate and precise UV Spectrophotometric method was developed for simultaneous estimation of Levocetrizine dihydrochloride and Ambroxol hydrochloride in bulk and in Syrup formulation. Determination of Levocetrizine dihydrochloride (LCTZ) and Ambroxol hydrochloride (AMB) in bulk and in syrup was conducted by using simultaneous equation method based on measurement of absorbance at 246 nm and 231 nm, λmax of LCTZ and AMB, respectively. The solvent used for proposed method was methanol: water (50:50). The linearity for LCTZ and AMB was in the range of 5-35 µg/ml and 5-50 µg/ml, respectively. The developed method was validated as per ICH guidelines. The % recovery was found to be in the range of 99.52- 99.82% and 100.37- 100.72% for LCTZ and AMB, respectively. Thus the proposed method was simple, rapid, reproducible and accurate and can be successfully applied for simultaneous estimation of Levocetrizine dihydrochloride and Ambroxol hydrochloride in bulk and in Syrup formulation.

KEYWORDS: Levocetrizine dihydrochloride, Ambroxol hydrochloride, Simultaneous Equation Method, UV Spectrophotometry, Syrup, Validation.

INTRODUCTION: Levocetirizine is a third generation non-sedative antihistamine. Chemically, levocetirizine is the active enantiomer of cetirizine. It is the L-enantiomer of the cetirizine racemate. Levocetirizine works by blocking histamine receptors. Levocetrizine is chemically, (R)-2-[2-[4-[(4- chlorophenyl)phenylmethyl] piperazin-1-yl] ethoxy] acetic acid dihydrochloride.

Chemical Structure of Levocetrizine dihydrochloride:

Ambroxol is an active N-desmethyl metabolite of mucolytic bromhexine. Ambroxol is used as a bronchosecretolytic and expectorant drug. Ambroxol has been shown to increase the quantity and decrease the viscosity of respiratory tract secretions. It enhances pulmonary surfactant production (surfactant activator) and stimulates ciliary motility. These actions result in improved mucus flow and transport (mucociliary clearance). Enhancement of fluid secretion and mucociliary clearance facilitates expectoration and eases cough. Ambroxol is chemically, trans-4-[(2-Amino-3,5-dibromobenzyl)amino] cyclohexanol hydrochloride.

Chemical Structure of Ambroxol hydrochloride:

Literature survey revealed that various analytical methods involving UV Spectrophotometry, HPLC and HPTLC have been reported for LCTZ and AMB alone and in Combination with other drugs. However there is no method developed for simultaneous estimation of Levocetrizine dihydrochloride and Ambroxol hydrochloride in liquid dosage form (Syrup). The objective of present work was to develop simple, rapid, economical accurate and precise analytical method for simultaneous estimation of Levocetrizine dihydrochloride and Ambroxol hydrochloride in bulk and in liquid dosage form (Syrup). The developed method was validated in accordance with ICH guidelines and successfully employed for the assay of LCTZ and AMB in combined liquid dosage form (Syrup).

MATERIALS AND METHODS

Chemicals and Reagents: Analytically, pure LCTZ and AMB were provided as gift sample by Bidwai Chemicals Pvt. Ltd. Nanded and Glenmark Pharmaceuticals, Mumbai, Maharashtra, respectively. Syrup of LCTZ and AMB, Mzic D manufactured by Monad Pharmaceuticals, were purchased from local pharmacy. Methanol AR grade (Molychem) and Distilled water was used throughout the experimental work.

Instruments: A UV-Visible Spectrophotometer: Shimadzu, Model: UV-1800 with 1 cm matched quartz cell and UV Probe 2.33 software was used. Calibrated analytical balance Anamed was used for weighing purpose. Digital Ultrasonic Cleaner (Sonicator): HMG India and Vaccum Pump Assembly: Millipore was used for the experimental work.

Preparation Standard Stock Solution:

Ambroxol hydrochloridre standard stock solution:

Accurately weighed reference standard of AMB (10mg) was transferred to 100ml volumetric flask and dissolved in 50 ml solvent (Water, methanol 1:1) and sonicate for 5 min. then volumes was made up to the mark with same solvent; to obtain standard stock solution (100µg/ml) of drug. For the preparation of working standards, suitable aliquots of stock solution were pipette out and volumes were made up to the mark with solvent (Water, methanol 1:1); so as to get required concentrations.

