Development and validation of HPLC method for simultaneous determination of Ketotifen Fumarate and Salbutamol Sulfate in bulk and tablets dosage forms

Abstract

The aim of the study was to develop and validate a new, rapid, sensitive, simple, accurate and reproducible HPLC method for simultaneous determination of ketotifen fumarate and salbutamol sulfate. Simultaneous HPLC method was developed using RP-C₁₈ stainless steel analytical column 4.6×150 mm C₁₈.Acetonitrile and phosphate buffer pH 4 (30 : 70) were used as mobile phase and wavelength was adjusted to 276 nm for detection of drugs. Developed method was validated for its specificity, accuracy, precision, linearity and robustness. Method was also applied to quantify drugs in commercial tablets. Chromatogram obtained by newly developed method for simultaneous determination of two anti-asthmatic drugs, having well distinguished peaks for both drugs. Retention time of ketotifen fumarate and salbutamol sulfate were 2.69 minutes and 9.47 minutes respectively. Total run time for both anti-asthmatic drugs was 12 minutes. Limit of quantification for ketotifen fumarate and salbutamol sulfate was 1 ng/ml and 1.50 ng/ml respectively. Limit of detection of ketotifen fumarate and salbutamol sulfate was 3.03 and 4.54 respectively. A simple, easy, precise and new method was developed for simultaneous quantification of frequently used anti-asthmatic drugs. Developed method may prove effective and beneficial in determination of ketotifen fumarate and salbutamol sulfate in bulk and other pharmaceutical dosage forms.

Keywords

HPLC anti-asthmatic Ketotifen fumarate Salbutamol sulfate simultaneous determination

1 Introduction

Ketotifen fumarate is mast cell stabilizer having additional properties of antihistamine. It is commonly used to treat the chronic condition of asthma, allergies like rhinitis and conjunctivitis. Dose of ketotifen fumarate is 1 mg twice daily with food and administration may continue for long period of time, up to years. Drug is completely absorbed from gastro intestinal track (GIT) followed by oral administration. Bioavailability of Ketotifen fumarate is limited to 50 %due to extensive first pass metabolism. Chemical name of ketotifen fumarate is 4, 9-Dihydro-4-(1-methylpiperidin-4-ylidene)-10H-benzo [4 –6] cyclohepta [1,2-b, 1,2-b] thiophen-10-one fumarate. Molecular weight of ketotifen fumarate is 425.50 amu; molecular formula of ketotifen fumarate is C₁₉H₁₉NOS.C₄H₄O₄ [1, 2].

Salbutamol sulfate is a more specific beta-2 selective adreno receptor agonist, having pronounced bronchodilatory effects. Broncho dilating effect of salbutamol sulfate depends upon types of dosage regimen to be used. Drug is completely absorbed from GIT, due to extensive first pass metabolism, bioavailability is limited only 50 %. Its dose is 4 mg two to three times a day. Chemically salbutamol sulfate is Bis [(1RS)-2-[(1,1-dimethylethyl)amino]-1-[4 hydroxy-3-(hydroxymethyl) phenyl] ethanol] sulfate, molecular weight of salbutamol sulfate is 288.4 amu and molecular formula is (C₁₃H₂₁NO₃)₂·H₂SO₄ [3, 7].

