Dabrafenib

LC-MS/MS studies for identification and characterization of new forced
degradation products of dabrafenib and establishment of their
degradation pathway
Parul Grovera,⁎,1
, Monika Bhardwaj
, Lovekesh Mehtac
a KIET School of Pharmacy, KIET Group of Institutions, Delhi-NCR, Ghaziabad 201206, India b Natural Product Chemistry Division, Indian Institute of Integrative Medicine, Canal Road, Jammu 180001, India c Amity Institute of Pharmacy, Amity University, Noida 201301, India
article info
Article history:
Received 30 April 2021
Received in revised form 8 August 2021
Accepted 27 August 2021
Available online 31 August 2021
Keywords:
Dabrafenib
Degradation pathway, HRMS/MS/TOF,
Characterization, Mass fragmentation pattern
Degradation products
abstract
Dabrafenib (Tafinlar) is used for the treatment of patients with BRAF V600 mutation positive unresectable
or metastatic melanoma. Forced degradation study of the drug product and drug substance is very much
important in drug development and drug discovery to establish the intrinsic stability and understand its
behaviors towards different stress conditions. In the current study, compressive stress testing of dabrafenib
has been performed as per the recommendation of ICH guidelines to identify and characterize all major
degradation products of dabrafenib (DPD) formed. Drug substances were exposed to different stressed
conditions as per ICH recommendations. The present study observed that the dabrafenib drug substance is
very much sensitive when exposed to oxidative degradation conditions at 80 °C temperature conditions and
also sensitive to photolytic degradation conditions. Dabrafenib is stable when treated in acidic, alkaline,
neutral and thermal degradation environments as there is no degradation observed in signification per￾centage under these stressed conditions. The best separation of eight degradation products and dabrafenib
drug substance was obtained in Waters BEH (Ethylene Bridge Hybrid) C-18 column (1.7 µm,
100 mm × 2.1 mm) having mobile phase composed of Formic acid (0.1%) and methanol as Eluent A and
Eluent B respectively using 225 nm wavelengths. The volume of injection (5 µL) and flow rate (0.3 mL/min)
was set throughout the study. Dabrafenib is highly unstable to oxidative stressed conditions as five major
degradation products (DPD-II, DPD-III, DPD-IV, DPD-V and DPD-VII) were obtained when exposed to hy￾drogen peroxide. When dabrafenib is treated under photolytic degradation conditions, three major DPs
were formed (DPD-I, DPD-VI and DP-VIII). These DPs were further identified and characterized on so￾phisticated HRMS/MS/TOF technique for accurate mass measurement. Characterization of all the de￾gradation products was carried out in the ESI positive mode of ionization. The establishment of the
degradation pathway of drug substance and fragmentation pathway of DPs were explained in the present
study which was never reported in any literature.
© 2021 Elsevier B.V. All rights reserved.
1. Introduction
The development of oral targeted anticancer drugs has increased
strongly in the past two decades and is expected to continue.
Dabrafenib (Tafinlar), is BRAF (v-raf murine sarcoma viral oncogene
homolog B1) inhibitor protein that is orally bio-available and is
approved for the management of nonresectable stage III or stage IV
metastatic melanoma that is responsible for mutation. BRAF, a
serine/threonine-protein kinase is stimulated by somatic point
mutations in human malignancy. This kinase is an important mo￾lecule of the RAS (rat sarcoma gene) that triggered the mitogen￾activated protein (MAP) kinase/extracellular signal-regulated kinase
(ERK) signaling pathway resulting in increased cell growth.
Approximate 7% of all cancers [1], including 60–70% of melanomas,
[2], 12% of colorectal cancers [3] and 15% of papillary thyroid carci￾nomas contributed due to mutation in BRAF gene. To a somewhat
lesser extent patients with hairy cell leukemia [4,5], papillary

https://doi.org/10.1016/j.jpba.2021.114351

0731-7085/© 2021 Elsevier B.V. All rights reserved.
