Estimation of Lapatinib, an Anticancer drug by HPLC (High-Performance Liquid Chromatography). This research would help treat women suffering from Breast Cancer.

1. DESIGN OF EXPERIMENTS TO DEVELOP AND VALIDATE NEW
ANALYTICAL METHODS FOR QUANTITATIVE ESTIMATION OF
LAPATINIB IN BULK DRUG AND PHARMACEUTICAL DOSAGE
FORM BY RP-HPLC
PRANJALI SINGH
Department of Pharmaceutical Analysis, Karnataka college of Pharmacy, Affiliation to Rajiv
Gandhi University of Health Sciences, Bengaluru, Karnataka, India
Address for correspondence:
Karnataka College of Pharmacy, Bangalore
Email ID: pranjalis7888@gmail.com

2. ABSTRACT: A new simple, specific, rapid, precise, accurate and economical Reverse Phase
High-Performance Liquid Chromatography (RP-HPLC) method for the quantitative estimation
of Lapatinib in bulk drug and pharmaceutical dosage form was developed and validated. The
analysis was carried out on a Water’s XBridge™ ODS C-18 (4.6*250 mm), particle size 5 pm
RP column with UV detection at 340nm, using a mixture of 60 volumes of acetonitrile and 40
volumes of Ammonium acetate buffer(pH 4.8) as the mobile phase at a flow rate of 1.0 ml/min
and the injection volume was 20 pl with a run time of 10 minutes. The method was validated by
using various validation parameters like accuracy, precision, linearity, robustness. The results
obtained were statistically evaluated using the Design of experiments(DOE) statistical tools.
These results showed the method could find practical application as a quality control tool for
analysis of Lapatinib in bulk drug and in its tablet dosage form in quality control laboratories.
Keywords: Lapatinib, RP-HPLC, Design of Experiments, IR, ANOVA, Method Validation
INTRODUCTION: Lapatinib is an anticancer drug mostly prescribed for treating advanced
and metastatic breast cancer but sometimes used in the treatment of lung cancer and other solid
tumours. Lapatinib is human epidermal growth factor receptor type 2 (HER2/ERBB2) and
epidermal growth factor receptor type 1(HER1/EGFR/ERBB1) tyrosine kinases inhibitor. It is
an orally active drug with pKa of 3.8 and molecular weight of 581.059 g/mol. It is pale yellow
in colour. It was approved by USFDA on March 13, 2007 1.
Quantitative analysis seeks to establish the amount of a given element or compound in a sample
2 Methods are developed for new products when no official methods are available. Alternate
methods for existing (non-pharmacopoeial) products are developed to reduce the cost and time
for better precision and ruggedness. Trial runs are conducted, method is optimized and validated
3.
The advantages of HPLC over other chromatographic techniques are improved resolution, faster
separation times and increased accuracy, precision and sensitivity with which the separated
substances may be estimated. The separated components in HPLC can be easily collected and
can be estimated both qualitatively and quantitatively 4.
The typical validation parameters for method validation are as follows: Specificity/Selectivity,
Precision, Accuracy, Linearity, Range, Limit of detection, Limit of quantitation, Robustness,
Ruggedness. The definitions of these parameters are given under Materials and methods in
Method Validation 5,6.
DOE(Design of experiments) is a powerful development tool for method validation to validate
the analytical method for a range of concentrations so that changes in formulation or
concentration will not require additional validation as they are changes within a characterized
design space. The tools like Analysis of variance(ANOVA) or multiple regression/analysis of
covariance (ANCOVA) software package that allows the DOE factors and any uncontrolled
variables to be correctly evaluated. ANCOVA consists of atleast one categorical independent
variable and atleast one interval natured independent variable. However, in this type of a
method only Analysis of variance(ANOVA) and Null hypothesis are suitable. When using
statistics from the method (e.g., CV, mean, standard deviation), rather than raw data, we should
weigh the analysis by the number of replicates or duplicates to assure statistical tests and
confidence intervals are meaningful 7.
During review of literature, It was found that one method was a Normal phase HPLC method 8
(which is quite expensive because of the expensive non polar solvents used and the retention

