INTRODUCTION:

Dacomitinib (CAS no. 1110813-31-4) is an irreversible tyrosine kinase inhibitor¹. In 2018, the Food and Drug Administration (FDA) has approved² Dacomitinib (Figure 1) for the first-line treatment of patients with metastatic non-small cell lung cancer in the form of tablets. Chemically Dacomitinib is (2E)-N- {4- [(3-Chloro-4-fluoro phenyl) amino]-7-methoxy-6-quinazolinyl}-4-(1-piperidinyl)-2-butenamide with molecular formula, C₂₄H₂₅ClFN₅O₂ and molecular weight 469.95 grams/mole.

Figure 1: Structure of Dacomitinib mono hydrate (C₂₄H₂₇ClFN₅O₃. H₂O)

Abdelhameed et al., developed a LC/MS-MS assay³ to quantify Dacomitinib in rat liver microsomes in presence of an internal standard, Lapatinib using a mobile phase mixture of 10 mM ammonium acetate (pH adjusted to 4.2 with formic acid) and acetonitrile. Beer-Lambert’s law was obeyed over the concentration range 2.0-500 ng/ml. Lutful Kabir et al. and Qiu X et al., developed a bioanalytical assay using UPLC/MS-MS methods^4-5 using Acetonitrile: 0.1% Formic acid in water for the quantitative determination of Dacomitinib in rat plasma. Kirti Kumari et al., developed a HPLC method⁶ for the quantification of Dacomitinib using Kromasil C₁₈ column with mobile phase mixture consisting of 0.2% tri ethyl amine solution (pH = 3.2 ± 0.1 adjusted with 85% orthophosphoric acid): acetonitrile (70:30) on isocratic mode (Detection wavelength 260 nm) and Beer-Lambert’s law obeyed over the concentration range 2.0-500 µg/ml. In the present study a new stability indicating HPLC and LC-APCI-MS methods have been proposed for the quantification of Dacomitinib and the method was validated as per ICH guidelines.

MATERIALS AND METHODS:

Instrumentation

HPLC Conditions

TSQ scientific Quantis LCMS with Thermo Vanquish model HPLC with PDA detector and Simpack C18 (250 mm x 4.6 mm x 5µm) column was employed for the present study. The injection volume was 10 µL and the total run time was 25 mins (Detection wavelength 259 nm). 0.1% Formic acid: Acetonitrile was used as mobile phase on gradient mode with flow rate was 1 ml/min and a mixture of Methanol: Water (50:50) was used as diluent.

MS Conditions

Ion Source type : APCI

Spray Voltage : Static

Positive Ion discharge current (V) : 4

Negative Ion discharge current (V) : 10

Sheath Gas (Arb) : 45

Aux Gas (Arb) : 10

Sweep Gas (Arb) : 2

Ion transfer tube temperature : 275

Vaporizer temperature : 400

Scan mode : Full scan Q1

Scan Range : 50-2000 m/z (Positive mode)

: 50-2000 m/z (Negative mode)

Preparation of stock solution

50 mg of Dacomitinib API was weighed accurately and transferred carefully into a 50 ml volumetric flask and was dissolved in HPLC grade Acetonitrile (1000 µg/ml) and the resulting solution was sonicated for 30 mins and dilutions were made further with the mobile phase and all the solutions were filtered.

Method validation⁷

Linearity, Precision, Accuracy and Robustness

2.0-200 µg/ml Dacomitinib solutions were prepared from the stock solution (1000 µg/ml) on dilution with the mobile phase and each solution was injected (n=3) into the LC system and the average peak area from the respective chromatograms was calculated. A calibration graph was drawn by plotting the concentration of the drug solutions on the x-axis and the corresponding peak area of the chromatograms on the y-axis. The intraday precision studies were conducted on the same day at different equal time intervals and the interday precision studies were conducted on three successive days (Day 1, Day 2 and Day 3) and the % RSD was calculated. Accuracy studies were performed by spiking the formulation solution with 50, 100 and 150% API solution and thereby the percentage recovery was calculated with the help of regression equation. The percentage relative standard deviation was calculated in all the validation parameters.

