UV Spectrophotometric estimation of Drotaverine hydrochloride by zero order and area under curve methods in bulk and Pharmaceutical Dosage Form

 

Rajan V. Rele*

Central Research Laboratory, D.G. Ruparel College, Matunga, Mumbai 400016.

 

ABSTRACT:

Simple and precise UV spectrophotometric methods by UV spectroscopy and area under curve [AUC] – in zero order derivative spectrum have been developed and validated for the estimation of drotaverine hydrochloride in pharmaceutical formulation. The standard and sample solutions of drotaverine hydrochloride were prepared in methanol. Drotaverine hydrochloride was estimated at 230 nm for the UV-spectrophotometric method (A), while in area under curve (AUC) method (B) the zero order spectrum of drotaverine hydrochloride was measured in between 225-235 nm. Beer’s law was obeyed in the concentration range of 1 to 24 μg / ml with coefficient of correlation value 0.002261 for zero order method. Similarly in AUC method, Beer’s law was obeyed in the concentration range of 5 to 50 μg / ml with coefficient of correlation value 0.6238. These methods were validated for various parameters according to ICH guidelines. The precision expressed as relative standard deviation were of 0.002261 % and 0.1403 % for the above two methods respectively. The proposed methods were successfully applied for the determination of drotaverine hydrochloride in pharmaceutical formulation. Results of the analysis were validated statistically and were found to be satisfactory. The proposed methods are simple, easy to apply, low-cost and require relatively inexpensive instruments.

 

 

 

INTRODUCTION:

Drotaverine hydrochloride is [(1-(3,4– diethoxybenzylidene)-6,7–diethoxy-1,2,3,4 tetrahydroisoquinoline) hydrochloride], a benzylisoquinoline derivative. It is a highly potent spasmolytic drug. It shows excellent properties of smooth muscle relaxant. Its antispasmodic activity is due to inhibition of phosphodiesterase enzyme IV. It causes smooth muscle relaxation by increasing intracellular levels of cyclic adenosine mono-phosphate (cAMP) secondary to inhibition of phosphodiesterase.

According to the literature review several methods has been developed for drug, like spectroscopy, methods^1-7.HPLC^8-11 and miscellaneous^12-14.

 

 

The proposed aim of the study was to develop simple, accurate, specific and precise UV spectrophotometric method for the estimation of drug in the bulk and pharmaceutical formulation.

 

Structure of drotaverine:

 

 

MATERIAL AND METHODS:

Shimadzu UV-1800 was used with 10 mm matched quartz cell to measure absorbance of solution.

A Shimadzu analytical balance with 0.01 mg was used.

Chemical and reagents:

Reference standard of drotaverine hydrochloride was obtained from reputed firm with certificate analysis.  All spectral absorbance measurements were made on Shimadzu UV-1800 with 10 mm matched cell.

 

Preparation of standard solution:

About 10 mg of standard drotaverine hydrochloride was weighed accurately and transferred in 100 ml of volumetric flask. About 30 ml of methanol was added and sonicated for 15 minutes. The volume was adjusted up to the mark with methanol to give concentration as 100 μg /ml.

 

Estimation from tablets:

Twenty tablets were weighed accurately and average weight of each tablet was determined. Powder equivalent to 10 mg of drotaverine hydrochloride was weighed and transferred in 100 ml of volumetric flask. A 30 ml of methanol was added and sonicated for 15 minutes and filtered. The filtrate and washing were diluted up to the mark with methanol to give concentration as 100 μg /ml. Such solution was used for analysis.

 

Experimental:

Method A: Zero order method:

For the selection of analytical wavelength, 10 μg /ml solution of drotaverine hydrochloride was scanned in the spectrum mode from 350 nm to 200 nm by using distilled water as blank. The zero order spectrum was obtained by UV probe 2.42 software. From the spectrum, the absorbance  of the spectrum was measured at 230 nm (Fig. 2).