3. time was also high) and only three RP-HPLC methods were available for Lapatinib 9,10,11 , out
of which only one for the estimation of Lapatinib which was used for the analysis of only
Lapatinib and not its impurities 2 , but an internal standard was used in this method which is too
expensive. The other two were either for the determination of Lapatinib impurities(retention
time of lapatinib was too high in this method) and costly organic solvents 11 or a combination
of RP-HPLC and Mass spectrometry method 10. Few other LC/MS-MS methods were found
12,13,14,15,16,17 . So, it is obligatory to develop a simple, specific, precise, rapid and economical RP-
HPLC method for the quantitative estimation of Lapatinib.
The main aim of this study was to develop a rapid and economical RP-HPLC method for the
estimation of Lapatinib in bulk and its tablet dosage form as no such method is available till
date. The accuracy, precision, recovery results were validated using Design of Experiments
(DOE) statistical tools like ANOVA, Null Hypothesis to double check whether the results
obtained were robust.
FIG. 1: CHEMICAL STRUCTURE OF LAPATINIB 1
MATERIALS AND METHOD: This experiment was conducted in Instrumentation Central
lab, Department of Pharmaceutical Analysis, Karnataka College of Pharmacy, Bengaluru, India
in the month of May-June, 2020.
HPLC Instrumentation and Conditions: The experiment was carried out on Agilent LC
compact 1120 and consisted of UV Detector(Variable wavelength detector), Isocratic pump,
Rheodyne manual injector with 20 pl fixed loop. The chromatographic analysis was performed
on Waters’s X Bridge™ 5pm(particle size) ODS C18(250mm X 4.6mm) at room temperature
using EZ-Chrome elite Software. The flow rate was set at 1 ml/min. Analysis was carried out at
a wavelength of 340 nm. The injection volume was 20 pl. The run time was of 10 mins. The
method used here was based on Reverse phase High Performance Liquid Chromatography(RP-
HPLC).
Materials: Lapatinib reference standard was obtained from Wuhan Vanz Pharm Inc., Wuhan,
China and the tablets which were marketed by Cipla Ltd., Mumbai were obtained from
Pharmeasy App.
Reagents and Chemicals: Acetonitrile and Methanol used were of HPLC grade purchased
from Sigma-Aldrich Chemicals Pvt. Ltd. and from Merck Lifesciences Pvt. Ltd. respectively.
HPLC water was obtained from Milli Q®3 Direct Water Purification system. Glacial acetic
acid used was of HPLC grade obtained from Merck Specialities Pvt. Ltd. All other chemicals
used were of analytical grade.
Preparation of Buffer: 20 mM Ammonium acetate buffer was prepared by dissolving
7.70825g of Ammonium acetate in 500 ml HPLC grade water and adjusting the pH to 4.8 by
using glacial acetic acid. This was filtered through a 0.45 pm membrane filter and degassed by
sonicating for about 10-15 minutes prior to use.
Preparation of Mobile phase: The mobile phase was prepared by mixing 40 volumes of
ammonium acetate buffer and 60 volumes of acetonitrile. The mobile phase was filtered

