| Research Article |
Open Access |
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| Development and Validation of Spectrophotometric and HPLC Method for
the Simultaneous Estimation of Salbutamol Sulphate and Prednisolone in
Tablet Dosage Form |
| Sohan S. Chitlange*, Kaushalendra K. Chaturvedi and Sagar B. Wankhede |
| Padmashri Dr. D.Y. Patil Institute of Pharmaceutical sciences and Research, Sant Tukaram Nagar, Pimpri, Pune-411018, Maharashtra, India |
| *Corresponding author: |
Dr. Sohan S. Chitlange
Padmashri Dr. D.Y. Patil Institute
of Pharmaceutical sciences and Research, Sant Tukaram Nagar
Pimpri, Pune-
411018, Maharashtra, India
Tel: +91-9922904305
E-mail: sohanchitlange@
rediffmail.com |
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| Received December 27, 2010; Accepted February 11, 2011; Published March
02, 2011 |
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| Citation: Chitlange SS, Chaturvedi KK, Wankhede SB (2011) Development
and Validation of Spectrophotometric and HPLC Method for the Simultaneous
Estimation of Salbutamol Sulphate and Prednisolone in Tablet Dosage Form. J Anal Bioanal Tech 2:117. doi:10.4172/2155-9872.1000117. |
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| Copyright: © 2011 Chitlange SS, et al. This is an open-access article distributed
under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the
original author and source are credited. |
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| Abstract |
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| Simple, accurate, and reproducible UV spectrophotometric and HPLC method for simultaneous estimation of
salbutamol (SAL) and prednisolone (PRE) was developed in the present work. The first developed method was
Simultaneous equation method, wavelength selected are 227 nm for salbutamol and 244 nm for prednisolone
respectively. Linearity was observed in concentration range of 6-20µg/ml for salbutamol as well as for prednisolone.
Second developed method was RP-HPLC method using Thermo C18 column (4.6 mm i.d × 250 mm) and acetonitrile:
0.025M potassium dihydrogen orthophosphate buffer (pH adjusted to 3.5 with orthophosphoric acid) in the ratio of
30:70% v/v as mobile phase. For HPLC method, linearity was observed in the concentration range of 20-100µg/ml
for salbutamol as well as for prednisolone and drugs was subjected to oxidation, hydrolysis, and heat to apply stress
condition for degradation studies. Results of analysis were validated stastically and by recovery studies. |
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| Keywords |
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| Salbutamol; Prednisolone; Simultaneous equation method;
RP-HPLC method |
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| Non-Standard Abbreviations |
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| SAL: Salbutamol sulphate; PRE:
Prednisolone |
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| Introduction |
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| Salbutamol sulphate (SAL), chemically known as bis [(1RS)-
2-[(1,1-dimethylethyl) amino]-1-[4-hydroxy-3-(hydroxymethyl)
phenyl] ethanol] sulphate, is beta-adrenocepter agonist used for the
relief of Broncho-spasm in conditions such as asthma and chronic
obstructive pulmonary disease and it is official in Indian pharmacopoeia
[1,2]. Chemically Prednisolone(PRE) is a glucocorticoid and
its IUPAC name is (8S,9S,10R,11S,13S,14S,17R)-11,17-dihydroxy-17-
(2-hydroxyacetyl)-10,13-dimethyl7,8,9,11,12,14,15,16-octahydro-6H
cyclopenta[a]phenanthren-3-one. Prednisolone is used as anti-inflammatory
or immunesuppressive agent and it is official in India pharmacopoeia
[3]. Both the drugs alone or in combination with other drugs
are reported to be estimated but do not involve simultaneous determination
of SAL and PRE. Detailed survey of literature for SAL revealed
several methods based on different techniques such as UV spectrophotometry
[4-8], RP-HPLC [9], and TLC [10] for its determination
from pharmaceuticals. Similarly survey of literature for PRE alone or in
combination with other drugs is reported to be estimated by UV spectrophotometry
[11], RP-HPLC [12], matrix solid phase dispersion liquid
chromatography [13], and LC-MS [14] for its determination from
pharmaceuticals. But no methods have been reported for simultaneous
determination of SAL and PRE. Hence in the present work a successful
attempt has been made to estimate both these drugs simultaneously by
UV spectrophotometric method (Simultaneous equation method [15,16] and HPLC method. To establish stability indicating nature [17] of
the LC method, forced degradation of drug substances was performed
under stress conditions (thermal, oxidation, acid and base hydrolysis).
