International Journal of Pharma Sciences Vol. 2, No. 4 (2012): 79-83 Research Article Open Access

ISSN: 2320-6810

Development and Validation of an HPLC method for the Simultaneous Estimation of the Salbutamol Sulphate and Ipratropium in Inhalation Dosage Forms N Jyothi1*, K VenuGopal2 and JVLN Seshagiri Rao2 1 2

Malla Reddy Institute of pharmaceutical sciences Secunderabad, Andhra Pradesh, India University College of Pharmaceutical Sciences, Andhra University, Visakhapatnam, Andhra Pradesh, India

* Corresponding author, email: [email protected]

Received: 18 October 2012

Revised: 15 November 2012

Accepted: 15 November 2012

ABSTRACT A simple, precise and sensitive reverse-phase high performance liquid chromatographic method was developed and validated for the simultaneous estimation of Salbutamol sulphate and Ipratropium in pharmaceutical formulations. Chromatographic separation was performed on a High performance liquid chromatography equipped with auto sampler and photo diode detector. Good sensitivity for all analytes was observed with UV detection at wavelength of 226 nm, Separation was performed on a Symmetry C18 (4.6 X 150mm) 5µm,using a mixture of 0.05M phosphate buffer pH 3.5 and methanol in the ratio of (400:600, v/v). The method results in excellent separation with good resolution between the two analytes. The within day variation was between Salbutamol and Ipratropium1.72 and 1.97 % and between day variation was between 1.99 and 1.82 %. The recovery was greater than 99.06 % with RSD less than 1.95 %. The method was validated according to ICH guidelines by performing linearity, accuracy, precision, limits of quantitation and selectivity. The results show the method is suitable for its intended use.

Keywords: Salbutamol sulfate and Ipratropium bromide, simultaneous estimation, Nebulizer solution, HPLC

INTRODUCTION Salbutamol is a short-acting β2-adrenergic receptor agonist used for the relief of bronchospasm in conditions such as asthma and chronic obstructive pulmonary disease, chemically described as alpha-[(tertButylamino)methyl]-4-hydroxy-m-xylene-alpha,alpha-diol. It has a molecular weight of 239.31 and the empirical formula is C13H21NO3. The tertiary butyl group in salbutamol makes it more selective for β2-receptors [1]. The drug is sold as a racemic mixture mainly because the (S)-enantiomer blocks metabolism pathways while the (R)enantiomer shows activity. Ipratropium bromide is an anticholinergic drug used for the treatment of chronic obstructive pulmonary disease and acute asthma [2]. It blocks the muscarinic acetylcholine receptors in the smooth muscles of the bronchi in the lungs, opening the bronchi. Chemically know as 8-azoniabicyclo octane, 3-(3-hydroxy-1-oxo-2phenylpropoxy)-8-methyl-8-(1-methylethyl)-, bromide

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monohydrate. It has a molecular weight of 430.4 and the empirical formula is C20H30BrNO3•H2O. So far, Literature survey shows that several LC-MS [3], conductometric [4], potentiometric [5], spectrofluorimetric [6], spectrophotometric methods [725] and HPLC [26-37] methods have been reported for estimation of racemic salbutamol sulphate in various sample matrixes. The aim of the present study was to develop a simple, isocratic, accurate and sensitive HPLC method for the simultaneous determination of salbutamol sulphate and Ipratripium in their fixed dose combination. MATERIALS AND METHODS Duoneb inhalation solution (3/0.5 mg/3ml vial) which contains Salbutamol sulphate and Ipratropium bromide were purchased from the local market. Methanol (HPLC grade), Ortho phosphoric acid (HPLC Grade) Potasium dihydrogen orthophosphate (GR Grade) were purchased 79

Jyothi et al. / HPLC simultaneous estimation of Salbutamol Sulphate and Ipratropium

from Merck and de-ionized water was used throughout the experiment.

