INTRODUCTION:

Male erectile dysfunction has been defined as the persistent inability to attain and maintain an erection adequate to permit satisfactory sexual performance. Although erectile dysfunction is regarded as a benign disorder, it has a medical and social impact due to its high prevalence, costs and implications for the quality of life for many men and their partners.

A recent review concludes that the prevalence of erectile dysfunction of all degrees is 52% in men 40 to 70 years old, with the incidence increasing with advancing age.

Normal erectile function requires the coordination of psychological, hormonal, neurological, vascular and anatomic factors. Alteration of any of these factors is sufficient to cause erectile dysfunction.¹

Tadalafil is reversible phosphodiesterase type 5 (PDE5) inhibitor approved for the treatment of erectile dysfunction (ED). As a category PDE5 inhibitors (including sildenafil and vardenafil), enhance erectile response to sexual stimulation by increasing penile blood flow. The duration of action of Tadalafil is longer than sildenafil or vardenafil.²

Tadalafil (CIALIS) is an orally administered PDE5 inhibitor has been developed for a treatment for erectile dysfunction. When sexual stimulation causes the local release of nitric oxide gas, which plays a central role within the vasodilation of erectile tissues by stimulating guanylyl cyclase activity, consequently raising intracellular concentrations of cyclic guanosine monophosphate (cGMP) and relaxing vascular smooth muscle. This leads in smooth muscle relaxation and inflow of blood into the penile tissues, thereby producing an erection. Thus tadalafil is indicated for the treatment of male erectile dysfunction. Tadalafil has no effect within the absence of sexual stimuli.¹

Tadalafil is a phosphodiesterase 5 inhibitor accustomed treats erectile dysfunction, benign prostatic hyperplasia, and pulmonary arterial hypertension. Tadalafil is practically insoluble in water. It does not possess any ionisable groups within the pH range of 1-11 and, subsequently, doesn’t demonstrate any changes in solubility in aqueous buffers in this range. It is freely soluble only in solvents like as dimethylsulfoxide and dimethylformamide.¹

This molecule has 2 chiral centers and thus four different stereoisomers are also found. The molecule obtained in the process described is within the RR form. Crystallization studies show that Tadalafil doesn’t exhibit polymorphism.¹

CHEMISTRY:

1. Synonyms: Adcirca, Cialis, GF196960, HSDB7370, IC351, ICOS351, Tadalafil, Tadalafil Lilly, UNII- 742SXX0ICT

2. IUPUC:(6R,12aR)-6-(1,3-benzodioxol-5-yl)-2-methyl-2,3,6,7,12,12a-hexahydropyrazino[1’,2’:1,6]pyrido[3,4-b]indole-1,4-dione

3. Formula: C22H19N3O4

4. Molar mass: 389.411 g·mol−1 ³

Figure 1: Chemical Structure of Tadalafil

This study is to develop an easy and accurate RP-HPLC method for the estimation of Tadalafil in tablet dosage form. The method validation is to demonstrate that the strategy is suitable for its intended purpose because it is stated in ICH guidelines. The strategy was validated for linearity, precision, accuracy, specificity, and limit of detection, limit of quantification, robustness and system suitability.⁴

MATERIAL AND METHOD:

Material:

Tadalafil pure drug sample obtain from Tadacip 20 tablet which is manufactured at Cipla Ltd, India. HPLC grade Acetonitrile was procured from E. Merck Ltd, India. SQ grade Potassium Hydrogen Orthophosphate was purchased from Fisher Scientific. AR grade Orthophosphoric Acid was purchased from Rankem. Milli-Q water was used throughout the experiment. Tadacip 20 tablets were purchased from local pharmacy.