The proposed methods were optimized and validated as per ICH guidelines. |
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| Experimental |
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| Chemicals |
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| Gift samples of Salbutamol sulphate and Prednisolone were provided by Macleods Pharmaceutical Pvt.Ltd Mumbai (Maharastra) and
Lupin laboratories Ltd. Pune (Maharastra) respectively. Acetonitrile
and Methanol for chromatography (Qualigens laboratory, Mumbai),
Doubled distilled water was used to prepare phosphate buffer solutions
for HPLC method. All solutions were prepared daily. |
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| Instrumentation and analytical conditions |
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| The UV method was performed on a Double-beam Shimadzu UVVisible
spectrophotometer, 1700, with spectral bandwidth of 2 nm,
wavelength accuracy ± 0.5 nm and a pair of 1-cm matched quartz cells
was used to measure absorbance of solution. Working wavelength for
UV method was 227nm (λmax of SAL) and 244nm (λmax of PRE). |
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| HPLC method was performed on HPLC system (Merck Hitachi)
consisting of quaternary gradient pump, column oven, and UV detector
(L-7400) was employed for analysis. Chromatographic data was acquired
using Winchrome software. Thermo C18 column (4.6 mm i.d ×
250 mm) was used as stationary phase. SAL and PRE was eluted isocratically
with a flow rate 1.0 ml/min using a mobile phase consisting
of 0.025M phosphate buffer (pH was adjusted to 3.5 using orthophosphoric
acid) and acetonitrile in a proportion of 70:30 v/v respectively.
The wavelength of UV detector was set to 207nm. The mobile phase
was prepared daily, filtered through 0.45µm membrane filter (Milipore)
and sonicated before use. The HPLC system was operated at 25± 1°C. |
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| Preparation of standard solutions |
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| UV method: Standard stock solution containing SAL and PRE
were prepared by dissolving quantity of Salbutamol sulphate equivalent
to SAL base 2.5mg and 2.5mg of PRE separately in 20ml of methanol in
separate 25ml volumetric flask and final volume of both solutions were
made up to 25ml with methanol to get stock solution containing each
of 100µg/ml of SAL and PRE. From these solutions, concentrations of
6-20 µg /ml were made in 10.0ml volumetric flasks. |
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| HPLC method: Standard stock solution of SAL and PRE were prepared
by dissolving quantity of Salbutamol sulphate equivalent to SAL
base 4.0mg and 4.0mg of PRE separately in 20ml of mobile phase in
separate 25ml volumetric flask and final volume of both solutions were
made up to 25ml with the same mobile phase and further dilution was
made to get 20-100 µg /ml in 10.0ml volumetric flasks. |
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| Preparation of the sample solutions |
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| UV method: Twenty tablets were weighed and average weight was
calculated. The tablets were crushed to obtain fine powder. Tablet powder
equivalent to 2.0 mg of PRE and 1.6mg of SAL was transferred to
10.0 ml volumetric flask; 5 ml methanol was added and sonicated for 10
min. The volume was then made up to the mark with methanol. The resulting
solution was filtered through Whatmann filter paper and filtrate
was appropriately diluted to get approximate concentration of 8µg/ml
of PRE and 6.4µg/ml of SAL. |
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| HPLC method: Twenty tablets were weighed and average weight
was calculated. The tablets were crushed to obtain fine powder. Tablet
powder equivalent to 4.0mg SAL and 5.0mg of PRE was taken and
dissolved in mobile phase and sonicated for 20min. and then volume
was made up to the mark with mobile phase. The resulting solution
was mixed and filtered through Whatmann filter paper and filtrate was
appropriately diluted to get approximate concentration of 32µg/ml of
SAL and 40µg/ml of PRE. The diluted solutions were filtered through
0.20µm membrane filter to get clear solutions. |
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| Procedure for forced degradation study |
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| Degradation studies were performed in solutions containing drug
at a concentration of 20µg/ml for SAL and 40µg/ml for PRE. Samples
were withdrawn and subjected to HPLC analysis, after suitable dilution.