⁎ Correspondence to: KIET School of pharmacy, KIET Group of institutions, Delhi￾NCR, Ghaziabad 201206, Uttar Pradesh, India.
E-mail address: [email protected] (P. Grover). 1 Both authors contributed equally.
Journal of Pharmaceutical and Biomedical Analysis 206 (2021) 114351
pharyngeal cancer [6,7] and 1.6–4.9% of lung cancer cells may be due
to these BRAF mutations in the gene. After understanding the very
crucial role of BRAF mutation in the case of melanoma, more focus is
on the advanced development of an inhibitor of BRAF mutation to
treat malignant melanoma. In this, Dabrafenib plays a vital role as it
is a competitive inhibitor of BRAF kinase that is responsible for
mutation in cell lines, Xenografts and kinase screening [8,9] as it is
potent adenosine triphosphate (ATP). The structure of dabrafenib is
given below in Fig. 1.
For treatment of metastaticBRAFV600E-positive melanoma, dab￾rafenib is approved in Europe and the USA. Dabrafenib drug has been
approved by different regulatory agencies like USFDA (The US Food
and Drug Administration) and EMA (European Medicines Agency)
either alone or in combination with other anticancer drugs like
trametinib for the treatment of metastatic melanomas.
Looking into the emergent applications of selective BRAF in￾hibitors, we decided to execute stress degradation studies of dab￾rafenib and to establish a highly sensitive and fast LC-HRMS method
for the identification and characterization of its degradation
products.
An LC-MS method to measure dabrafenib and its six metabolites
are described by Vikingsson et al. in human plasma [10]. Another
LC-MS assay method of dabrafenib is reported on plasma samples of
the mouse [11]. Few LC-MS recent methods on simultaneous quan￾tification of dabrafenib along with other drugs like trametinib,
vemurafenib, niraparib, etc. in human plasma are also reported.
[12–15]. Separation, identification and characterization of degrada￾tion products of few anticancer drugs using the UPLC-MSMS tech￾nique have been reported [16–18]. Based on the recommendations of
ICH guidelines Q3A (R2) and Q3B (R2), identification and char￾acterization of all the degradation products or process-related
impurities of dabrafenib have been carried out [19,20].
But to date, no study is reported on the degradation and stability
of drugs under varied conditions such as hydrolytic, oxidative,
thermal and photolytic. Herein we report (i) A comprehensive forced
degradation study of dabrafenib under hydrolytic, oxidative, thermal
and photolytic conditions as prescribed by ICH guidelines; (ii) to
separate the degradation products by UPLC (eight degradation pro￾ducts were separated) (iii) structural characterization of degradation
products through HR-MS-TOF; and (iv) most plausible mechanisms
of dabrafenib degradation.
2. Experimental
2.1. Drugs and reagents
Dabrafenib drug substance having purity of more than 99.80%
was kindly provided by MSN laboratory limited, Hyderabad as a gift
sample. Ultra-pure LC-MS grade acetonitrile (CH3CN) and methanol
(CH3OH) were procured from JT baker (Bangalore, India). Sodium
hydroxide (NaOH) pallets and formic acid (HCOOH) of Analytical
Reagent (AR) grade were procured from Merck (Mumbai, India).
Finar chemicals (Hyderabad, India) provided the analytical grade
Hydrochloric acid (HCl, 37%). Hydrogen peroxide (H2O2, 30%) was
procured from S.D Fine chemicals (New Delhi, India). Milli-Q water
purification system (Millipore, USA) used for purification of water.