Instrumentation:

Analysis was performed on a Summit HPLC chromatographic system, Low Pressure Quaternary Gradient Dionex manufacturer equipped with ASI-100 Automated sample injector, LPG-4 HPLC Pump, programmable variable wavelength PDA-3000 detector. Chromatographic separation was achieved by Agilent eclipse C₁₈column (4.6 x 250mm, 5um). The HPLC system was equipped with “Chromeleon 6.8 SR 11” software to acquire and process the data. Peak purity was checked the PDA detector.⁵

METHODOLOGY:

Standard solutions:

Weigh accurately and transfer about 40mg Tadalafil standard in 200ml volumetric flask. Add about 150ml of Mobile phase. Sonicate for 5 minutes. Allow the solution to attend room temperature and dilute up to mark with mobile phase.

Mobile phase:

Phosphate Buffer pH 4.0: 1.360gm. of Potassium Di-hydrogen Orthophosphate dissolved and diluted in 1000 ml water. Adjust the pH to 4.0 with dilute ortho phosphoric acid. Phosphate buffer pH 4.0: Acetonitrile (50:50). Mix, Sonicate and filter through 0.45 micron nylon filter paper.

Chromatographic Conditions:

An isocratic condition HPLC analysis was performed an Agilent Eclips C18 (150 x 4.6mm, 5um) maintained at conditions (30°C). Chromatographic separation was achieved with mobile phase and mixture of at flow rate of 1.0ml/min and injection volume of 20µl and also the run time is 6 min. the Tadacip 20 was scanned under conditions and from the spectra maxima of 284nm was observed.

Assay of formulation:

Procedure:

Weigh 20 Tablet at a time. Calculate Average weight. Crush Mix to make uniform and perform assay from it.

Sample Preparation:

Weigh and transfer sample powder containing about 40 mg of Tadalafil in 200ml volumetric flask. Add about 150ml of mobile phase. Sonicate for 20 minutes. Allow to attend room temperature. Dilute up to mark with mobile phase. Filter with whatman 41 or equivalent filter paper.

Inject blank, standard and Sample preparation as sequence given below,

(1) Blank (2) Standard preparation (5 replicates) (3) Blank (4) Sample preparation (5) Standard preparation (Bracketing standard)

Perform calculation for system suitability parameters –% RSD of retention time and area of standard replicates, cumulative %RSD of retention time and area of replicate standards with bracketing standard.

System suitability criteria:

1. %RSD for retention time of replicates of standard preparation should not be more than 1.0%

2. %RSD for area of replicates of standard preparation should not be more than 2.0%

3. Theoretical plates for all the standard injections should not be less than 2000. Report Theoretical plate of first replicate of standard preparation.

4. Tailing factor for all the standard injections should not be less than 2.0. Report tailing factor of first replicate of standard preparation.

5. Cumulative %RSD for retention time of replicates of standard preparation with retention time of bracketing standard should not be more than 1.0 %

6. Cumulative %RSD for area of replicates of standard preparation with area of bracketing standard should not be more than 2.0%

If system suitability found within limit, perform calculations for content of Tadalafil Tablet by using formula, Calculate Content of Tadalafil (mg/Tablet) for sample preparations independently by using formula described below.

(Area of Tadalafil in sample preparation) W1 (mg) 200 P1

= ----------------------------------------------------x------------x---------x-----xW3 (gm.)

(Avg. area of Tadalafil Standard preparation) 200 W2 (g) 100

Where,

W1 = Weight of Tadalafil standard taken for preparation of standard preparation (in mg)

W2 = Weight of Tadalafil sample taken for preparation of sample preparation (in g)

W3 = Average weight of Tablet in gm.

W = Weight of sample in gm.

P1 = % Purity of Tadalafil Standard

Acceptance Criteria:

Each film coated tablet content:

Tadalafil per tablet = 18.00mg to 22.00mg

% Assay = 90.00% to 110.00%

Method validation:

A. Specificity and system suitability:

· Experimental design:

1. Inject mobile phase to observe interference at interested peaks.

2. Prepare and inject std preparation individually (as per final conc. Of mixed std preparation).

· Acceptance criteria:

1. There should not be interference of any peak at interested peaks.

2. System suitability parameter should meet and assay for tadalafil 20mg should be within limit.

B. Precision

a) Repeatability:

· Experimental design:

Perform assay in six individual replicates of sample. Perform % RSD of assay.