The stress conditions were as follows: |
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| Stress degradation by hydrolysis under acidic conditions: For
acid degradation study 1ml of 0.1M HCl was added to final drug solution.
And it was refluxed for 2hr at 80° C. After 2hr this solution was
injected in stabilized chromatographic condition. |
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| Stress degradation by hydrolysis under alkaline conditions: For
alkali degradation study 1ml of 0.1M NaOH was added to final drug
solution. And it was refluxed for 2hr at 80° C. After 2hr this solution was
injected in stabilized chromatographic condition. |
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| Oxidative degradation: For oxidation study 1ml of 3% v/v H2O2
was added to final drug solution. And it was refluxed for 2hr at 80°
C. After 2hr this solution was injected in stabilized chromatographic
condition. |
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| Temperature stress studies: For temperature stress studies final
drug solution was refluxed for 2hr at 80°C. And then after 2hr this
solution was injected in stabilized chromatographic condition. |
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| Method validation |
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| The methods were validated according to International Conference on Harmonization guidelines for validation of analytical procedures
[18]. |
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| Linearity: The calibration curve was obtained with concentrations
of the standard solutions (6-20µg /ml) for UV method and 20-100µg
/ml for HPLC method). The solutions were prepared in triplicate. The
linearity was evaluated by regression analysis, which was calculated by
the least square regression method. |
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| Precision: Precision of these methods was checked by analyzing
the samples at three different time intervals of the same day (intraday
precision) as well as on different days (interday precision). |
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| Accuracy: The accuracy of proposed method was determined by
recovery studies. It was determined by recovery of known amounts of
SAL and PRE reference standard added to the sample at the beginning
of the process and all solutions were prepared in triplicate. For recovery
studies proportion of SAL and PRE in in-house preparation was made
1:1 by adding reference standard SAL in to tablet powder. |
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| Robustness: Robustness for HPLC method was determined by
analysis of samples under deliberately changed chromatographic conditions.
The flow rate of the mobile phase was changed from 1ml/min
to 0.9ml/min and 1.1ml/min while the ratio of the mobile phase was
changed by ± 2%. The effect on retention time and peak parameter were
studied. |
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| Limit of detection and limit of quantitation: LOD and LOQ are
calculated by using the values of slopes and intercepts of the calibration
curves for both the drugs. |
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| Results and Discussion |
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| UV method |
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| The proposed UV method allows a rapid and accurate quantitation
of SAL and PRE in in-house tablet preparation without any time-consuming
sample preparation. Moreover, the spectrophotometric methods
involve simple instrumentation compared with other instrumental
techniques. The absorption spectra of SAL and PRE in methanol are
shown in Figure 1. Wavelengths selected for analysis are 227nm (λmax
of SAL) and 244nm (λmax of PRE). Calibration curves were constructed
in the concentration range of (6-20 µg/ml). Beer's law was obeyed
over this concentration range, and the coefficient of regression for both the drugs was found to be nearer to 1 (Table 1). Precision was calculated
as interday and intraday variations for both the drugs. Percent relative
standard deviations for estimation of SAL and PRE under intraday and
interday variations were found to be less than 1. Two simultaneous
equations (in two variables CSAL and CPRE) were formed using absorptivity
coefficient values and these are as follows. |
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Figure 1: Overlain spectra of Salbutamol (SAL) and Prednisolone (PRE) in
methanol. |
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Table 1: Results of regression analysis of data for the quantitation of
salbutamol(SAL) and prednisolone(PRE) by he proposed methods. |
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| A1(Absorbance at 227nm) = CX (0.03542) + CY (0.0296) (I) |
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| A2 (Absorbance at 244nm) = CX (0.0007) + CY (0.0457) (II) |
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| By solving above simultaneous equation the concentration CSAL
