1.0 INTRODUCTION:

Tadalafil is a PDE5 inhibitor, used in the treatment of erectile dysfunction. Tadalafil is (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. The empirical formula is C₂₂H₁₉N₃O₄ and its molecular weight is 389.404. It is a crystalline solid practically insoluble in water and very slightly soluble in ethanol^1-8. The chemical structure is given below

It is not official in any pharmacopoeiaand till now, few liquid chromatography procedures have been reported for the determination of Tadalafil and their metabolites in biological fluids ^9-10. There is still need of simple, sensitive method for analyzing the same in plasma. This paper describes a method of analyzing Tadalafil in plasma, which is very simple, inexpensive, sensitive, rapid, and accurate method for analysis of Tadalafil in human plasma with reliable reproducibility suitable for pharmacokinetic studies.. The method was validated according to procedures and acceptance criteria based on FDA guidelines and recommendations of ICH.

2.0 EXPERIMENTAL:^11-13

2.1 INSTRUMENTATION:

The waters LC system equipped with 2489 pump and 2487 UV detector for which the output signal monitored and integrated using waters Empower 2 software was used for the determination of tadalafil in human plasma

2.2 PREPARATION OF REAGENT AND SOLVENTS:

2.2.1 Solution A: Preparation of pH 2.5 buffer solution:

2mL of Triethylamine was taken in 1000mL of milli-Q water and mixed well , pH of the solution was adjusted to 2.5±0.05 with ortho Phosphoric acid.

2.2.2 Solution B: Preparation of 95% Acetonitrile:

Add 50 ml of Milli-Q-Water in to 950ml of Acetonitrile in a suitable mobile phase bottle and mix well. Filter through 0.45µ nylon membrane filter and degas for about 10 min.

2.2.3 Preparation of Mobile phase:

Mix Solution A and Solution B in the ratio of 66:34 respectively and degas for about 10 min.

2.2.4 Preparation of 10% Methanol :

Add 10 mL of methanol to a suitable container and dilute to 100mL with water.

2.2.5 Preparation of 50% Methanol :

Add 50ml of methanol to 50 ml of Milli-Q-water in a suitable container and mix well..

2.2.6 Preparation of Reconstitution solution (0.5%NH3 in 50% methanol):

Add 0.5 ml of Ammonia to 100ml of 50% methanol in a suitable container and mix well.

2.2.7 Preparation of Standard Stock solution (1mg/ml):

Weigh and transfer Tadalafil working standard equivalent to about 10.0mg of tadalafil into a 10mL volumetric flask. Add about 5 ml of Acetonitrile, sonicate to dissolve the material completely and made up to the volume with Acetonitrile and mix.

2.2.8 Preparation of Intermediate Standard solution:

Transfer about 1mL of the above 1mg/mL stock in to 10 mL volumetric flask and dilute with Acetonitrile: water-50:50

2.2.9 Preparation of Tadalafil spiking solution:

Volume of Standard/quality control stock or Intermediate solutions	Make up Volume of Acetonitrile: water(50:50) (50: 50)	Conc of Working /spiking solutions
40	10	0.4
100	10	1.0
120	10	1.2
200	10	2.0
400	10	4.0
800	10	8.0
1000	10	10.0
1200	10	12.0
1500	10	15.0
1600	10	16.0
2000	10	20.0

2.2.10 Preparation of Internal Standard solution (1mg/ml):

Weigh and transfer phenytoin standard equivalent to about 10mg of phenytoin in to 10ml volumetric flask , add about 5ml of acetrontrile ,sonicate to dissolve the material completely and made up to the volume with acetontrile and mix. Pipette 1.0mL of above solution into a 10mL volumetric flask and make up the volume with Acetonitrile:water-50:50 to get 100 µg/mL concentration.

