ANALYTICAL METHOD DEVELOPMENT AND VALIDATION FOR THE SIMULTANEOUS ESTIMATION OF SALBUTAMOL SULPHATE, GUAIFENESIN, AND AMBROXOL HYDROCHLORIDE BY RP-HPLC METHOD IN COMMERCIAL ORAL LIQUID DOSAGE FORM

ANALYTICAL METHOD DEVELOPMENT AND VALIDATION FOR THE SIMULTANEOUS ESTIMATION OF SALBUTAMOL SULPHATE, GUAIFENESIN, AND AMBROXOL HYDROCHLORIDE BY RP-HPLC METHOD IN COMMERCIAL ORAL LIQUID DOSAGE FORM

Prabha Thangavelu ^*, Arul Caroline Grace and Jagadeeswaran Murugesan

Department of Pharmaceutical Analysis, Nandha College of Pharmacy, Koorapalayam Pirivu, Pitchandam Palayam Post, Erode - 638052, Tamil Nadu, India.

ABSTRACT: Salbutamol is a bronchodilator, Guaifenesin is an expectorant and Ambroxol is a mucolytic. Combination of these drugs is used in the formulation of cough syrups. In the literature, there is no method reported for the simultaneous estimation of the drugs in oral liquid dosage form. Hence, the present work is aimed to develop reverse phase HPLC method for the simultaneous determination of Salbutamol sulfate (SAL), Guaifenesin (GUA) and Ambroxol hydrochloride (AMB) in oral liquid dosage form and validation of the developed method. The chromatographic separation of the drugs was achieved with the mobile phase system sodium dihydrogen phosphate buffer pH 3.0: acetonitrile: methanol in the ratio of 65:10:25 with the flow rate of 1 mL/min and injection volume 10 µL. An Inertsil C8-3 (250 × 4.6 mm, 5 µm) column was used, and the detection wavelength was 276 nm. This system produced sharp peaks with good resolution, minimum tailing and satisfactory retention times of SAL, GUA and AMB were found to be 3.157, 9.949 and 11.883 min respectively indicating the suitability of the system. The developed method was validated for various parameters accuracy, precision, linearity, robustness, and specificity as per ICH guidelines.

Salbutamol, Ambroxol, Guaifenesin, Oral liquid dosage form, RP-HPLC

INTRODUCTION: Salbutamol sulfate (SAL) is a selective beta2-adrenergic receptor agonist. It works by binding of Salbutamol to beta (2) receptors in the lungs results in relaxation of bronchial smooth muscles ¹. Salbutamol is chemically designated as (RS)- 4-[2-(tert Butyl amino)-1-hydroxyethyl)-2-(hydroxymethyl) phenol Fig. 1 and its molecular formula is (C₁₃H₂₁NO₃)₂.

Guaifenesin (GUA) chemically designated as 3-(2-methoxy phenoxy) propane-1, 2-diol Fig. 1 and its molecular formula are C₁₀H₁₄O_4., mainly used as an expectorant which will reduce the viscosity of sputum. It may act as an irritant to the gastric vagal receptor, and recruit efferent parasympathetic reflexes that cause glandular exocytosis of a less viscous mucus mixture ².

Ambroxol hydrochloride (AMB) is a mucolytic agent chemically designated as Trans-4-[(2-amnio-3, 5-dibromo benzyl) amino] cyclohexyl Hydro-chloride Fig. 1 and its molecular formula is C₁₃H₁₉Br₂ClN₂O. Ambroxol produces their action by inhibiting the NO-dependent activation of soluble guanylate cyclase can suppress the excessive mucus secretion. Therefore it lowers the phlegm viscosity and improves the mucociliary transport of bronchial secretions. It promotes the removal of tenacious secretions in the respiratory tract and reduces mucus stasis ³.