Chromatographic separation was performed using a Symmetry C18 (150X4.6)5µ with isocratic elution. The mobile phase consisted of a mixture of 0.05M phosphate buffer pH 3.5 and methanol in the ratio of (40:60, v/v) with flow rate as 0.6 ml/min.wave length 226nm, Peak identity was confirmed by retention time comparison and the HPLC system was operated at room temperature. Diluent preparation

Figure 1. Chemical structure of Salbutamol sulphate

Mobile phase was used as diluent Preparation of standard solution

A stock standard solution containing 600 mg/ml salbutamol and 100 mg/ml Ipratropium was prepared by dissolving 30 mg each of salbutamol and 5mg of ipratropium in mobile phase in 50 ml volumetric flask and raising the volume up to the mark. To prepare the working standard solution (60 mg/ml salbutamol and 10 mg/ml ipratropium), the stock standard solution was diluted with diluent.. Preparation of sample solution

Figure 2. Chemical structure of Ipratropium bromide Preparation of mobile phase

A mobile phase was prepared by mixing 0.05M phosphate buffer pH 3.5 and methanol in the ratio of 40:60, v/v. The mobile phase was filtered using 0.45µ nylon filters (Millipore, USA) and was degassed by sonication before use. Apparatus and chromatographic conditions

HPLC apparatus consisting of waters system 2695, variable wavelength detector (set at 226nm), software Empower and a injection volume with a 15 µL was used for development and evaluation of this method.

Twenty vials each containing (3mg of salbutamol sulphate and 0.5mg of Ipratropium in 3ml of solution) were weighed squeeze the content into a beaker to get the average weight and then they were homogenized by mixing the solution. An accurately weighed quantity of homogenized solution equivalent to 30 mg of Salbutamol was placed in 50 ml volumetric flask. 30 ml diluent was added and the flask was shaken for 5 minutes, so as to completely extract all the drugs. The volume was then made up to the mark with mobile phase to get a solution containing 600 mg/ml salbutamol and 100 mg/ml ipratropium. Solution was then filtered using 0.45µ PVDF filters and diluted with diluent to obtain a final concentration of 60 mg/ml salbutamol and 10 mg/ml ipratropium.

Figure 3. Typical chromatogram of sample solution.

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Jyothi et al. / HPLC simultaneous estimation of Salbutamol Sulphate and Ipratropium

RESULTS AND DISCUSSION The developed chromatographic method for the simultaneous determination of Salbutamol sulphate and Ipratropium was validated using ICH guidelines. Assessed validation parameters include linearity, limit of quantitation, selectivity, accuracy and repeatability. Linearity

Linearity of the proposed method was done by analyzing seven solutions in the range LOQ to 150% of the target concentration for both Salbutamol sulphate and Ipratropium. Good linearity was observed over the above range for both Salbutamol sulphate and Ipratropium. The calibration curve was made using concentration of the analytes versus peak area (Figure 3). The correlation coefficient from the linear regression analysis was calculated and found to be 0.9994 and 0.9998 for both the analytes. This indicates that there exists a good linear relationship between concentration of drugs and the peak area. The linear regression graph is given in Figures 4 and 5. LINEARITY GRAPH FOR SALBUTAMOL SULPHATE

Average area co u n ts(µ V*S ec)

Two types of solutions i.e. blank and spiked with known progressively decreasing concentrations of each analyte were prepared and analysed. The limit of quantification (LOQ) was then established by evaluating the minimum level at which the analyte can be readily quantified with accuracy (signal to noise ratio of 10:1). The limits of quantitation were found to be 0.03 µgml-1 and 0.19 µgml1 for Salbutamol sulphate and Ipratropium respectively. Accuracy

The accuracy of the method was performed by adding known amounts of Salbutamol sulphate and Ipratropium to placebo solution and then comparing the added amount with the observed amount. Three levels of solutions were made which correspond to 80, 100 and 120 % of the nominal analytical concentration. Each level was made in triplicate. The recovery range and the relative standard deviation for each of the analytes were found to be 99.3-101.34 , 99.06-101.03 % and 0.16-1.06, 0.22-1.95 % respectively (Table 1). Precision

Precision was assessed at two levels i.e. Method precision(repeatability) and intermediate precision. The repeatability was determined as intra-day variation whereas intermediate precision was determined by measuring inter-day variation in the assay of the drug in six replicate preparations (n=6). The assay results for repeatability and intermediate precision are 2.99 and 2.95 for Salbutamol and 0.50 and 0.49 mg/vial for Ipratropium respectively (Table 2).

