Stability Indicating Fast LC Method for Determination of Tadalafil and Its Intermediates in Bulk and Pharmaceutical Formulation
GVH Raju*, S Ganapathy, DG Sankar and PY Naidu
*Corresponding Author E-mail: gadepalli_raj@yahoo.com
ABSTRACT:
Fast LC method has been developed and subsequently validated for the determination of Tadalafil and its intermediates in bulk and pharmaceutical formulation. Separation was achieved in Gradient mode using Peerless HT gold, C18, 50 x 4.6 mm, 1.8”m column with mobile phase A containing Potassium Dihydrogen Phosphate buffer (pH adjusted to 3.0±0.05 with Orthophosphoric acid) and mobile phase B containing Methanol 100% at different time intervals as eluent at a flow rate 0.8ml/min. UV detection was performed at 220nm.The method is simple, rapid, selective and stability indicating .The described method is linear over a range of 12.5748”g/mL to 76.4548”g/mL.The method precision for the determination of assay was below 2.0% RSD .The Percentage recoveries of Active Pharmaceutical Ingredient(API) from dosage forms ranged from 101.0 to 102.1 for all available strengths of Tadalafil in market. LOD and LOQ of all related impurities of Tadalafil was established and ranged from 0.006”g/ml - 0.011”g/ml for LOD and 0.018”g/ml - 0.033”g/ml for LOQ .The method is useful in the quality control of bulk manufacturing and also in pharmaceutical formulations.
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 ethanol1-6. The chemical structure is given below
It is not official in any pharmacopoeia and few liquid chromatography procedures have been reported for the determination of Tadalafil in Bulk and Pharmaceutical dosage forms. However there are no publications concerning the analysis of Tadalafil in presence of its intermediates in bulk and Pharmaceutical dosage forms7-10. So it is felt necessary to develop a Fast LC method which would serve as a rapid and reliable method for the determination of Tadalafil in respective related impurities [Fig 1] in bulk and pharmaceutical dosage forms. In the proposed method, related impurities were well separated and eluted before 20min .Finally the method was thoroughly validated for the assay and it is related impurities.
EXPERIMENTAL:11-14
Instrumentation: Agilent 1100 series equipped with binary pump and DAD detector was used .The output signal was monitored and integrated using waters Empower 2 software
Solutions:
Mobile Phase A:
Preparation of pH 3.0 Buffer solution:
Weigh and dissolve 2.72 g of potassium dihydrogen orthophosphate in 1000 mL of milli-Q water in a suitable container, adjust the pH of the solution to 3.0 ± 0.05 with dilute ortho phosphoric acid.
Mobile phase B:
Methanol 100%
Fig-1 TADALAFIL RELATED IMPURITIES
Impurity-1
Impurity-2
Impurity-3
Impurity-4
Impurity-5
Impurity-6
Impurity-7
Impurity-8
Diluent:
Preparation of pH 2.9 buffer:
1. Add about 1.6mL of Orthophosphoric acid in 1000 mL of milli Q water and mix. Adjust the pH of the solution to 2.9 ± 0.05 with Triethylamine.
2. Mix pH 2.9 buffer and Acetonitrile in the Ratio 40:60(v/v)
Preparation of Standard solution: About 50mg of Tadalafil working standard was accurately weighed and transferred in to a 100mL volumetric flask and dissolved in 70ml of diluent and diluted to volume with diluent and mixed well. Solution was filtered through 0.45”m nylon membrane filter prior to use.
Preparation of Test Solution: Twenty tablets were taken weighed and powdered .Sample equivalent to about 50mg of Tadalafil was accurately weighed and transferred in to a 100mL volumetric flask 70ml of diluent was added and sonicated for 20min and diluted to volume with diluent and mixed well. Filtered through 0.45”m nylon membrane filter prior to use.
Preparation of Degradation samples for Specificity Study:
For Acid degradation Tadalafil sample was refluxed with 1N HCl at 70șC for 1hr and then neutralized by adjusting pH to 7.0 with 1N NaOH .The Solution was further diluted to required concentration with diluent.
For basic degradation Tadalafil sample was refluxed on water bath at 70șC with 1N NaOH for 30min and then neutralized by adjusting pH to 7.0 with 2N HCl .The Solution was further diluted to required concentration with diluent.
For Oxidative degradation Tadalafil sample was refluxed 1%H2O2 by heating on water bath at 40șC for
1hour .The Solution was further diluted to required concentration with diluent.
For Photolightic Stress the samples were exposed to UV at 254nm for 47 hrs and visible light for 166 hrs meeting the specification of ICH i.e UV(200watt/m2) and Visible(1.2million Lux hours).