4. through a 0.45 gm membrane filter and degassed by sonicating for about 10-15 minutes prior to
use.
Preparation of Diluent: It was prepared by mixing 40 volumes of ammonium acetate buffer
and 60 volumes of acetonitrile. The mobile phase was filtered through a 0.45 gm membrane
filter and degassed by sonicating for about 10-15 minutes prior to use.
HPLC Estimation of Lapatinib:
Standard Stock solution: Accurately weighed 50 mg of Lapatinib was transferred into 50 ml
volumetric flask which was dissolved in few ml of diluent and sonicated until the solution was
clear and the volume was made up to the mark with the diluent. This gave the concentration of
1000 gg ml-1 of Lapatinib. This solution was then filtered using 0.45 gm membrane filter paper.
Standard solution: From the above Standard stock solution, further dilutions were made to get
the concentration of 10 gg ml-1 of Lapatinib with the diluent.
Sample Stock solution: 50 mg of Lapatinib drug was weighed for further study(the powder
equivalent to 50 mg of Lapatinib was accurately weighed and transferred into 50 ml volumetric
flask, which was then dissolved in few ml of diluent and sonicated until the solution became
clear and then the volume was made upto the mark with the diluents. The above solution filtered
using 0.45 gm membrane filter paper. This gave us the concentration of 1000 gg ml-1 of
Lapatinib.
Sample solution: From the above Sample stock solution, further dilutions were made to get the
concentration of 10 gg ml-1 of Lapatinib with the diluent.
Procedure: The final dilution of standard and sample solutions(10 gg ml-1) were injected to the
system and analyzed at 340 nm, and both eluted at the retention time of 5.40 ± 0.03 minutes.
Six injections of standard solution were given.
Method Development and Optimisation of HPLC method: For the development and
optimisation of HPLC method, wavelength was set at 340 nm after carrying out the analysis at
various wavelengths 227 nm, 253 nm, 262 nm, 309 nm, 332 nm, 340 nm, 342 nm, 362 nm, out
of which 340 nm gave the maximum, best peak height with no impurities. Also carried out the
UV Spectrophotometric analysis for the detection of kmax and got the same results.
The mobile phase selection was made after carrying out the analysis with different ratios of
different organic solvents with the ammonium acetate buffer. The different combinations and
the different trials made were as follows - Ammonium acetate buffer : Acetonitrile pH 4.8
(50:50), Ammonium acetate buffer : Acetonitrile pH 4.8 (60:40), Ammonium acetate buffer :
Acetonitrile pH 4.8 (40:60), Ammonium acetate buffer : Acetonitrile pH 4.8 (30:70),
Ammonium acetate buffer : Methanol pH 4.8 (50:50), Ammonium acetate buffer : Methanol pH
4.8 (60:40), Ammonium acetate buffer : Methanol pH 4.8 (40:60), Ammonium acetate buffer :
Methanol pH 4.8 (30:70), out of which the best combination(40 volumes of buffer : 60 volumes
of acetonitrile) was selected
For the selection of diluents, various solvents were tried including methanol, acetonitrile alone
and 40 volumes of buffer : 60 volumes of acetonitrile, out of which the last one was selected.
The flow rate was set at 1 ml/min, injection volume was 20 gl, analysis was carried out at room
temperature and the run time was for 10 mins.

