INTRODUCTION:

Asthma, a common chronic inflammatory disease of the airways characterized by variable and recurring symptoms, reversible airflow obstruction, and bronchospasm. It’s symptoms include wheezing, coughing, chest tightnes and shortness of breath. It is thought to be caused by a combination of genetic and environmental factors. Treatment of acute symptoms is usually done with an inhaled short-acting beta-2 agonist (such as Salbutamol Sulphate) and by avoiding triggers, such as allergens, irritants and by inhaling corticosteroids (such as Theophylline)[1].

Salbutamol Sulphate (SAL) is generally used for acute episodes of bronchospasm caused by bronchial asthma, chronic bronchitis and other chronic bronchopulmonary disorders such as Chronic Obstructive Pulmonary Disorder (COPD) (Fig 1).

It is also used prophylactically for exercise-induced asthma. Salbutamol Sulphate is a beta (2)-adrenergic agonist and thus it stimulates beta (2)-adrenergic receptors, which results in relaxation of bronchial smooth muscles. In addition to bronchodilation, it inhibits the release of bronchoconstricting agents from mast cells, inhibits microvascular leakage, and enhances mucociliary clearance [2].

Theophylline (THEO) is a methyl xanthine derivative obtained from tea with diuretic and smooth muscle relaxant, bronchial dilation, cardiac and central nervous system stimulant activities (Fig 1). It inhibits the 3',5'-cyclic nucleotide phosphodiesterase that degrades cyclic AMP thus potentiates the actions of agents that act through adenylate cyclase and cyclic AMP. It relaxes the smooth muscle of the bronchial airways and pulmonary blood vessels and reduces airway responsiveness to histamine, methacholine, adenosine, and allergen [3].

The combined effect of SAL and THEO was observed greater than the sum of their individual effects. It was observed that in peripheral airways THEO potentiates the action of Salbutamol both directly via smooth muscle relaxation and indirectly via inhibition in mediator release [4].

Literature survey revealed that few analytical methods like RP-HPLC method using UV and fluorescence detector, stability indicating HPLC method [5,6,7], Capillary electrophoresis[8], spectrophotometric method [9,10], Titrimetric method [11],Bromimetric method [12], Voltammetric method [13] are reported for Salbutamol Sulphate and Theophylline as an individual drug or in combination with other drugs. As there is no analytical method reported for the simultaneous estimation of these prescribed drugs, the present research work was aimed for the development of spectroscopic method using Multicomponent mode for analysis of SAL and THEO without prior separation in pharmaceutical dosage form

Salbutamol Sulphate

Theophylline

Fig 1; Structures of Salbutamol Sulphate and Theophylline

MATERIALS AND METHODS:

Reagents and Materials:

SAL and THEO in the form of gift samples were kindly supplied by, Micro Labs Ltd, Bangalore. Double distilled water was used as solvent throughout the study. A combination of SAL (2 mg) and THEO (100 mg) in tablet formulation was procured from local pharmacy (Theoasthalin, Cipla).

Instrument:

A Shimadzu UV-Visible double beam spectrophotometer model 1700 (Japan) with 1 cm matched quartz cells connected to a PC computer running UV-Probe 2.32 software for absorbance measurements and treatment of data was used. Sartorius digital balance for weighing and PCI analytics sonicator for extracting the drugs from the marketed formulation was used.

Method:

The mixed stock solution of analytes was prepared by transferring 8 mg of SAL and 400 mg of THEO in 100 ml volumetric flask. The drugs were dissolved in few ml of Double Distilled Water and volume was made upto the mark with the same solvent to get the concentration of 80 µg/ml of SAL and 4000 µg/ml of THEO. Five mixed standards of two drugs were prepared so as to contain 2-10 µg/ml of THEO and 0.04-0.2 µg/ml of SAL in double distilled water. All mixed standard solutions were scanned over the range of 320 nm to 200 nm in multi component mode of spectrophotometer at medium scanning speed where measuring wavelength interval of 40 nm was selected. An overlain spectrum of mixed standard solutions is as shown in Fig 2. The spectral data of these scans were stored in the instrument and used to determine the concentration of SAL and THEO in the sample solution.

Fig 2: Overlain Spectra of Mixed Standards of SAL and THEO

Analysis of commercial formulation

Twenty tablets (Theoasthalin, Cipla) were accurately weighed and crushed to fine powder. The tablet powder equivalent to 10mg of THEO (i.e. 0.2 mg of SAL) was accurately weighed, transferred to 100ml volumetric flask. Small quantity of double distilled water was added and sonicated for 10 min and finally using the same solvent the volume was made up to mark to get the concentration of 100 µg/ml of THEO and 2 µg/ml of SAL. This solution was filtered through Whatman filter paper No. 41. The filtrate was further diluted with double distilled water to get concentration of 5 µg/ml of THEO and 0.1 µg/ml SAL.

The sample solution was scanned over the range of 320 nm to 200 nm in Multicomponent mode immediately after scanning of serial dilution of mixed standard solution and concentration of each component was estimated by analysis of spectral data of sample solution with respect to that of mixed standards by the instrument. The spectrum of sample solution is given in Fig 3 and the results of assay of marketed formulation are given in Table no. 1.