For Thermal Degradation Samples were Exposed to Temperature at 150șC for 10hrs 30min.
The above stressed samples i.e Photolightic and Thermal stress samples solutions were prepared to required concentration with diluent.
Chromatographic Conditions:
A Peerless HT C18 (50 x 4.6mm;5”m packing) column was used for analysis at column temperature 35 ș C. The mobile phases was pumped through the column as per the gradient composition given below at a flow rate of 0.8mL/min.
|
Time in minutes |
% of Mobilephase-A |
% of Mobilephase-B |
|
0.0 |
70.0 |
30.0 |
|
15.0 |
40.0 |
60.0 |
|
20.0 |
40.0 |
60.0 |
|
21.0 |
70.0 |
30.0 |
|
25.0 |
70.0 |
30.0 |
The Sample injection Volume was 5”L.The photodiode array detector was set to a wavelength of 220nm for the detection
RESULTS AND DISCUSSION:
Method development:15
3.1.1 Separation of Known degradant impurities:
To develop a suitable and robust Fast LC method for the determination of Tadalafil different mobile phases and columns were employed to achieve the best separation and resolution. The method development was started with a Peerless basic C18 250 x 4.6mm ,5” column using a mobile phase of pH 2.9 buffer: Acetonitrile in the ratio 62:38.In the above condition elution was late for Tadalafil and its related impurities . Early elution with little separation was observed with mobile phase consisting of pH 3.0 buffer: Acetonitrile in the ratio 30:70 using column Peerless HT C18 50 x 4.6mm, 1.8”. Finally the mobile phase consisting of pH 3.0 buffer [Mobile phase A]: Methanol 100%[Mobile phase B]in different ratios pumped through the column was found to be appropriate ,allowing good separation and symmetrical peak at a flow rate of 0.8mL/min using Peerless HT C18 50 x 4.6mm ,1.8”,. The Chromatogram of Tadalafil sample spiked with the related compounds using the proposed method is shown in Fig.2.In the proposed method the resolution is more than 1.5 between the Tadalafil and Impurity-6 .System suitability results of the method are presented in Table 1.Tadalafil and its related compounds show significant UV absorbance at Wavelength 220nm .Hence this wavelength has been chosen for detection in the analysis of Tadalafil.
TABLE-1 SYSTEM SUITABILITY REPORT
|
Compound |
Tailing Factor a |
Resolution |
% RSDa |
Theoretical Plates |
|
Tadalafil |
1.2 |
---- |
0.1 |
36912 |
|
Impurity 6 |
1.0 |
1.7 |
0.5 |
33414 |
a Number of samples analyzed are six
Column Selection:
Based on the retention time and separation of the compounds Peerless HT C18 (50 x 4.6mm, 1.8”) column was selected as suitable column for the analysis of Tadalafil.
Method Validation: 16-17
The developed Fast LC method of Tadalafil is extensively validated for assay and its Related impurities using the following parameters.
Specificity:
Placebo Interference:
A study to establish the interference of placebo was conducted. Assay was performed on Placebo in triplicate equivalent to about the weight of placebo in portion of test preparation as per test method. Chromatograms of placebo solutions showed no peaks at the retention time of Tadalafil peak. This indicates that the excipients used in the formulation do not interfere in estimation of Tadalafil in Tadalafil tablets.
Interference from degradation products:
A study was conducted to demonstrate the effective separation of degradants from Tadalafil peak. Separate portions of Drug product, Drug substance and Placebo were exposed to following stress conditions to induce degradation.Stressed samples were injected into the RRLC system with diode array detector by following test method conditions. All degradant peaks were resolved from Tadalafil peak in the chromatograms of all samples. The chromatograms of the stressed samples were evaluated for peak purity of Tadalafil using Empower software. In all forced degradation samples, Tadalafil peak Purity angle is less than purity threshold. All degradant peaks were well separated from the Tadalafil Peak .The results are given under Table-2. The method can be used for determining the stability of Tadalafil as bulk and pharmaceutical formulations.
TABLE -2 TABLE RESULTS FOR SPECIFICITY
[INTERFERENCE FROM DEGRADATION PRODUCTS]
|
Stress Condition |
% Degradation |
Purity Angle |
Purity Threshold |
Purity Flag |
|
Acid Stress |
5.52 |
0.242 |
2.640 |
No |
|
Base Stress |
22.29 |
1.343 |
7.834 |
No |
|
Oxidation Stress |
Nil |
0.946 |
3.016 |
No |
|
Photolightic Stress |
Nil |
1.113 |
3.202 |
No |
|
Thermal Stress |
Nil |
1.659 |
4.067 |
No |
Limit of detection and limit of quantitation:
A study to establish the Limit of detection and limit of quantitation of Tadalafil related impurities were conducted.
