INTRODUCTION:

High Performance Liquid Chromatography (HPLC)¹

A ‘Regulatory Analytical Procedure’²is used to evaluate a defined characteristic of the drug substance or drug product. An ‘alternative analytical procedure’ is proposed by the applicant for use other than regulatory analytical procedure.¹A tyrosine kinase inhibitor called lapatinib prevents breast cancer stem cells from developing into tumours. American women are more likely to develop breast cancer than any other type of cancer, making it the most prevalent malignancy.²Lapatinib inhibits both the EGFR and erbB-2 receptors. These receptors are a component of the Type I receptor kinase, which is involved in growth factor-induced cell proliferation, differentiation, and anti-apoptotic signalling.

Uncontrolled cell growth is prevented in cells with inactive Type I receptors; an inhibitor of erbB kinase 2 is thought to be helpful in controlling uncontrolled cell development by inducing stasis or cell death (QPS).³A number of lapatinib-resistant, HER2-positive, estrogen-positive breast cancer clones produced from lapatinib-sensitive BT474 cells have been identified using long-term lapatinib exposure. This dual tyrosine kinase inhibitor is used in addition to Herceptin 3 as a follow-up medication.⁴ This stops receptor phosphorylation and activation as well as the subsequent downstream signalling cascades.⁵Tykerb and Herduo were the brand names used in its advertising.⁶ Lapatinib is a synthetic chemical that prevents a natural product made from a natural substrate from being produced. Lapatinib has been approved by GSK as a first-line treatment for patients with triple positive (hormone receptor, EGFR, and HER2) breast cancer based on recent research.⁷Lapatinib, a very hydrophobic chemical, is made from the quinazoline core present in other tyrosin kinase inhibitors. The rate of dissolution in water is 0.007 mg/mL.⁸N-f3-chloro4-[(3-flurobanzyl)oxyl] is its chemical formula. phenylg-6-[5-(f[2-(methylsulfonyl)ethyl] aminogmethy] phenylg-6-[5-(f[2-(methylsulfonyl)ethyl] aminogmethy] phenylg-6-[5-(f[2-(methylsulfonyl)ethyl] aminogmethy] phenylg-6-[5-(f[2-(methylsulfonyl)ethyl] aminogmethy] -2-fury] -4-quinazolinamine.⁹ In the indicated techniques, the impurity peak shapes were broad with weak peak morphologies, and there was no apparent resolution between the impurities.¹⁰ The purpose of this research is to develop and test an HPLC approach for detecting stability.

Figure 1: Chemical Structure of Lapatinib

METHODS:

Instrumentation:

Shimadzu LC-20 AD RP-HPLC equipment with UV detector SPD-20A column Hypersil BDS C18 (250mm x 4.6 mm, particle size: 5µm) and LC Solution software, along with a pump and manual injector, were used to construct the method. ELICO L 1127 pH metre, BT ultra sonicator, Shimadzu (AUW220D) digital weighing balance, and Millipore vacuum filter pump (XI 5522050). The experiment used a 0.45µm Nylon filter from Merck Millipore.

REAGENTS AND MATERIALS:

A free sample of the pure drug Lapatinib was supplied by Hetero Drugs limited in Hyderabad, India. We utilised acetonitrile, methanol, and HPLC grade water. Trifluoroacetic acid of the AR grade was used.

Conditions for liquid chromatography:

Column Hypersil BDS C18 (250mm x 4.6mm, particle size: 5µm), 1.1mL/min flow rate, 20µL injection volume, 50ppm sample concentration, ambient temperature, 262nm wavelength, and 10min run time. Mobile phase water, methanol, and trifluroacetic acid (30:70:0.1) v/v

Mobile Phase Preparation:

prepared water, methanol, and trifluoroacetic acid (30:70:0.1) v/v, mixed, and sonicated. They were employed as a mobile phase.

Preparation of standard stock solutions:

A working standard stock solution of lapatinib was made by dissolving 50mg of standard in a 100ml volumetric flask, adding 30ml of diluent, and sonicating for 10 min. Before dilution to volume with diluent to produce a concentration of 500ppm, allow the solution to cool to room temperature.

Standard Diluted Preparation:

2mL of the standard stock solution were correctly mixed and diluted to a volume of 20mL using diluent. The solution's concentration was 50ppm.

Sample Solution Preparation:

Weighed 10 tablets, determined their average weight, then ground them into a fine powder with a mortar and pestle. 10mg of Lapatinib in crushed form should be sonicated in a 100ml volumetric flask for 10 min with periodic shaking in an ultrasonic bath with a temperature no higher than 20°C. Let the flask cool to room temperature before diluting with diluent to the necessary volume. Use a nylon membrane filter with a 0.45µm diameter to filter the fluid.

Validation of the Method:

System suitability, linearity, accuracy, precision, sensitivity (LOQ and LOD), robustness, and a forced degradation study were among the validation factors used in the method's evaluation in accordance with ICH guidelines.^11,12,13

1. System suitability:

It was established with three injections of a 10ppm Lapatinib solution. A 10ppm Lapatinib working standard solution was created by pipetting 1 ml of the stock solution into a 10ml volumetric flask and diluting it to the proper concentration with mobile phase. When establishing the application of the system, the study takes into account factors like theoretical plates, peak area, retention time, and asymmetry factor.¹⁴

2. Linearity:

With concentrations ranging from LOQ to 150 percent of the specification threshold, linearity of the detector response was shown for all known contaminants, including Lapatinib (0.15%). The test procedure recommended was used to evaluate the samples. The correlation coefficient and Y-intercept at 100% response were obtained from a linearity graph by plotting the impurity responses (Y-axis) vs the actual concentration in ppm (X-axis).¹⁵

3. Precision:

According to ICH Q2 (R1), the degree of agreement for a measurement process carried out on repeated samples is the definition of precision of an analytical method. The percent RSD of each individual impurity for precision and intermediate precision was computed and reported in accordance with the recommendations, and method precision and intermediate precision were determined on a homogenous sample.^16,17

10mg of lapatinib were used to create the sample solutions. Lapatinib was tested three times on the same day at three different concentrations (10µg/ml, 30 µg/ml, and 60µg/l), and the %R.S.D. was calculated. It is frequently described using the standard deviation or relative standard deviation.

Preparation of standard stock solution:

In a 100ml volumetric flask, the volume of the mentioned concentration was made up with diluent after 50mg working standards were weighed and placed there. This solution was diluted up to 20mL with diluent.

4. Accuracy:

Recovery studies, which involved mixing standard drug solution with reanalyzed sample solution at three major concentrations: 80%, 100%, and 120% spiked levels, were used to evaluate the correctness of the most recent research. The correctness of an analytical approach is determined by how closely the real value and the experimental value coincide. Five levels, ranging from LOQ to 150% of the impurity's specification level in relation to the test concentration level, were used to evaluate the accuracy of the three contaminants. By comparing the impurity level of the spiked sample to that of the control sample at each level, the percent recovery was computed.

Standard Drug Solution Preparation:

A precisely weighed quantity of 50mg of lapatinib was dissolved in diluent and the volume was increased to 50 ml in order to create stock solutions containing 500 µg/ml.

Standard solution in work:

To diluted the standard stock solution in a 20ml volumetric flask, pipette out 1.6ml for 80%, 2ml for 100%, and 2.4ml for 120% of the solution.

5. LOD and LOQ:

The linearity technique was used to calculate the detection limit (LOD) and quantification limit for each of the three components (LOQ). Five distinct impurity injection levels, ranging from 0.1 to 10 parts per million (ppm), were made. The lowest concentration of each impurity that may be measured and detected was computed based on the impurity reaction and STEYX value.