2.2.11 Preparation of calibration standards and quality control standards of tadalafil :

ID.	Concentration of Spiking solution, in µg/ml	Volume of spiking solution to be taken, in µl	Volume of plasma to be taken, in ml	Final concentration in µg/ml
CS1	0.4	500	9.5	20
CS2	1.0	500	9.5	50
CS3	1.2	500	9.5	60
CS4	2.0	500	9.5	100
CS5	4.0	500	9.5	200
CS6	8.0	500	9.5	400
CS7	10.0	500	9.5	500
CS8	12.0	500	9.5	600
LQC	15.0	500	9.5	750
MQC	16.0	500	9.5	800
HQC	20.0	500	9.5	1000

2.2.12 Preparation of Sample :

Sample was prepared by using Solid phase extraction technique.25µl of internal standard solution was admixed with 0.5ml of sample in ria vial to it 10µl of Ammonia is added .1ml of this premixed sample was loaded onto each strata extraction cartridge which was kept for equilibration .After loading sample on to cartridge, cartridge was washed with 1ml of 10% methanol and drained out by applying vacuum. Finally the sample was collected in to a suitable vessel by selecting 100% methanol as eluent. Evaporate 1ml of collected eluent at 50°C for 15 minutes using nitrogen evaporator. Reconstitute the dried residue with 250µL of 0.5% NH3 in 50%methanol and mix well.

2.2.8 Chromatographic Conditions:

A Grace Genesis C18 (150 x 4.6 mm, 5µ) column was used for analysis at column Temperature 40ºC .The mobile phase was pumped through the column at a flow rate of 1.2mL/min. The Sample injection Volume was 50µL.The UV detector was set to a

wavelength of 225 nm for the detection.

3.0 Method Validation: ^14-15

The developed bioanalytical method for determination of Tadalafil in Human plasma was extensively validated as per FDA guidelines and recommendations of ICH. This validated HPLC method can be used in determination of tadalafil in human plasma for bioequivalence and bioavailability study of tadalafil.

3.1. CALIBRATION CURVE:

Stock solutions of tadalafil and internal standard were prepared in acetonitrile and kept at -20°. Working solutions were prepared by diluting the stock solutions of standard with Acetonitrile:water-50:50. For calibration curve eleven different concentrations (20, 50, 100, 200, 400, 600, 800, 1000ng/ml, 60, 500 and 750 ng/ml) in plasma were prepared by adding required volume of working solution of analyte to blank plasma. For internal standard the final concentration in plasma was 25µg/ml. The plasma samples were subjected to the sample preparation procedure and injected onto HPLC. Plasma calibration curve was prepared by taking area ratio of analyte to internal standard as Y-axis and concentration of analyte (ng/ml) as X-axis. Data are presented in Table 1. A typical calibration curve is presented in Figure 1

Table-1 Summary of Calibration Curve Parameters

S No	Y-Intercept	Slope	Determination Coefficient (r²)
1	-0.00070	0.00101	0.999
2	0.00141	0.00098	0.999
3	-0.00150	0.00104	0.999
4	-0.00009	0.00101	0.999
5	-0.00123	0.00096	0.999
6	0.00073	0.00107	0.999

3.2 Extraction recovery:

The extraction recovery of analyte was determined by measuring the peak areas of the drug from the prepared plasma quality control samples. 60 ng/ml, 500 ng/ml and 750 ng/ml plasma samples were taken as LQC (low quality control), MQC (medium quality control) and HQC (high quality control) samples respectively. The peak areas of extracted LQC, MQC and HQC were compared to the absolute peak area of the unextracted samples containing the same concentration of the drug as 100%. To obtain good extraction efficiency the extraction recovery of tadalafil was determined using five replicates of each QC samples. The results are provided in Table 2 .For the internal standard ,mean peak responses of twelve extracted samples was compared to the mean peak responses of twelve appropriately diluted internal standard solutions. Results are presented in Table 3

3.3 Between –Run Accuracy and precision and Within-Run Accuracy and Precision:

Precision and accuracy were also determined from LQC (60 ng/ml), MQC (500 ng/ml) and HQC (750 ng/ml). Five replicates of each concentration were analyzed on the same day to determine the within-run accuracy and precision of the method. To confirm the between-run accuracy and precision five replicates of each concentration on the first day and four replicates of each concentration on second day and third day were analyzed. The Results are presented in Table 4 and Table 5

3.4 Stability study:

The stability of tadalafil in plasma was evaluated with four studies; a short-term stability study, a long-term stability study, a freeze thaw study and stability in processed sample. Plasma blank samples were spiked with tadalafil at concentration of 60 ng/ml (LQC), 500 ng/ml (MQC) and 750 ng/ml (HQC) and each concentration was carried out for six times. Plasma samples were extracted and subsequent HPLC analysis was carried out as described previously.