Limit of detection and limit of quantitation were established based on signal to noise ratio. A series of solutions having Tadalafil related impurities were injected. Limit of detection for related impurities were established by identifying the concentration which gives signal to noise ratio about 3. Limit of quantitation was established by identifying the concentration which gives signal to noise ratio about 10.
Fig -2 TYPICAL CHROMATOGRAM OF TADALAFIL AND ITS RELATED IMPURITIES
Precision of Tadalafil related impurities at about Limit of Quantitation were conducted. Six test preparations of Tadalafil Tablets having related impurities at about Limit of quantitation was prepared and injected into the RRLC system. The %RSD at LOQ level was calculated for all known impurities and found to be less than 5.0%.
Accuracy of Tadalafil related impurities at about Limit of Quantitation was conducted. Test solutions spiked with related impurities at about Limit of Quantitation was prepared in triplicate and injected into RRLC system and calculated the % recovery. The mean recovery of Tadalafil related impurities at about Limit of Quantitation was ranged from 96.5 to 103.8% .The results are given under Table-3
TABLE-3 TABLE RESULTS FOR LOD AND LOQ OF TADALAFIL IMPURITIES
|
IMPURITY |
Limit of detection (LOD) |
Limit of Quantification (LOQ) |
% RSD* |
% Recovery*
|
|
Conc. ”g/mL |
Conc. ”g/mL |
|||
|
Impurity 1 |
0.008 |
0.026 |
4.7 |
101.5 |
|
Impurity 2 |
0.006 |
0.018 |
3.5 |
97.4 |
|
Impurity 3 |
0.01 |
0.029 |
4.2 |
98.2 |
|
Impurity 4 |
0.007 |
0.025 |
4.4 |
103.8 |
|
Impurity 5 |
0.01 |
0.029 |
3.4 |
99.7 |
|
Impurity 6 |
0.011 |
0.033 |
3.8 |
96.5 |
|
Impurity 7 |
0.008 |
0.021 |
4.6 |
102.0 |
|
Impurity 8 |
0.0098 |
0.031 |
4.1 |
102.9 |
a Number of samples analyzed are six
Linearity of Detector Response:
a) Active Ingredient:
Linearity of detector response for Tadalafil was established by plotting a graph to concentration versus average area and determining the correlation coefficient. A series of solutions of Tadalafil standard were prepared in the concentration range of about 12.5748”g/mL to 76.4548”g/mL .A graph was plotted to concentration in ”g/mL on X- axis versus response on Y-axis. The detector response was found to be linear with a correlation coefficient of 0.999. Linearity graph is shown in Fig-3.
FIG-3 LINEARITY OF DETECTOR RESPONSE GRAPH OF TADALAFIL
b) Related impurities:
Linearity of detector response of all known Tadalafil Related impurities is established by plotting a graph to concentration versus area of Tadalafil related impurities and determining the correlation coefficient. A series of solutions of Tadalafil related impurities in the concentration ranging from Limit of Quantitation level to about 150% of target concentration level of Tadalafil known impurities were prepared and injected into the RRLC system.
The detector response was found to be linear from Limit of quantitation to 150% of target concentration level of Tadalafil known Impurities. Linearity graph is Shown in Fig-4
Precision of test Method:
a) Active Ingredient:
The precision of test method for Active Substance was conducted by assaying six samples of Tadalafil tablets. The Average % assay of Tadalafil in Tadalafil tablets was found to be 101.0%, 101.3% and 102.1% respectively for 5mg, 10mg and 20mg tablets and RSD was found to be 0.6%, 0.5% and 0.3% respectively. The results were given in Table-4.
b) Related impurities:
The precision of test method of all known impurities of Tadalafil was evaluated by spiking all known impurities at target concentration level on Tablets. The Relative standard deviations of all known impurities were calculated and found to be less than 1.0%. The results were given in Table-4.