2.2. Instrumentation/Equipments
LC studies were performed using Waters H-Class system which
consists of a quaternary pump, Quartnary solvent Manager (QSM),
photodiode array detector (PDA), injector (Rheodyne sample loop of
50 µL) and controlled column oven temperature. Data were in￾tegrated and processed using Empower 2.0 software. Waters BEH
(Ethylene Bridge Hybrid) C-18 column was used to resolve all the
closely eluted DPs and their peak purity was confirmed with the
help of a photodiode array (PDA) detector. A highly precise calibrated
semi microbalance (Mettler Toledo, India) was used for weighing all
the samples throughout the study. Photo-stability studies were
performed in a photostability chamber (Thermolab, 95 Th-400 G
Mumbai, India) set at 40 ± 1 C/75 ± 3% RH comprised of black UV
lamps and White fluorescent lamps as per ICH recommended
guidelines. A highly precise calibrated water bath (Thermostatic
classic scientific Pvt Ltd, Ahmadabad, India) was used to perform the
degradation study at 80 c. A hot air oven (NSW, New Delhi) was used
to perform the thermal degradation study of the drug substance. For
preparing the sample solution and dilutions, pipettes (Eppen-Dorf,
Hamburg, Germany) were used. The optimized developed conditions
of chromatography are shown in Table 1.
2.3. Stress degradation studies
Stress degradation study was performed out as per ICH Q1A (R2)
and Q1B recommended guideline [21,22]. Different stressed condi￾tions were given to drug substances to define the chemical stability
of the drug substance. Dabrafenib was exposed forcefully in an
acidic, basic, neutral, oxidative, photolytic and thermal environment
at different temperature conditions to monitor the changes in the
purity concerning the control sample. A stock solution of Dabrafenib
having a concentration of 1 mg/mL was prepared with diluent. Then
further this stock solution was diluted while doing the stress study
to get the final nominal concentration of 50 µg/mL. To execute the
hydrolytic degradation study, dabrafenib was exposed to water at
80 °C (Neutral), 1 N HCl for 24 Hrs at 80 °C (Acidic) and 1 N NaOH
kept at 80 °C for 24 hrs (Alkaline). Peroxide degradation study was
executed by treating drug substance with 10% H2O2 for 8 hrs at 80 °C
Thermal degradation study was carried out by keeping the dabra￾fenib powder in a dry oven for 48 hrs at 105 °C Dabrafenib powder as
well as a solution was kept in Petri dish and exposed in the photo￾lytic chamber. Also, the control sample (Dark) was kept in a photo￾lytic chamber. In the chamber, drug was exposed to 1.2 million lux
hrs of fluorescent light and 200 W h/m2 of UV light. Stressed sample
solutions of acid and base were neutralized with base and acid re￾spectively before final dilution. A blank solution was also prepared in
Fig. 1. Structure of Dabrafenib.
Table 1
Optimized chromatographic conditions.
Eluent A 0.1% Formic acid in water
Eluent B Methanol
Column ACQUITY BEH C-18
(100 *2.1 mm, 1.8 µm)
Injection volume 5 µL
Flow Rate 0.3 mL/min
Wavelength 225 nm
Auto sampler Temperature 10 °C
Column Temperature 40 °C
Diluent Acetonitrile: Water (1:1)v/v
Run time 15 min
Sample/Nominal concentration 50 µg/mL
Elution type Gradient
P. Grover, M. Bhardwaj and L. Mehta Journal of Pharmaceutical and Biomedical Analysis 206 (2021) 114351
each degradation condition in the same way without using a drug
substance.
2.4. Sample preparation for LC-MS analysis
Stock solution (1.0 mg/mL) was diluted in the case of every
stressed parameter to get the final nominal concentration of 50 µg/mL.
All the treated sample solutions were filtered with low micron filter
paper before being injected into UPLC. The control sample (where no
stressed treatment was given to the sample) was also injected into the
UPLC system. A blank solution belongs to every stressed environment
was also injected. The percentage of degradation was obtained by
comparing the purity of the degraded sample with that of the control
sample. Dabrafenib and all degradants were mixed and injected to get
the well-resolved UPLC method and also used to perform the
LC-MSMS study.
