INTRODUCTION:

Tadalafil (TDF), chemically (6R-trans)-6-(1,3-benzodioxol-5-yl)-2,3,6,7,12,12a- hexahydro-2-methyl-pyrazino (1’,2’:1,6.) pyrido (3,4-b) indole 1,4-dione, belongs to Phospho diesterase – 5 inhibitor (PDA -5) class of drugs¹, which stimulates the synthesis of cGMP in the smooth muscle cells leading to vasodilatation.

The structure of Tadalafil is shown in (Figure 1)².

Figure 1: Structure of TDF

The chemical stability of the molecule in the pharmaceutical field is of a major concern, as it affects the safety and efficacy of the drug product. Moreover, stress testing has been recognized as a pivotal part of the drug development process. The ICH guideline states that stress testing is a tool that furnishes the prediction of stability problems, development of analytical methods, as well as, the identification of the degradation products and pathways^3,4.

The FDA and ICH guidelines clearly state the requirement of stability testing data to recognize how the quality of a drug substance and drug product changes with time under the ascendancy of various environmental factors like temperature, UV radiation, water etc⁵. Knowledge of the stability of molecule which helps in selecting proper formulation and package as well as providing proper storage conditions and shelf life is crucial for regulatory documentation. Forced degradation is a process that involves degradation of drug products and drug substances at conditions more severe than accelerated conditions and thus generates degradation products that can be studied to determine the stability of the molecule⁶.

Tadalafil is used for treatment against erectile dysfunction. Now a day it is also used in pulmonary arterial hypertension, combination with Endothelin receptor antagonist. The literature review of Tadalafil reveals that various analytical methods which are available for the determination of Tadalafil individually and in other combination with other. Various analytical methods like RP-HPLC^7-19, spectrophotometric methods^20-29, stability indicating HPLC^30-35 method for single drug and combination and LC-MS^36-37 method, UPLC-MS method³⁸, GC-MS³⁹ and QbD⁴⁰ methods are also available. However, no stress degradation kinetic study by UV was reported so far. Thus, in present study it was decided to carryout stress degradation kinetic study using UV Spectrophotometry for estimation of Tadalafil in bulk and tablet dosage form, and method was validated in compliance with ICH guideline (Q2 R1)⁴¹.

MATERIAL AND METHODS:

Chemicals and instruments:

Tadalafil was provided by B.K. Mody Govt. Pharmacy College, Rajkot. HPLC grade methanol, AR grade hydrochloric acid, AR grade sodium hydroxide, AR grade hydrogen peroxide were used of Finar pvt. Ltd. Tablet formulation of Tadalafil was purchased from market as TADACIP (Cipla Pharmaceutical Ltd.) tablets containing TDF 20mg.

Instrumentation:

The proposed work was carried out using UV/VIS spectrophotometer (UV-1800 Shimadzu) and data were processed using UV probe (version 2.6) software. All weighing was done on electronic balance (MAB 220 Wensor).

Preparation of Standard stock solution of TDF:

50mg of TDF was accurately weighed it was taken in 50 ml volumetric flask and dissolved in 30ml of methanol. Ultra sonicated it about 5 min. Then the volume was made up to the mark using methanol to get 1000µg/ml standard stock solution of TDF.

Preparation of working standard solution:

5ml of solution was taken from standard stock solution and diluted up to 50ml with methanol to get 100µg/ml of working standard solution of TDF.

Wavelength Selection for detection:

4ml of working standard solution was taken and transferred into 10ml volumetric flask, the volume was made upto the mark with methanol to get 40µg/ml of TDF. Solution was scanned between 200-400nm using UV spectrophotometer and methanol was taken as a blank. The detection wavelength was obtained 284nm. Wavelength selection spectrum is shown in figure no. 2.

Figure 2: Wavelength selection spectrum of TDF

Stress degradation study:

TDF was kept in 0.01 N HCl for acid hydrolysis and in 0.01 N NaOH for alkali hydrolysis, after the samples were neutralized and diluted with methanol and filtered. For photolytic degradation, thermal degradation and oxidative degradation TDF samples were kept at 1.2* 106 LUX h, 105°C and in 1% H₂O₂, all degradation kinetic studies were carried out at room temperature except thermal degradation study from 0 to 24 hrs. TDF sample solutions were prepared with concentration 16 μg/ml.

METHOD VALIDATION:

Linearity:

From the standard stock solution was diluted appropriately to obtain concentration of 4, 8, 12, 16, 20, 24, 28, 32, 36 and 40μg/ml of TDF in 10ml volumetric flask with methanol.

LOD and LOQ:

The limit of detection and limit of quantification were computed to establish method sensitivity. The LOD and LOQ were determined from the standard deviation of the intercept and the slop of the calibration curve by using the equation LOD=3.3*σ/C and LOQ= 10*σ/C respectively.⁴¹

Specificity:

Specificity was performed under 6 replicates at concentration of 16μg/ml of TDF with and without addition of excipients to check the interference of excipients. The specificity of the method was evaluated by calculating percentage interference.

Accuracy:

The accuracy of the method was confirmed by recovery study was performed using drug to drug spiking at three different amounts of analytes as level of 75%, 100%, 125%. Concisely, a recovery study was performing by spiking of 12μg/ml, 16μg/ml and 20μg/ml of TDF to the prepared mixture containing 16μg/ml of TDF.