· Acceptance criteria:

% RSD for assays should be within less than 2%

b) Intermediate precision:

· Experimental design:

1. Perform assay in six individual replicates of sample by other person on other day with other set of chemicals.

2. Perform %RSD of six sample preparations.

3. Also calculate % variation of average assay values obtained via repeatability and intermediate precision.

· Acceptance criteria:

1. Assay for all samples should be within limit.

2. % RSD for assays for intermediate preparation should be less than 2%.

3. % variation of average assay values obtained via repeatability and intermediate precision should be less than 3%.

C. Accuracy:

· Experimental design:

1. Prepare samples in triplicate by taking weight equivalent to 80%, 100% and 120% of working levels and perform analysis.

2. Calculate % recovery by factors amount added vs. amount recovered.

3. Calculate %RSD of recovery at each level for triplicate preparations.

· Acceptance criteria:

1. % recoveries of individual preparation should be 98 to 102%.

2. % RSD at each level should not be more than 3%

D. Linearity and range:

· Experimental design:

1. Prepare standard preparations at each 80%, 100% and 120% of working level and inject them.

2. Determine co-relation coefficient by plotting linearity graph. Calculate % y intercept

· Acceptance criteria:

1. Graph should be linear and co-relation coefficient should be not less than 0.999

2. % y intercept should be within ±2%

E. LOD AND LOQ

· Experimental design:

1. 5ppm solution to be prepared and signal to noise ratio is determined.

2. LOD is determined by preparing diluted solutions with signal to noise ratio about 3.

3. Level of concentration at which peak got detected repeatedly but not necessary to be precise is LOD.

4. LOQ is determined by preparing diluted solutions with signal to noise ratio above 3 and about 10.

5. Level of concentration at which peak got detected with precise area (%RSD NMT 2%) is LOQ.

· Acceptance criteria:

1. For LOD Single to noise ratio is about 3.

2. For LOQ Single to noise ratio is about 10.

3. For LOQ, %RSD for replicate injections should be less than 2 %

F. Robustness:

· Experimental design:

1. Perform analysis of sample by changing flow rate as 0.8 and 1.2ml per minutes.

2. Perform analysis of sample by changing wavelength 282 nm and 286nm.

3. Calculate % cumulative RSD of assay obtained at repeatability and by changing parameters.

· Acceptance criteria:

1. % cumulative RSD of assay obtained at repeatability and by changing parameters should not be more than 3 %

Non-conformance:

Specify the non-conformance (if any) observed during method validation.

RESULT AND DISCUSSION:

Method development and optimization:

In order to optimize the LC conditions for the estimation of Tadalafil in tablet the following trials were performed. Initially a mobile phase consisting of 50mM Potassium Di-hydrogen Orthophosphate (pH 5.0): Acetonitrile (80: 20 %v/v) at a flow rate of 1.0mL/min was used on an Agilent Eclipse C18 column (250 x 4.6) column at ambient temperature using mobile phase as diluent, Tadalafil did not elute under these conditions,

In the next trial, same column was employed but the mobile phase was changed to mobile phase consisting of 10 mM Potassium Di-hydrogen Orthophosphate (pH 5.0): Acetonitrile (70:30% v/v) at a flow rate of 1.0 mL/min was used on an Agilent Eclipse C18 column (250 x 4.6) column at ambient temperature.

Tadalafil eluate with at 12.331 with theoretical plates 4234 (limit NLT 2000 and Tailing factor 1.58 (NMT2) which well within the limit but as retention time is more and will time consuming during analysis therefore focusing on reduction of retention time drastically. pH of buffer solution reduced with dilute Orthophosphoric acid solution. Organic Solvent content in mobile phase composition increased and also temperature of column increased.