2.5. HRMS and LC-HRMS studies
A highly sensitive, advanced and accurate Triple Quadrupole
mass spectrometer (Agilent, Q-TOF LC/MS 6510 series) coupled with
an electrospray ionization source was used to identify and char￾acterize all the DPs accurately. These DPs were separated using C-18
UPLC column having Ethylene Bridge Hybrid (BEH)technology with a
mobile phase having the combination of Eluent A (0.1% formic acid)
and Eluent B (Methanol). Flow rate (0.3 mL/min), detector wave￾length (225 nm), Column temperature (40 °C) and sample tempera￾ture (10 °C) was used throughout the analysis. ESI positive mode of
ionization was used for carrying out the analysis. Mass hunter
workstation software was used to process all the mass spectrum
data of dabrafenib and its degradation products. All the optimized
conditions of mass analysis are as follows: capillary voltage, 3500 V;
collision energy, 25; the skimmer, 50 V, Nebulizing gas, 40 psi. Since
HRMS can give the results up to the fourth decimal, it helped in the
accurate mass measurement of all the results.
3. Results
3.1. Stress decomposition behavior
Overlaid UPLC chromatogram of the degradation behavior of
dabrafenib drug substance under different stress conditions was
depicted in Fig. 2. A total of 8 novel degradation products were
formed under different stress conditions that were never reported
earlier. As per the elution pattern, these degradation products were
denoted from DPD-I to DPD-VIII. The formation of DPD-I and DPD￾VIII was formed due to photolytic degradation conditions. Other
degradation products (DPD-II, DPD-III, DPD-IV, DPD-V and DPD-VII)
were formed in oxidative degradation conditions at 80 °C tempera￾ture conditions whereas DPD-VII was commonly formed in photo￾lytic well as oxidative degradation conditions which indicate that
dabrafenib is highly sensitive to oxidative and photolytic degrada￾tion conditions. There was no major degradation observed in acidic,
alkaline, neutral and thermal degradation conditions indicating that
the drug is highly stable in these degradation conditions.
3.2. Dabrafenib mass fragmentation pathway
The LC-MS/MS line spectrum of the dabrafenib is depicted in
Fig. 3. The fragmentation pattern observed for dabrafenib is shown
Fig. 2. Representative UPLC Overlay chromatograms of Dabrafenib (50 µg/mL) control
(A), neutral (B), acidic (C), alkaline (D), oxidative (E), photolytic (F), UV (G) and
thermal (H) degradations.
Fig. 3. Dabrafenib line spectrum acquired in MS study.
P. Grover, M. Bhardwaj and L. Mehta Journal of Pharmaceutical and Biomedical Analysis 206 (2021) 114351
Fig. 4. Plausible ESI/MS/MS fragmentation pattern of protonated dabrafenib.
P. Grover, M. Bhardwaj and L. Mehta Journal of Pharmaceutical and Biomedical Analysis 206 (2021) 114351
Table 2
Elucidation of MSMS data of fragments of dabrafenib.
Peak No Experimental mass Best probable molecular formula Theoretical mass RDB Difference from parent ion Probable losses corresponding to
difference
0 520.1073 C23H21F3N5O2S2
+ 343.0770 14.5
1 343.1239 C15H14F3N2O2S+ 343.0770 8.5 176.9834343 C8H7N3S
2 342.1175 C16H15F3NO2S+ 342.0770 8.5 1.0064 H
3 327.1042 C15H14F3N2OS+ 327.0773 8.5 16.0197 [O]
4 307.1011 C15H16FN2O2S+ 307.0911 8.5 36.0228 2F
5 296.1078 C14H12F2NO2S+ 296.0551 8.5 46.0097 C2H3F
6 292.0755 C15H15FNO2S+ 292.0802 8.5 15.0256 NH
7 286.0545 C15H13FN3S+ 286.0809 10.5 234.0528 C8H8F2N2O2S
8 277.0455 C14H17N2O2S+ 277.1005 7.5 66.0784 C, 3F
9 240.0455 C13H10N3S+ 240.0590 10.5 46.009 C2H3F
RDB: Ring plus double bond.
