Spectrophotometric Simultaneous Estimation of Salbutamol and Prednisolone in Bulk and Formulation.

 

Parag A. Kulkarni* and Pramod V. Kasture

ABSTRACT:

Combination of Salbutamol and Prednisolone was found effective for the treatment of acute and chronic asthma. A fast, simple and accurate method for the estimation of this combination is necessary so, two UV Spectrophotometric methods for simultaneous estimation of salbutamol (SAL) and prednisolone (PRE) have been developed in the present work. First method is Q value analysis based on measurement of absorptivity at 230nm (iso-absorptive point) and 244nm (λmax for Prednisolone) as two analytical wavelengths for both drugs in methanol. Second method involves multicomponent mode of analysis, wavelength selected are 227nm (λ_max for Salbutamol) and 244 nm (λ_max for Prednisolone).  All the methods show linearity in the concentration range of 6-20µg/ml for salbutamol as well as for prednisolone. The accuracy and precision of the methods were determined and validated stastically which shows no significant difference between the results obtained by the two methods. The proposed methods are rapid, simple and accurate and can be used for its intended purpose.

 

 

 

INTRODUCTION:

Combination of Salbutamol and Prednisolone was found effective for the treatment of acute and chronic asthma¹. A fast, simple and accurate method for the estimation of this combination is necessary so, two UV Spectrophotometric methods for simultaneous estimation of Salbutamol (SAL) and Prednisolone (PRE) have been developed in the present work. Salbutamol sulphate (SAL), chemically known as bis [(1RS)-2-[(1, 1-dimethylethyl) amino]-1-[4-hydroxy-3-(hydroxymethyl) phenyl] ethanol] sulphate, is beta-adenocepter 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^2-4. 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-dimethyl-7,8,9,11,12,14,15,16-octahydro-6H-cyclopenta[a]phenanthren-3-one. Prednisolone is used as anti-inflammatory or immunosuppressive agent and it is official in India pharmacopoeia⁵.

 

Salbutamol in combination with other drugs is reported to be estimated by UV spectrophotometry^6-10, RP-HPLC^11,12, TLC¹³. Prednisolone is also in combination with other drugs is reported to be estimated by UV spectrophotometry¹⁴, RP-HPLC¹⁵, matrix solid phase dispersion liquid chromatography¹⁶, LC-MS¹⁷. Since no spectrophotometric methods are reported for the simultaneous estimation of Salbutamol and Prednisolone in combination therefore, in the present work a successful attempt has been made to estimate both these drugs simultaneously by two simple UV spectrophotometric methods (Q value analysis, multicomponent mode analysis)^18,19. The proposed methods were optimized and validated as per ICH guidelines.

 

MATERIAL AND METHODS:

Instrument: A double-beam UV- Visible spectrophotometer (Shimadzu –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.

 

Materials: Gift samples of Salbutamol sulphate and Prednisolone were provided by Macleods Pharmaceutical Pvt. Ltd and Lupin laboratories Ltd. respectively.

 

Solvent: Methanol of analytical reagent grade.

 

Preparation of stock solutions: Standard stock solution containing Salbutamol sulphate and Prednisolone were prepared by dissolving quantity of Salbutamol sulphate equivalent to Salbutamol base 2.5mg and 2.5mg of Prednisolone separately in 20ml of methanol in separate 25ml volumetric flask and final volume of both solution were made up to 25ml with methanol to get stock solution containing each of 100µg/ml of Salbutamol and Prednisolone in two different 25ml volumetric flasks.

 

Determination of Absorption Maxima:

By appropriate dilution of two standard drug solutions with methanol, solutions containing       12 µg/ml of Salbutamol and 12 µg/ml of Prednisolone were scanned separately in the range of 200- 400 nm to determine the wavelength of maximum absorption for both the drugs showed absorbance maxima at 227nm for Salbutamol and 244nm for Prednisolone and isoabsorptive point at 230nm (Fig 1).

 

Fig.1: Overlain UV spectra of Salbutamol and Prednisolone

 

Method I (Q method analysis) ^{18, 19}

Two wavelengths selected for this method are 230nm and 244nm that are iso-absorptive point of both drugs and maxima of Prednisolone respectively in methanol. The stock solutions of both the drugs were further diluted separately with methanol to get a series of standard solutions of 6-20 µg /mL concentrations. The absorbances were measured at the selected wavelengths and absorptivities (A 1%, 1 cm) for both the drugs at both wavelengths were determined. The calibration curves for Salbutamol and Prednisolone were plotted in the concentration range of 6-20µg/ml. The method employs Q values and the concentrations of drugs in sample solution were determined by using the following formula,

 

Method II: Multicomponent mode method analysis ^{18, 19}

In this method, six mixed standard solutions with concentration of Salbutamol and Prednisolone in the ratio of 6:8 μg/ml were prepared in methanol. All the standard solutions were scanned over the range of 400-200 nm, in the multicomponent mode, using two working wavelength 227 nm (λ_max of Salbutamol) and 244 nm (λ_max of Prednisolone). The data from these scans was used to determine the concentrations of two drugs in tablet solutions.