Ketotifen fumarate and salbutamol sulfate are frequently prescribed together to treat the complications associated with chronic asthma. Combination of ketotifen fumarate and salbutamol sulfate is enhancing the anti-asthmatic action particularly in case of allergic asthma [4]. Both of the drugs are being combined in single tablets by East West Pharma, Haridwar. India and marketing at commercial name of Mastifan-s. Tablets contained 1 mg of ketotifen fumarate and 2 mg of salbutamol sulfate. Survey of literature revealed that methods are available for analysis of both anti-asthmatic drugs separately or in combinations with some other drugs [5]. Kimura et al developed HPLC method for quantification of ketotifen fumarate in ophthalmic dosage form [8]. Inoue developed HPLC method for quantitative analysis of ketotifen fumarate in pressure sensitive adhesive patches [9]. Muralidharan et al developed a simple and easy RP-HPLC method for quantification of ketotifen fumarate [10]. Maithani developed a stability indicating (HPLC) method to analyze simultaneously salbutamol sulfate and theophylline in tablets dosage forms [11]. Patel et al elaborated a UV-spectrophotometric method for quantification of salbutamol and ambroxol simultaneously in pharmaceutical tablets dosage form [12]. Joshi et al. designed the method for simultaneous determination of Salbutamol Sulfate and Ketotifen Fumarate in tablet dosage form, by using spectrophotometric method. In another study Mohammed et al. successfully designed HPLC method in order to quantify the ketotifin fumarate in syrup dosage form [13]. Salih et al developed and determined HPLC method for Ketotifen Fumarate in syrup dosage [14]. Literature survey reveals that still no HPLC method have been developed for quantification of ketotifen fumarate and salbutamol sulfate simultaneously in pharmaceutical dosage forms. However, Abdoon et al developed and validated UV-Visible spectrophotometric method for simultaneous quantification of ketotifen fumarate and salbutamol sulfate in pharmaceutical tablets forms [7].

In the present study, a new, simple, precise and easy method was developed and validated for simultaneous determination of two anti-asthmatic drugs ketotifen fumarate and salbutamol sulfate. Developed method was applied to determine the active pharmaceutical ingredients in commercial tablets dosage form.

2 Experimental

2.1 Material and reagents

Salbutamol sulfate (SS) was obtained as gift sample from GlaxoSmithKline (Karachi, Pakistan). Ketotifen fumarate (KF) was supplied as gift sample from Barrett Hodgson (Karachi, Pakistan). Chloroform was purchased from BDH (UK). Potassium dihydrogen phosphate (KH2PO4), acetonitrile (CH₃CN) HPLC grade, methanol (CH₃OH) HPLC grade, perchloric acid (HClO₄) and glacial acetic acid were purchased from Merck (Germany). All other chemicals used were of analytical grade. Water was distilled at water distillation assembly, in research laboratory of the Islamia of University Bahawalpur, Pakistan.

2.2 Chromatographic conditions

Samples were analyzed at high performance liquid chromatographic system (Agilent Technologies, 1200 Series, USA), equipped with RP-C₁₈ stainless steel analytical column 4.6×150 mm (Agilent Technologies, USA). UV Detector (Agilent technology, USA). Rheodyne injector with 20μl loop and quaternary pump was used. System was operated at ambient temperature. Mobile phase had a composition of acetonitrile and 0.1 M potassium dihydrogen phosphate buffer 30 : 70, pH 4 adjusted with glacial acetic acid. Flow rate was adjusted 0.5 ml/minute and injection volume was 20μl.

2.3 Evaluation of wavelength

The wavelength maxima of ketotifen fumarate and salbutamol sulfate were determined by UV scans of both drugs on UV-Vis Spectrophotometer (Shimadzu-1601, Germany). On the basis of λ_max obtained from scanning of both drugs separately, a common λ_max for simultaneous determination of both anti-asthmatic drugs was adjusted by hit and trial method.

3 Analytical procedure

3.1 Preparation of mobile phase

Mobile phase for the simultaneous determination of ketotifen fumarate and salbutamol sulfate consisted of 30 : 70 mixture of acetonitrile and 0.1 M potassium dihydrogen phosphate buffer, pH was adjusted at 4 by glacial acetic acid. Prior to application, mobile phase was sonicated at sonicator (Elmasonic Elma E 30 H) and filtered by cellulose acetate filter 0.45μm pore size (Sartorius Goettingen, Germany).

3.2 Preparation of stock solutions

Stock solutions of ketotifen fumarate and salbutamol sulfate were prepared separately, by dissolving 100 mg of each drug in 100 ml of double distilled water to give a final concentration of 1 mg/ml of each drug. Working solutions of both drugs were prepared and combined in mobile phase by dilutions of stock solutions ranging from 1.56–100 ng/ml. The prepared solutions were stored at a temperature of –20 °C and protected from light.