6. Robustness:

By altering the experimental conditions and examining the impact on system appropriateness, the method's resilience was evaluated to ascertain its capabilities. Robustness was tested using variations in process variables such the mobile phase, flow rate, and column temperature. Small and deliberate changes to the experimental tests, such as (i) a cooling of the column to ± 5°C (ii) a flow rate of ±1ml/min (iii), and (iii) a length of approximately ±2nm, were made to verify the resilience of the procedure (iv) Organic composition of the mobile phase's content five % to see how the modification will impact the process, it was made. By calculating percent RSD and percent recovery for each case, the resulting data was evaluated.¹⁸

7. Force Deterioration Study:

Accelerated disintegration, another name for forced degradation, is a method where additional stress is used to speed up the natural degradation rate of a product or material. To find reactions that might lead to the degradation of a given product, forced degradation studies are utilised. Prior to final formulation, forced deterioration is typically performed, and it entails imposing external stress conditions and speedy material stability testing.^19,20

The following formula was used to calculate the % degradation:

(Area of Unstressed-Area of Stressed)/(Area of Unstressed) *100 = % Degradation

· Acid degradation

· Basic degradation

· Peroxide degradation

· Thermal degradation

· Photolytic degradation

8. Analytical Solution Stability:

Byanalysing the standard and sample preparations at 0 h, 1 day in the refrigerator, and 30°C ambient room temperature, it was possible to assess the stability of analytical solutions. The average peak and RSD from three injections of each solution were calculated.^21,22

RESULTS:

Method development and experimental parameter optimization:

A few of Lapatinib's physical and chemical characteristics were discovered in the literature during the creation and optimization of the technology. Preliminary reversed phase HPLC chromatographic parameters, including the detection wavelength, mobile phase, stationary phase, and sample preparation method, were selected using the analytical approach. On the Hypersil BDS C18 (250 x 4.6mm) 5µm column, several experiments were run in an effort to optimise the chromatographic conditions and improve the methanol and water ratios. Summarizing the results of method optimization (Table 1).The best chromatographic conditions for the entire study were the mobile phase of Methanol and water in the ratio of 90:10 v/v, flow rate of 1.0mL/min, injection volume of 20µL, run time of 15 min, column temperature of 30°C at wavelength (λ) 262, where Lapatinib was eluted forming symmetrical peak shape, resolution, and suitable analysis time with retention time of 4.15min (Figure 2). The optimum alternative was ultimately determined to be the mobile phase of water, methanol, and trifluoroacetic acid at a ratio of 30:70:0.1 v/v, a flow rate of 1.1ml/min, and a column temperature of 30°C because it produced a peak of lapatinib that was clearly defined and well-resolved. The optimal wavelength for identifying and measuring Lapatinib was determined to be 262nm.Under optimal chromatographic conditions, the retention time for lapatinib was reported to be 4.15min. (Figure 2) displays an example of a chromatogram.

Figure 2: Chromatogram of Lapatinib obtained by using water, Methanol and trifluoroacetic acid (30:70:0.1)

Validation of the Method:

System suitability:

The peak area, retention period, tailing factor, and theoretical plate were compared to determine system suitability parameters. Table 2 shows the result. The % RSD of peak area and retention duration were found to be within acceptable limits. Both the theoretical plate and tailing factor are within acceptable bounds. This means that the parameters of system suitability meet acceptable standards.

Table 2: Result of System suitability for Lapatinib

Sr. No.	Peak Area	Retention time	Tailing factor	Theoretical plate
1	1225.7698	4.43	1.38	7282
2	1223.2367	4.42	1.37	7259
3	1247.2739	4.40	1.36	7361
Mean	1232.0935	4.42	-------	-------
SD	13.2075	0.0153	-------	-------
RSD	1.07	0.35	-------	-------

Linearity:

By plotting the peak area vs. concentration of Lapatinib working standard solutions, the suggested method's linearity was examined. Lapatinib peak area vs corresponding concentrations was shown to be linear in the 25–75 µg/ml concentration range. The research results are displayed in Figure 3 and Table 3. The regression analysis produced the following linear equation: y= 23.53x + 54.005, suggesting a linear relationship between analyte concentration and area under the peak, with a goodness of fit (R²) of 0.9992.