Assay of Dacomitinib tablets

Dacomitinib is available as tablets with different brand names such as DACOPLICE (Label claim: 45 mg) (Pfizer), VIZIMPRO (Label claim: 15, 30, 45 mg) (Pfizer) and DACONIB (Label claim: 45 mg) (Everest) in India. Two different brands of Dacomitinib were collected and extracted with acetonitrile after sonication with the mobile phase. The resulting solution was filtered through 0.24 μm membrane filter and 10 μL of these formulation solutions were injected in to the HPLC system. The peak area of the chromatogram (n =3) was noted and the percentage purity was determined.

Stress degradation studies⁸

During the acidic degradation study Dacomitinib solution was treated with 0.1N HCl and immediately neutralized with 1ml 0.1N NaOH solution. The contents were diluted with mobile phase and the resultant solution was injected into HPLC and LCMS system and the peak area as well as the mass spectrum was recorded. During the thermal degradation study Dacomitinib solution was heated at 60ºC and the contents were diluted with mobile phase and the resultant solution was injected into HPLC and LCMS system and the peak area as well as the mass spectrum was recorded. During the basic degradation study Dacomitinib solution was treated with 0.1N NaOH for about 30 mins and then neutralized with 1ml 0.1N HCl solution. The contents were diluted with mobile phase and the resultant solution was injected into HPLC and LCMS system and the peak area as well as the mass spectrum was recorded. During the oxidative degradation study Dacomitinib solution was treated with hydrogen peroxide for about 30 mins and then diluted with mobile phase and the resultant solution was injected into HPLC and LCMS system and the peak area as well as the mass spectrum was recorded.

RESULTS AND DISCUSSION:

A new stability-indicating RP-HPLC and LC-MS methods have been developed for the quantification of Dacomitinib. The earlier reported methods were discussed with the present proposed method and the details were given in Table 1.

Method	Mobile phase (v/v)	Linearity (μg/ml)	Reference
RP-HPLC and LC-APCI-MS	0.1% Formic acid: Acetonitrile (Gradient mode)	2.0-200	Present method

Linearity, Precision, accuracy and robustness

Dacomitinib obeys Beer-Lambert’s law over the concentration range 2.0-200 µg/ml (Table 3) and the linear regression equation was found to be y = 157.45x - 37.03 (R² = 0.9999) Figure 3). The LOD and LOQ values were found to be 0.6112 µg/ml and 1.8741 µg/ml respectively. The % RSD in intraday precision (0.6593), interday precision (0.6586-1.6489) (Table 4) was found to be less than 2.0% stating that the method is precise. In the accuracy study the % RSD was found to be 0.67-0.94 (<2) (Table 5) with a recovery of 99.54-99.83 indicating that the method is accurate.

Table 3: Linearity

Conc. (µg/ml)	*Mean peak area
0	0
2	372.598
5	786.329
10	1569.254
25	3854.325
50	7725.241
75	11789.649
100	15634.613
150	23384.157
200	31659.324

*Mean of three replicates

Figure 3: Calibration curve

Table 4: Precision study

Intraday precision study
Conc. (µg/ml)	Mean peak area		*Mean peak area ± SD (% RSD)
10	1569.254		1553.9953 ± 10.2448 (0.6593)
10	1541.984
10	1552.142
10	1549.857
10	1547.514
10	1563.221
Interday precision study
Conc. (µg/ml)	Day 1	Day 2	Day 3	Mean peak area ± SD (% RSD)*
10	1569.254	1549.864	1553.749	1557.6223 ± 10.2589 (0.6586)
50	7725.241	7891.258	7801.365	7805.9547 ± 83.1036 (1.0646)
100	15634.613	15684.352	15671.548	15663.5043 ± 25.8267 (1.6489)

*Mean of three replicates

Table 5: Accuracy study

Spiked conc. (µg/ml)	Formulation (µg/ml)	% Recovery	% RSD
10 (50 %)	20	99.83	0.81
20 (100 %)	20	99.54	0.94
30 (150 %)	20	99.72	0.67

*Mean of three replicates

Assay of Dacomitinib tablets

The assay of Dacomitinib tablets was performed using the proposed liquid chromatographic method with the optimized chromatographic conditions. The percentage of purity of Dacomitinib was found to be 99.40-99.80 (Table 6).

Table 6: Assay of Dacomitinib ophthalmic solution

S. No.

Brand name

Label claim

(mg)

*Observed amount (mg)

% Recovery*

Brand I

44.91

99.80

Brand II

44.73

99.40

*Mean of three replicates

Stress degradation studies

Dacomitinib (100 µg/ml) was exposed to different stress conditions under the optimized chromatographic conditions and then injected in to the system. During the acidic degradation, Dacomitinib was eluted at Rt 11.908 min and the total drug (99.62 %) has undergone decomposition with the elution of degradant peak at 12.475 min. From the mass spectrum (Figure 4) it was observed that peaks were observed at Rt 12.01 min (m/z 471.19) and 12.54 min (m/z 404.20).

Acid blank

Representative chromatogram of Dacomitinib(Rt 11.908 min) during acidic degradation

LC-MS Chromatograms of Dacomitinib during acidic degradation

Mass spectrum of Dacomitinib during acidic degradation (Rt 12.01 min; MH⁺: m/z 471.19)

Mass spectrum of Dacomitinibduring acidic degradation (DP: Rt 12.54 min; m/z 404.20)

Figure 4: Representative chromatograms and mass spectra of Dacomitinib during acidic degradation

During the thermal degradation, Dacomitinib was eluted at Rt 11.908 min and about (82.39 %) has undergone decomposition with the elution of degradant peaks at 12.467 min. From the mass spectrum (Figure 5) it was observed that peaks were observed at Rt 12.01 min (m/z 471.22), 12.04 (m/z 473.21) and 12.60 min (m/z 470.16).

Representative chromatogram of Dacomitinib (Rt 11.908 min) during thermal degradation

LC-MS Chromatograms of Dacomitinib during thermal degradation

Mass spectrum of Dacomitinib during thermal degradation (DP: Rt 12.01 min; MH⁺: m/z 471.22)

Mass spectrum of Dacomitinibduring thermal degradation (Rt 12.04 min; m/z 473.21)

Mass spectrum of Dacomitinibduring thermal degradation (Rt 12.60 min) (M⁺ m/z = 470.16)

Figure 5: Representative chromatograms and mass spectra of Dacomitinib during thermal degradation

During the basic degradation, Dacomitinib was eluted at Rt 11.917 min and almost 84.29 % of the total drug has undergone decomposition with the elution of degradant peak at 12.475 min. From the mass spectrum (Figure 6) it was observed that peaks were observed at 11.98 min (m/z 473.23), Rt 12.01 min (m/z 471.23) and 12.60 min (m/z 470.16).

Base blank

Representative chromatogram of Dacomitinib (259 nm) during basic degradation

LC-MS Chromatogram of Dacomitinibduring basic degradation

Mass spectrum of Dacomitinib (Rt 11.98 min) (MH²⁺: m/z 473.23) during basic degradation

Mass spectrum of Dacomitinib during basic degradation(Rt 12.01 min; m/z 471.23)

Mass spectrum of Dacomitinib during basic degradation

(DP: Rt 12.60 min; m/z 470.16)

Figure 6: Representative chromatograms and mass spectra of Dacomitinibduring basic degradation

During the oxidative degradation, Dacomitinib was eluted at Rt 12.108 min and about 20.51 % drug has undergone decomposition with the elution of degradant peaks at 2.277, 11.575, 11.925 and 12.217 min. From the mass spectrum (Figure 7) it was observed that peaks were observed at Rt 11.98 min (m/z 473.23), Rt 12.01 min (m/z 489.24) and Rt 12.31 min (m/z 487.19).

Peroxide blank

Representative chromatogram of Dacomitinib (Rt 12.108 min) during peroxide degradation

LC-MS chromatogram of Dacomitinib (259 nm) during peroxide degradation

LC-MS chromatogram of Dacomitinib during peroxide degradation

Mass spectrum of Dacomitinib (DP: Rt 11.98 min; m/z 473.23) during oxidative degradation

Mass spectrum of Dacomitinib(DP: Rt 12.01 min) during oxidative degradation

Mass spectrum of Dacomitinib (Rt 12.17 min; MH⁺: m/z 489.24) during oxidative degradation

Mass spectrum of Dacomitinib (Rt 12.21 min; M⁺: m/z 487.19) during oxidative degradation

Mass spectrum of Dacomitinib (Rt 12.31 min) during oxidative degradation

Mass spectrum of Dacomitinib(DP: Rt 12.34 min) during oxidative degradation

Figure 7: Representative chromatograms and mass spectra of Dacomitinib during oxidative degradation

The details of the stress degradation studies of Dacomitinib were shown in Table 7. It is observed that Dacomitinib is highly sensitive towards acidic and basic degradation conditions.