Fig. 5. DPD-I line spectrum acquired in LC-MS/MS studies.
Fig. 6. DPD-II line spectrum acquired in LC-MS/MS studies.
P. Grover, M. Bhardwaj and L. Mehta Journal of Pharmaceutical and Biomedical Analysis 206 (2021) 114351
5
in Fig. 4. A total of eight fragments were observed. The loss of amine
substituted pyrimidine ring from DBR then thiazole ring opening
with the formation of amine substituted propene chain formed the
molecular ion owing m/z 343.1239. Further elimination of amine
with methyl substitution formed the molecular ion having m/z
342.1175. Removal of one sulfonyl oxygen and loss of two substituted
fluorine atoms on benzene ring from the molecular ion having m/z
343.1239 gave other two fragments having m/z 327.1042 and m/z
307.1011. Further, the loss of one fluorine atom and dimethyl from m/
z 342.1175 gave the other fragment with m/z 296.1078. The elim￾ination of primary amine from m/z 307.1011 formed the molecular
ion having m/z 292.0755. The removal of all the fluorine atoms and
methyl from m/z 343.1239 gave the molecular ion having m/z
277.0528. Other fragments formed from DBR by the loss of 2,6-
difluorobenzenesulfonamide, primary amine and dimethyl resulting
in molecular ion owing m/z 286.0545 and further removal of a
fluorine atom and ethyl group formed the molecular ion with m/z
240.0455. The above fragments confirmed the dabrafenib structure.
The elucidation of MS/MS data of fragments of the dabrafenib is
shown in Table 2.
3.3. HR-MS/MS analysis of the samples degraded
Figs. 5−12 presented the line spectral pattern of all the eight
degradation products (DPD-I to DPD-VIII) obtained when dabrafenib
is treated in different stress conditions. The most attainable ex￾perimental mass, molecular formulae, theoretical mass, ring double
Fig. 7. DPD-III line spectrum acquired in LC-MS/MS studies.
Fig. 8. DPD-IV line spectrum acquired in LC-MS/MS studies.
P. Grover, M. Bhardwaj and L. Mehta Journal of Pharmaceutical and Biomedical Analysis 206 (2021) 114351
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bond value and major fragments and also the chemical formulae of
all the eight DPs (DPD I-DPD VIII) are shown in Table 3.
3.4. Identification/Characterization of degradation product
The determination of all the eight Degradation Products (I-VIII)
was carried out with the help of the fragmentation patterns ob￾served and HRMS/MS studies. The fragmentation order of all the DPs
were also compared with the fragmentation pattern of dabrafenib as
obtained in HRMS/MS studies.
3.4.1. DPD-I (m/z 307.0911)
In the LC–MS/MS line spectral data, the mass of Degradation
Product-1was observed as m/z 307.0989 as [M+H] +
, represented in
Fig. 5, which was obtained by the elimination of pyrimidine ring and
thiazole ring-opening along with the removal of disubstituted
fluorine atoms from DBR. The plausible fragmentation order of DPD￾I is represented in Fig. 13. The other fragment losses amine resulting
in a molecular ion having m/z 292.0749. The removal of one sul￾phonamide oxygen from the ion having m/z 307.0989 gave the other
molecular ion owing m/z 291.0673. Further methyl elimination gave
the fragment with m/z 277.0516. Then the methyl amine loss formed
the molecular ion having m/z 250.0301. With the above information,
the structure of DPD-I could be characterized as N-(3-(1-aminoprop-
1-en-1-yl)-2-fluorophenyl) benzenesulfonamide ion.