5. Method Validation:
Linearity: The linearity of an analytical method is its ability to elicit test results that are
directly proportional to the concentration of the analytes in samples within a given range or
proportional by means of well-defined mathematical transformations. The concentrations e.g.,
10 pg/ml, 20 pg/ml, 30 pg/ml, 40 pg/ml, 50 pg/ml, 60 pg/ml of Lapatinib were prepared and the
Area of Lapatinib measured at 340 nm. Area was plotted graphically as a function of analyte
concentration.
Preparation of standard Stock of Lapatinib: 50 mg of Lapatininb was taken into 50 ml
volumetric flask and dissolved in few ml of diluent and sonicated at room temperature for about
05 minutes until the solution becomes clear. Finally the volume was made up with the diluent.
This solution gave a concentration of 1000 pg/ml of Lapatinib. The above stock-I solution was
diluted serially to get the concentrations e.g., 10 pg/ml, 20 pg/ml, 30 pg/ml, 40 pg/ml, 50 pg/ml,
60 pg/ml of Lapatinib. 20pl of these solutions of Lapatinib were injected and the
chromatograms were recorded.
Precision: Precision of a method is the extent to which the individual test results of multiple
injections of a series of Standards agree. Method reproducibility was demonstrated by
repeatability and intermediate precision measurements of peak area and peak symmetry
parameters of HPLC method for Lapatinib. The repeatability (within-day in duplicates) and
intermediate precision (for 2 days) were carried out at single concentration level for Lapatinib.
Six injections were made and the obtained results within and between the days of trials were in
acceptable range. The precision is expressed as % RSD.
Accuracy: Accuracy was performed in three different levels, each level in triplicate for
Lapatinib using standards at 80%, 100% and 120%. Each sample was analyzed in triplicate for
each level. From the results, % recovery was calculated. The accuracy of estimation for
Lapatinib by using HPLC method using the mobile phase Ammonium acetate : Acetonitrile
(40:60) was determined by adding known amount of the analytes.
Preparation of Lapatinib solution for accuracy: Accurately weighed 80%, 100%, & 120% of
Lapatinib was transferred into three 50 ml volumetric flasks separately along with the
formulation, which is equivalent to amount of 200, 250, 300 mg of Lapatinib respectively.
These were dissolved in 20ml diluent and sonicated for 05 minutes at room temperature & the
final volumes were made up with the diluent. The solutions were filtered using 0.45 pm and
collect the filtrate. From the above filtrates appropriate final concentrations were prepared and
the volumes were made up with the diluent. Areas of Lapatinib were measured at 340 nm. 20 pl
of accuracy solutions containing Lapatinib were injected and the chromatograms were recorded.
Robustness : Robustness of the method was determined to ensure its capacity to remain
unaffected by small deliberate variation in the method parameters like change in flow rate of
mobile phase, change in wavelength.
RESULTS AND DISCUSSION:
Optimization and Method Development: In order to achieve proper optimized HPLC
conditions, several mobile phase combinations, flow rates as well as wavelength were properly
examined. Finally, a mobile phase consisting of Ammonium acetate and Acetonitrile mixed in
the ratio of 40:60 v/v and stationary phase of Waters’s X Bridge™ 5pm(particle size) ODS
C18(250mm X 4.6mm) were monitored, and they were found to be extremely suitable for
analyzing Lapatinib.
The flow rate and detection wavelength were adjusted to 1 ml/min and 340 nm respectively at
room temperature. The summary of optimized method is shown below in Table 1.

6. TABLE 1: OPTIMIZED CHROMATOGRAPHIC CONDITIONS FOR PROPOSED
HPLC METHOD
Parameter Chromatographic conditions
Instrument Agilent LC compact 1120
Column Waters’s X Bridge™ 5 pm ODS C18(250mm X 4.6mm)
Detector UV Detector(Variable wavelength detector
Mobile phase Ammonium acetate buffer : Acetonitrile (40:60 v/v)
Flow rate 1 ml/min
Detection wavelength UV at 340 nm
Run time 10 min
Temperature Room temperature
Injection volume 20 pl
Retention time (Rt) 5.40 minutes
Method Validation: Once the chromatographic and the experimental conditions were
accomplished, the method was validated by the determination of the following parameters:
system suitability, specificity, linearity, precision, accuaracy, robustness, limit of
detection(LOD), limit of quantitation(LOQ) following the ICH guidelines Q@(R1).
System Suitability(SST): System suitability test was appropriate for a representative
chromatogram to validate the various parameters such as retention time, theoretical plates,
capacity factor and tailing factor. The system suitability test results for the proposed method are
shown in TABLE 2. Hence, the system follows suitable criteria.
TABLE 2: SYSTEM SUITABILITY TEST RESULTS FOR LAPATINIB
S.No. Parameters Results
1 Theoretical plates 8041
2 Tailing factor 1.24431
3 Capacity factor 0.00309
4 S/N (6 sigma) 125.358
S/N = Signal/Noise
FIG. 2: CHROMATOGRAM OF LAPATINIB DRUG ASSAY

7. FIG. 3: CHROMATOGRAM OF THE BLANK SOLUTION
Specificity: The method specificity was determined by comparing the chromatograms of
Lapatinib and blank solution. By mixing the most commonly used excipients in the mobile
phase devoid of the drug. Fig. 2 and Fig. 3 show the chromatograms of the drug and the blank
respectively.
FIG. 5: CALIBRATION GRAPH OF LAPATINIB BY RP-HPLC

8. Linearity: The linearity of the method is demonstrated by the concentrations 10 gg/ml, 20
gg/ml, 30 gg/ml, 40 gg/ml, 50 gg/ml, 60 gg/ml of Lapatinib that were prepared and the area of
Lapatinib was measured at 340 nm. Area was plotted graphically as a function of analyte
concentration. A calibration curve is shown in Fig. 5. The corresponding chromatograms for
linearity of Lapatinib are shown in Fig. 6 to Fig. 11. The correlation coefficient was found to
be 0.999. Thus, the HPLC method was found to be linear.
FIG. 6: CHROMATOGRAM OF LINEARITY 10 ^g/ml
FIG. 7: CHROMATOGRAM OF LINEARITY 20 ^g/ml
FIG. 8: CHROMATOGRAM OF LINEARITY 30 ^g/ml

9. FIG. 9: CHROMATOGRAM OF LINEARITY 40 ^g/ml
FIG. 10: CHROMATOGRAM OF LINEARITY 50 ^g/ml
FIG. 11: CHROMATOGRAM OF LINEARITY 60 ^g/ml
Precision: The precision of the method was determined by repeatability (within-day in
duplicates or intra-day) and intermediate precision (for 2 days or inter-day) that were carried out
at single concentration level for Lapatinib. Six repeated injections were made and the obtained
results within and between the days of trials were in acceptable range. The precision is
expressed as % RSD and the results are tabulated in Tables 3 and 4. It was noted that the % RSD
values of precision for intra-day and inter-day were 0.008333 and 0.0084745 respectively. Also
the DOE statistical analysis using DOE tool: One - way ANOVA(Analysis of Variance) was
performed. The results obtained in the ANOVA table (shown in Table 5) shows P-value is

10. not less than F value and also not less than Alpha error value : 0.05 or 5% significance level. F
value is much lesser than F critical value this indicates that we accept Null Hypothesis or we do
not reject Null hypothesis. Sample to sample precision were evaluated using six samples of one
concentration, which was prepared and analyzed on same day. Hence, the assay method is
robust against the results obtained. The corresponding chromatograms are shown in Fig. 12 to
15.
FIG. 12: CHROMATOGRAM OF MORNING PRECISION(INTRA-DAY)
FIG. 13: CHROMATOGRAM OF AFTERNOON PRECISION(INTRA-DAY)
FIG. 14: CHROMATOGRAM OF DAY 1 PRE CISION(INTER-DAY)

11. FIG. 15: CHROMATOGRAM OF DAY 2 PRE CISION(INTER-DAY)
TABLE 3: INTRA-DAY PRECISION
S. No. Injections Morning peak area Afternoon peak area
1 Injection 1 18424607 18427546
2 Injection 2 18426401 18426521
3 Injection 3 18426812 18424812
4 Injection 4 18423417 18423452
5 Injection 5 18423450 18423506
6 Injection 6 18424820 18425423
Average 18424918 18425210
SD 1435.331 1635.455
% RSD 0.007790 0.008876
SD = Standard Deviation, % RSD = % Relative Standard Deviation
TABLE 4: INTER-DAY PRECISION
S. No. Injections Morning peak area Afternoon peak area
1 Injection 1 18423503 18423545
2 Injection 2 18424503 18424512
3 Injection 3 18425418 18421235
4 Injection 4 18426982 18424236
5 Injection 5 18424265 18426523
6 Injection 6 18423562 18425463
Average 18424706 18424252
SD 1317.479 1805.212
% RSD 0.007151 0.009798
SD = Standard Deviation, % RSD = % Relative Standard Deviation
TABLE 5: ANOVA(ANALYSIS OF VARIANCE) ONE WAY FOR PRECISION

12. Source of
variation
SS df MS F P-value F crit
Between days 2925514.167 3 975171.3889 0.400916 0.75386 3.098391212
Within days 48647153.67 20 2432357.683
Total 51572667.83 23
df = Degrees of Freedom, SS = Sum of Squares, MS = Mean of Squares
Accuracy: Accuracy was performed in three different levels, each level in triplicate for
Lapatinib using standards at 80%, 100% and 120%. From the results, % recovery was
calculated. The result obtained in this method was within the limit of 98.0.0% to 102%. The %
Recovery was found to be 99.663%. The % RSD is less than 2.0%. The results obtained in the
ANOVA table(Table 7) indicate that P-value is not lesser than that the standard alpha error =
0.05 and this shows that the results are significant. Since F value is lesser than F critical value,
we accept Null Hypothesis at 5% level of significance. The assay method is robust against the
results obtained. Day to day variability was assessed using single concentration analyzed on two
different days. These results show the accuracy and reproducibility of the assay. Table 6 shows
the accuracy results of Lapatinib. The corresponding chromatograms are shown in Fig. 16 to 18.
FIG. 16: CHROMATOGRAM OF ACCURACY 80%
FIG. 17: CHROMATOGRAM OF ACCURACY 100%

13. FIG. 18: CHROMATOGRAM OF ACCURACY 120%
TABLE 6: ACCURACY RESULTS
S. No. Area of the % level concentrations, mAU
80% 100% 120%
1 18425685 18425623 18425389
2 18424256 18424263 18425483
3 18424633 18425667 18426423
Average 18424858 18425184 18425765
SD 740.5937 798.2013 571.7796
% RSD 0.004019 0.004332 0.003103
SD = Standard Deviation, % RSD = % Relative Standard Deviation
TABLE 7: ANOVA(ANALYSIS OF VARIANCE) ONE WAY FOR ACCURACY
Source of
variation
SS df MS F P-value F crit
Between % conc 1266316 2633158.111 1.255821 0.350279 5.143253
Within % conc 3025073 6504178.778
Total 4291389 8
df = Degrees of Freedom, SS = Sum of Squares, MS = Mean of Squares
Recovery: The result obtained in this method was within the limit of 98.0.0% to 102%. The %
Recovery was found to be 99.663%. The results obtained in the ANOVA table(Table 9) shows
P-value is not less than F value and also not less than Alpha error value : 0.05 or 5%
significance level. F value is much lesser than F critical value this indicates that we accept Null
Hypothesis or we do not reject Null hypothesis and this shows that the results are significant.
Since F value is lesser than F critical value, we accept Null Hypothesis at 5% level of
significance. The assay method is robust against the results obtained. The results are shown in
Table 8.
TABLE 8: RECOVERY RESULTS
S. No. Label claim Concentration added Concentration recovered %Recovery % RSD
1 250 200 199.3222 99.6610.00402
2 250 250 249.1573 99.6630.00433
3 250 300 298.998 99.6660.00310

14. TABLE 9: ANOVA(ANALYSIS OF VARIANCE) ONE WAY FOR RECOVERY
Source of
variation
SS df MS F P-value F crit
Between amt 1.06050 1 1.060501 1.255821 0.350279 5.143253
added & recovered
Within amt 9967.63 42491.90814
added & recovered
Total 9968.69 5
df = Degrees of Freedom, SS = Sum of Squares, MS = Mean of Squares
Limit of Detection(LOD) and Limit of Quantitation(LOQ): It is calculated according to ICH
recommendations where the approach is based on the signal-to-noise ratio. Chromatogram
signals obtained with known low concentrations of analyte were compared with the signals of
blank samples. A signal-to-noise ratio 3:1 and 10:1 was considered for calculating LOD and
LOQ respectively. LOD and LOQ were obtained from the slope and the standard deviation of
the intercept from the calibration curve determined by a linear regression line. The results of
LOD and LOQ are shown in the Table 10. The limit of detection and limit of quantitation were
found to be 0.7804 qg/ml and 2.3648 qg/ml respectively.
TABLE 10: LOD & LOQ RESULTS
Parameter Lapatinib
LOD(qg/ml) 0.7804
LOQ(qg/ml) 2.3648
Robustness: Robustness of the method was determined to ensure its capacity to remain
unaffected by small deliberate variation in the method parameters like change in flow rate of
mobile phase, change in wavelength. The corresponding chromatograms are shown in Fig. 19 to
22. There were no significant changes in the chromatographic pattern when these modifications
were made in the experimental conditions, therefore showing that the method is robust.
FIG. 19: CHROMATOGRAM OF ROBUSTNESS AT 1.1 ml FLOW RATE

15. FIG. 20: CHROMATOGRAM OF ROBUSTNESS AT 0.9 ml FLOW RATE
FIG. 21: CHROMATOGRAM OF ROBUSTNESS AT 338 nm
FIG. 22: CHROMATOGRAM OF ROBUSTNESS AT 342 nm
INFRA-RED SPECTRUM: This test was done for Identification of the pure drug to compare
the drug with the sample(Tablet). The corresponding spectrums of Standard drug and the
sample(tablet) are shown in the Fig. 23 and Fig. 24.

16. FIG. 23: IR SPECTRUM OF LAPATINIB STANDARD
ToisJXC
35CO 3000 2630 2003 1500 1000 500
WavBnumbar cm-'i
FIG. 24: IR SPECTRUM OF LAPATINIB SAMPLE(TABLET)
TRIAL CHROMATOGRAMS:
Mobile phase composition : Amm. Acetate : Acetonitrile (50:50), Wavelength : 262 nm, Base
line is not proper, impurities observed, Peak tailing, Peak height/area reduced, Retention time
high : 9.503 minutes

17. Mobile phase composition : Amm. Acetate : Acetonitrile (50:50), Wavelength : 253 nm,
Baseline is not proper, Lots of Impurities observed, Retention time high : 7.87 minutes
Mobile phase composition : Amm. Acetate : Acetonitrile (50:50), Wavelength : 309 nm,
Impurities observed, Retention time high : 7.893 minutes
Mobile phase composition : Amm. Acetate : Acetonitrile (50:50), Wavelength : 332 nm,
Impurities reduced but still present, Retention time high: 7.933 minutes

18. Mobile phase composition : Amm. Acetate : Acetonitrile (50:50), Wavelength : 227 nm,
No peak observed
Mobile phase composition : Amm. Acetate : Acetonitrile (40:60), Wavelength : 227 nm,
No peak observed
Mobile phase composition : Amm. Acetate : Acetonitrile (50:50), Wavelength : 362 nm,
Impurities reduced, Retention time still high: 7.903 minutes
Mobile phase composition : Amm. Acetate : Acetonitrile (40:60), Wavelength : 340 nm,
Very Less Impurities observed with acetonitrile alone as diluent, Retention time quite less :
4.833 minutes

19. TABLE 11: SUMMARY OF HPLC METHOD
Validation Parameters Results
Mobile phase Flow rate
Detection wavelength Rt
Run time Theoretical plates
Linearity(R* 1 2 3 4 5) Correlation
Coefficient Limit of Detection(LOD)
Limit of Quantitation(LOQ)
Assay(%w/w)
Inter-day Precision(% RSD) Intra-
day Precision(% RSD) Accuracy(%
RSD)
Ammonium acetate : Acetonitrile (40:60)
1 ml/minute
340 nm
5.40 minutes
10 minutes
8041
0.999
0.7804 pg/ml 2.3648
99.663% w/w 0.0084745 0.008333
0.003818
CONCLUSION: The Proposed RP-HPLC method was suitable, precise and accurate for the
determination of Lapatinib without any interference. All the parameters for the drug met the
criteria of ICH guidelines for method validation. DOE(Design of Experiments) Statistical
analysis for these results clearly demonstrates that the method is suitable for the determination
of Lapatinib in bulk drug and tablet dosage form without any interference. The results of the
assay were of the pharmaceutical dosage form of the developed method were highly
reproducible, accurate and reliable. From this study, it is concluded that the novel RP-HPLC
method for the determination of Lapatinib in tablet dosage form is convenient, inexpensive,
simple, specific, accurate, effective and robust for research studies, quality control(QC) and
routine analysis of Lapatinib in bulk & tablet dosage forms.
ACKNOWLEDGEMENT: I acknowledge the support from the Department of Pharmaceutical
Analysis, Cell Biology lab, Karnataka College of Pharmacy, Bengaluru, India. I would like to
thank my lecturers - Professor & Head of Department - Dr. C. Sreedhar, Assistant Professor -
Mr. Srinivas, Karnataka College of Pharmacy, Bengaluru for their immense support and
constant encouragement.
CONFLICT OF INTERESTS: I declare that there are no conflict of interests.
REFERENCES:
1. Lapatinib - Drug bank. drugbank.ca
2. Brereton RG: Introduction to multivariate calibration in analytical chemistry. Analyst J
2000; 125(11): 2125-2154.
3. Berry RI and Nash AR: Pharmaceutical Process Validation and Analytical method
validation. Marcel Dekker Inc 1993; 57: 411-428.
4. Wold S, Martens H and Wold H: The Multivariate Calibration Problem in Chem Solved
by PLS method. Heidelberg Springer 1983: 120-125.
5. ICH Harmonized Tripartite Guideline Validation of Analytical Procedures: Text and
Methodology Q2 (R1). 2005.