Short-term stability test was performed at room temperature. Plasma samples spiked with tadalafil were kept at room temperature for 6 h, extracted and then analyzed. The long term stability study was carried out with plasma blank samples spiked with tadalafil, which were stored -20° and they were analyzed periodically 2months against a standard curve prepared on the analysis day. For freeze thaw stability spiked samples were analyzed immediately after preparation and on a daily basis after repeated freeze thaw cycles at -20° on three consecutive days. Finally the stability in the processed sample ready for injection was determined at two level of concentration, 60 ng/ml (LQC), 500 ng/ml (MQC) and 750 ng/ml (HQC). The processed QC samples ready for injection were kept for 6h before HPLC analysis. Results are presented in Table 6 to Table 9

Table 3 Recovery of Internal Standard from Biological Matrix

S. No	Internal Standard
S. No	Un Extracted	Extracted
1	633996	628841
2	626831	623545
3	631586	611667
4	639072	624149
5	617355	630014
6	646425	643251
7	630593	618421
8	623397	637549
9	630466	633456
10	637101	622078
11	634313	619783
12	634613	628825
Mean	632145.67	626798.25
±SD	7505.9463	8725.3473
%CV	1.187	1.392
%Mean Recovery		99.15

TABLE 4: BETWEEN-RUN ACCURACY AND PRECISION OF THE ANALYTICAL METHOD FOR TADALAFIL

Q. C. Samples	LQC	MQC	HQC
Concentration spiked (ng/ml)	60	500	750
Concentration found	58.431	498.098	745.214
S.D.	1.218	1.119	4.935
C.V.%	2.085	0.225	0.662
% Nominal	97.39	99.62	99.36

LQC, MQC, HQC are low quality control, medium quality control, high quality control samples of 60, 500 and 750 ng/ml. Data obtained are average of 6 observations (n= 6) where SD means Standard Deviation, CV% (precision) means Coefficient of Variation calculated as (SD/Mean concentration found) ×100 and % nominal means accuracy which is calculated as (Concentration found/ Concentration spiked) ×100.

TABLE 5: WITHIN-RUN ACCURACY AND PRECISION OF THE ANALYTICAL METHOD FOR TADALAFIL

Q. C. Samples	LQC	MQC	HQC
Concentration spiked (ng/ml)	60	500	750
Concentration found	58.085	494.38	741.549
S.D.	1.52	8.217	6.725
C.V.%	2.617	1.662	0.907
% Nominal	96.81	98.88	98.87

LQC, MQC, HQC are low quality control, medium quality control, high quality control samples of 60, 500 and 750 ng/ml. Data obtained are average of six observations (n= 6) where SD means Standard Deviation, CV% (precision) means coefficient of variation calculated as (SD/Mean concentration found)×100 and% nominal means accuracy which is calculated as (Concentration found/ Concentration spiked)×00.

Table 6 Short term stability of Analyte in Biological Matrix stored at Room Temperature

S.NO	Comparison samples		Stability Samples
S.NO	LQC	HQC	LQC	HQC
Nominal Concentration	60	750	60	750
1	57.116	750.116	55.023	737.124
2	59.215	737.164	57.116	739.891
3	59.087	732.881	56.213	731.564
4	61.103	747.476	57.667	739.547
5	59.022	733.046	57.021	729.821
6	59.114	739.661	57.331	734.246
Mean	59.11	740.057	56.729	735.366
±SD	1.262	7.286	0.965	4.183
%CV	2.135	0.985	1.701	0.569
%Mean Stability			95.97	99.37