Tadalafil eluate with at 3.447 with theoretical plates 3431 (limit NLT 2000 and Tailing factor 1.55 (NMT 2) which well within the limit. Thus further injection of same std is done and to conclude precision. Corresponding area, RT and system suitability parameters observed.

Lambda max is determined before conducting next trial and found 284nm.Next trial conducted with mobile phase consisting of 10mM Potassium Di-hydrogen Orthophosphate (pH 4.0): Acetonitrile (50:50% v/v) at a flow rate of 1.0mL/min was used on an Agilent Eclipse C18 column (250 x 4.6) column at 30°C.

METHOD VALIDATION:

A. Specificity And System Suitability:

Specificity demonstrated by observing interference of mobile phase (Diluent). System suitability parameters (% RSD of area, Retention time, Theoretical Plates and Tailing factor) demonstrated by injecting standard preparation in replicate.

Table no1: Specificity and System Suitability

Std Inj No	Retention Time	Area	Theoretical Plates	Tailing Factor
1	2.507	80482.079	3436	1.59
2	2.507	81838.312	3359	1.59
3	2.508	80682.928	3412	1.58
5	2.507	80378.131	3398	1.57
5	2.509	80181.986	3422	1.58
% RSD	0.04 (Limit NMT 1 %)	0.81 (Limit NMT 2 %)	Limit: NLT 2000	Limit: NMT 2

No interference observed from Mobile phase (Diluent).

Conclusion – Method found specific and capable to achieve System suitability.

B. PRECISION:

A. Repeatability:

The repeatability was demonstrated by preparing the standard solution at 200ppm concentration and six independent consecutive sample preparations at 200 ppm. System suitability found within limit. Relative standard deviation of assay value for six preparations found within 2 %.

Table no 2: Repeatability Suitability System

Std Inj No	Retention Time	Area	Theoretical Plates	Tailing Factor
1	2.507	80550.869	3436	1.59
2	2.508	80234.452	3392	1.58
3	2.507	81223.21	3414	1.57
4	2.505	80877.456	3433	1.58
5	2.508	80281.714	3390	1.58
% RSD	0.05 (Limit NMT 1 %)	0.52 (Limit NMT 2 %)	Limit: NLT 2000	Limit: NMT 2

Average std area = 80633.5402

Sample	RT	Area	% Assay
1	2.508	81050.398	101.35
2	2.507	81124.947	101.16
3	2.507	81038.768	101.38
4	2.508	81098.332	100.71
5	2.509	81168.295	101.06
6	2.509	81068.679	100.84
% RSD (NMT 2 %)			0.27

B. Intermediate Preparation:

The Intermediate Precision was demonstrated by preparing the standard solution at 200ppm concentration and six independent consecutive sample preparations at 200ppm. By other person on other day with other set of chemicals. System suitability found within limit. Relative standard deviation of assay value for six preparations found within 2%. % variation of average assay values obtained via repeatability and intermediate precision found within 3%

Table no 3: Intermediate Preparation System Suitability

Std Inj No	Retention Time	Area	Theoretical Plates	Tailing Factor
1	2.507	84508.997	3369	1.58
2	2.507	84122.505	3278	1.57
3	2.504	83453.121	3245	1.56
5	2.505	84467.887	3314	1.56
5	2.504	83567.886	3243	1.57
% RSD	0.06 (Limit NMT 1 %)	0.59 (Limit NMT 2 %)	Limit: NLT 2000	Limit: NMT 2

Average std area = 84024.0792

Sample	RT	Area	% Assay
1	2.507	84235.965	100.47
2	2.507	84156.235	100.52
3	2.509	84578.691	100.92
4	2.509	84269.523	101.28
5	2.508	84638.419	100.84
6	2.507	84398.651	100.86
% RSD (NMT 2 %)			0.29
% RSD with repeatability (NMT 3 %)			0.30

Conclusion – Method found Precise.

C. ACCURACY:

To determine the accuracy of the method, recovery studies were carried out in triplicate by using different concentrations of pure drug in the pre analyzed samples with 3 different concentrations of sample that consists of 80 %, 100 % and 120 % of the pure drug. The accuracy was expressed as the percentage analytes recovered.

