INTRODUCTION:

Salbutamol sulphate is an anti-asthmatic drug. It possess pharmacological actions for the treatment of asthma attacks, bronchoconstriction, chronic obstructive pulmonary disease. Salbutamol sulphate is a highly selective β₂receptor agonist.

It possess sympathomimetic actions. It exhibits its mechanism of action by binding to β₂receptors (a type of G–Protein coupled receptors), which are located on tracheobronchial smooth muscle.

After stimulation of β₂receptor it causes cAMP release. cAMP production increases which causes activation of cAMP dependent protein kinase A and nucleotide exchange factor Epac, which ultimately leads to relaxation of bronchial smooth muscles (bronchodilation). Salbutamol sulphate is available in various dosage forms such as oral tablets, intravenous solution, inhaler and Transdermal patches^1,2,3,4,5. Asthma is a long term chronic inflammatory disease of the airways of the lungs. Asthma can be acute or chronic. Clinical features of asthma are wheezing, coughing, chest tightness and shortness of breath, dyspnea^6,7. Bronchial asthma based on measurement of immunoglobulin E is of two types extrinsic(atopic) and intrinsic(non-atopic)⁸.

Salbutamol sulphate is white or almost white, crystalline powder. Its molecular formula is C₂₆H₄₄N₂O₁₀S. Molecular weight is 576.7. Chemically outlines as Bis[(1RS)-2-[(1, 1-dimethylethyl)amino]-1-[4-hydroxy-3- (hydroxymethyl)phenyl]ethanol] sulphate.It is freely soluble in water, insoluble in ethanol and methylene chloride. It exhibits optical rotation of -0.10º to + 0.10º in water⁹.

Principle involved in this colorimetric method is as follows. Brady’s reagent is well known as 2, 4-Dinitrophenylhydrazine.This reagent is used for colorimetric determination of chemical compounds containing carbonyl groups (aldehydes, ketones) as functional group. Brady’s reagent reacts with carbonyl groups under alkaline conditions and results in formation of colored hydrazones-yellow colored for aliphatic carbonyl and red colored for aromatic carbonyl compound. Brady’s reagent undergoes nucleophilic addition-elimination reaction. Nucleophilic addition of –NH₂ group to the C=O carbonyl group occurs, in presence of oxidizing agent and under alkaline conditions water molecule gets eliminated, this results in formation of red colored hydrazone product. Salbutamol sulphate contains aromatic aldehyde as a functional group, hence red colored hydrazone is formed upon reacting it with Brady’s reagent^10,11.

Girish Pai et.al. evaluated Salbutamol sulphate syrup¹². Mr. Salve developed sustained release beads of Salbutamol sulphate and evaluated for in-vitro dug release¹³. Vaseeha Banu et.al. prepared Transdermal film of Salbutamol sulphate and determined drug content using buffer¹⁴. Tasnuva Haque et.al. performed in-vitro release testing of Salbuatmol sulphate matrix tablets¹⁵. Anubha khale at.al. performed drug testing on Liposomal nebulising solutions of Salbutamol sulphate¹⁶. Somnath Kedar et.al. evaluated sustained release Salbutamol sulphate tablets¹⁷.

Figure 1 Chemical structure of Salbutamol sulphate⁹

Arifa Begum et.al. formulated and evaluated Pediatric Oral soft jellies of Salbutamol sulphate¹⁸. Various analytical techniques such as ultra violet spectrophotometric, HPLC and HPTLC were developed, as per literature survey^19,20,21,22.

MATERIAL AND METHODS:

Chemicals and reagents:

Distilled water was used for analysis. Various chemicals such as Brady’s reagent (Merck Grade), Concentrated sulphuric acid (Rankem Grade), Potassium bromate solution (Merck Grade) and Sodium hydroxide (Labogens Grade) of analytical grade were used in this method. Salbutamol sulphate API was gifted sample from Aizant labs. Salbutamol sulphate tablets-Asthalin Tablets (Dose-4mg) was procured from market, manufactured by Cipla Limited.

Instrumentation:

UV Visible Shimadzu spectrophotometer of UV 1800 model was used for analysis. Glass quartz Cuvettes were used. Other instruments used were Analytical Electronic Balance, calibrated (Mettler Toledo), Ultra Sonicator (Lab line), Hot Air Oven (Accumax India) and Porcelain made Mortar and pestle were used. Calibrated glass wares used were made up of borosilicate glass of Class-A grade.

Preparation of Brady’s Reagent Solution:

0.08% W/V Brady’s reagent solution was prepared. About 0.08gm of Brady’s reagent was weighed and transferred into a 100mL of volumetric flask. 2mL of concentrated sulfuric acid was added carefully. To this carefully about 15mL of warm water was added and mixed well. Then volume was made upto 100mL with water. The contents were mixed well and finally the solution was filtered through a Whatman filter paper.

