1. INTRODUCTION:

The crystalline powder of tadalafil is white to off-white in color. It dissolves somewhat in methanol and somewhat in water. Tadalafil's chemical formula is C22H19N3O4, and its molecular weight is 438.4g/mol. After oral treatment, tadalafil is quickly absorbed. It takes 30 to 120 minutes to attain the maximal plasma concentration. The cytochrome P450 enzyme system breaks down tadalafil in the liver. 17.5hours is the elimination half-life. One PDE5 inhibitor is tadalafil. One of the enzymes that breaks down cGMP is PDE5. One chemical that is involved in erectile function is called cGMP. Tadalafil aids in boosting cGMP levels in the penis, which causes an erection, by preventing the breakdown of cGMP.^1,2

IUPAC-Tetraene-4,7-dione(2R-8R)-2-(1,3-benzodioxol-5-yl)-6-6methyl-3,6,17-triazatetracyclo [8.7.0.03,8.011.16] heptadeca-1(10).11,13,15.

2. METHODS:

MATERIALS AND METHODS:

2.1Chemicals and solvents:

Merck Lifesciences Pvt. Ltd., Mumbai, India produces HPLC grade methanol (LichrosolR), and HPLC water 2487 MPOWER 2. Sigma-Aldrich (USA) kindly provided the working standards for sildenafil and tadalafil. We got the frozen human plasma from the Red Cross blood bank.

2.2 Instrumentation:

Throughout this work, an Alliance model 2996 with a PDA detector-equipped UV spectrophotometer was utilized to determine the medicines' λ max values. For the method development, a ZORBAX C18 (250 x 4.6,5 µ) column was used. Empower software was used to keep an eye on the chromatographic apparatus. Analytes were measured at 284nm by UV detection using a methanol:water ratio of 15:85, %v/v), at pH 2.5, and a flow rate of 0.8ml/min maintained by a binary gradient system. at 1.00ml/min, the movement amount was adjusted, and the effluent was measured at 284 nm. At 37°C and 8 minutes, respectively, the temperature and run time were maintained.^3,4

2.3 Standard Stock Solution and Internal:

Standard Preparation:

25mg of Tadalafil were dissolved in 25ml of solvent to create a standard stock solution of 1.0 mg/ml By diluting the aforesaid stock solution to the necessary concentration (10μg/ml), the standard working solution was created. Three levels of concentration were used to prepare quality control (QC) sample: lower (LQC), Middle (MQC), and Higher (HQC). The final concentration range of 0.4-6.4μg/ml was obtained by creating linearity standards. 10milligrams of the solution was mixed in ten milliliters of volumetric flask, and the volume was changed with the solvent to create the internal standard for tadalafil. Pour 0.3ml of the original solution into a flask with a 10ml capacity using the tip of a pipette. Add solvents to the volume to achieve 30 μg/ml.

Plasma Extraction Procedure:

Plasma that had been frozen first thawed. The centrifuge tube received 2 milliliters of plasma after it was extracted. Equivalent to the needed concentration, stock solution was added. After 3 to 4 minutes, the mixture was vortexed. The extraction solvent was then added, 2 milliliters of ethyl acetate. After another three to four minutes, the mixture was vortexed. The extracted sample was finally separated from the mixture by centrifuging it and removing the clear non-polar solvent supernatant. The leftover material was mixed with mobile phase, a non-polar solvent (ethyl acetate), and the resulting solution was injected for additional analysis after the solvent had evaporated.^5,6

Selection of Wavelength

After scanning the initial solution with a amount of 10 μg/ml throughout a range of 200–400nm, 284nm was chosen as the wavelength based on the recorded spectra.

Fig No 1: Uv Spectra for Tadalafil

Fig No 2: Ftir Spectra for Tadalafil

Table No 2: FTIR Spectra for Tadalafil

No.	Functional Group	Normal absorption (s) range (cm-1)	Recognized in this study (cm-1)
1	Aromatic C-H stretch	2950-2850	2918.53
2	C=C bond	1750-1500	1427.59
3	N-H stretch	3500-3300	3317.06
4	Benzene C=C bond	1600-1500	1645.36
5	C=O stretch	1750-1680	1675.45

2.4 Validation Parameter:

Linearity:

The procedure of calibration was carried out using various standard solutions that fell between the Concentration range of 0.4-6.4μg/ml of Tadalafil. linearity of suggested approach was assessed using the least squares method. A standard curve is deemed verified if the coefficient of correlation (r ² value) is near to one.

