INTRODUCTION:

Tadalafil (TDF)^1-5 is chemically (6R-trans)-6-(1,3-benzodioxol -5-yl) -2,3,6,7,12 a-hexahydro -2 methyl- pyrazino [1’,2’,1,6] pyrido [3,4-b] indole -1,4-dione used in erectile dysfunction. TDF is a selective reversible inhibitor of cyclic guanosine monophosphate (cGMP)-specific phosphodiestrase type 5 (PDEF5). When sexual stimulation causes the local release of nitric oxide, inhibition of PDEF5 by TDF produces increased levels of cGMP in the corpous cavernosum, this result in smooth muscle relaxation and in flow of blood into the penile tissues, there by producing an erection. Literature survey revealed that, determination of TDF in plasma by HPLC – Tandem mass spectrometry ^6-7 and HPLC-UV ⁸ detection. But there is no method reported for the estimation of TDF by UV-Visible spectrophotometry. Hence, the aim of the present study is to develop and validate simple, precise and accurate UV-Visible spectrophotometric methods for the estimation of TDF in bulk and in tablet dosage form.

MATERIALS AND METHODS:

Shimadzu–1700 double beam UV-Visible spectrophotometer was used; TDF was generously gifted by Aurobindo pharma Ltd., Hyderabad. Formulations Forzest (Ranbaxy laboratories limited, New Delhi) and Tazzle (Dr. Reddys Laboratories, Hyderabad) were procured from the local the market. All the chemicals used were of analytical grade.

Method A: UV – spectroscopic method:

An accurately weighed quantity of 50 mg pure TDF was transferred into a 100 ml volumetric flask and dissolved in the mixture of methanol: water (80:20) and made up to the volume to produce standard stock solution (500µg /ml). Aliquots of standard stock solution were pipette out and suitably diluted to get a final concentration of 5- 30 µg / ml. 10 µg/ ml solution was scanned between 200 nm to 400 nm and the spectrum was recorded. From the spectra, 284.5 nm was selected for the analysis at which TDF has maximum absorbance. The above solutions of different concentrations were measured at the selected wavelength and the calibration graph was plotted using concentration against absorbance

Method B: Colorimetric method:

The method is based on reduction of ferric ions into ferrous ions by the drug, which further in presence of potassium ferricyanide as oxidizing agent produces bluish green colored complex measured at 828 nm against reagent blank. Aliquots of 0.2 to 1.0 ml of 100 µg/ ml standard solution using a mixture of acetonitrile: water (40:60) as solvent was transferred into a series of 10 ml volumetric flasks to that 0.2 ml of ferric chloride and 0.8 ml potassium ferricyanide were added and made up to the volume with a mixture of acetonitrile : water (40:60). The intensity of the colour was determined by measuring the absorbance at 828 nm against reagent blank. The calibration curve was plotted using concentration and absorbance.

Figure 1: UV Spectrum of TDF in Methanol:Water (80:20)

Figure 2: Spectrum of Tadalafil by Colorimetric Method

Analysis of pharmaceutical dosage forms:

In method A, twenty tablets were weighed; their average weight was determined and was finely powdered. The weight equivalent to 20 mg of TDF was transferred into a 100 ml volumetric flask, added methanol: water (80:20) and sonicated for 5 mins and made up to 100 ml. After sonication the solution was filtered and 1 ml of the filtrate was transferred into 10 ml volumetric flask to produce the nominal concentration of 20µg/ ml from the calibration curve. The absorbance measurements were made six times for each formulation at 284.5 nm. In method B, twenty tablets of two formulations (Forzest and Tazzle) were weighed, powdered and the powder equivalent to 25 mg of TDF were transferred in to 50 ml volumetric flasks separately. Acetonitrile: water (40:60) mixture was added to dissolve the substance by sonication for 5 minutes and filtered and 4 ml of filtrate was transferred to a 100 ml volumetric flask and made up to the required volume with the same solvent. To 3 ml of each test solution, 0.2 ml of ferric chloride solution and 0.8 ml of potassium ferricyanide solution were added in to a 10 ml volumetric flask and heated for two minutes. Cooled and final dilution was made with distilled water to get 6 µg/ ml solution. The absorbance of these solutions was measured at 828 nm against reagent blank. The concentrations of test solution were determined by using slope and intercept values from the calibration graph.

Table 1: Optical Characteristics of TDF

RECOVERY STUDIES:

Recovery studies was carried out to determine the accuracy of the method and to study the interference of formulation additives. It was done by adding known concentration of raw material to the pre-analyzed formulations. In method A, a series of five 100 ml volumetric flasks were taken and 20mg equivalent of each formulation were weighed and 1 - 5 ml of TDF standard stock solution was added to get a concentration of 2000 µg/ ml using methanol: water (80: 20). The absorbance of the resulting solutions was measured at 284.5 nm against blank and the amount of drug recovered from the formulation was calculated by using slope and intercept values. The procedure was repeated for six times. In method B, tablet powder equivalent to 25mg of TDF was weighed in to five 50 ml standard flasks and added 1 - 5 ml to produce 2500 µg/ ml using acetonitrile: water (40:60). The procedure was repeated as per the formulation. The amount of drug recovered from each test solution was determined.

