STABILITY INDICATING UPLC METHOD FOR SIMULTANEOUS ESTIMATION OF ALBUTEROL SULPHATE, THEOPHYLLINE AND BROMHEXINE IN BULK AND COMBINED DOSAGE FORM

STABILITY INDICATING UPLC METHOD FOR SIMULTANEOUS ESTIMATION OF ALBUTEROL SULPHATE, THEOPHYLLINE AND BROMHEXINE IN BULK AND COMBINED DOSAGE FORM

V. Padmaja ^* and M. Prasanthi

Department of Pharmaceutical Analysis, Nirmala College of Pharmacy, Atmakuru, Mangalagiri - 522503, Andhra Pradesh, India.

ABSTRACT: A new simple, precise, accurate and selective UPLC method has been developed and validated for stability indicating UPLC method for simultaneous estimation of Albuterol sulfate, Theophylline and Bromhexine HCl in the tablet dosage form. The method was carried out on a Hibra C18, 250mm x 4.6mm, 5µm. a column with a mobile phase consisting of buffer and acetonitrile and buffer in the ratio of (55: 45 v/v/v) and flow rate of 1.0 ml/ min. The detection was carried out at 260nm. The retention time for Albuterol sulfate, Theophylline, and Bromhexine HCl were found to be 5.8, 2.3 and 9.7 min respectively. The method was validated according to the ICH guidelines for specificity, LOD, LOQ, precision, accuracy, linearity and robustness. The method showed good reproducibility and recovery with %RSD less than 2. So the proposed method was found to be simple, specific, precise, accurate and linear than the methods reported earlier. Hence, the method is economical, and it can be applied for routine analysis of Albuterol sulphate, Theophylline and Bromhexine HCl in bulk drug and pharmaceutical preparations. When applied for tablet assay, drug content was within the limits of the labelled content.

UPLC, Albuterol sulfate, Theophylline, Bromohexine hydrochloride, ICH guidelines

INTRODUCTION: Albuterol is a beta (2)-adrenergic agonist. It stimulates beta (2)-adrenergic receptors. Binding of Albuterol to beta (2)-receptors in the lungs results in the relaxation of bronchial smooth muscles. Albuterol increases cAMP production by activating adenylate cyclase, and the actions are mediated by cAMP. Increased intracellular cyclic AMP increases the activity of cAMP-dependent protein kinase A, which inhibits the phosphorylation of myosin and lowers intracellular calcium concentrations.

A lowered intracellular calcium concentration leads to smooth muscle relaxation. Increased intracellular cyclic AMP concentrations also cause an inhibition of the release of mediators from mast cells in the airways.

FIG. 1: ALBUTEROL SULPHATE

Theophylline, also known as 1,3-dimethylxanthine, is a methylxanthine drug used in therapy for respiratory diseases such as chronic obstructive pulmonary family, it bears structural and pharmacological similarity to theobromine and caffeine. Disease (COPD) and asthma under a variety of brand names. As a member of the xanthine.

FIG. 2: THEOPHYLLINE

Bromhexine is an oral mucolytic agent with a low level of associated toxicity. Bromhexine acts on the mucus at the formative stages in the glands, within the mucus-secreting cells. Bromhexine disrupts the structure of acid mucopolysaccharide fibers in mucoid sputum and produces less viscous mucus, which is easier to expectorate.

FIG. 3: BROMHEXINE HYDROCHLORIDE

According to the literature survey, few HPLC methods ^{1 - 8} UV methods ^{9, 10} and UV differential ¹¹ method and UV-Visible spectrophotometric methods ¹² are developed individually or with another combination of drugs. The proposed method aimed to developed and validate a stability indicating a method for the estimation of Albuterol sulfate, Theophylline and Bromohexine hydrochloride in pharmaceutical dosage form by UPLC.

MATERIAL AND METHODS:

Selection of Wavelength (For Detection): In setting up the conditions for the development of assay method, the choice of detection wavelength was based on the scanned absorption spectrum for Albuterol sulfate, Theophylline, and Bromhexine HCl. The UV-spectrum of Albuterol sulfate, Theophylline, and Bromhexine HCl was obtained separately by scanning the sample over the wavelength range 200 - 400 nm against blank as methanol. After a thorough examination of the spectra, the wavelength of 260 nm was selected for further analysis. The Overlay spectrum for Albuterol sulfate, Theophylline, and Bromhexine HCl was shown in Fig. 4.

Optimized Method:

HCl Preparation of Buffer: Taken 1 ml of triethylamine in 1L distilled water to this adjusts pH-2.5 with OPA. Filter through a 0.45μ membrane filter.

Mobile Phase: A mixture of buffer and acetonitrile in the ratio of 45:55% v/v was sonicated to degas and filtered through a 0.45µm nylon membrane filter.

Preparation of Diluent: Acetonitrile: Buffer (55: 45v/v).