System Suitability Parameters:

The results were compared to approximative standard values using system suitability parameters. It consists of the asymmetry factor, theoretical plates, and resolution. Every necessary value was discovered within the parameters.

Precision and Accuracy:

The degree of agreement between the repeated person who measures the analyte was termed as precision. It is given as a coefficient of variation (CV) expression. The proximity of the actual charge spiked with a well-known quantity of analyte was used to define accuracy. It has a percentage (%) as its expression. By introducing a known concentration of analyte into the sample matrix, it may be illustrated. For each of the three concentrations (LQC, MQC, and HQC), the percent deviation should be less than 15%.

Stability Studies:

Analysis of stability studies was done under various circumstances. Samples of quality control that have been stored in deep freeze at -4⁰C for extended stability can be evaluated in comparison to newly made stock solutions. ^7,8,9

2.5 RESULT AND DISCUSSION:

Optimization Conditions:

The mobile phase used in the HPLC study was found to provide superior separation and resolution when it was methanol: water in a 15:85, %v/v ratio at pH 2.5. Using a UV spectrophotometer to scan the solution, the wavelength was chosen, and the maximum absorbance of 284 nm was confirmed. In spiked human plasma, sildenafil serves as an internal standard. HPLC was used in the development and validation of the bioanalytical approach employing internal standard. Tadalafil and internal standard, tadalafil and plasma spiked with drug sample, and plasma chromatogram are shown in Figures 5.

2.6 Method Validation:

Linearity:

Validation samples were created by injecting Tadalafil and internal standard into plasma at several concentration range of 0.4-6.4ug/ml of Tadalafil. The calibration curve was produced (Fig. 2) with the area on and the concentration on the X-axis. As seen in Fig.2, the correlation coefficient was determined to be linear.

Specificity and Sensitivity:

The blank chromatogram (Fig. 3) demonstrated that neither the drug's retention period nor the internal standard were impacted. Sensitivity results were discovered to be exact and reliable. Results showed that the limits of quatiton (LOQ) and detection (LOD) were, respectively, 0.69 and 0.21. It was confirmed by the approach that the blank plasma showed no carryover effect. In the generated chromatogram, the X-axis represents time, while the Y-axis represents area.

Accuracy and Precision:

For the LQC, MQC, and HQC QC samples, respectively, accuracy and precision studies were carried out. Based on Table 6, it was determined that the percent SD and RSD were within specification. There was a range of 98.4 to 101.3% for percentage mean accuracy.^10,11

Table 3. Linearity Curve Data of Tadalafil

Ratio of Conc.(μg/ml)	Ratio of area
0.4	0.5025
0.8	0.9950
2.4	2.9950
4.8	6.0078
6.4	6.8708
slope	1.1056
intercept	0.2016
R²	0.9861

Fig No 3: Linearity curve of Tadalafil using internal standard

Fig No 4: A blank plasma chromatogram

Fig no 5: Tadalafil-spiked plasma chromatogram

Fig no 6: chromatogram for internal standard sildenafil

Table 4. Recovery of analyte from biological fluide

Sr. No	LQC		MQC		HQC
Sr. No	Unextracted	Extracted	Unextracted	Extracted	Unextracted	Extracted
1	38112.608	36897.654	327910.510	321089.450	439081.527	437081.427
2	37211.558	37197.652	334914.721	331190.650	428081.624	426082.524
3	37999.659	36597.644	337911.423	321291.950	437091.725	429991.925
4	38011.224	37210.554	328922.723	331199.350	425071.423	429071.823
5	37640.230	36997.765	337911.456	321389.550	434082.220	438085.520
MEAN	37795.06	36980.2538	333514.1666	325232.19	432681.7038	432062.6438
±SD	332.82	225.3085829	4315.458079	4869.577	5318.732511	4700.168949
% RSD	0.88	0.609	1.293935464	1.497262	1.229248305	1.087844325
%Mean Recovery		97.30		98.90		97.40

Table 5 Recovery of Internal standard from biological matrix

Sr. No	Internal standard
Sr. No	Unextracted	Extracted
1	623997	633987
2	643989	633784
3	633995	643996
4	643892	642892
5	622997	632991
6	644997	634949
7	633992	643932
8	643891	633891
9	643992	633982
10	633992	643993
11	643891	633993
12	633791	643797
MEAN	637284.6667	638015.5833
±SD	8010.056467	5062.809218
% RSD	1.256904	0.793524382
%Mean Recovery		99.10