20. 6. Ludwig H: Validation and Qualification in Analytical Laboratories. Second Edition
2007.
7. Little TA: Design of Experiments for Analytical Method Development and Validation.
Biopharm International 2014; 27(3): 1-3.
8. Babu H, Krishna BM and Krishnaveni R: Development and validation of HPLC
method for the estimation of lapatinib in bulk drugs and pharmaceutical formulations.
International Journal of Research Reviews in Pharmacy and Applied Sciences 2011;
1(4): 207-214.
9. Kumar KK, Nagoji KEV and Nadh RV: A Validated RP-HPLC Method for the
Estimation of Lapatinib in Tablet Dosage form using Gemcitabine Hydrochloride as an
Internal Standard. Indian Journal of Pharmaceutical Sciences 2012; 74(6): 580-583.
10. Saadat E, Kelishady PD, Ravar F, Kobarfard F and Dorkoosh FA: Development and
Validation of rapid stability-indicating RP-HPLC-DAD method for the Quantification of
Lapatinib and Mass spectrometry Analysis of degraded products. Journal of
Chromatographic Science 2015; 53(6): 932-939.
11. Ivaturi R, Sastry MT and Satyaveni S: Development and Validation of stability
indicating HPLC method for the determination of Lapatinib impurities in bulk and
finished formulations. International Journal of Pharmaceutical Sciences and Research
2017; 8(7): 3081-3091.
12. Bai F, Freeman BB, Fraga CH, Fouladi M and Stewart CF: Determination of lapatinib
in human plasma by liquid chromatography electrospray tandem mass spectrometry.
Journal of Chromatography B Analytical Technologies in the Biomedical and Life
Sciences 2006; 831(1-2): 169-175.
13. Haouala A, Zanolari B, Rochat B, Montemurro M, Zaman K and Duchosal MA:
Therapeutic Drug Monitoring of the new targeted anticancer agents imatinib, nilotinib,
dasatinib, sunitinib, sorafenib and lapatinib by LC tandem mass spectrometry. Journal of
Chromatography B Analytical Technologies in the Biomedical and Life Sciences 2009;
877: 1982-1996.
14. Bouchet S, Chauzit E, Ducint D, Castaing N, Canal-Raffin M and Moore N:
Simultaneous determination of nine tyrosine kinase inhibitors by 96-well solid-phase
extraction and ultra performance LC/MS-MS. Clinica Chimica Acta 2011; 412(11-12):
1060-1067.
15. Couchman L, Birch M, Ireland R, Corrigan A, Wickramasinghe S and Josephs D: An
automated method for the measurement of a range of tyrosine kinase inhibitors in
human plasma or serum using turbulent flow liquid chromatography-tandem mass
spectrometry. Analytical and Bioanalytical Chemistry 2012; 403(6): 1685-1695.
16. Gotze L, Hegele A, Metzelder SK, Renz H and Nockher WA: Development and
clinical application of a LC-MS/MS method for simultaneous determination of various
tyrosine kinase inhibitors in human plasma. Clinical Chimica Acta 2012; 413(1-2): 143-
149.
17. Sandra R, Gillian McM, Martin C and Robert OC: Development and application of
novel analytical methods for molecularly targeted cancer therapeutics. Journal of
Chromatography B 2009; 877: 3982-3990.