Chromatographic Conditions:

Column           :  Hibra C18, 250mm × 4.6mm, 5µm

Mobile phase  : Acetonitrile: Buffer (55:45% v/v)

Flow rate         :           1.0 ml/min

Detection        :           260 nm

Wavelength

Injection          :           5 µl

Volume

Temperature    :           Ambient

Run time         :           10 min

The retention time of Albuterol sulfate is about 5.8 min. The retention time of Theophylline is about 2.3 min. The retention time of Bromhexine is about 9.7 min.

Preparation of Standard Stock Solution:

Solution A:

Albuterol Sulphate: Weighed accurately about 4mg Albuterol sulfate working standard into a 100 ml volumetric flask. Added 70 ml of diluent, sonicated to dissolve and diluted to volume with diluents.

Solution B:                 

Theophylline: Weighed accurately about 200 mg of Theophylline working standard into a 100 ml volumetric flask. Added 70 ml of diluent, sonicated to dissolved and diluted to volume with diluent.

Solution C:

Bromhexine HCl: Weighed accurately about 8mg Bromhexine HCl working standard into a 100 ml volumetric flask. Added 70 ml of diluent, sonicated to dissolve and diluted to volume with diluent. Further diluted every 5 ml of solution-A, B and C to 50 ml with the diluent.

Preparation of Sample Solution: Weighed accurately 10 tablets and powdered then taken 5 tablets equivalent of the sample into a 250 ml volumetric flask. Added 200 ml of diluent, sonicated to dissolve and diluted to volume diluent and further diluted 5 ml to 100 ml with the diluent and filtered through a 0.45μ nylon syringe filter.

Procedure: Injected 5µL of standard preparation five times and sample preparation in the chromatograph. Recorded the chromatograms and measured the peak responses for Albuterol sulfate, Theophylline, Bromhexine HCl. The system suitability parameters should be met. From the peak responses, calculated the content of Albuterol sulfate, Theophylline, Bromhexine HCl in the sample. The assay calculations were shown in Table 1. And the Fig. is shown in 2, 3 and 4.

Method Validation:

System Suitability: The UPLC system was stabilized for thirty minutes. by following the chromatographic conditions to get a stable baseline. One blank followed by six replicates of a standard solution was injected to check the system suitability. The system suitability parameters were evaluated from standard chromatograms obtained, by calculating the retention times, tailing factor, theoretical plates, and %RSD peak areas from six replicate injections. The results are shown in the below Table 2, 3 and 4.

Linearity: A series of solutions were prepared using Albuterol sulfate, Theophylline and Bromhexine HCl working standard at concentration levels from 0-5µg/mL, 32-272µg/mL and 1-11 µg/mL respectively, the solutions were injected into the system as per test procedure. Measure the peak area response of the solution. The calibration graph was plotted with peak area in the Y-axis and concentration of standard solutions in the X-axis. The data is given in below Table 5, 6 and 7 and the calibration curve is shown in the below Fig. 8.

Accuracy: The accuracy of the developed method was determined by assay and recovery studies. Recovery studies were carried out at three different levels. The pre-analysed samples were spiked with 50%, 100% and 150% of the mixed standard solution. The mixtures were analyzed by the proposed method. The study was carried out in triplicate. The obtained recovery results are given in Table 8, 9 and 10.

Precision:

System Precision (Repeatability): System precision was carried out using six replicates of the same standard concentration. The chromatograms were recorded and mean, standard deviation and % RSD was calculated. The data of the system precision is given in below Tables 11, 12 and 13.

Method Precision (Intraday): Method precision was carried out using six different samples of Albuterol sulfate, Theophylline and Bromhexine HCl drug substance were prepared with a target concentration of about Albuterol sulfate 4.5 ppm, Theophylline 200.2 ppm, and Bromhexine HCl 8.3ppm. Preparations from the same homogenous blend of the marketed sample. The data of the method precision is given in below Table 14, 15 and 16.

Intermediate Precision (Inter day): Six sample solutions are prepared and injected on the next day into the HPLC system as per the test procedure. The observations of Intermediate precision were given in below Table 17, 18 and 19.

Robustness: The method can remain unaffected by small, deliberate variations in the method parameters. In the case of liquid chromatography examples of typical variations are: influence of variations in wavelength detectors (+/5nm), influence of variations in column temperature (+/-5nm), influence of variations in mobile phase compositions (+/-5%), influence of variations in flow rate (+/0.2%), influence of variations of p in mobile phase (+/-5%), all observed values are summarised in Table 20.

The Limit of Detection (LOD) and Limit of Quantification (LOQ): LOD and LOQ of the developed methods were determined by injecting progressively low concentrations of standard solutions using the developed UPLC method. The LOD is the smallest concentration of the analyte that gives a measurable response (signal to noise ratio of 3). The LOD for Albuterol sulfate was found to be 0.133mg/ml, for Theophylline 0.336 mg/ml and for Bromohexine 0.18 mg/ml. The LOQ is the smallest concentration of the analyte, which gives a response that can be accurately quantified 9 signals to noise ratio of 10). The LOQ of Albuterol sulphate 0.40mg/ml, 19.20 mg/ml for Theophylline and 0.57mg/ml of Bromohexine HCl. It was concluded that the developed method is sensitive and the results are shown in Table 21.