(Salbutamol) and CPRE (Prednisolone) was calculated. Table 2 shows the
experimental values obtained for the determination of SAL and PRE
in samples. A good accuracy of the method was verified with a mean
percent recovery in the range of 98.18-99.63% (Table 3). |
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Table 2: Results of the determination of SAL and PRE in formulation by the
proposed methods. |
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Table 3: Experimental values obtained in the recovery test for SAL and PRE in
formulation by proposed methods. |
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| HPLC method |
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| Different proportions of acetonitrile and 0.025M phosphate buffer
(pH was adjusted to 3.5 using orthophosphoric acid) was tried for selection
of mobile phase. Ultimately, 0.025M phosphate buffer (pH was
adjusted to 3.5 using orthophosphoric acid) and acetonitrile in a proportion
of 70:30 v/v respectively was finalized as the mobile phase. Figure
2 shows typical chromatogram obtained from the analysis of standard
solution of SAL and PRE using the proposed method. The elution
order was SAL (Rt = 2.76 min) and PRE (Rt = 6.12), at a flow rate of 1.0
ml/min. The chromatogram was recorded at 207nm as the overlain UV
spectra of SAL and PRE showed maximum response at this wavelength. |
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Figure 2: Chromatogram of standard SAL and PRE. |
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| The calibration curves for SAL and PRE were constructed by plotting
concentration versus peak area and showed good linearity in the
20-100 µg/ml, and the coefficient of regression for both the drugs was
found to be nearer to 1 (Table 1). The accuracy of proposed method
was determined and the mean percent recovery was found in the range
of 98.66-101.56% (Table 3) indicating an agreement between the true
value and found value. Precision was calculated as interday and intraday
variations for both the drugs. Percent relative standard deviations
for estimation of SAL and PRE under intraday and interday variations
were found to be less than 2. And for robustness studies in all deliberately
varied conditions, the RSD were found to be less than 2%. The
experimental values obtained for the determination of SAL and PRE in
samples are showed in (Table 2). |
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| SAL and PRE was underwent acid hydrolysis, but the rate of hydrolysis
was slower as compared to that in alkali. It was depicted in Figure
3. The degradation reaction was more intense and quicker in alkaline
condition. And decrease in peak area of both the drug was observed and it was shown in Figure 4. The drug showed lability to hydrogen
peroxide. SAL was decomposed to an extent of 13.97% while PRE was
decomposed to an extent of 16.72% Additional peak was observed and
it was shown in Figure 5. Upon refluxing the drug for 2hr at 80°C fall
in the original peak area of both the drugs and additional peaks was
observed and it was shown in Figure 6. The percent amount of drug
degraded after degradation studies are given in (Table 4). |
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Figure 3: Chromatogram of SAL and PRE in 0.1M HCl. |
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Figure 4: Chromatogram of SAL and PRE in 0.1M NaOH. |
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Figure 5: Chromatogram of SAL and PRE in 3% v/v H2O2. |
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Figure 6: Chromatogram of SAL and PRE in temperature stress condition. |
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Table 4: Result of stress degradation studies of SAL and PRE. |
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| Conclusion |
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| The two proposed methods based on the spectrophotometry and
HPLC were developed and validated as per ICH guidelines. The standard
deviation and % RSD calculated for the proposed methods are
low, indicating high degree of precision of the methods. The results of
the recovery studies performed show the high degree of accuracy for
the proposed methods. Hence, it can be concluded that the developed
spectrophotometric and chromatographic methods are accurate, pre- cise and selective and can be employed successfully for the estimation
of SAL and PRE in bulk and formulation. |
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| Acknowledgement |
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| The authors gratefully acknowledge Dr. P. D. Patil, Chairman, Dr. D. Y. Patil
Vidya Pratishthan Society and Dr. A. D. Deshpande, Director of Pharmacy for providing
excellent infrastructure facility to carryout this research work. Thanks also go
to Macleods Pharmaceutical Pvt. Ltd. Mumbai and Lupin Laboratories Ltd. Pune for
providing pure drug samples. |
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