 

Application of proposed method for the determination of Salbutamol and Prednisolone in tablets:

For the estimation of drugs in formulations, twenty tablets were weighed and average weight was calculated. The tablets were crushed to obtain fine powder. Tablet powder equivalent to 2 mg of Prednisolone and 1.6mg of Salbutamol 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 Prednisolone and 6.4μg/ml of Salbutamol. In method I, the concentration of both Prednisolone and Salbutamol were determined by measuring absorbances of sample solutions at 244 nm (λmax of Prednisolone) and 230 nm (Iso-absorptive point) using equations (I) and (II). For method II, the same tablet sample solutions were subjected to analysis in the multicomponent mode of instrument the concentration of both Prednisolone and Salbutamol were determined by analysis of spectral data of the sample solution with reference to the mixed standards at 244 nm (λmax for Prednisolone) and 227 nm   (λmax for Salbutamol). Results of tablet analysis are shown in Table No. 1

 

Table No – 1: Result of marketed formulation analysis

Method	Tablet content	Label claim (mg/tab)	Amount found*		± S.D*
			(mg)	( %)
I	SAL	4.0	3.9245	98.11	0.3538
	PRE	5.0	4.9262	98.52	0.5418
II	SAL	4.0	3.9974	99.93	0.8826
	PRE	5.0	5.010	100.20	0.8259

* Mean of six estimation. SAL= Salbutamol, PRE= Prednisolone

 

VALIDATION:

The method was validated according to ICH guidelines for validation of analytical procedures in order to determine the linearity, accuracy, precision and specificity for the analyte^19-20.

Table No – 2: Result of recovery studies

Level of Recovery	Amt. of pure drug added µg/ml	Drug	Method I		Method II
			Recovery (%)*	± S.D*	Recovery (%)*	± S.D*
80%	6.4	SAL	99.72	0.4734	98.62	0.5103
	6.4	PRE	99.00	0.8660	99.55	0.7623
100%	8.0	SAL	101.03	0.5718	99.26	0.6995
	8.0	PRE	98.75	0.0461	99.49	0.4479
120%	9.6	SAL	100.90	0.0300	99.84	0.4110
	9.6	PRE	97.77	0.3315	99.93	0.3339

* Mean of three estimations. SAL= Salbutamol, PRE= Prednisolone

 

Linearity:

A set of eight solutions of Salbutamol and Prednisolone at concentrations ranging from 6µg/ml to 20μg/ml were prepared. Each sample was analyzed and calibration curve was constructed by plotting the absorbance against concentration using linear regression analysis. The correlation coefficient was found to be 0.9940 at 227nm and 0.9940 at 244nm for Salbutamol and Prednisolone respectively. The results show that an excellent correlation existed between absorbance and concentration of each drug within the concentration range tested.

 

Accuracy:

To ascertain the accuracy of the proposed methods, recovery studies were carried out by standard addition method at three different levels (80%, 100% &120%).

For recovery analysis of drug, a standard addition method was used. The results of recovery studies were satisfactory and are presented in Table No.2

 

Precision:

The reproducibility of the proposed methods was determined by performing tablet assay at different time intervals on same day (Intra-day assay precision) and on three different days (Inter-day assay precision).

 

Specificity:

The specificity of the method was confirmed by comparing the λ max of standard with that of Salbutamol and Prednisolone for in house preparation. There is no interference from the excipients commonly present in the tablets. Hence the developed method is specific and selective.

 

RESULTS AND DISCUSSION:

For all the methods linearity was observed in the concentration range of 6-20 μg/ml for Salbutamol and Prednisolone, respectively. Formulations containing Salbutamol and Prednisolone were analyzed by the proposed methods. Six replicate analysis of formulation were carried out and the mean assay values were found in the range of 98.11-100.20 % .The proposed methods were validated as per the ICH guidelines. The accuracy of the proposed method was determined by recovery studies. Pure Salbutamol and Prednisolone was added to the preanalyzed tablet powder at three spiking levels viz 80, 100, 120 %. Three replicate analyses were carried out at each level. The mean percent recovery was found in the range of 97.77-101.03% for all the methods. Precision is calculated as interday and intraday variations for both the drugs. Percent relative standard deviations for estimation of Salbutamol and Prednisolone under intraday and interday variations were found to be less than 1.

 

CONCLUSION:

The two proposed methods based on the Spectrophotometry, 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 methods are accurate, precise and selective and can be employed successfully for the estimation of Salbutamol and Prednisolone in bulk and formulation.

 

ACKNOWLEDGEMENT:

The authors gratefully acknowledge 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 as a gift samples.

 

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Received on 07.12.2010        Modified on 15.12.2010

Accepted on 30.12.2010        © AJRC All right reserved