Preparation of Standard Solutions -Procedure:

Preparation of Salbutamol sulphate Stock Solution (about 1000µg/mL):

Weighed and transferred 100mg of Salbutamol sulphate into a dried 100mL volumetric flask. To this about 40 mL of distilled water was added and sonicated for 25 min. Then the volume was made upto 100mL with distilled water and finally mixed well.

Preparation of Salbutamol sulphate Standard Solution-I (about 100µg/mL):

10 mL of Salbutamol sulphate stock solution was pipette out and transferred to a 100mL volumetric solution, finally volume was made upto 100mL with distilled water.

Preparation of Salbutamol sulphate Serial Standard Solutions (about 10µg/mL, 20µg/mL, 30µg/mL, 40 µg/mL and 50µg/mL):

In a series of five 10mL volumetric flasks were labeled appropriately. To each flask 2mL of Brady’s reagent solution and 1.5mL of potassium bromate solution were pipette out and transferred to a 10mL volumetric flask. From Salbutamol sulphate standard solution-I 1mL, 2mL, 3mL, 4mL and 5mL was pipette out and transferred into each 10mL volumetric flask containing Brady’s reagent solution and potassium bromate. Then 0.8mL of 10M sodium hydroxide solution was added, finally volume was made upto 10mL with distilled water to produce about 10µg/mL, 20µg/mL, 30µg/mL, 40 µg/mL and 50µg/mL respectively. The flask was kept aside undisturbed for 15 min. to develop red color intensity solution. Absorbances of red colored solution was recorded at lambda max against a blank solution.

Preparation of Blank:

In a 10mL volumetric flasks 2mL of Brady’s reagent solution and 1.5mL of potassium bromate solution were pipette out and transferred to a 10mL volumetric flask. 6.25mL of distilled water was added. Then 0.8mL of 10 M sodium hydroxide solution was added, finally volume was made upto 10mL with distilled water to produce blank and the flask was kept undisturbed aside for 15 min.

Determination of Lamda Max:

10µg/mL Salbutamol sulphate prepared solution was scanned from 800 to 400nm, for determination of lambda max.

Procedure for Quantitative Estimation of Salbutamol Sulphate in Tablets:

Weighed and transferred 20 Asthalin tablets into dried mortar. Tablets were crushed to a fine powder by pestle.

Stock Solution:

Tablet powder containing an amount equivalent to 4mg of Salbuatmol sulphate was weighed and transferred into a 100mL volumetric flask. To this about 40mL of distilled water was added, sonicated for 25 min and finally volume was made upto 100mL with distilled water. The contents were mixed well and Salbutamol sulphate sample solution was filtered through a Whatman filter paper.

Sample Solution:

2 mL of Brady’s reagent solution and 1.5mL of potassium bromate solution were pipette out and transferred to a 10mL volumetric flask. From above tablet stock solution, 6.25mL of filtrate solution was added. Then to this 0.8mL of 10M sodium hydroxide solution was added, finally volume was made upto 10 mL using distilled water. The flask was kept aside undisturbed for 15 min. to develop red color intensity solution. Absorbance of red colored sample solution was recorded at 540nm against a blank solution. The concentration of Salbutamol sulphate in tablets sample solution was obtained from linearity graph. Then the amount of the drug and percent label claim of Salbutamol sulphate in tablets were calculated.

Limits:

Salbutamol sulphate tablets contain Not less than 90% and Not more than 110% of the stated amount of Salbutamol sulphate²³.

Validation of Developed Method:

Linearity:

Salbutamol sulphate solution were prepared in a concentration range of 10 to 50µg/mL as mentioned above under standard solutions preparations. Absorbances of the solutions were checked at λ max. of 540nm. Based on values calibration graph was plotted (as depicted in Figure 2) and correlation coefficient value was reported.

Accuracy:

Accuracy was performed at three levels. Samples in a replicate of three were prepared at three levels of 80%, 100% and 120% with respect to target concentration (25 µg/mL). % Recoveries were calculated and reported in table 1.

Precision:

Repeatability:

Six samples were prepared as described under procedure for quantitative estimation of Salbutamol sulphate in tablets. Absorbances were measured, concentrations were determined, and percent label claim or percent assay were calculated. From this Mean, Standard deviation and percent relative standard deviation (% RSD) were calculated and checked whether it passes ICH limits as tabulated in table 2.

Intermediate Precision:

Samples in a replicate of six were prepared on three different days. Percent label claim were calculated and % RSD were reported as shown in table 3.

Ruggedness:

Ruggedness was performed on different analyst. Samples in a replicate of six were prepared by two different analyst. % RSD were calculated and reported in table 4.

Robustness:

Robustness was performed on different wavelength. Six samples were prepared and analysed at a wavelength of λmax ± 2nm i.e. 538nm, 540nm and 542nm. %RSD were calculated and reported in table 5.

LOD and LOQ:

LOD and LOQ were determined using slope and standard deviation from linearity graph.

Physical Solution Appearance:

Sample solutions prepared were kept aside undisturbed upto 24 hrs. at room temperature.