Precision:

Repeatability was performed under 6 replicates at concentration of 16µg/ml of TDF. Intra-day and inter-day variations of TDF were performed in triplicate at three different concentration levels 75, 100, 125% (12, 16, and 20µg/ml) for TDF. Results from determination of repeatability and intermediate precision, are expressed in the form of RSD.

Robustness:

Robustness of method was established by introducing small change in experimental condition like wavelength. The changes made in wavelength ± 1 nm (283, 284, 285 nm). The robustness of the method was evaluated by calculating RSD.

Preparation of sample solution:

Twenty tablets were weighed and powdered to obtain the average weight, then weigh powder equivalent to drug was weighed and transferred into a 100ml volumetric flask, to which 50ml of diluent was added and sonicated for 10 min. Then it was diluted up to the mark with diluent and mixed well. After that, the solution was filtered through whatmann filter paper. First 5ml of the filtrate was discarded, and by appropriate dilution, 16 μg/ml solution was prepared from the sample solution.

RESULTS AND DISCUSSION:

Linearity:

The calibration curve was obtained for TDF in the range of 4-40μg/ml. The correlation coefficient of TDF was found to be 0.9994. Data for results are shown in table no 1 and in figure no. 3.

Table 1: Calibration data table

Concentration (μg/ml)	Absorbance
4	0.148
8	0.269
12	0.401
16	0.561
20	0.67
24	0.809
28	0.945
32	1.083
36	1.203
40	1.323

Forced degradation study of TDF:

TDF was kept in different stress condition as per the guideline, percentage degradation is shown in Table no 2, which is complies the standard limit and obtain results are satisfactory. Percentage degradation is shown in Figure no 4.

Table 2: Summary of force degradation

Stress condition	Time	% Degradation
Acidic (0.01 N HCl)	24 hrs	27.93
Basic (0.01 N NaOH)	24 hrs	23.99
Photolytic	24 hrs	36.63
Oxidative (1% H₂O₂)	24 hrs	18.11
Thermal (105 ℃)	24 hrs	7.93

Figure 4: Percentage degradation graph of TDF

Precision:

Repeatability and intermediate precision expressed in term of RSD. Absorbances were determined and RSD was found less than 2 for intra-day and inter-day precision and including repeatability each. The result of repeatability study and intermediate precision is shown in table no 3 and 4 respectively.

Table 3: Repeatability study of TDF

Drug	Concentration (μg/ml) (n=6)	Mean concentration found (μg/ml) ± SD (n=6)	RSD
Tadalafil	16	16.04 ± 0.0156	0.097

LOD and LOQ:

LOD and LOQ were determining using formula given in ICH Q2 (R1) guideline. LOD and LOQ were found to be 0.313µg/ml and 0.950µg/ml respectively.

Accuracy:

Accuracy of the recommend method was evaluated by adding known amount of standard drug into the placebo at three contradictory levels in three replicates. Percentage recovery for TDF was found in range of 97.06 to 99.56. Thus the recovery of drug is acceptable as shown in table no 5.

Table 5: Accuracy data for TDF

%Recovery level	Amount taken(μg/ml)	Amount added (μg/ml)	% Recovery range (n=3)
75	16	28	97.06 – 99.56
100	16	32
125	16	36

Robustness:

Deliberate change in wavelength was made and RSD of absorbance found less than 2, it specifies that the method is robust. Result remained uninfluenced by small variation in this parameter. Data is shown in table no 6.

Table 6: Robustness data for TDF

Concentration (µg/ml)	Absorbance at different wavelength (n=3)
Concentration (µg/ml)	283 nm	284 nm	285 nm
	0.555	0.561	0.559
16	0.553	0.56	0.558
	0.556	0.563	0.56
Mean ± SD	0.5546 ± 0.00152	0.5613 ± 0.00152	0.559 ± 0.001
RSD	0.2753	0.2721	0.1788

Specificity:

Percentage interference was found less than 0.5%. Thus the method is specific.

Assay of marketed formulation:

Percentage assay of TADACIP (Cipla Pharmaceutical Ltd.) tablet was calculated and result was found to be satisfactory. Result is expressing in table no 7.

Table 7: Assay of marketed formulation

Drug name	Label claim (mg)	Amount found (mg/tablet)	% Assay (n=6) ± SD
Tadalafil	20 mg	19.95 mg	99.75 ± 0.15

CONCLUSION:

A rapid and simple stress degradation kinetic study of TDF in tablet dosage form has been developed and Validated by UV Spectrophotometry. Stress degradation study was performed under acid base hydrolysis, oxidative degradation, thermal and under photolytic condition. Study of stress degradation kinetic showed that TDF was more stable under thermolytic and oxidative condition rather than under other degradation conditions. The method was fully validated, showing satisfactory data for all the parameters tested. Thus the proposed stability indicating method can be applied in pharmaceutical analysis for drug as well pharmaceutical dosage form stability monitoring and quality control.

CONFLICT OF INTEREST:

Author’s has declared that there is no conflict of interest.

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Precision		Intra-day (n=3)		Inter-day (n=3)
Drug	Level (%)	Absorbance (mean ±SD)	RSD	Absorbance (mean ±SD)	RSD
Tadalafil	75.00	0.5618 ± 0.0012	0.208	0.5616 ± 0.0008	0.149
	100.00	0.5614 ± 0.0002	0.034	0.5619 ± 0.0004	0.069
	125.00	0.5616 ± 0.0007	0.124	0.5619 ± 0.0007	0.137