Table 7 Long term stability of Analyte in Biological Matrix stored at -20ºC

S.NO	Comparison samples		Stability Samples
S.NO	LQC	HQC	LQC	HQC
Nominal Concentration	60	750	60	750
1	56.456	747.661	54.569	741.963
2	58.665	741.402	56.101	735.442
3	57.133	739.811	56.231	736.205
4	60.153	736.021	58.564	731.559
5	56.144	739.135	55.154	730.587
6	57.704	741.556	56.789	740.987
Mean	57.709	740.931	56.235	736.124
±SD	1.499	3.86	1.392	4.684
%CV	2.598	0.521	2.475	0.636
%Mean Stability			97.45	99.35

Table 8 Stability of Analyte in Biological Matrix after Freeze-Thaw Cycles at -20ºC

S.NO	Comparison samples		Stability Samples
S.NO	LQC	HQC	LQC	HQC
Nominal Concentration	60	750	60	750
1	57.891	749.054	56.012	735.141
2	59.514	747.551	55.787	737.115
3	60.213	750.198	57.563	732.589
4	60.987	751.223	57.021	741.564
5	58.114	749.561	56.001	747.514
6	59.602	751.447	57.214	741.227
Mean	59.387	749.839	56.6	739.192
±SD	1.197	1.453	0.755	5.354
%CV	2.016	0.194	1.334	0.724
%Mean Stability			95.31	98.58

Table 9 Stability of Analyte Following Sample Processing at at -20ºC

S.NO	Comparison samples		Stability Samples
S.NO	LQC	HQC	LQC	HQC
Nominal Concentration	60	750	60	750
1	59.661	750.119	57.844	741.256
2	60.251	749.223	59.117	747.564
3	58.478	748.874	56.055	740.235
4	61.097	750.991	57.884	739.457
5	59.621	747.564	58.161	749.665
6	58.747	750.564	56.225	736.472
Mean	59.643	749.556	57.548	742.442
±SD	0.964	1.26	1.185	5.084
%CV	1.616	0.168	2.059	0.685
%Mean Stability			96.49	99.05

3.5 BLANK MATRIX SPECIFICITY :

Randomly selected blank human EDTA plasma sources were carried through the extraction procedure and chromatographed to determine the extent to which endogenous human EDTA plasma components may contribute to chromatographic interference with the analyte and internal standard .No significant interferences were observed in 8 different lots of Human EDTA samples.

3.6 DILUTION INTEGRITY:

Dilution integrity experiment was carried out at six replicate of two times diluted (1 in 2 dilution ) and four times diluted of approx 1.5 × ULOQ (1 in 4 dilution) samples were prepared and concentrations were calculated including the dilution factor against the freshly prepared calibration curve. Results are presented in Table 10.

Table 10 Dilution Integrity

S.NO	Dilution integrity Spiked Std Conc (2ULOQ) ng/ml (2000 ng/ml)
	DI ½ Sample (1000ng/ml)		DI ¼ Sample (500ng/ml)
	Without DF	With DF	Without DF	With DF
1	996.145	981.561	496.521	491.456
2	997.567	970.801	494.254	487.234
3	1012.054	994.117	490.897	483.221
4	1011.987	996.214	499.214	491.465
5	998.164	978.21	497.255	492.115
6	1054.56	997.802	501.541	496.403
Mean	1011.746	986.451	496.614	490.316
±SD	22.191	11.133	3.734	4.533
%CV	2.193	1.129	0.752	0.925
%Nominal		97.5		98.73

3.7 Evaluation of Lower limit of quantitation and limit of detection:

The lower limits of detection and quantitation were evaluated by comparing the mean peak responses of six extracted samples of the lowest calibration standard(CS1) and serial dilution of CS1(1/2,1/4,1/8,1/16) to the mean peak responses of six replicates of blank matrix sample. The lower limit of quantitation must demonstrate a signal to noise ratio higher than or equal to 10. The lower limit of detection must demonstrate a signal to noise ration higher than or equal to 3.The lower limit of Quantitation is 50.13ng/ml with a signal to noise ratio of 52 .The lowest concentration tested is 3.13ng/ml which exhibits signal to noise ratio of 3.2.Results are presented in Table 11

Table 11 Lower limit of Detection (LLOD) and Lower Limit of Quantitation(LLOQ)