Table 6: Accuracy and Precision of the Tadalafil determination technique

Intra assay (n=5)				Accuracy	Precision
S. N	Conce.	Conce added in μg/ml	Conce.found in μg/ml	%	% RSD
1	LCQ	12	11.6	99.2	0.08
2	MQC	60	61.23	101.1	O.42
3	HQC	120	118.9	99.80	0.06

Table 7: Recovery studies for Identification of Tadalafil from spike dhuman plasma

Sr.no.	QC sample	Area Of Standard	Area of Sample	% Recovery
1	LQC	269991	269991	98.60
2	MQC	2959612	2959612	100.88
3	HQC	3728881	3728881	100.20

Table 8: Long term stability studies of Tadalafil

Sr No	Concentration in ug/ml (QC Sample)	Measured conc. + SD (ug/ml)	% RSD
1	0.3	0.179±0.002	1.35
2	0.6	0.371+-0.003	1.98
3	1.8	1.221±0.191	1.34

Recovery:

Tadalafil standard spiked with retrieved blank plasma was used to perform and measure the study. The results of the recovery investigations (HQC, MQC, and LQC) ranged from 98.60 to 100.19%. The outcomes are displayed in Table 7.

Sample Stability:

For three QC samples, the long-term matrix sample stability of Tadalafil was assessed by deep freezing them at -40 C for a duration of 60 days. It was discovered that Tadalafil's percent RSD fell within an acceptable limit. The findings are reported in Table 8.

Blank Matrix Specificity:

To assess potential interference from endogenous components in human EDTA plasma, randomly selected blank EDTA plasma samples were processed through the extraction procedure and subjected to chromatography. The purpose was to evaluate whether any plasma components could interfere with the analyte or internal standard. No notable interferences were detected across eight different lots of human EDTA plasma samples.

Dilution Integrity:

A dilution integrity test was performed using six replicate samples at two different dilution levels: 1:2 (two times) and 1:4 (four times), prepared at approximately 1.5 times the upper limit of quantitation (ULOQ). The concentrations of the diluted samples were determined by applying the dilution factors, and the results were compared with those obtained from a freshly prepared calibration curve.^10,11

Table no 9: Dilution Integrity

S.No	Dilution Integrity Spiked std concentration 500 ng/ml
	DI ½ sample 250 ng/ml		DI 1/4 sample 125 ng/ml
	Without DF	With DF	Without DF	With DF
1	886.134	872.234	440.421	435.234
2	896.534	877.221	445.345	438.235
3	890.23	875.567	438.230	428.980
4	895.123	860.345	449.342	425.890
5	896.139	865.230	442.345	423.456
6	890.234	874.356	445.567	435.567
MEAN	892.399	870.8255	443.5416667	431.227
±SD	4.174877531	6.617900657	4.009544297	5.965311928
% RSD	0.467826335	0.759957151	0.903983684	1.383334515
%Mean Reovery		97.60		98.56

Fig no 8: Chromatogram for LQC of Tadalafil

Fig no 9: Chromatogram for MQC of Tadalafil

Fig no 10: Chromatogram for HQC of Tadalafil

2.7 CONCLUSION:

High-performance liquid chromatography was used to quantitatively assess tadalafil-stripped plasma (HPLC). Using an internal standard that is affordable and easily accessible, the suggested method improves upon the existing method in terms of simplicity, sensitivity, reliability, and accuracy. In storage conditions, the analyte demonstrated good stability. Therapeutic drug monitoring and pharmacokinetic research can benefit from the new method's simple sample preparation and short chromatographic retention period. The USFDA requirements were followed in validating the procedure, which is within specification

ETHICAL APPROVAL:

It is not applicable

ACKNWOLEDGEMENTS:

The writers would like to thank Sigma-aldrich (USA) for supplying the samples of Tadalafil and Sildenafil within the required time so as to enable us to complete this research paper quickly. Special thanks to KPGU University Vadodara.

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Received on 24.12.2024 Revised on 08.03.2025

Accepted on 16.04.2025 Published on 06.05.2025

Available online from May 10, 2025

Asian Journal of Pharmaceutical Analysis. 2025; 15(2):85-90.

DOI: 10.52711/2231-5675.2025.00014

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