FIG-4 LINEARITY OF DETECTOR RESPONSE GRAPH OF TADALAFIL RELATED IMPURITIES
|
Test No |
% Assay |
||
|
5mg |
10mg |
20mg |
|
|
01 |
100.6 |
101.0 |
101.8 |
|
02 |
100.4 |
101.4 |
101.8 |
|
03 |
101.6 |
100.8 |
102.4 |
|
04 |
101.0 |
100.9 |
102.4 |
|
05 |
100.8 |
101.7 |
101.9 |
|
06 |
101.8 |
102.0 |
102.4 |
Average |
101.0 |
101.3 |
102.1 |
|
% RSD |
0.6 |
0.5 |
0.3 |
|
S. No |
%Impurity |
|||||||
|
Imp 1 |
Imp 2 |
Imp 3 |
Imp 4 |
Imp 5 |
Imp 6 |
Imp7 |
Imp8 |
|
|
1 |
0.5057 |
0.5041 |
0.4453 |
0.4663 |
0.4351 |
0.4948 |
0.5728 |
0.4867 |
|
2 |
0.5059 |
0.5048 |
0.4446 |
0.4658 |
0.4344 |
0.4956 |
0.5716 |
0.4882 |
|
3 |
0.5048 |
0.5042 |
0.4426 |
0.4648 |
0.4343 |
0.4944 |
0.5693 |
0.4859 |
|
4 |
0.5023 |
0.4987 |
0.4444 |
0.4623 |
0.4325 |
0.4926 |
0.5679 |
0.4855 |
|
5 |
0.5056 |
0.5039 |
0.4461 |
0.4663 |
0.4356 |
0.4969 |
0.5724 |
0.4875 |
|
6 |
0.5047 |
0.5022 |
0.4455 |
0.4629 |
0.4385 |
0.4975 |
0.5710 |
0.4868 |
|
Average |
0.5048 |
0.5030 |
0.4448 |
0.4647 |
0.4351 |
0.4953 |
0.5708 |
0.4868 |
|
%RSD |
0.3 |
0.5 |
0.3 |
0.4 |
0.5 |
0.4 |
0.3 |
0.2 |
TABLE-5 ACCURACY IN THE ASSAY DETERMINATION OF TADALAFIL
|
Spike level |
Average mg of Tadalafil added |
Average mg of Tadalafil Recovered |
Average % Recovery |
|
2.5% |
1.27 |
1.28 |
100.8 |
|
10% |
5.04 |
5.08 |
100.8 |
|
25% |
12.39 |
12.34 |
99.6 |
|
50% |
24.79 |
24.75 |
99.8 |
|
75% |
37.21 |
37.88 |
101.8 |
|
100% |
49.56 |
49.96 |
100.8 |
|
150% |
74.36 |
74.78 |
100.6 |
|
SLOPE |
1.0077 |
||
|
INTERCEPT |
-0.0179 |
||
|
CORRELATION COEFFCIENT |
0.999 |
||
|
|
|
||
a Number of samples analyzed at each spike level are three
Accuracy:
a) Active Ingredient:
A study of recovery of Tadalafil from spiked placebo was conducted at five different Spike levels i.e. 2.5%, 10%, 25%, 50%, 75%, 100%, and 150%.
Samples were prepared by mixing placebo with Tadalafil raw material equivalent to about of the target initial Concentration of Tadalafil. Sample solutions were prepared in triplicate for each spike level and assayed as per proposed method. The Slope, intercept, % recovery and Correlation Coefficient were calculated and given in Table-5.The mean Recoveries of Tadalafil from spiked were found to be in the range of 99.6-101.8%.
TABLE-6 ACCURACY IN THE DETERMINATION OF TADALAFIL RELATED IMPURITIES
|
Spike level |
IMPURITY 1 |
IMPURITY 2 |
IMPURITY 3 |
||||||
|
”g/ml added |
”g/ml found |
Avg % Recovery |
”g/ml added |
”g/ml found |
Avg % Recovery |
”g/ml added |
”g/ml found |
Avg % Recovery |
|
|
50 % |
1.2891 |
1.3625 |
105.7 |
1.1852 |
1.1556 |
97.5 |
1.2339 |
1.2191 |
98.8 |
|
75 % |
1.9336 |
2.0398 |
105.5 |
1.7779 |
1.7068 |
96.0 |
1.8509 |
1.7991 |
97.2 |
|
100 % |
2.5782 |
2.7071 |
105.0 |
2.3705 |
2.3302 |
98.3 |
2.4678 |
2.3765 |
96.3 |
|
150% |
3.8672 |
4.1247 |
106.7 |
3.5557 |
3.4704 |
97.6 |
3.7018 |
3.5389 |
95.6 |
|
Spike level |
IMPURITY 4 |
IMPURITY 5 |
IMPURITY 6 |
||||||
|
”g/ml added |
”g/ml found |
Avg % Recovery |
”g/ml added |
”g/ml found |
Avg % Recovery |
”g/ml added |
”g/ml found |
Avg % Recovery |
|
|
50 % |
1.1432 |
1.0895 |
95.3 |
1.0722 |
1.0240 |
95.5 |
1.0256 |
1.0108 |
98.6 |
|
75 % |
1.7148 |
1.6959 |
98.9 |
1.6083 |
1.5408 |
95.8 |
1.5385 |
1.4980 |
97.4 |
|
100 % |
2.2864 |
2.2338 |
97.7 |
2.1444 |
2.0415 |
95.2 |
2.0513 |
1.9968 |
97.3 |
|
150% |
3.4295 |
3.2820 |
95.7 |
3.2166 |
3.0783 |
95.7 |
3.0769 |
3.0042 |
97.6 |
|
Spike level |
IMPURITY 7 |
IMPURITY 8 |
|
||||||
|
”g/ml added |
”g/ml found |
Avg % Recovery |