S. NO	CS1	Dilution 1/2	Dilution 1/4	Dilution 1/8	Dilution 1/16
S. NO	Conc (ng/ml)
1	19.787	9.767	4.902	2.317	1.201
2	20.014	9.668	4.991	2.481	1.259
3	19.654	9.901	5.025	2.509	1.227
4	18.747	10.112	4.871	2.467	1.194
5	17.98	9.701	5.112	2.554	1.176
6	19.119	9.884	4.961	2.524	1.221
Mean	19.217	9.839	4.977	2.475	1.213
±SD	0.763	0.164	0.087	0.084	0.029
S/N Ratio	61.987	29.441	13.045	7.054	2.801

RESULTS AND DISCUSSION:

Representative chromatogram of blank plasma and plasma spiked with Tadalafil and internal standard are shown in Figures. 2 and 3, respectively.

ü The retention times of Tadalafil and internal standard are 10.058 and 7.666 min respectively.

No interfering peaks at these times were found in the chromatogram obtained from blank plasma. Good separation and baselines with low background noise were observed.

ü The overall chromatographic run time is 20min.

The linearity of the calibration curve was evaluated by calculating the r²(regression coefficient) value. The standard curves of Tadalafil in human plasma were linear over the ranges of 20 to 750ng/ml and the regression coefficients (r²) were over 0.999 from each standard curve of six separate runs.

The limit of detection defined as three times the base noise was 1.213ng/ml for this analytical method and the lower limit of quantitation defined as ten times the base noise was 4.997ng/ml.

Between and Within -run precision and accuracy of the method was assessed by analyzing the QC samples spiked with known amount of Tadalafil according to the procedure described in the previous section. Results are shown in Table 4 and 5. The accuracy of this bioanalytical method for between - and within -run was from 97.39 to 99.36% and from 96.81 to 98.88%, respectively. The %RSD between and within--run precision ranged from 0.225 to 2.085. and from 0.907 to 2.617%, respectively.

The recoveries (mean) of Tadalafil from plasma were found to be 97.38% at 60ng/ml, 98.94% at 500ng/ ml and 97.45% at 750ng/ml.

The stability of Tadalafil in plasma was determined under various conditions. Short-term stability test performed at room temperature showed that QC samples were stable for 48h (mean recoveries were 95.97%, and 99.37 % at LQC and HQC, respectively).

The long- term stability results indicated that Tadalafil samples were stable for a period of 2 months , with an average recovery of 97.45% and 99.35 % at LQC and HQC, respectively. No significant decrease of Tadalafil concentration in plasma was detected after exposing samples to three freeze/thaw cycles and mean recovery was found to be 95.31% and 98.58% at LQC and HQC, respectively. Finally, the stability in the processed sample ready for injection was also determined. Result showed that three QC samples were stable at least for 6h with loss not higher than 10%.

From the above discussion it is found that the analytical method for analysis of Tadalafil in plasma is simple, rapid and sensitive. The main advantage of this method is the use of soild extraction procedure for sample preparation, which shows less interference from plasma.. It can be used as a reliable assay method in the study of Pharmacokinetics of Tadalafil as well as bioavailability / bioequivalence study.

ACKNOWLEDGEMENTS:

The authors would like to acknowledge the Orchid Healthcare in providing the samples and to conduct the study

S. No	LQC		MQC		HQC
	Un Extracted	Extracted	Un Extracted	Extracted	Un Extracted	Extracted
1	37214.564	36987.664	327912.615	321186.555	446061.621	440995.787
2	38115.610	36951.294	322951.201	320198.334	440668.747	427761.671
3	37921.661	37021.61	327016.634	324159.661	439864.638	431789.603
4	38015.226	37715.361	322054.551	330542.521	451462.456	444389.154
5	37645.229	36351.292	327745.338	320179.627	449668.740	429657.104
Mean	38767.451	36691.564	328464.456	319115.229	454456.989	439181.205
±SD	516.603	451.111	2780.994	4272.609	5907.884	6789.112
%CV	1.361	1.221	0.853	1.325	1.322	1.558
%Mean Recovery		97.38		98.94		97.45

REFERENCES: