Introduction
Erlotinib, N-(3-ethynylphenyl)-6,7-bis (2-methoxyethoxy)-4-quinazolinamine (Figure 1), is a new drug for the treatment of lung cancer.1 Simultaneous quantification of Erlotinib, Gefitinib and Imatinib in human plasma by liquid chromatography tandem mass spectrometry, therapeutic drug monitoring. The mechanism of action involved is an epidermal growth factor receptor inhibitor. It specifically targets the epidermal growth factor receptor (EGFR) tyrosine kinase, which is highly expressed and occasionally mutated in various forms of cancer. It binds in a reversible fashion to the adenosine triphosphate (ATP) binding site of the receptor. For the signal to be transmitted, two members of the EGFR family need to come together to form a homo dimer. These then use the molecule of ATP to autophosphorylate each other, which causes a conformational change in their intracellular structure, exposing a further binding site for binding proteins that cause a signal cascade to the nucleus. By inhibiting the ATP, autophosphorylation is not possible and the signal is stopped.2
Simultaneous quantification of erlotinib, gefitinib and imatinib in human plasma by liquid chromatography, tandem mass spectrometry.3 High performance thin layer chromatographic method for estimation of erlotinib hydrochloride as bulk drug.4 A Simple HPLC –UV method for the simultaneousquantification of gefitinib and erlotinib in human plasma.5 Separation and determination of process- related impurities of Erlotinib using reverse phase HPLC with a photodiode array detector.6 New derivative spectrophotometric methods for the determination of erlotinib hydrochloride (A tyrosine kinase inhibitor).7 Development and validation of a stability indicating HPLC method for the quantification of impurities in erlotinib hydrochloride dosage forms.8 Validation of HPLC method for determination of erlotinib related substance in pharmaceutical dosage form.9 Analytical method development and validation of erlotinib hydrochloride in bulk and pharmaceutical dosage form by RP-HPLC.10 So we planned to develop UV and RP- HPLC method and to validate developed method according to ICH norms. 11, 12, 13
Materials and Methods
Equipment and Apparatus used in HPLC
Analytical balance (Metller Toledo AG-245), Waters LC system equipped with 2695 pump and UV visible detector, Chromatographic data software : EMPOWER, ColumnC18 , 150mm×4.6mm. 5.0µ column, Vacuum filter pump, Ultrasonicator (sonarex), Membrane filter (0.45 and 0.2microns), pH-Meter (Lab India), Model: LC 2010 (Shimadzu).
Chemicals and Reagents used for HPLC
Acetonitrile (HPLC grade), Orthophosphoric acid (HPLC grade), Potassium dihydrogen phosphate.-buffer. (HPLC grade), Water (HPLC grade), Methanol (HPLC grade), Hydrochloric acid (HPLC grade),Sodium hydroxide (HPLC grade), Hydrogen peroxide (HPLC grade). Purchased from Sd fine-chem Ltd., Mumbai, India. High purity deionized water was obtained by double distillation and purification through milli-Q water purification system. The 0.45 μm nylon filter was purchased from advanced micro device Pvt Ltd., Chandigarh.
Equipment and Apparatus used in UV
Analytical balance (metller toledo AG-245), Shimadzu UV visible detector, Model: UV 1700, Ultrasonicator (sonarex), pH meter (lab India).
Chemicals and reagents used for UV
Methanol, Water, Hydrochloric acid.
Buffer preparation
Potassium dihydrogen orthophosphate (2.72 g) was dissolved in 100 mL of HPLC grade water and pH 5.0 was adjusted with orthophosphoric acid. It was filtered through 0.45 μm nylon membrane filter and degassed. It was used as a diluent for the preparation of sample and standard solution.
Method
Selection of wavelength
Accurately weighed quantity of the sample equivalent to 50 mg of Erlotinib was taken in a 50 mL volumetric flask. 25 mL of methanol was added, sonicated for 10 min and filtered through 0.45 μm nylon filter. The above solution was scanned between 200 and 400 nm by UV spectroscopy (Figure 2).