FIG. 1: (A) SALBUTAMOL (B) GUAIFENESIN (C) AMBROXOL

There are few methods have been reported for simultaneous estimation of SAL, AMB, and GUA and in combination with some other drugs ^{4, 5}. Among those, the only one method is available which is a simple and precise Liquid Chromatography method for the simultaneous estimation of SAL, GUA, and AMB in the combined tablet dosage form and was developed, validated by Mukesh et al., Several methods such as HPLC ^6-22, Spectrophotometric ^{23, 24}, Gas Chromatography ^{25, 26, 27} and stability indicating LC Method ²⁸ have been reported for the analysis of SAL, GUA and AMB as an individual drug or in combination, either in pure or in pharmaceutical forms as well as in biological fluids and tissues.

However, there is no method reported for the simultaneous estimation of SAL, GUA and AMB in the oral liquid dosage form. So it was felt that there is a need to develop the RP-HPLC method for the determination of SAL, GUA and AMB simultaneously in a single step process. Hence the present work is aimed to develop and validate a simple RP-HPLC method for the simultaneous estimation of SAL, GUA and AMB in the liquid oral dosage form.

MATERIALS AND METHODS: The chromato-graphic technique performed on HPLC Agilent separation module 1200 with UV detector with reversed phase inertsil C₈ column (250 × 4.6 mm, 5 µ) as stationary phase, Jasco double beam UV-VIS spectrophotometer was used as a detector for this study. CRMS Syrup containing 500 mg of Guaifenesin, 150 mg of Ambroxol and 25 mg of Salbutamol was procured from local market. All other AR and LR grade chemicals such as sodium dihydrogen phosphate, acetonitrile, methanol and, triethylamine were procured from Rankem Limited, New Delhi.

Chromatographic Conditions: The sample separation was achieved on an Inertsil C₈ column (250 mm × 4.6 mm, 5 µ) with ambient température, the injection volume was set as 10 µL, UV detection at 276 nm, with the flow rate of 1.0 mL/min, the mobile phase aided by mobile phase mixture of Buffer: acetonitrile: methanol (65:10: 25% v/v).

Preparation of Phosphate Buffer: An amount of 1.56 gm of NaH₂PO₄ taken into a 1000 mL flask, dissolved and made up to 1000 mL with water and added 3 mL of triethylamine and adjusted the pH to 3.0 with ortho-phosphoric acid.

Preparation of Mobile Phase: Mixed the mixture of above buffer 650 mL (65%), 100 mL of Acetonitrile (10%) and 250 mL of Methanol (25%) and degassed in ultrasonicator for 5 min.

Finally, filtered through 0.45 µ nylon membrane filter under vacuum filtration.

Standard Solution (Preparation of SAL, GUA and AMB Stock Solution): Accurately weighed quantity of 500 mg of Guaifenesin, 150 mg Ambroxol and 25 mg of Salbutamol was transferred to three different 100 mL volumetric flask, dissolved in 25 mL of mobile phase, and sonicated for 5 min and the volume was made up with mobile phase.

Preparation of Working Standard: Working standard for Guaifenesin, Ambroxol and Salbutamol were prepared by pipetting out 10 mL, 10 mL, and 4 mL respectively from each of the above stock solutions into a 100 mL volumetric flasks and the volume was made up with the mobile phase.

Sample Preparation: An amount equivalent to 500 mg of Guaifenesin, 150 mg of Ambroxol and 25 mg of Salbutamol from liquid formulation was accurately weighed and taken in three different 100 mL volumetric flask, and 25 mL of mobile phase was added. The mixture was subjected to sonication for 15 min with frequent shaking for the complete dissolving of drugs.

Cooled to room temperature and the solution was made up with mobile phase and filtered through a 0.45µ membrane filter. From the above of each SAL, GUA and AMB solution, 4 ml, 10 ml, and 10 ml respectively were pipetted out into 100 ml volumetric flask, and the volume was made with mobile phase. Then 10 μl of this solution was injected for HPLC analysis.

Determination of Working Wavelength (λ_max): The stock solution of SAL, GUA, and AMB, each 10 µg/mL in methanol, were scanned over the wavelength range of 200 - 400 nm against blank. After a thorough examination of the spectra, the wavelength of 276 nm was chosen for further analysis.