3.3 Calibration curve

Seven dilutions containing various concentrations of each ketotifen fumarate and salbutamol sulfate were prepared in different portions of mobile phases. Solutions had a final concentration of 1.56, 3.125, 6.25, 12.5, 25, 50, 100 ng/ml of each drug. Prepared samples were sonicated for 2 minutes to remove the entrapped air. Analytical column was washed for 30 minutes with mobile phase before injecting the drug sample. About 20μl of prepared sample was injected and noted down the obtained peak area. Three injections for each concentration were evaluated and averaged. Calibration curve was constructed by plotting obtained area at Y-axis and drug concentrations at X-axis.

Signal to noise ratio of 3 : 1 and 10 : 1 was used to estimate the LOD and LOQ, respectively by following expressions

(i) and (ii):

LOD = 3.3 s / S

LOQ = 10 s / S

In above mentioned expression “s” represents the standard deviation of blank readings or standard deviation of the regression line and “S” is stand for slope of calibration curve.

3.4 Application of developed method in analysis of commercial tablets

Newly developed method for simultaneous determination of anti-asthmatic drugs was applied to quantify ketotifen fumarate and salbutamol sulfate in marketed tablets. In order to analyze drugs in marketed tablets, 50 tablets of Ventolin^® (salbutamol sulfate 2 mg) by GlaxoSmithKline Karachi, Pakistan, and 100 tablets of Asthotifen^® (ketotifen fumarate 1mg) by ZAFA pharmaceutical laboratories Karachi, Pakistan were crushed. Powder of pulverized tablets were separately dissolved in mobile phase in 100 ml of conical flask, in order to make a solution of 100 mg/ml. Solution was sonicated for 30 minutes in order to dissolve the drugs properly and solutions was then filtered through Whatman filter paper No. 42. 10 ml of each solution i.e., ketotifen fumarate and salbutamol sulfate were combined in a conical flask and diluted to make a working solution containing 100μg/ml of each drug. The working solution was further diluted up to required concentrations to analyze. About 20μl of prepared sample solution was injected in Rheodyne of HPLC. Area of chromatogram obtained from sample solution was compared with calibration curve.

3.5 Analytical validation of the method

Simultaneous chromatographic method for determination of ketotifen fumarate and salbutamol sulfate was validated according to FDA guidelines, i.e. specificity, accuracy, precision, linearity, robustness and ruggedness.

3.6 Specificity

The specificity of the newly developed method for simultaneous determination of anti-asthmatic drugs was determined by comparing the chromatograms obtained from spiked plasma sample incorporating anti-asthmatic drugs with chromatograms of blank plasma sample.

3.7 Accuracy

The accuracy of developed method was estimated by conducting the recovery studies (80, 100 and 120%) of both anti-asthmatic drugs from commercial tablets form (Asthotifen^® (1 mg ketotifen fumarate) by ZAFA pharmaceutical laboratories Karachi, Pakistan and Ventolin^® (2 mg salbutamol sulfate) by GlaxoSmithKline Karachi, Pakistan) by standard addition method. Mentioned amount of drugs in tablets was compared with measured concentration of drugs in tablets.

3.8 Precision

In order to ensure the reproducibility of method developed for simultaneous determination of ketotifen fumarate and salbutamol sulfate, three different concentrations of ketotifen fumarate and salbutamol sulfate (40 ng/ml, 80 ng/ml and 120 ng/ml) were analyzed in triplicates. Assay was performed at different time points in same day (Intra-day precision) and experiments were also performed for three consecutive days (Inter-day precision).

3.9 Linearity

Linearity of newly developed method for simultaneous determination of multiple anti-asthmatic drugs was confirmed from seven concentrations of each, ketotifen fumarate and salbutamol sulfate (1.56, 3.125, 6.25, 12.5, 25, 50, 100 ng/ml). Three samples of each strength were analyzed and averaged.