Figure 3: Optimization of 1%Ferric Chloride with Different Volumes

Table – 2: Analysis of Pharmaceutical Formulations

Formulations	Methods	Amount found (mg/tablet)*	% label claim	SD	%RSD	SE
Forzest	A	19.95	99.24	0.513	0.517	0.210
Forzest	B	20.01	100.03	0.218	0.218	0.089
Tazzle	A	19.96	99.77	0.411	0.412	0.168
Tazzle	B	19.98	99.89	0.244	0.244	0.100

*Mean ± SD of six observations

Table – 3: Intra Day and Interday Analysis of Formulation

Formulations	Methods	Amount found *		%RSD
Formulations	Methods	Inter day	Intra day	Inter day	Intra day
Forzest	A	19.76	19.91	0.554	0.447
Forzest	B	19.94	20.05	0.812	0.257
Tazzle	A	19.98	20.02	0.076	0.313
Tazzle	B	19.98	20.05	0.100	0.256

*Mean of three observations

Table 4: Recovery studies for TDF

Formulatios	Methods	Amount present (µg /ml)	Amount added (µg/ml )	Amount found (µg /ml)	Amount recovered (µg /ml)	% recovery*	SD	% RSD	SE
Forzest	A	19.95	2.003	21.98	2.03	101.35	1.363	1.358	0.609
		19.95	6.009	26.07	6.12	101.85
		19.95	10.015	29.98	10.03	100.15
	B	5.998	0.600	6.597	0.599	99.83	0.488	0.487	0.199
		5.998	1.800	7.818	1.820	101.11
		5.998	3.000	8.999	3.001	100.03
Tazzle	A	19.97	2.000	21.97	2.000	100.00	0.345	0.345	0.154
		19.97	6.000	26.00	6.030	100.50
		19.97	10.00	29.93	9.96	99.60
	B	6.00	0.599	6.597	0.597	99.67	0.535	0.534	0.219
		6.00	1.797	7.818	1.818	101.17
		6.00	2.995	8.999	2.999	100.13

*Mean ± SD of six observations

RESULTS AND DISCUSSION:

Two simple, precise, accurate and economical methods were developed. Method A was a UV spectroscopic method in which methanol and water (80:20) was used as a solvent and the λ max was found to be as 284.5 nm. This is shown in figure1. Stability of TDF at this selected λ max was determined and it was found that, the drug was stable for three hours. Method B is a colorimetric method which is based on the reduction of ferric ions to ferrous ion by the drug in the presence of potassium ferricyanide as an oxidising agent produces bluish green colored complex exhibiting the maximum absorbance at 828 nm. (Figure2) The reagents used were optimized and it was found to be the addition of 0.2 ml of ferric chloride and 0.8 ml of potassium ferric cyanide showed the maximum absorbance when it is forming as bluish green colored complex. This is shown in figure 3 and 4. The methods were validated as per ICH guidelines^9,10. The TDF was linear in the concentration range of 5 - 30 µg/ ml and 2 - 10 µg/ ml for method A and B, respectively. The optical characteristics such as molar absorptivity, Sandell’s sensitivity, correlation coefficient, slope and intercept were calculated for both the methods as shown in table 1.The results were found to be satisfactory.

The nominal concentration from calibration curve was selected for the quantification of TDF in formulation. The amount of Forzest and Tazzle, were found to be 19.85 ± 0.103 mg and 19.95 ± 0.082 mg, respectively for method A and 19.98 ± 0.049 mg and 20.01 ± 0.44 mg, respectively for method B. To confirm the precision of the method repeatability and intermediate precision was performed. To perform repeatability, the analysis of formulation was done for six times and the %RSD was calculated and was found to be 0.517 and 0.412 for Forzest and Tazzle, respectively for method A and for method B, it was found to be 0.218 and 0.244 for Forzest and Tazzle, respectively. The results were listed in table 2.

The intermediate precision was confirmed by the analysis of formulation was performed for three times in the same day and on three consecutive days. The %RSD values were found to be less than 2% indicating that the developed methods were found to be precise and the instruments used for the analysis were working properly.(table3) The accuracy of the method was confirmed by the recovery studies, a known amount of pure drug was added to the previously analyzed formulation and it was analyzed by the proposed method, the amount of raw material recovered was calculated, the percentage recovery for method A was found to be 100.34 ± 1.363 (Forzest) and 100.04 ± 0.345 (Tazzle) with the %RSD of 0.487 and 0.534 for Forzest and Tazzle respectively. The results are shown in table 4 .The low %RSD values indicating that there was no interference due to excipients used in the formulation. Hence the method was found to be accurate.

CONCLUSION:

Two simple, precise, accurate and selective methods were developed for the estimation of TDF in bulk and in tablet dosage form. These methods are economical, rapid and do not require any sophisticated instruments contrast to chromatographic method. Hence, these methods can be effectively applied for the routine analysis of TDF.

REFERENCE:

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Parameters	Method A	Method B
λ max (nm)	284.5	828
Beers law limit (µg/ml)	5 - 30	2 - 10(µg /ml)
Sandells sensitivity (µg/ml) cm²/0.00/A.V)	0.027598	0.00466
Molar absorptivity (Lmol^-1cm^-1	1.4429x10⁴	8.3529x10⁴
Correlation coefficient (r)	0.9999	0.9999
Regression equation (y =m x+c)	Y = 0.03624 x +0.008176	Y = 0.2213 x + 0.00184
Slope (m)	0.03624	0.2213
Intercept (c )	0.008176	0.00184
LOD (µg/ml)	0.0799	0.0322
LOQ (µg/ml)	0.2423	0.0976