RESULT AND DISCUSSION:

Method Validation (System Suitability): The system suitability parameters like resolution, retention time, plate number (N), peak asymmetry factor (Tailing) were evaluated with the help of standard chromatogram and showed in Table 1.

TABLE 1: RESULTS FOR SYSTEM SUITABILITY PARAMETERS

FIG. 2: CHROMATOGRAM FOR SAL, GUA AND AMB

Accuracy: The recovery study was carried out, and the contents were determined from the respective chromatogram. Recovery studies did the accuracy at 50%, 100%, and 150% concentration level. The recoveries at three different concentrations were found to be within the range of 98.4 to 101.6%, and the results were shown in Table 2.

TABLE 2: RESULTS FOR RECOVERY STUDY FOR SAL, GUA AND AMB

Precision: System Precision, Method Precision, and Intermediate Precision / Ruggedness: The system precision was performed by injecting standard solution for six times to the analytical column and the peak area was measured then the % RSD for the area was calculated.

The method precision was done by performing an assay on six replicate determination of sample preparation at test concentration level (as per the method of analysis), and the relative standard deviation of % assay of results was calculated.  To evaluate the intermediate precision of the method, the precision was performed on a different day by using different column make of same dimensions. The below Table 3 shows the overall data precision.

Linearity and Range: SAL showed linearity in the range of 5-15 (µg/mL), GUA showed linearity in the range of 250-750 (µg/mL) and AMB showed linearity in the range of 75-225 (µg/mL). The calibration graphs were plotted with peak area in the Y-axis and concentration of the standard solution in the X-axis Fig. 3. The degree of linearity was estimated by calculating the correlation coefficient. The correlation coefficient values for SAL, GUA and AMB, were found to be 0.9994, 0.9999 and, 0.9998 respectively and shown in Table 4.

TABLE 3: DATA FOR PRECISION OF SAL, GUA AND AMB

TABLE 4: RESULTS FOR LINEARITY DATA

FIG. 3: LINEARITY PLOT FOR SAL, GUA AND, AMB

Robustness: The robustness of a method is evaluated by varying method parameters such as flow rate, wavelength, etc., and determining the effect (if any) on the results of the method. The overall percentage relative standard deviation in the various parameters was found to be less than the acceptance limit of % RSD which is within 2%. The result indicated that the method was robust and is represented in Table 5 and 6.

Solution Stability: Solution stability study was performed at different days against the freshly prepared standard, and sample solution and the results were represented in Table 7 and 8.

TABLE 5: RESULTS FOR EFFECT OF FLOW RATE AND WAVELENGTH

TABLE 6: RESULTS FOR CHANGE IN FLOW RATE AND WAVELENGTH

TABLE 7: RESULTS FOR STANDARD SOLUTION STABILITY

TABLE 8: RESULTS FOR SAMPLE SOLUTION STABILITY

CONCLUSION: The developed method for simultaneous estimation of Salbutamol sulfate, Guaifenesin and Ambroxol hydrochloride said to be rapid, simple, accurate, precise, sensitive, robust and specific that can be successfully applied for the routine analysis of Salbutamol sulfate, Guaifenesin and Ambroxol hydrochloride in their marketed oral liquid dosage form.

ACKNOWLEDGEMENT: The authors are highly thankful to the Principal and the Management of Nandha College of Pharmacy, Erode, Tamil Nadu for providing the necessary facilities to carry out this research.

CONFLICT OF INTEREST: The authors declare no conflicts of interest.

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    How to cite this article:

    Thangavelu P, Grace AC and Murugesan J: Analytical method development and validation for the simultaneous estimation of Salbutamol sulphate, Guaifenesin and Ambroxol hydrochloride by RP-HPLC method in commercial oral liquid dosage form. Int J Pharm Sci & Res 2019; 10(1): 260-65. doi: 10.13040/IJPSR.0975-8232.10(1).260-65.

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