RESULTS AND DISCUSSION:

Lamda max Determination:

Upon scanning Salbuatmol sulphate solution (10 µg/mL), from 800 to 400nm, λ max was found to be 540 nm.

Specificity:

Blank solution was analysed at 540nm. No blank interference was found.

Linearity:

Figure 2: Linearity graph of Salbutamol sulphate solutions

Linearity was found to be passed as correlation coefficient was found to be 0.999.

Accuracy:

Table 1: Results of Accuracy

S. No.	Recovery Level	Sample	Theoretical Concentration, µg/mL	Obtained concentration, µg/mL	% Recovery	Average % Recovery
1	80 %	Sample-1	20.00	19.66	98.30	100
		Sample-2	20.00	20.22	101.10
		Sample-3	20.00	20.12	100.60
2	100 %	Sample-1	25.00	24.89	99.56	99.56
		Sample-2	25.00	24.69	98.76
		Sample-3	25.00	25.49	101.96
3	120 %	Sample-1	30.00	29.56	98.53	98.53
		Sample-2	30.00	30.45	101.50
		Sample-3	30.00	30.01	100.0

% Recovery were found to be within acceptance criteria, as per acceptance criteria of ICH guidelines for assay of tablets²⁴.

Precision:

Repeatability:

Table 2: Results of Assay-Repeatability

Sample	Concentration obtained, μg/mL	Amount of the drug, mg	% Label Claim
Sample-1	24.50	3.92	98.0
Sample-2	25.03	4.00	100.1
Sample-3	25.48	4.08	101.9
Sample-4	24.93	3.99	99.7
Sample-5	25.13	4.02	100.6
Sample-6	25.49	4.08	102.0
Mean	-	-	100.38
SD	-	-	1.50
% RSD	-	-	1.49 %

Based on % RSD, repeatability was found to be passed.

Intermediate Precision:

Table 3: Results of Intermediate Precision

Day	% Label Claim
Day	Sample-1	Sample-2	Sample-3	Sample-4	Sample-5	Sample-6	Mean	SD	%RSD
Day-1	98.00	100.10	101.90	99.70	100.60	102.00	100.38	1.50	1.49
Day-2	99.80	102.10	100.10	99.60	98.90	99.60	100.02	1.09	1.09
Day-3	101.00	100.80	100.60	98.40	101.30	102.60	100.78	1.37	1.36

Based on tabulated results, intermediate precision was found to be passed.

Ruggedness:

Table 4: Results of Ruggedness-Different Analyst:

Analyst	% Label Claim
Analyst	Sample-1	Sample-2	Sample-3	Sample-4	Sample-5	Sample-6	Mean	SD	%RSD
Analyst-1	100.60	100.20	101.60	101.20	99.60	99.80	100.50	0.79	0.78
Analyst-2	103.0	102.60	100.01	99.90	98.90	101.60	101.00	1.64	1.63

Robustness:

Table 4: Results of Robustness-Change in λmax.

Sample	Wavelength
Sample	538 nm (λmax -2 nm)	540 nm (λmax)	542 nm (λmax + 2 nm)
Sample-1	99.98	100.10	102.00
Sample-2	98.67	99.99	101.90
Sample-3	99.99	101.20	102.99
Sample-4	100.10	102.10	104.10
Sample-5	97.90	98.60	100.10
Sample-6	98.30	99.60	102.40
Mean	99.16	100.27	102.25
SD	0.98	1.23	1.33
% RSD	0.99	1.23	1.30

Robustness was found to be passed at λmax± 2nm, as per ICH limits.

LOD and LOQ:

LOD and LOQ for the proposed were found to be 2.40 µg/mL and 7.26µg/mL respectively.

Physical Solution Appearance:

Samples were checked visually after 24hrs., no precipitates or crystallization of solution were observed.

CONCLUSION:

Salbutamol sulphate is a widely used anti-asthmatic drug. Brady’s reagent solution was used for quantitative estimation of Salbutamol sulphate in tablets. This colorimetric assay method developed is a simple visible spectrophotometric method. The developed method was subjected to various validation parameters. Hence the developed method for assay of Salbuatmol sulphate by using Brady’s reagent solution was found to be simple, accurate, linear, precise, robust and rugged as per ICH guidelines. Developed method can even detect and quantitates on lower concentrations. It is less sophisticated compared to other hyphenated analytical techniques. Method developed involves low-cost equipment and is economical. Percent label claim of Salbutamol sulphate in tablets were found to be within Pharmacopeial limits. Sample preparation is simple and uncomplicated. Hence we conclude that, the developed and validated method for assay of Salbuatmol sulphate tablets can be implemented for routine analysis of tablets.

ACKNOWLEDGEMENT:

We are thankful for the management to our college management for providing us necessary requirements for performing this work.

CONFLICT OF INTEREST:

We declare no conflicts of interest.

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Received on 12.10.2020 Modified on 16.11.2020

Res. J. Pharma. Dosage Forms and Tech.2021;13(1):1-6.

DOI: 10.5958/0975-4377.2021.00001.X