Table no 4: Accuracy

Std Inj No	Retention Time	Area	Theoretical Plates	Tailing Factor
1	2.506	84122.127	3312	1.55
2	2.505	84613.560	3214	1.55
3	2.504	83948.654	3344	1.54
4	2.508	84026.334	3370	1.56
5	2.509	84386.329	3351	1.55
% RSD	0.08 (Limit NMT 1 %)	0.33 (Limit NMT 2 %)	Limit: NLT 2000	Limit: NMT 2

Sample	% Recovery	Average % recovery	% RSD
80 %	99.66	99.54	0.36
80 %	99.13
80 %	99.82
100 %	98.94	99.39	0.41
100 %	99.73
100 %	99.51
120 %	99.15	99.25	0.55
120 %	98.76
120 %	99.84
Limit		Limit 98 to 102 %	NMT 3 %

Conclusion – Method found Accurate.

D. LINEARITY AND RANGE:

From the standard stock solution, the various dilutions of Tadalafil in the concentration of 160.0, 200.0, 240.0 ppm three level standard solutions of each were prepared. The solutions were injected using 20 μL injection volumes in to the chromatographic system at the flow rate of 1.0 ml/min and the effluents were monitored at 284nm, chromatograms were recorded. Calibration curve of Tadalafil was obtained by plotting the peak area ratio versus the applied concentrations of Tadalafil by using average of each sample. The linear correlation coefficient (R2) was found to be 1.000 and %y intercept is 0.0035%

Table No 5: Linearity and Range

Sr. No.	Conc. ppm	Area	Average
1	160.0	66710.597	66689.670
2	160.0	66638.432
3	160.0	66719.981
4	200.00	83676.153	83672.5675
5	200.00	83698.982
6	200.00	83642.568
7	240.00	100407.985	100652.8887
8	240.00	100598.458
9	240.00	100952.223

Conclusion – Method found Linear in the range 80 % to 120 % of working level

E. Limit of Detection and Limit of Quantitation (LOD and LOQ):

The limit of detection and limit of quantification means the lowest concentration of analytes in the sample are detected and quantified. LOD and LOQ was found as listed below

Table no 6: LOD and LOQ

Parameter	Obtained value
LOD	0.00035 ppm
LOQ	0.00523 ppm

F. ROBUSTNESS:

Robustness of the method was determined by intentionally changing some operating conditions such as flow rate and wavelength. The flow rate as per the developed method is 1.0ml/min. It has been purposely changed to 0.8ml/min and 1.2ml/min and the chromatogram was developed as well as the wavelength of developed method is 284nm. It has been purposely changed to 282nm and 286nm and the chromatogram was developed.

Table No 7: Robustness system suitability flow rate = 0.8 ml

Std Inj No	Retention Time	Area	Theoretical Plates	Tailing Factor
1	3.128	100735.480	3388	1.59
2	3.126	100123.563	3321	1.58
3	3.124	99131.118	3339	1.57
4	3.125	99790.264	3350	1.59
5	3.128	99520.003	3297	1.59
% RSD	0.06 (Limit NMT 1 %)	0.61 (Limit NMT 2 %)	Limit: NLT 2000	Limit: NMT 2

Parameter	Condition	RT	Area	% Assay	% Cumulative RSD with Repeatability
Change in flow rate	0.8 ml /min	3.131	99300.247	100.11	0.38

Table no 8: Robustness system suitability flow rate = 1.2 ml /min

Std Inj No	Retention Time	Area	Theoretical Plates	Tailing Factor
1	2.098	68319.994	3335	1.56
2	2.097	67222.965	3334	1.55
3	2.098	68210.003	3330	1.54
5	2.099	68427.675	3323	1.56
5	2.096	69276.998	3245	1.56
% RSD	0.05 (Limit NMT 1 %)	1.07 (Limit NMT 2 %)	Limit: NLT 2000	Limit: NMT 2