Table 7: Stress degradation studies

Condition	R_t (min)	Mean peak area*	% Recovery*	% Drug degradation
Standard drug	11.967	15634.613	100	-
Acidic hydrolysis	11.908 12.475	59.226	0.38	99.62
Thermal degradation	11.908 12.467	2753.074	17.61	82.39
Alkaline hydrolysis	11.917 12.475	2456.819	15.71	84.29
Oxidative degradation	2.733 11.575 11.925 12.108 12.217	12428.443	79.49	20.51

*Mean of three replicates

CONCLUSION:

The authors have established a new stability indicating RP-HPLC as well as LC-MS method coupled with APCI and triple quadrupole analyser for the estimation of Dacomitinib. The method is simple, precise and accurate and used for the routine analysis of Dacomitinib in pharmaceutical formulations and no interference of excipients was observed during the assay.

ACKNOWLEDGEMENT:

The authors are grateful to MSN Laboratories Pvt. Ltd. (India) for providing the gift samples of Dacomitinib and the authors declare no conflict of interest.

REFERENCES:

1. Liao B-C, Lin C-C, Yang JC-H. Second and third-generation epidermal growth factor receptor tyrosine kinase inhibitors in advanced non-small cell lung cancer. Current Opinion in Oncology. 2015; 27(2): 94-101.

2. U.S. Food and Drug Administration (FDA). Vizimpro^® (dacomitinib) tablets. In: Center for Drug Evaluation and Research, editor. MD, USA 27th September 2018.

3. Abdelhameed AS, Kadi AA, Attwa MW, AlRabiah H. Validated LC-MS/MS assay for quantification of the newly approved tyrosine kinase inhibitor, Dacomitinib, and application to investigating its metabolic stability. PLoS ONE. 2019; 14 (4): e0214598.

4. Md. Lutful Kabir, Frederick Backler, Andrew H. A. Clayton and Feng Wang. Quantitative bioanalytical assay for the human epidermal growth factor receptor (HER) inhibitor dacomitinib in rat plasma by UPLC-MS/MS. Journal of Pharmaceutical and Biomedical Analysis 2019; 166: 66-70.

5. Qiu X, Lin Q, Ning Z, Qian X, Li P, Ye L, et al. Quantitative bioanalytical assay for the human epidermal growth factor receptor (HER) inhibitor dacomitinib in rat plasma by UPLC-MS/MS. Journal of Pharmaceutical and Biomedical Analysis. 2019; 166: 66-70.

6. Kirti Kumari, Pankaj Thakur, Sayali Warde, Vijay M and Raman Mohan Singh. HPLC method development and validation for quantitative estimation of Dacomitinib in pharmaceuticals dosage form. Human Journals Research Article October 2021; 22(3): 606-620.

7. ICH Validation of analytical procedures: Text and methodology Q2 (R1), International Conference on Harmonization (2005).

8. ICH Stability testing of new drug substances and products Q1A (R2), International Conference on Harmonization (2003).

Received on 17.07.2023 Modified on 23.08.2023

Research J. Pharm. and Tech 2023; 16(9):4391-4398.

DOI: 10.52711/0974-360X.2023.00718

Time (minutes)	Mobile phase A%	Mobile phase B%
0.0	95	5
5.0	95	5
15.0	10	90
20.0	10	90
20.1	95	5
25.0	95	5


Blank	Representative HPLC chromatogram of Dacomitinib (Rt 11.967 min)

LC-MS Chromatograms of Dacomitinib (API)

Mass spectrum of Dacomitinib Rt 12.04 min (MH₂⁺ m/z = 473.22)	Mass spectrum of Dacomitinib Rt 12.08 min (MH⁺ m/z = 471.22)

Mass spectrum of Dacomitinib (Rt 12.57 min) (M⁺ m/z = 470.21)	Mass spectrum of Dacomitinib (Rt 12.54 min) (MH₂⁺ m/z = 468.22)

*Corresponding Author E-mail: mmukthin@gitam.edu