Fig 3: Spectrum of Sample Solution

Table 1: Results of Assay of Marketed Formulation (Theoasthelin tablets) (*n=6)

Analyte

Label claim per tablet

( mg)

Mean amount found per tablet ( mg ) *

Mean

amount

found (%) *

% R.S.D*

SAL

2.015

100.50

1.987

THEO

100

100.49

100.79

1.653

Validation of Method:

The proposed method of analysis for SAL and THEO in combination was validated as per the recommendation of ICH guideline [14] for accuracy and precision. To study accuracy of the method recovery studied were carried out by standard addition method at 80,100,120%. A known amount of SAL and THEO was added simultaneously in to the preanalysed sample solution and percentage recoveries were calculated. The result of recovery studied were found satisfactory and reported in Table-2. While precision of the method was determined by repeatability (intra-day) and intermediate precision (inter-day) and expressed as % Relative Standard Deviation (%RSD).Intra-day precision was evaluated by analyzing concentration of SAL (0.1μg/ml) and THEO(5μg/ml) of standard and sample solutions at three different time intervals under the same experimental conditions on the same day. Intermediate precision (inter-day precision) was determined by analyzing above mentioned concentration of solutions on three consecutive days. (Table 3)

Table 2: Results of Recovery Studies *( n=3 )

Analyte	LEVEL OF RECOVERY
Analyte	80% (±RSD) *	100% (±RSD)*	120% (±RSD)*
SAL	97.54 ±1.63	94.76 ±1.56	94.34% ±1.30
THEO	102.96 ±0.598	99.84 ±0.70	98.50% ±1.18

Table 3: Results of Precision Studies (Intra-day and Inter-day) *(n=3 )

Analyte

Concentration of sample solution (µg/ml)

Intraday Precision (%) R.S.D.*

Interday Precision (%) R.S.D.*

SAL

0.100

0.586

0.9881

THEO

0.575

1.188

RESULTS AND DISCUSSION:

Considering the common solubility of drugs, the stock solution was prepared in water while further dilutions were made in different solvents like 0.1 N Hydrochloric acid, 0.1N Sodium hydroxide, Methanol, Dimethyl Formamide, Dichloro methane and Glacial Acetic Acid. Results obtained were found to be satisfactory in water. Hence water was selected as solvent.

As the proposed method is specific to instrument having software for provision of such determination, selection of proper sampling wavelength and concentration of mixed standard are critical. Hence overlay spectra of analytes were studied carefully. SAL was found to be absorbing prominently at 277 nm while THEO absorbed at 270 nm (λmax) and scanning range of 320nm to 200 nm was selected for the Multicomponent analysis as both the drug showed absorbance in this range .

The mean content of analytes in the marketed formulation were found as 100.50% and 100.79% for SAL and Theo respectively, while results of recovery study was found in the range of 93.26-99.24% for SAL and 97.39-103.615% for THEO. The values of relative standard deviations of inter -intraday studies were found to be less than 2%. The assay and validation results confirmed that the contents of SAL and THEO estimated in the tablet dosage form were free from the interference of excipients.

CONCLUSION:

Hence it can be concluded that the developed multi component spectroscopy method for simultaneous estimation of Salbutamol Sulphate and Theophylline in combined tablet dosage form is simple, economical, accurate and reproducible and will be conveniently adopted for the routine quality control analysis from its pharmaceutical formulations and bulk drug.

REFERENCES:

1) en.wikipedia.org/wiki/Asthma#cite_note_lemanske-9 (access date Dec 6,2011)

2) www.drugbank.ca/drugs/DB01001 (access date Dec 6, 2011).

3) www.drugbank.ca/drugs/DB00277 (access date Dec 6, 2011).

4) www.ncbi.nlm.nih.gov/pubmed/90618 (access date Nov 16, 2011)

5) Maithani M, Singh R. Development and validation of a stability-indicating HPLC method for the simultaneous determination of Salbutamol Sulphate and Theophylline in pharmaceutical dosage forms. Journal of Analytical and Bioanalytical Techniques.2011; 1:116

6) Chitlange SS, Chaturvedi KK, Wankhede SB. Development and validation of spectrophotometric and HPLC Method for the simultaneous estimation of Salbutamol Sulphate and Prednisolone in tablet dosage form. Journal of Analytical and Bioanalytical Techniques.2011;2:117.

7) Shidhaye S, Malke S, Kadam V. Validated stability indicating HPLC method for estimation of Theophylline from a novel microsphere formulation. Asian Journal of Pharmaceutics.2009;3(1):13-7

8) Chen C, Li H, Fan Y, Detemination of Salbutamol Sulphate in medicaments by capillary electrophoresis with contactless conductivity detection. China Journal of Chromatography/ SE PU. 2011;28(2):137-40

9) Patel PA, Dole MN, Shedpure PS, Sawant SD. Spectrophotometric simultaneous estimation of Salbutamol and Ambroxol in bulk and formulation. Asian Journal of Pharmaceutical and Clinical Reserch.2011; 4(3):42-5.

10) Mishra AK, Kumar M, Mishra A, Verma A, Chattopadhyay P. Validated UV spectroscopic method for estimation of Salbutamol from tablet formulations. Archive of Apllied Science Reserch.2010;2(3):207-11.

11) Kanakapura B, Bankavadi C.S, Veeraiah R, Rapid Titrimetric and spectrophotometric method for Salbutamol Sulphate in pharmaceutical using N-Bromosuccimide. Acta Pharmaceutica.2007:57(1); 87-98

12) Basavaiah K, Prameela H.C., Bromimetric methods for the determination of Salbutamol Sulphate. Analytical and Bioanalytical Chemistry, July2003: 376(6); 879

13) Niyazi Y, Sibel A, Ozakan, Bengi U, Zuhre S, Inci B, Voltammetric determination of Salbutamol based on electrochemical oxidation at platinum and glassay carbon electrodes. Turkish Journal of Chemistry.1998:22,175-182

14) ICH Guidance on Analytical Method Validation, in: Proceedings of the International Convention on Quality for the Pharmaceutical Industry, Toronto, Canada, and September, 2002.

Received on 21.01.2013 Modified on 04.02.2013

Asian J. Research Chem. 6(3): March 2013; Page 200-203