Parameter	Condition	RT	Area	% Assay	% Cumulative RSD with Repeatability
Change in flow rate	1.2 ml /min	2.097	68411.243	101.43	0.24

Table no 9: Robustness system suitability detection wavelength = 282 nm

Std Inj No	Retention Time	Area	Theoretical Plates	Tailing Factor

1	2.097	80130.345	3287	1.58
2	2.096	80023.776	3186	1.56
3	2.093	80211.543	3233	1.57
5	2.095	81235.867	3295	1.58
5	2.097	80425.971	3231	1.58
% RSD	0.08 (Limit NMT 1 %)	0.61 (Limit NMT 2 %)	Limit: NLT 2000	Limit: NMT 2

Parameter

Condition

Area

% Assay

% Cumulative RSD with Repeatability

Change in wave length

282nm

2.096

77812.828

100.11

0.38

Table no 10: Robustness system suitability detection wavelength = 286 nm

Std Inj No	Retention Time	Area	Theoretical Plates	Tailing Factor
1	2.097	79145.885	3212	1.56
2	2.095	78230.870	3198	1.55
3	2.092	79431.114	3243	1.54
5	2.094	79786.240	3230	1.57
5	2.096	79555.476	3211	1.55
% RSD	0.09 (Limit NMT 1 %)	0.76 (Limit NMT 2 %)	Limit: NLT 2000	Limit: NMT 2

Parameter

Condition

Area

% Assay

% Cumulative RSD with Repeatability

Change in wave length

286nm

2.097

80160.9116

100.78

0.23

% Cumulative RSD of % assay observed for changing parameters calculated and found within limit i.e. below 3 %.

Conclusion – Method found Robust.

Non-conformance: Specify the non-conformance (if any) observed during method validation.

CONCLUSION:

The proposed RP-HPLC method was simple, sensitive, precise and accurate for determination of Tadalafil n tablet dosage form. The results obtained for all validated parameters were within the limits; hence the proposed method can be easily applied for the quantification of Tadalafil in routine quality control pharmaceutical laboratories. The analytical method used for determination of assay of Tadalafil Tablet is within acceptance criteria for the analytical parameters such as Specificity and system suitability, Linearity and Range, Precision, Accuracy and Robustness. Hence method stands validated.

ACKNOWLEDGEMENT:

Author are thankful to Insta Vision Laboratory and Services, Satara for providing gift sample of tadalafil drug and as well as technical support and also thankful to Management and Principal Dr. V. K. Redasani of YSPM, YTC Faculty of Pharmacy (B. pharm), Satara, for providing necessary facilities to carry out this work.

REFERENCES:

1. Eli Lilly Nederland BV. Cialis-epar- scientific discussion. European Medicine Agency.2005

2. John A. Noviasky, Asif Masood, Vincent Lo. Tadalafil (Cialis) for Erectile Dysfunction. American Family Physician. 2014 Jul. 15. Volume: 70. Issue: 2. Page No.359- 360

3. Tadalafil. Wikipedia. Chemistry of Tadalafil

4. Saroj Kumar Raut, B.V.V. Ravi Kumar, Ajaya Kumar Pattnaik. A RP- HPLC method development and validation for the estimation of Tadalafil bulk and pharmaceutical Dosage Form. Research Journal of Pharmacy and Technology.2012. Volume: 5. Issue: 2. Page No.1573- 1576

5. Aziz Unnisa, Yogesh Babu, Santosh Kumar, Siva Chaitanya. RP-HPLC-PDA method development and validation for the analysis of tadalafil in bulk, pharmaceutical dosage form and in-vitro dissolution samples. Journal of Applied Pharmaceutical Science. Vol.4 (12)12. Dec, 2014.

Received on 10.07.2021 Modified on 28.07.2021

Asian J. Research Chem. 2021; 14(5):380-388.

DOI: 10.52711/0974-4150.2021.00065