Solution Stability: The solution stability of Albuterol sulfate, Theophylline, and Bromhexine HCl in diluents were determined by storing sample solution in a tightly capped volumetric flask at room temperature for 24 h. The amount of Albuterol sulfate, Theophylline, and Bromhexine HCl were measured at different time intervals like 12 and 24 h and results obtained were compared with Albuterol sulfate, Theophylline, and Bromhexine HCl freshly prepared solution. The results are shown in below Table 22, 23 and 24.

RESULTS AND DISCUSSION: The main aim of the study was to develop a stability indicating UPLC method for the estimation of Albuterol sulfate, Theophylline and Bromohexine hydrochloride in bulk and tablet dosage form and to validate the method. Initially, various mobile phase compositions were tried to elute the drugs. Mobile phase ratio and flow rate were selected based on peak parameters and retention time. Standard solution of 40 μg/ml, 200 μg/ml and 8 μg/ml were prepared and scanned in the range of 200-400 nm for detecting the maximum absorption wavelength, and it was found to be 260 nm Fig. 4.

FIG. 4: SELECTION OF WAVELENGTH

TABLE 1:   ASSAY CALCULATIONS   

TABLE 2: SYSTEM SUITABILITY FOR ALBUTEROL SULPHATE

TABLE 3: SYSTEM SUITABILITY FOR THEOPHYLLINE

TABLE 4: SYSTEM SUITABILITY FOR BROMHEXINE HCl

After considering the entire system suitability parameters mobile phase buffer and acetonitrile in the ratio of (45:55% v/v) run in the isocratic mode and flow rate 1.0ml/min were selected. The retention time of Albuterol sulfate, Theophylline, and Bromohexine hydrochloride was found to be 5.7, 2.3, 9.7 min. The system suitability parameters are calculated from standard chromatograms Fig. 6.

TABLE 5: LINEARITY DATA OF ALBUTEROL SULPHATE

TABLE 6: LINEARITY DATA OF THEOPHYLLINE

TABLE 7: LINEARITY DATA OF BROMHEXINE

TABLE 8: ACCURACY RESULTS OF ALBUTEROLSULPHATE BY UPLC

TABLE 9: ACCURACY RESULTS OF THEOPHYLLINE BY UPLC

TABLE 10: ACCURACY RESULTS OF BROMHEXINE HCl BY UPLC

TABLE 11: SYSTEM PRECISION VALUES OF ALBUTEROL SULPHATE BY UPLC

TABLE 12: SYSTEM PRECISION VALUES OF THEOPHYLLINE BY UPLC

TABLE 13: SYSTEM PRECISION VALUES OF BROMHEXINE HCl BY UPLC

TABLE 14: METHOD PRECISION FOR ALBUTEROL SULPHATE BY UPLC

TABLE 15: METHOD PRECISION FOR THEOPHYLLINE BY UPLC

TABLE 16: METHOD PRECISION FOR BROMHEXINE HCl BY UPLC

TABLE 17: INTERMEDIATE PRECISION VALUES FOR ALBUTEROL SULPHATE BY UPLC

TABLE 18: INTERMEDIATE PRECISION VALUES FOR THEOPHYLLINE BY UPLC

TABLE 19: INTERMEDIATE PRECISION VALUES FOR BROMHEXINE HCl BY UPLC

TABLE 20: ROBUSTNESS

TABLE 21: LOD AND LOQ

TABLE 22: SOLUTION STABILITY OF ALBUTEROL SULPHATE

TABLE 23: SOLUTION STABILITY OF THEOPHYLLINE

 TABLE 24: SOLUTION STABILITY OF BROMHEXINE HCl

CONCLUSION: The developed and validated UPLC method was found to be rapid, accurate, precise and robust, thus can be used for routine analysis of Albuterol sulfate, Theophylline and Bromohexine HCl in the combined dosage form.

ACKNOWLEDGEMENT: I express my sincere thanks to Nirmala College of Pharmacy for encouragement throughout my research work. And also thankful to ICON labs Vijayawada for providing the Albuterol sulfate, Theophylline, and Bromohexine hydrochloride pure drugs.

CONFLICT OF INTEREST: Nil

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    How to cite this article:

    Padmaja V and Prasanthi M: Stability indicating UPLC method for simultaneous estimation of Albuterol sulphate, Theophylline and Bromohexine in bulk and combined dosage form. Int J Pharm Sci Res 2019; 10(5): 2403-11. doi: 10.13040/IJPSR.0975-8232.10(5).2403-11.

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