 

 

Fig. 2. spectra of Zero order spectrum of drotaverine hydrochloride (20 μg/ml) showing  absorbance at  230 nm

 

 

Fig. 3. Calibration curve for drotaverine hydrochloride at 230 nm by zero order   Spectroscopy

 

Into series of 10 ml graduated flask, varying amount of standard solutions of drotaverine hydrochloride was pipette out and volume was adjusted with  distilled water as solvent. Solutions were scanned between 350 nm to 200 nm in spectrum mode. Absorbances of the resulting solutions were measured at between 230 nm by using distilled water as blank. The calibration curve was prepared in the concentration range of  1 to 24 μg/ml.  (Fig. 3)

 

Method B: Area under curve (AUC) method

Area under curve method involves the calculation of integrated value of absorbance with respect to the wavelength between two selected wavelengths such as λ₁ and λ₂. The area under curve between λ₁ and λ₂ were calculated by UV probe 2.42 software. In this method, 10 μg/ml solution of drotaverine hydrochloride was scanned in the spectrum mode from 300 nm to 200 nm. From zero order spectrum the AUC calculation was done. The AUC spectrum was measured between 225 nm to 235 nm (Fig. 4).

 

 

Fig. 4. Area under curve spectrum of drotaverine hydrochloride ( 20 μg/ml) showing area from 225  nm to 235  nm.

 

Into series of 10 ml graduated flask, varying amount of standard solutions of drotaverine hydrochloride were pipette out and volume was adjusted with distilled water. Solutions were scanned between 300 nm to 200 nm in spectrum mode. The AUC calculations were done and the calibration curve for drotaverine hydrochloride was plotted in the concentration range of 5 to 50 μg/ml (Fig. 5).

 

 

Fig. 5. Calibration curve for drotaverine   hydrochloride by area under curve spectroscopy

 

Results of analysis are given in table 1.

 

Table 1: Values of results of optical and regression of drug

Parameter	Zero order method	Area under curve (AUC) method
Detection Wavelength (nm)	230	225-235
Beer Law Limits (µg/ml)	1-24	5-40
Correlation coefficient(r2)	0.9999	0.9999
Regression equation (y=b+ac)
Slope (a)	0.0046	0.0004
Intercept (b)	0.00004	0.00004

 

Validation:

Accuracy:

Accuracy of the proposed methods was carried as on the basis of recovery studies. It is performed by the standard addition method. Recovery studies were performed by adding standard drug at different levels to the pre-analyzed tablets powder solution and the proposed method was followed. From the amount of the drug estimated, the percentage recovery was calculated. The results of the analysis are shown in table (2, 3).

 

 

Table 2: Results of recovery of drotaverine hydrochloride for zero order method

Amount of Sample Added in (µg/ml)	Amount of Standard Added in (µg/ml)	Total amount recovered	Percentage recovery(%)	Standard deviation	Percentage of relative standard deviation (C.O.V.)
2	0	1.9981	99.90744	0.001943	0.097254
2	2	4.000617	100.0154	0.002403	0.060072
2	6	7.999691	99.99614	0.001943	0.024292
2	10	12.00031	100.0026	0.002624	0.021867
				Mean=0.002228	Mean=0.05087

 

 

 

Table 3: Results of recovery of drotaverine hydrochloride for area under curve (AUC) method

Amount of Sample Added in (µg/ml)	Amount of Standard Added in (µg/ml)	Total amount recovered	Percentage recovery(%)	Standard deviation	Percentage of relative standard deviation (C.O.V.)
10	0	10.02256	100.2256	0.023677	0.23624
10	10	20.0188	100.094	0.20982	1.048116
10	20	30.03759	100.1253	0.181596	0.604563
10	30	40.41353	101.0338	0.256816	0.63547
				Mean=0.1403	Mean=0.6238

 

Precision:

The method precision was established by carrying out the analysis of homogenous powder blend of tablets. The assay was carried out of drug by using proposed analytical method in six replicates. The values of relative standard deviation lie well within the limits indicated the sample repeatability of the method. The results obtained are tabulated in table 4.

 

Table 4: Precision- method precision

Experiment no.	drotaverine hydrochloride taken in μg/ ml	Drotaverine hydrochloride in μg/ ml
		Zero order method	Area under curve method
1	20	20.00649	20.26316
2	20	20.00648	19.76316
3	20	20.00650	20.02632
4	20	20.00647	19.76316
5	20	20.00865	20.02632
6	20	20.00869	20.28947
	Standard deviation	0.001128	0.229666
	%RSD	0.00564	1.147071

 

Inter-day and intra-day precision:

An accurately weighed quantity of tablets powder equivalent to 10 mg of drotaverine hydrochloride was transferred to 100 ml of volumetric flask. A 30 ml of methanol was added and sonicated for 15 minutes and filtered. The filtrate and washing were diluted up to the mark with methanol to give concentration as 100 μg /ml. Such solution was used for analysis.