3.4.2. DPD-II (m/z 536.1082)
As represented in Fig. 6, the mass of DPD-II was observed as
536.1040 as [M+H]+ which was built by N-oxide formation at thia￾zole nitrogen in DBR. Five fragments were formed in total. The
Fig. 9. DPD-V line spectrum acquired in LC-MS/MS studies.
Fig. 10. DPD-VI line spectrum acquired in LC-MS/MS studies.
P. Grover, M. Bhardwaj and L. Mehta Journal of Pharmaceutical and Biomedical Analysis 206 (2021) 114351
7
probable fragmentation order of DPD-II is presented in Fig. 14. The
fragment owing m/z 483.0758 formed from DP-II by pyrimidine ring￾opening forming imino-propene-amine. Sulfanone oxygen loss and
loss of pyrimidine and thiazole ring forming diamine propene
formed the fragment owing m/z 342.1175. Then the elimination of a
fluorine atom gave the molecular ion owing m/z 322.1116. Removal
of one amine gave the molecular ion owing m/z 307.0991. Another
amine loss gave the fragment with m/z 292.0672. With the above
information, the structure of DPD-II could be characterized as 5-(2-
aminopyrimidin-4-yl)-2-(tert-butyl)-4-(3-((2,6- difluorophenyl)sul￾fonamido)-2-fluorophenyl)thiazole 3-oxide ion.
3.4.3. DPD-III (m/z 470.1115)
The LC–MS/MS line spectrum in Fig. 7, shows the mass of DPD-III
to be m/z 470.0816 as [M+H]+
which was built from DBR by the loss of
difluoro atoms from benzene ring and a methyl molecule from t-butyl
substituted on thiazole ring. Seven fragments were observed in total.
The plausible fragmentation order of DPD-III is presented in Fig. 15.
The loss of pyrimidine ring and thiazole ring-opening leaving ethene
amine formed the molecular ion owing m/z 293.0988. The other ion
owing m/z 291.0622 obtained from DPD-III by the removal of pyr￾imidine ring, thiazole ring-opening leaving N-methylethenamine and
one sulfanone oxygen atom. From m/z 293.0988 loss of fluorine atom
Fig. 11. DPD-VII line spectrum acquired in LC-MS/MS studies.
Fig. 12. DPD-VIII line spectrum acquired in LC-MS/MS studies.
P. Grover, M. Bhardwaj and L. Mehta Journal of Pharmaceutical and Biomedical Analysis 206 (2021) 114351
8
formed a new fragment owing m/z 275.0639. Then the removal of
fluorine from m/z 291.0622 gave the other ion with m/z 273.0939. Loss
of methylamine from fragment with m/z 275.0639 resulted in a new
molecular ion having m/z 248.0542. The elimination of benzene ring
from m/z 293.0988 and hydrogenation of double bond gave the mo￾lecular ion having m/z 233.0191. Further sulfanone oxygen atom and
methyl loss gave the ion having m/z 217.0299. With the above in￾formation, the structure of DPD-III could be characterized as N-(3-
(5-(2-aminopyrimidin-4-yl)-2-isopropylthiazol-4-yl)-2-fluorophenyl)
benzenesulfonamide ion.
3.4.4. DPD-IV (m/z 552.0982)
The DPD-IV depicted the mass value to be m/z 552.0990 as [M
+H]+ which was built from DBR by N-oxide formation at thiazole and
pyrimidine nitrogen as presented in LC–MS/MS line spectrum in
Fig. 8. Eight fragments were formed in total as shown in Fig. 16. The
first fragment formed due to the loss of N-oxides resulting back to m/
z 520.0682. Then thiazole substituted trimethyl were lost resulting
in a molecular ion having m/z 478.0614. From the ion with m/z
520.0682 loss of pyrimidine amine and thiazole substituted di￾methyl gave the molecular ionowing m/z 477.0514. Then the loss of
pyrimidine and thizole ring along with a fluorine atom forming
propene amine at benzene ring gave the ion having m/z 307.0640.