Levocetrizine dihydrochloride standard stock solution:

Accurately weighed reference standard of LCTZ (10mg) was transferred to 100ml volumetric flask and dissolved in 50 ml solvent (Water, methanol 1:1) and sonicated for 5 min. then volumes was made up to the mark with same solvent; to obtain standard stock solution (100µg/ml) of drug. For the preparation of working standards, suitable aliquots of stock solution were pipette out and volumes were made up to the mark with solvent (Water, methanol 1:1); so as to get required concentrations.

Selection of analytical wavelength:

Sample solutions were scanned over the wavelength range of 200 nm to 400 nm, λmax for AMB and LCTZ were found at 231 nm and 246 nm. Representative absorption spectra of AMB and LCTZ are shown in fig. 1 & 2.

Fig. 1 : Absorption Spectra of LCTZ

Fig. 2 : Absorption spectra of AMB

Preparation of calibration curve of AMB and LCTZ

By appropriate dilution of standard stock solution, different dilutions were prepared ranging from 5µg/ml to 35µg/ml for LCTZ and 5 µg/ml to 50 µg/ml for AMB. Absorbance of all the dilutions was plotted against the respective concentrations to obtain the calibration curves. The absorption overlain spectrum of AMB and LCTZ is shown in fig. 3

Fig. 3 : Overlay Spectra of LCTZ & AMB

Fig. 4 : Calibration Curve of LCTZ

Fig. 5 : Calibration Curve of AMB

Analysis of Marketed Syrup Formulation:

Ten syrup bottles were weighed accurately, average weight was determined and poured in calibrated measuring cylinder to check extractable volume. As per label claim syrup contain LCTZ 5mg/5ml and AMB 30mg/5ml, so 5ml syrup was pipette out in 100 ml volumetric flask and dissolved in water: methanol (1:1) solution, sonicated for 10 min and filtered. Then different concentrations of syrup sample were prepared by serial dilution method and used for analysis. Prepared syrup sample was analysed on UV spectrophotometer using Simultaneous Equation Method.

Method Validation

The method validation parameters linearity, precision, accuracy, repeatability, limit of detection and limit of quantitation were checked as per ICH guidelines.

Precision: Precision of the method was verified by using syrup stock solution, the repeatability indicates the performance of the UV instrument. Interday and intraday precision was determined by repeating the assay three times in same day for intraday precision and on different day for interday precision studies.

Accuracy (Recovery Study): To check the accuracy of proposed method, recovery studies were carried out at 80%, 100% and 120% of the test conc. As per ICH guidelines.

To perform recovery study at 100%, 5ml syrup pipette out in volumetric flask and to this standard 30mg AMB and 5mg LCTZ was added. The volume as made up to the mark with water: methanol (1:1) solution and further dilution with same solvent to obtain sample solution Similarly, for 80% recovery study, 26mg AMB and 4mg LCTZ were added, and for 120% recovery study, 36mg AMB and 6mg LCTZ were added respectively.

RESULTS AND DISCUSSION

Precision

Intraday Precision:

Table 1 : Intra-day Precision data of marketed formulation

* Indicates average of three determinations

Table No. 2 : Statistical validation of intra-day precision data

Inter-day Precision:

Table 3 : Inter-day Precision Data

* Indicates average of three determinations

Table 4 : Statistical validation of inter-day precision data

Accuracy (Recovery study):

Table 5 : Recovery study data

Table 6 : Statistical validation of recovery study data

Limit of Detection and Limit of Quantitation:

Table 7 : LOD & LOQ

CONCLUSION:

The method used is simple and rapid and does not involve the use of complex instrument, low value of Standard deviation showed that the method is precise and high percentage of recovery of as shown in table shows that the method is accurate. Thus from the results it can be concluded that above developed UV method is suitable for estimation of AMB and LCTZ in syrup formulation. The estimated method is sensitive, precise, accurate, specific and economic. Hence, the method can be used successfully for quality control and routine analysis of finished pharmaceutical dosage form.

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