Method development by UV-spectroscopy
The identification method for erlotinib hydrochloride in erlotinib hydrochloride tablets by UV-VISIBLE spectrophotometer test has been checked for specificity with respect to placebo interference and also to confirm the identification of sample by comparison with maxima obtained from reference standard.
Methology
Preparation of Diluent (0.1M Hydrochloric acid)
Dissolve 8.5ml of hydrochloride acid in 1000ml of water and mix.
Preparation of standard solution
Weigh and transfer accurately about 54.8mg of erlotinib hydrochloride into 100ml clean, dry volumetric flask, add 10 ml of methanol and add diluent 0.1M HCL to makeup the volume upto 100ml.
Method validation by UV-spectroscopy
The analytical procedure are validated for the following parameters as per ICH Guidelines and reported.
Validation parameters
System Suitability
To verify the analytical system is working properly and can give accurate precise results, the system suitability parameters are to be set. Calculation shown in (Table 4).
The percentage relative standard deviation should not more than 2.
Linearity
Linearity shall be established by demonstrating that the absorbance obtained is directly proportional to the concentration of the standard solution. The standard solutions are to be prepared at 5 different concentration levels ranging of working concentration and finding the response at each concentration level for assay.
System precision
54.8 mg of erlotinib powder is dissolved in 10ml of methanol. Then makeup the volume up to 100ml. Calculation shown in (Table 5). The percentage relative standard deviation of Erlotinib was found to be less than2.0
Recovery studies
The stock solution was diluted with mobile phase. Further to obtain a concentration ranging from 80%, 100%, 120%. Calculation shown in(Table 6) The percentage recovery of Erlotinib was found to be 78.39 to 117.83.
Chromatographic conditions
The mobile phase containing Acetonitrile: Potassium dihydrogen phosphate (buffer 0.02M) PH 4.5 with methanol in the ratio of 50:50v/v were filtered through 0.45μm nylon filter ,degassed and were pumped from the solvent reservoir in the column. The flow rate of mobile phase was maintained at 1ml/min and detection wavelength was set at 248nm with a run time of 7min. The column and the HPLC system were kept in 30ᵒC temperature.
Preparation of diluents
The mixture of acetonitrile and potassium dihydrogen phosphate (0.02M) in the ratio of 50:50(v/v) used as diluents for the study. The chromatogram of the blank is presented.
Preparation of standard stock solution
About 55 mg of working standard of Erlotinib Hydrochloride was weighed and transferred into a clean and dry 50 ml standard flask, the sample was dissolved in a small volume of mobile phase by sonication for about 10 min and the volume was made up with the mobile phase filtered through 0.45µ filter.then.5 ml of the stock solution was pipetted into a 50 ml standard flask and diluted to mark with mobile phase. The retention time of Erlotinib Hydrochloride was found to be 6.545 min when injected and chromatograms are shown.
Preparation of sample solution
Average weight of the tablet was computed from the weight of 10 tablets. The tablets were powdered. The tablet powder equivalent to 170.9mg of Erlotinib was accurately weighed and transferred into a clean and dry 100 ml standard flask. The sample was dissolved in a small volume of mobile phase by sonication for about 10 min and the volume was made up with the mobile phase. The solution was filtered by using Whatmann filter paper. (Concentration 1000µg/ml).5 ml of the stock solution was pipetted into a 50 ml standard flask and diluted to mark with mobile phase. It was filtered through 0.45µ filter (Concentration-50 mcg/ml). The sample was injected and chromatograms were recorded and shown. The amount of Erlotinib present in each tablet formulation was calculated by comparing the peak area of the test with that of the standard.
Method validation by HPLC
Validation of analytical method is a process to establish that the performance characteristics of the developed method meet the requirement of the intended analytical application.