Table 3: Standard linearity Calibration curves of Lapatinib

Conc. (µg/ml)	Area
25.10	632.4937
37.65	952.5480
50.20	1248.2606
62.75	1516.7096
75.3	1827.2126
Correlation coefficient (r²)	0.9996
Intercept	54.005
Slope	23.53

Figure 3: Standard Calibration Curve for Lapatinib

Precision:

The system and technique's precision results showed that the method's accuracy is within reasonable bounds. For both solutions, the RSD, tailing factor, and total number of theoretical plats were calculated; all of the results fell within acceptable bounds. According to Tables 4 and 5, there were fewer than 1000 plates and acceptable accuracy for the RSD and tailing factor was less than 2.0 %.

Table 4: System precision data from the standard solution of the proposed HPLC method.

Sr. No.	RT	Peak Area	Theoretical Plate	Tailing Factor
1	4.13	1247.2739	7361	1.36
2	4.14	1252.2548	7324	1.37
3	4.14	1230.8429	7343	1.37

Table 5: Method precision data from the sample solution of the proposed HPLC method

Sr. No.	RT	Peak Area	% Assay
1	4.14	1253.4678	100.13
2	4.14	1231.4185	99.83

Accuracy:

The accuracy of an analytical procedure reveals how true to the original value the results it produces are. According to accuracy data, at all three levels, percentage recovery was between 99.33 and 101.10%, and %RDS values were between 1.35 and 1.41%, as demonstrated in (Table 6). The approach may be utilised for regular drug analysis, as indicated in, because the % recovery and %RSD values were both within acceptable ranges of 98.0 to 102.0% and not more than 2.0% (Table 7).

Table 7: Statistical Validation of Recovery Study

Recovery at	Mean	SD	%RSD
80%	99.39	0.6815	0.69
100%	99.465	1.8685	1.88
120%	99.40	0.9393	0.9450

Robustness:

Under precisely controlled circumstances, six sample solutions were created and examined while the flow rate, mobile phase ratio, buffer pH, and detecting wavelength were adjusted at three levels. From 0.99ml/min earlier, the mobile phase flow rate was raised to 1.2ml/min. The wavelength has changed from 261nm to 263nm. To determine their retention time and tailing factor, a sample was injected. Additionally, the mobile phase's Methanol-to-buffer balance changed. To evaluate the method's resilience, minor changes in variables like wavelength and flow rate were made. The wavelength changes were 2nm and 1ml/min, respectively, as was the flow rate. By calculating percent RSD values, the method's robustness was assessed, as shown in (Table 8).

Table 8: Robustness data of the proposed HPLC method.

Investigated Parameter	RT	Peak Area	Plate Count	Tailing
Flow-1	3.70	1242.7618	7285	1.34
Flow-2	4.55	1249.6549	7168	1.38
Temp.-1	4.61	1250.6849	7162	1.40
Temp.-2	4.61	1206.1654	7218	1.37
Wavelength-1	4.16	1192.6421	7268	1.35
Wavelength-2	4.18	1176.6578	7260	1.37

Limits of detection and limits of quantification (LOD and LOQ):

LOD and LOQ were determined using the slope and standard deviation. The limit of quantification (LOQ) is the lowest amount of analyte in a sample that can be quantitatively determined with adequate accuracy. The limit of detection (LOD) is the lowest amount of analyte in a sample that can be detected but not necessarily quantified. The LOD and LOQ for lapatinib tartrate were established to be 0.45 and 1.35µg, respectively.

Solution Stability:

The percent of recovery ranged from 98.0 and 102.0%, with an RSD of less than 2.0%, indicating that the sample and standard solutions were stable for 24 hours under both conditions. The percent RSD was less than 2.0%, and the percentage of recovery was within acceptable norms. The theoretical plate number and tailing parameters were also found to be within acceptable limits.

Force Degradation Study:

Table 9 shows the % degradation and % area after degradation results.

Table9: % degradation and % area after degradation results

Stress condition	Min	% Area Lapatinib observed after degradation	% of degradation
Acidic	30min	84.91	5.12
Basic	30min	80.12	4.91
Peroxide	12hr	88.45	2.45
Thermal	1hr	99.78	3.69
Photolytic	1hr	99.15	7.60

Figure 4: Chromatogram of acid degraded sample.