3.10 Robustness

Robustness of newly developed method was studied according to International Conference on Harmonization guidelines (ICH) for determination of robustness, sample with known concentrations of ketotifen fumarate and salbutamol sulfate was analyzed after a little and deliberate changing in operational parameters of developed method like mobile phase composition, pH of mobile phase and wave length applied to detect both drugs simultaneously.

4 Results and discussion

4.1 HPLC method

Quantification of both drugs in collected samples were carried out by high performance liquid chromatography technique (HPLC). For detection of both drugs simultaneously, a new HPLC method was developed. The wavelength maxima results from scanning of both anti-asthmatic drugs on spectrophotometer was 300 nm for ketotifen fumarate and 276 nm for salbutamol sulfate. On the basis of λ_max obtained from scanning of both drugs separately, a common λ_max of 276 nm was selected for simultaneous determination of ketotifen fumarate and salbutamol sulfate. Developed method showed considerable sensitivity for the detection of both anti-asthmatic drugs in commercial tablets form. Ketotifen fumarate and salbutamol sulfate showed a retention time of 2.4 minutes and 9.2 minutes, respectively. Various parameters of chromatogram for both anti-asthmatic drugs are given in Table 1. The significant difference in the retention times of both drugs reduced the chances of peaks merging. Total run time for both drugs was 12 min. Figure 3 represents typical chromatogram of ketotifen fumarate and salbutamol sulfate.

Table 1
Computed parameters of chromatogram for ketotifen fumarate and salbutamol sulfate

Peak No Retention Time (min) Peak name Peak type Peak area mAU (sec) Peak height mAU Peak area %

1 2.694 Ketotifen fumarate PB 205.274 8.32 15.81

2 9.47 Salbutamol sulfate BB 1092.66 23.025 84.18

Peak No	Retention Time (min)	Peak name	Peak type	Peak area mAU (sec)	Peak height mAU	Peak area %
1	2.694	Ketotifen fumarate	PB	205.274	8.32	15.81
2	9.47	Salbutamol sulfate	BB	1092.66	23.025	84.18

Fig. 1

Chemical Structure of Ketotifen Fumarate.

Fig. 2

Chemical Structure of Salbutamol Sulfate.

Fig. 3

Typical chromatogram of ketotifen fumarate and salbutamol sulfate.

4.2 Calibration curves

Calibration curves for both drugs were constructed by analyzing different concentrations of both drugs like 1.56, 3.125, 6.25, 12.5, 25, 50 and 100 ng/ml. Concentrations of both drugs have been measured in ng/ml due to high sensitivity of method, low dose and low amounts of both drugs expect to absorbed in systemic circulation at various time intervals. Peak areas of calibration curves were placed at Y-axis and concentrations of the drugs were taken at X-axis. Table 2 shows the parameters of regression line. Figure 4 represents the calibration curve of ketotifen fumarate and Fig. 5 gives the calibration curve of salbutamol sulfate. A good linearity (r² = 0.999) was found over above mentioned concentration range of both the drugs.

Table 2
Parameters of regression analysis of calibration line

Serial No Parameters Ketotifen fumarate Salbutamol sulfate

1 Number of samples 7 7

2 Concentration range (ng/ml) 1.56–100 1.56–100

3 Regression equation y = ax + b y = ax + b

4 Slope (a) 5.635 23.28

5 Intercept (b) 0.317 24.43

6 Regression coefficient (r²) 0.999 0.999

Serial No	Parameters	Ketotifen fumarate	Salbutamol sulfate
1	Number of samples	7	7
2	Concentration range (ng/ml)	1.56–100	1.56–100
3	Regression equation	y = ax + b	y = ax + b
4	Slope (a)	5.635	23.28
5	Intercept (b)	0.317	24.43
6	Regression coefficient (r²)	0.999	0.999

Fig. 4

Calibration curve of Ketotifen Fumarate.

Fig. 5

Calibration curve of Salbutamol Sulfate.

4.3 LOD and LOQ

Limit of detection for ketotifen fumarate was 3.03 ng/ml and of salbutamol sulfate was 4.54 ng/ml. Limits of quantification of ketotifen fumarate and salbutamol sulfate were 1 ng/ml and 1.5 ng/ml, respectively.