Elimination of sulfanone oxygen atom pyrimide and thiazole ring
from m/z 477.0514 gave the molecular ion having m/z300.0692, also
further loss of difluorine atom gave the ion owing m/z 264.0853.
Then from molecular ion owing m/z 307.0640 loss of methyl amine
resulted in molecular ion owing m/z 280.0643. Further elimination
of ethylbenzene gave a small fragment having m/z 176.8770. With
the above information, the structure of DPD-IV could be character￾ized as5-(2-amino-1-oxidopyrimidin-4-yl)-2-(tert-butyl)-4-(3-((2,6-
difluorophenyl)sulfonamido)-2-fluorophenyl)thiazole 3-oxide ion.
3.4.5. DPD-V (m/z 471.1056)
As represented in LC–MS/MS line spectral data in Fig. 9, the mass
of DPD-V was observed as m/z 471.0659 as [M+H]+ formed from DBR
by the loss of a fluorine atom, primary amine, a methyl group and
then the cyclization of pyrimidine ring with a benzene ring. The
fragmentation order of DPD-V is presented in Fig. 17. Further loss of a
fluorine atom gave the molecular ion having m/z 453.0527. Conver￾sion of phenyl sulfanedione into thiohydroxylamine, pyrimidine
ring-opening leaving amine and loss of a methyl gave the molecular
ion owing m/z 294.0810. Displacement of terminal amine with a
methyl group formed the fragment having m/z 293.0776. Loss of
phenyl sulfanone, pyrimidine ring and fluorine atom from fragment
having m/z 453.0527 gave a new fragment having m/z 291.0574.
From the ion with m/z 294.0810 removal of a fluorine atom resulted
in a molecular ion having m/z 276.0705. Again displacement of
amine with methyl group formed the ion with m/z 275.0628. Further
loss of ethyl and methyl group with the hydrogenation of thiazole
ring gave molecular ion having m/z 235.0686. The reduction of the
thiazole ring then formed the molecular ion having m/z 233.0165.
Two small fragments were formed due to the formation of N-(3-(1-
aminovinyl)phenyl) thiohydroxylammonium ion and 3-(1-amino￾vinyl)benzenaminium ion having m/z 167.0816 and 135.0467
respectively. With the above information, the structure of DPD-V
could be characterized as2-fluoro-N-(11-fluoro-2-isopropylbenzo[f]
thiazolo[4,5-h]quinazolin-10-yl)benzenesulfonamide ion.
3.4.6. DPD-VI (m/z 500.1021)
In Fig. 10, LC MS/MS line spectrum presented the mass of DP-VI as
m/z 500.1013[M+H] +
, formed by the cyclization of pyrimidine ring
with a benzene ring and loss of one fluorine atom resulting in m/z
500.1013, then this fragment dimerized to form new fragment
having m/z 999.1981. The plausible fragmentation order of DPD-VI is
represented in Fig. 18. The molecular ion owing m/z 323.1179 was
able 3
The LC-MS/MS data analysis of DPs (DPD I-DPD VIII) along with their major fragments and plausible molecular formulae.