 

For zero order method:

Solution was scanned between 300 nm to 200 nm in spectrum mode. Absorbance of the resulting solution was measured at 230 nm by using distilled water as blank. The absorbance of final solution was read after 0 hr., 3 hrs. and 6 hrs. in 10 mm cell  at 230 nm for zero order (method A). Similarly the amplitude of the same solution was read on 1^st, 2^nd and 5^th day. The amount of drotaverine hydrochloride was estimated by comparison with standard at 230  nm for zero order, table 5.

 

For area under curve method:

Solution was scanned between 350 nm to 200 nm in spectrum mode. The area under curve of resulting solutions was measured at between 225 nm to 235 nm by using distilled water as blank. The area under curve of final solutions was read after 0 hr., 3 hrs. and 6 hrs. in 10 mm cell at 225 nm to 235 nm (method B).  Similarly area under curve of the same solution was read on 1^st, 2^nd and 5^th day. The amount of drotaverine hydrochloride was estimated by comparison with standard at 225 nm to 235 nm, table 5.

 

Table 5: Summary of validation parameter for intra-day and inter-day

Sr. no.	Parameters	Zero order method	Area under curve (AUC) method
(A)	Intra-day precision (n=3) Amount found ±% RSD	100.0154   0.060072	100.3008 %   0.594997
(B)	Inter-day precision ( n=3) Amount found ± % RSD	99.90744   0.097254	100.1253%   0.604563
(c)	Ruggedness Analyst to analyst ( n= 3) %RSD	0.012145	0.63547

 

Limit of Detection (LOD) and Limit of Quantification (LOQ):

The limit of detection (LOD) is defined as the lowest concentration of an analyte that an analytical process can reliably differentiate from back-ground levels. In this study, LOD and LOQ were based on the standard deviation of the response and the slope of the corresponding curve using the following equations-

 

LOD = 3.3 σ/S    and   LOQ = 10 σ/S

 

Where σ   is the standard deviation of the signal to noise ratio of the sample and S is the slope of the related calibrations graphs.

 

The limit of quantification (LOQ) is defined as the lowest concentration of the standard curve that can be measured with an acceptable accuracy, precision and variability .The values of LOD and LOQ are given in table 6.

 

Table 6: Values of results of LOD and LOQ

parameters	Zero order method	Area under curve (AUC) method
Limit of Detection (μg/ml)	0.006455	0.078473
Limit of Quantification (μg/ml)	0.019559	0.237797

 

 

Ruggedness:

The ruggedness of the method is defined as degree of reproducibility of results obtained by analysis of drotaverine hydrochloride sample under variety of normal test conditions such as different laboratories, different analysts and different lots of reagents. Quantitative determination of drotaverine hydrochloride was conducted spectrophotometrically on one laboratory. It was again tested in another laboratory using different instrument by different analyst. The assays obtained in two different laboratories were well in agreement. It proved ruggedness of the proposed methods.

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RESULT AND DISCUSSION:

The zero order and area under curve UV-spectroscopic methods are useful for routine analysis of drotaverine hydrochloride in bulk drug and formulation. The method was validated according to International Conference on Harmonization guidelines for validation of analytical procedures. Drotaverine hydrochloride has the absorbance maxima at 230 nm (method A) and in the AUC spectrum method areas were measured between 225 nm to 235 nm (method B). The polynomial regression data for the calibration plots showed good linear relationship in the concentration range of 1 to 24 μg/ml for zero order spectroscopy and 5 to 40 µg / ml and given in table1. Recovery studies were carried out by adding the pure drug to the previously analyzed tablet powder sample and shown in table 2, 3. The percentage recovery value indicates non interference from excipients used in formulation. The reproducibility and accuracy of the method were found to be good, which was evidenced by low standard deviation.

 

CONCLUSION:

The most striking features of two methods are its simplicity and rapidity, not requiring tedious sample solutions preparations which are needed for other instrumental methods.  From the results obtained it can be concluded that the proposed methods are fully validated and found to be simple, sensitive, accurate, precise, reproducible, rugged and robust and relatively inexpensive. So, the developed methods can be easily applied for the routine quality control analysis of drotaverine hydrochloride in pharmaceutical formulation.

 

ACKNOWLEDGMENT:

Authors express sincere thanks to the Principal, Dr. Tushar M. Desai of D. G. Ruparel college.

 

 

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Accepted on 31.08.2018        © RJPT All right reserved

Research J. Pharm. and Tech 2018; 11(12): 5576-5580.