4.4 Analysis of commercial tablets

Newly developed method for simultaneous determination of anti-asthmatic drugs was applied to determine the contents of ketotifen fumarate and salbutamol sulfate in commercially available tablets. Percent drug content was calculated in comparison of claimed strength of active pharmaceutical ingredients in tablets. Label of Asthotifen^® claimed to have 1 mg ketotifen fumarate and Ventolin^® claimed to have 4 mg of salbutamol sulfate. Result of percent recovery study revealed that Asthotifen^® tablets had 0.997 mg (0.997%of claimed quantity) of ketotifen fumarate and Ventolin^® tablets had 3.968 mg (992%claimed quantity) of salbutamol sulfate. Results of percent drugs in marketed tablets are given in Table 3. Obtained values of drugs contents are very closer to the actual quantity of drugs present in tablets dosage form for both anti-asthmatic drugs, closeness of finding with true values support the accuracy of developed method for quantification of drugs in bulk and pharmaceutical dosage forms.

Table 3
Contents (%) of ketotifen fumarate and salbutamol sulfate in commercial tablets

Tablets content Label claim(mg/tab) Amount found ±RSD

mg (%)

Ketotifen fumarate 1 mg 0.997±0.02 99.7 0.856

Salbutamol sulfate 4 mg 3.968±0.09 99.2 0.721

Tablets content	Label claim(mg/tab)	Amount found	±RSD
Ketotifen fumarate	1 mg	0.997±0.02	99.7	0.856
Salbutamol sulfate	4 mg	3.968±0.09	99.2	0.721

4.5 Analytical validation of developed method

Developed method for simultaneous determination of ketotifen fumarate and salbutamol sulfate was validated for their specificity, accuracy, precision, linearity, robustness,

4.6 Specificity

The specificity for newly developed simultaneous method revealed that well shaped peaks indicate that the method is specific for combined drugs to be studied. Large difference in retention times between two peaks distinguished well from each other.

4.7 Accuracy

In order to study the accuracy of developed method, recovery of both drugs from formulations was evaluated in term of percentage. The mean %recoveries for both anti-asthmatic drugs were in the range between 98.0–102.0 %. Percent recoveries of ketotifen fumarate and salbutamol sulfate within acceptable limit ensured the accuracy of simultaneously developed method.

4.8 Precision

Precision of newly developed method was determined by intra-day and inter-day variations in results of assays for ketotifen fumarate and salbutamol sulfate in tablets dosage form. The samples assay revealed that results of both drugs lies in range of 98.50 %to 101.7 %. RSD results found were also in satisfactory range (0.31%and 0.19%).

4.9 Linearity

Linearity of newly developed method for simultaneous determination of multiple anti-asthmatic drugs was confirmed from seven concentrations to be analyzed for standard curve. Developed method showed a linear response over range of analyzed concentrations (1.56, 3.125, 6.25, 12.5, 25, 50, 100 ng/ml). Further, goodness of fit was supported by values of r² (0.999) for both anti-asthmatic drugs.

4.10 Robustness

Robustness of newly developed method was studied by little change in mobile phase composition, pH of mobile phase and wave length applied to detect both drugs. The results of analysis obtained after small variation indicated that the method was robust for operating parameters, as the results remained same by change in composition of mobile phase up to 5 %. Variation in pH±0.5 showed no effects on the results of HPLC evaluations. The method showed similar results at wavelength in the range of 274–277 nm. Therefore, from results it can be assumed that developed method having suitable robustness for the variation usually expected in analyzing conditions.

5 Conclusion

On the basis of analytical results it can be concluded that by newly developed method ketotifen fumarate and salbutamol sulfate can be analyzed simultaneously and developed method could be used successfully to determine the drugs content in bulk and tablets dosage form. Validation of method further revealed that developed method is simple, easy and precise for simultaneous determination of two anti-asthmatic drugs.

Expression of conflict of interest

All the authors declare that for publication of this research article they have no conflict of interests.

Funding disclosure statement

There is no funding received by any author for this write-up, preparation and submission.

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