4000000 3500000 3000000 2500000 2000000 1500000 1000000 500000 0 0

10

20

30

40

50

60

70

80

90

100

Concentration(µg/mL)

Selectivity

Selectivity of the proposed method was checked by making a synthetic mixture of both the analytes with commonly occurring excipients that are found in most formulations and then measuring the percentage recovery of each component along with chromatographic parameters. The results show no interference from the excipients.

Figure 4. Linearity regression graph for Salbutamol Sulphate LINEARITY GRAPH OR IPRATROPIUM BROMIDE 120000 Av erag e area co unts(µ V *S ec )

Limit of Quantitation

100000 80000

Stability

60000 40000 20000 0 0.00

2.00

4.00

6.00

8.00

10.00

12.00

14.00

16.00

18.00

The stability of each component in the presence of other was assessed by analyzing the samples after 26 hrs. The relative standard deviation of peak area was less than 0.44 %. The results are presented in Table 3 which indicates good stability for each drug.

Concentration(µg/mL)

Figure 5. Linearity regression graph for Ipratropium bromide

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Jyothi et al. / HPLC simultaneous estimation of Salbutamol Sulphate and Ipratropium

Table 1. Accuracy of the Method Salbutamol sulphate

Ipratropium bromide

Recovery Levels

Amoun t added (mg)

Amount recovered (mg)

% Recovered

Amount added (mg)

Amount recovered (mg)

% Recovered

80% Rec-1

48.34

48.11

99.52

8.92

8.76

98.21

80% Rec-2

48.16

47.96

99.58

8.47

8.39

99.06

80% Rec-3

48.33

47.75

98.80

8.31

8.37

100.72

100% Rec-1

60.14

61.05

101.51

10.94

10.81

98.81

100% Rec-2

60.84

61.63

101.30

10.37

10.29

99.23

100% Rec-3

60.51

61.24

101.21

10.64

10.55

99.15

120% Rec-1

72.81

72.83

100.03

13.19

13.24

100.38

120% Rec-2

72.91

71.81

98.49

13.34

13.27

99.48

120% Rec-3

72.67

73.04

100.51

13.86

14.31

103.25

Mean % recovery

% RSD

99.30

0.44

101.34

0.16

99.68

1.06

Mean % recovery

% RSD

99.33

1.29

99.06

0.22

101.03

1.95

Table 2. Precision of the method Sample no

Precision

Precision

Salbutamol sulphate (3 mg/Vial )

Ipratropium (0.5mg/Vial)

Method precision

Intermediate precision

Method precision

Intermediate precision

1

2.98

2.94

0.51

0.49

2

2.91

2.89

0.49

0.50

3

3.06

2.91

0.49

0.48

4

2.96

2.99

0.50

0.49

5

2.99

2.89

0.51

0.49

6

3.02

2.87

0.49

0.49

Mean

2.99

2.92

0.50

0.49

SD

0.05

0.04

0.01

0.01

%RSD

1.72

1.50

1.97

1.29

Overall Mean

2.95

0.49

Overall SD

0.06

0.01

Overall RSD

1.99

1.82

Table 3. Bench top stability for Standard and test solutions % Assay Salbutamol sulphate

Time

Standard

Ipratropium

Formulation

Standard

Formulation

Trial– 1

Trial – 2

Trial– 1

Trial – 2

0 hrs

100.94

101.1

101.8

98.8

100.21

101.02

At 26 hrs

99.61

100.9

101.4

99.2

99.52

99.64

Difference

0.94

0.14

0.28

0.29

0.49

0.97

CONCLUSION Simultaneous determination of Salbutamol sulphate and Ipratropium in their pharmaceutical formulation has been successfully achieved by the use of a validated analytical method. The method is accurate and precise for reliable quality control evaluation of drugs with good accuracy and precision. From these values it is concluded that the new HPLC method is suitable for the simultaneous http://ijps.aizeonpublishers.net/content/2012/4/ijps79-83.pdf

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