”g/ml added |
”g/ml found |
Avg % Recovery |
|
|||
|
50 % |
1.1986 |
1.2698 |
105.9 |
1.4160 |
1.4457 |
102.1 |
|
||
|
75 % |
1.7978 |
1.9052 |
106.0 |
2.1240 |
2.1962 |
103.4 |
|
||
|
100 % |
2.3971 |
2.5583 |
106.7 |
2.8320 |
2.9396 |
103.8 |
|
||
|
150% |
3.5957 |
3.8793 |
107.9 |
4.2481 |
4.3416 |
102.2 |
|
||
a Number of samples analyzed at each spike level are three
TABLE-7 STABILITY DATA OF TADALAFIL IN STANDARD AND TEST SOLUTIONS
|
BENCH TOP STABILITY |
||||||||
|
Time in Days |
% Assay of Standard preparation |
Difference from Initial |
% Assay of test preparation |
Difference from Initial |
||||
|
Test - 1 |
Test - 2 |
Test - 1 |
Test 2 |
|||||
|
Initial |
99.8* |
NA |
100.6 |
100.4 |
NA |
NA |
||
|
1 |
100.3 |
0.5 |
101.4 |
101.5 |
0.8 |
1.1 |
||
|
2 |
99.7 |
0.1 |
100.3 |
100.5 |
0.3 |
0.1 |
||
|
REFRIGERATOR STABILITY |
||||||||
|
Time in Days |
% Assay of Standard preparation |
Difference from Initial |
% Assay of test preparation |
Difference from Initial |
||||
|
Test - 1 |
Test - 2 |
Test - 1 |
Test 2 |
|||||
|
Initial |
99.8* |
NA |
100.6 |
100.4 |
NA |
NA |
||
|
1 |
100.0 |
0.2 |
102.3 |
102.1 |
1.7 |
1.7 |
||
|
2 |
100.5 |
0.7 |
101.6 |
101.2 |
1.0 |
0.8 |
||
* Potency of Tadalafil on as is basis
b) Related impurities:
A study of recovery of Tadalafil related impurities in spiked samples of Tadalafil test preparation was conducted. Samples were prepared in triplicate by spiking of all known impurities in test preparation at 50%, 75%, 100% and 150% of the target concentration level of Tadalafil known Impurities.
The average %recovery for Tadalafil Related Impurities was Calculated and given in Table-6. Tadalafil related impurities from spiked were found to be in the range of 95.2-107.9%
Ruggedness:
A study to establish the stability of Tadalafil in standard was conducted on bench top and Refrigerator at Initial, 1 day and 2 days. The assay of Tadalafil in standard solution was estimated against freshly prepared standard each time. The difference in % assay of Standard from initial to 1 day and 2 days was calculated and given in Table-7. From the above study, it was established that the standard preparation was stable for a period of 2days on bench top and Refrigerator.
A study to establish the stability of Tadalafil in Test Solution was conducted on bench top and Refrigerator at
Initial, 1 day and 2 days. The assay, of Tadalafil in test solutions was estimated against freshly prepared standard each time. The difference in % assay of test from initial to 1 day and 2 days was calculated and given in Table-7. From the above study, it was established that the Test Solution was stable for a period of 2days on bench top and Refrigerator.
Robustness:
A study to establish the effect of variation in mobile phase composition, Flow, Temperature and pH of buffer in mobile phase was conducted. Standard and test solutions spiked with known related impurities of Tadalafil prepared as per proposed method were injected into RRLC system. The System suitability parameters, % Assay and RRT of all individual known impurities were evaluated. From the above study the proposed method was found to be Robust.
ACKNOWLEDGEMENTS:
The authors wish to thank the Orchid Healthcare for providing the samples of Tadalafil.
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Received on 04.02.2010 Modified on 09.03.2010
Accepted on 17.04.2010 © AJRC All right reserved
Asian J. Research Chem. 3(2): April- June 2010; Page 447-453