Validation parameters
System suitability
System suitability of the method was performed by calculating the parameters namely, tailing factor and number of theoretical plates on replicate injection of standard solution.
Tailing factor = 1.00
No. Of. Theoretical plate = 11269.63
Acceptance criteria
Tailing factor should be not more than 2.
No of theoretical plate should be not less than 2000.
Linearity report for erlotinib
Linearity was assessed by performing measurement at several analyte concentrations. A minimum five concentrations were recommended for linearity studies. The linearity of an analytical method is its ability to show test results that is directly proportional to the concentration of analyte in sample with in a given range. The linearity of an analytical method was determined by mathematical treatment of test result obtained by analysis of samples with analyte concentration across claimed range of peak area Vs concentration is plotted and percentage curve fitting is calculated.Calculation shown in (Table 6)
System precision for Erlotinib
The standard solution was injected for five times and measured the area for all five injections in HPLC. The %RSD for the area of five replicate injections was found to be within the specified limits. Calculation shown in (Table 5)
Method precision of Erlotinib
The sample solution was injected for five times and measured the area for all five injections in HPLC. The %RSD for the area of five replicate injections was found to be within the specified limits. Calculation shown in (Table 8)
Recovery studies
The stock solution was diluted with mobile phase. Further to obtain a concentration ranging from 50%, 100%, 150%.Calculation shown in (Table 9).
Robustness
Effect of variation of pH: A study was conducted to determine the effect of variation in +0.2 pH and -0.2 pH. Sample solution was prepared and injected into the HPLC system. The values were measured and shown in(Table 10)
Effect of variation of temperature: A study was conducted to determine the effect of variation in temperature Standard solution was prepared and injected into the HPLC system. The effects of variation in temperature were measured and shown in (Table 11).
The percentage relative standard deviation was found to be not more than 2.
Degradation Studies
5ml of standard stock solution was mixed with 5ml of degradation agents then make up the volume with 50ml of the mobile phase. Actual amount of erlotinib was found to be 98.3. Calculation shown in (Table 12)
Addtion of 3% hydrogen peroxide.
Degradation limit 5 to 10%.
Results and Discussion
Table 0
Table 1
Method validation report by RP-HPL
Table 2
System suitability in UV method
|
Sample name |
%RSD |
|
Blank( diluent) |
------ |
|
Standard solution |
1.23 |
.
Table 3
Linearity in UV method
|
Concentration |
Absorbance |
Mean |
SD |
%RSD |
|
5.48 |
0.475 |
0.4868 |
0.0012 |
0.2465 |
|
8.22 |
0.689 |
0.6401 |
0.0041 |
0.6405 |
|
10.96 |
0.918 |
0.9063 |
0.0102 |
1.1254 |
|
13.7 |
1.142 |
1.1293 |
0.01616 |
1.4309 |
|
16.44 |
1.331 |
1.297 |
0.0231 |
1.7810 |
Table 4
Percentage Recovery data forErlotinib in UV method.