Figure 5: Chromatogram of base degraded sample.

Figure 6: Chromatogram of Peroxide degraded sample

Figure 7: Chromatogram of Thermal degraded sample

Figure 8: Chromatogram of photolytic degraded sample.

Discussion:

In this approach, lapatinib eluted at 4.15 min. The current method was created using water, methanol, and trifluoroacetic acid (30:70:0.1) v/v at a flow rate of 1.1 ml/min at room temperature. The new strategy was more tactful and economical. The stability indicating RP-HPLC is responsible for the peculiar relationship between active medicinal components and any degradation products created under the specified conditions. The chemical and its degradants were successfully separated using the suggested method.

Analysis of a commercial sample:

The suggested method can be used to examine Lapatinib in marketed tablet dosage form of 250 mg. The assay results were found to be 98.57 % correct, and the amount identified was up to 246.41 mg. The work is summarised in Table 10.

Table10: The proposed method's assay results

Formulations	Label Claim	Amount found in mg	% Assay
Herduo	250 mg	246.41	98.57%

Conclusions:

A quick, easy, precise, exact, and linear stability-indicating HPLC technique for lapatinib tartrate was created and validated in this study; it may now be used for standard quality control analysis. The mobile phase solvents and analytical technique parameters for lapatinib show acceptable resolution. The suggested approach also has a brief run time and a retention time of nearly 4.15 mins. The method was validated in compliance with ICH recommendations. The technique is reliable enough to reproduce exact results in a range of chromatographic setups.

ABBREVIATIONS LIST:

ICH: International Conference on Harmonization; reverse phase high-performance liquid chromatography (RP-HPLC); AR: analytical reagent; RT: retention time; percent RSD: relative standard deviation. MeOH: methenol, OPA: orthophosphoric acid, Trifluroacetic Acid: TFA, whereas Sodium Hydroxide: NaOH. LOD and LOQ: "limit of detection" and "limit of quantitation," respectively. Hydrochloric acid: HCL, and hydrogen peroxide: H₂O₂.

COMPETING INTERESTS:

The authors declare that they have no competing interests.

AUTHORS’ CONTRIBUTIONS:

Swati Dattuji Vidhate, Apurva Raju Gattewar and Apeksha Premlal Motghare designed the study and was a major contributor in writing the manuscript. Nikita Gaydhane performed the experiment. Guided by Dr. Parimal Pradipkumar Katolkar and supervised it. All authors have read and approved the final manuscript.

ACKNOWLEDGEMENTS:

The authors are thankful to Principal and Guide, Kamla Nehru College of Pharmacy, Butibori, Nagpur, for the support.

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Received on 22.12.2022 Modified on 08.02.2023

Research J. Pharm. and Tech 2023; 16(7):3125-3131.

DOI: 10.52711/0974-360X.2023.00514

Column used	Mobile phase	Flow rate	Wavelength	Observation	Result
HypersilBDSC18 (250 mm x 4.6 mm, Particle size: 5 µm)	Methanol: Water (90:10)	1.0 ml/min	262 nm	Poor resolution	Method rejected
HypersilBDSC18 (250 mm x 4.6 mm, Particle size: 5 µm)	Ammonium methanol: dihydrogen phosphate diluted with orthophosphoric acid (20:80)	1.2ml/min	262 nm	Poor resolution	Method rejected
HypersilBDSC18 (250 mm x 4.6 mm, Particle size: 5 µm)	water, Methanol and trifluoroacetic acid (30:70:0.1)	1.2 ml/min	262 nm	Good resolution	Method accepted

Recovery level	Area	Amount added µg/ml	Amount found µg/ml	Amount recovered µg/ml	% recovery
Accuracy. 80%	1245.4218	40.0000	89.6443	47.6037	99.39
Accuracy. 100%	1253.6428	50.0000	100.8110	60.6594	99.465
Accuracy. 120%	1249.5246	60.0000	109.6185	70.6527	99.40