|
Concentration in % |
Absorbance |
%Recovery |
|
80% |
0.899 |
78.39 |
|
100% |
0.840 |
99.18 |
|
120% |
0.998 |
117.83 |
RP-HPLC method. (Table 6, Table 7, Table 8)
Table 5
Linearity Report forErlotinib
|
Parameters |
Results |
|
Slope |
38.56 |
|
Intercept |
78.40 |
|
Correlation co-efficient |
0.999 |
|
Percentage curve fitting |
99.9% |
Table 6
Precision data of the system
|
Injection no |
Peak area |
%Recovery |
|
1 |
5490217 |
98.52 |
|
2 |
5485395 |
98.02 |
|
3 |
5497965 |
98.04 |
|
4 |
5496878 |
98.22 |
|
5 |
5496512 |
98.34 |
|
6 |
5490079 |
98.20 |
Table 7
Method precision ofErlotinib
|
Injection no |
Peak Area |
% Recovery |
|
1 |
5022126 |
98.28 |
|
2 |
5015948 |
98.16 |
|
3 |
5046105 |
98.75 |
|
4 |
5018685 |
98.22 |
|
5 |
5017221 |
98.19 |
|
6 |
5039165 |
98.62 |
Recovery studies.(Table 9, Table 10, Table 11, Table 12)
Table 8
Percentage Recovery data forErlotinib
Table 9
Robustness (change in pH)
|
Changes |
Peak area |
% Recovery |
SD |
Mean |
%RSD |
|
+0.2PH |
5091247 |
99.64 |
|
|
|
|
+0.2PH |
5076577 |
99.35 |
0.03535 |
99.49 |
0.035 |
|
-0.2PH |
5018994 |
98.22 |
|
|
|
|
-0.2PH |
5028789 |
98.41 |
|
|
|
|
-0.2PH |
5053805 |
98.90 |
0.5419 |
98.74 |
0.5488 |
Table 10
Robustness (change in temperature)
Table 11
Degradation studiesAddtion of 3% hydrogen peroxide.
Discussion
The observations and results obtained for HPLC validation parameter including Linearity and Range, Precision (System precision, Method precision) and Robustness within the acceptance criteria. Since the results are within acceptance criteria for all validation parameters, therefore, the method is considered as validated and suitable for intended use.
For routine analytical purpose it is desirable to establish methods capable of analyzing huge number of samples in a short time period with good robustness and precision without any prior separation step. HPLC method generates large amount of quality data, which serve as highly powerful and convenient analytical tool.
Erlotinib was slightly soluble in methanol and very slightly soluble in water. Methanol and Mixture of Buffer was chosen as the mobile phase. The run time of the HPLC procedure was 10 minutes. The method was validated for system suitability, linearity, precision, robustness, LOD and LOQ. The system suitability parameters were within limit, hence it was concluded that the system was suitable to perform the assay. The method shows linearity between the concentration range of 88.32-132.48 µg /ml.
The correlation co-efficient of the Erlotinib was found to be 0.999. The system precision of Erlotinib was found within the limit. The percentage relative standard deviation was found to be 0.0913. The method precision of Erlotinib was found to be within the limit. The relative standard deviation was found to be 0.2546.
The % recovery of Erlotinib was found to be in the range of 50%, 100%, 150%. As there was no interference due to excipients and mobile phase, the method was found to be specific.50% recovery of Erlotinib- percentage relative standard was found to be 0.0507.100% recovery of Erlotinib -percentage relative standard was found to be 0.0057.150% recovery of Erlotinib – percentage relative standard was found to be 0.8813.
The percentage degradation should be in 5 to 10%. The degradation studies of Erlotinib was found to be within the limits. The method was robust as observed from insignificant variation in the results of analysis by changes in pH and temperature separately and analysis being performed .The observation and results obtained for UV validation parameters including system suitability, linearity, precision, recovery within the acceptance criteria.
In system suitability, the percentage relative standard deviation was found to be 1.23.The correlation of coefficient of Erlotinib was found to be 0.998. In precision, percentage relative standard deviation was found to be less than 2. (0.2465 to 1.7810). The acceptance criteria of percentage recovery was 80% to 120%. The percentage recovery of Erlotinib was found to be the range of 78% to 117%.
Summary and Conclusion
A HPLC and UV method for Erlotinib hydrochloride was developed and Validated in tablet dosage form as per ICH guidelines. This condition is applied only for tablet dosage form. Good agreement was seen in the assay results of pharmaceutical formulation by developed method. Hence it can be concluded that the proposed method was a good approach for obtaining reliable results and found to be suitable for the routine analysis of Erlotinib in the pharmaceutical formulation.
Acknowledgement
The authors are thankful to Nebulae Hi-tech Laboratories, porur,Chennai, for providing the gift sample of erlotinib hydrochloride drug and providing the facilities necessary to carry out research work.