Degradation
products (DPs)
Experimental mass Most probable molecular
formulae
Theoretical mass RDBa Major fragments (Chemical formulae)
DPD-I 307.0989 C15H16FN2O2S+ 307.0911 8.5 292.0802 ( C15H15FNO2S+), 292.0692 (C15H16FN2OS+), 277.0805 (C14H14FN2OS+), 250.0696 (C13H13FNOS+)
DPD-II 536.1025 C23H21F3N5O3S2+ 536.1032 14.5 483.0767 (C20H18F3N4O3S2
+), 342.0882 (C15H15F3N3OS+), 322.1020 (C15H17FN3O2S+), 307.0911 (C15H16FN2O2S+),
292.0802 ( C15H15FNO2S+)
DPD-III 470.0787 C22H21FN5O2S2+ 470.1115 14.5 293.0755 (C14H14FN2O2S+), 291.0962 (C15H16FN2OS+), 275.0849 (C14H15N2O2S+), 273.1056 (C15H17N2OS+), 248.0740
(C13H14NO2S+), 233.0755 (C9H14FN2O2S+), 217.0805(C14H14FN2O2S+)
DPD-IV 552.0953 C23H21F3N5O4S2+ 552.0982 14.5 520.1083 (C23H21F3N5O2S2
+), 478.0614 (C20H15F3N5O2S2
+), 477.0661(C21H16F3N4O2S2
+), 307.0911 (C15H16FN2O2S+),
300.0664 (C14H13F3NOS+), 280.0802 (C14H15FNO2S+), 264.0853 (C14H15FNOS+), 176.0176 (C6H7FNO2S+)
DPD-V 471.0621 C22H17F2N4O2S2+ 471.0756 15.5 453.0850 (C22H18FN4O2S2
+), 294.0529 (C13H13FN3S2
+), 293.0577 (C14H14FN2S2
+), 291.0620 (C14H15N2OS2
276.0624 (C13H14N3S2
+), 275.0671 (C14H15N2S2
+), 235.0358 (C11H11N2S2
+), 233.0202 (C11H9N2S2
+), 167.0637
(C8H11N2S+), 135.0917 (C8H11N2
DPD-VI 500.1013 C23H20F2N5O2S2+ 500.1021 15.5 323.0963 (C14H16FN4O2S+), 322.1020 (C15H17FN3O2S+), 306.1071 (C15H17FN3OS+), 290.1122 (C15H17FN3S+), 266.1322
(C13H20N3OS+), 240.1495 (C15H18N3
+), 208.1154 (C12H18NS+)
DPD-VII 358.1132 C15H15F3N3O2S+ 358.0832 8.5 328.0624 (C15H13F3NO2S+), 327.0773 (C15H14F3N2OS+), 321.1068 (C16H18FN2O2S+), 312.0664 (C15H13F3NOS+),
309.1068 (C15H18FN2O2S+), 307.0911 (C15H16FN2O2S+), 295.0911 (C14H16FN2O2S+), 292.0802 (C15H15FNO2S+),
277.0805 (C14H14FN2OS+), 271.1263 (C16H19N2S+), 218.0634 (C12H12NOS+), 212.0740 (C10H14NO2S+)
DPD-VIII 518.0913 C23H19F3N5O2S2+ 518.0927 15.5 341.1231 (C18H18FN4S+), 326.0428 (C13H13FN3O2S2
+), 314.0870 (C15H13FN5S+), 285.0605 (C14H10FN4S+), 1035.1781
(C46H37F6N10O4S4
built from DPD-VI by the loss of fluoro benzene and 2-(tert-butyl)
thiazole ring with cyclohexene ring opening. The molecular ion with
m/z 322.1109 was formed by the displacement of the amine group at
the pyrimidine ring with a methyl group. Further, the loss of a sul￾fonyl oxygen atom gave the molecular ion having m/z 306.0918. The
other oxygen atom loss from the sulfinyl group formed the fragment
with m/z 290.1387. Further elimination of methyl sulfane gave the
molecular ion having m/z 240.0450. The molecular ion with m/z
266.0476 formed from fragment having m/z 306.0918 by pyrimidine
ring-opening giving diamine substituted propene chain. Further, a
small fragment formed by the conversion of methyl sulfinyl into
thiohydroxylamine and loss of amine and methyl amine having m/z
208.0740. With the above information, the structure of DPD-VI could
be characterized as N-(5-amino-2-(tert-butyl)-11-fluorobenzo[f]
thiazolo[4,5-h]quinazolin-10-yl)-2-fluorobenzenesulfonamide ion.
3.4.7. DDP-VII (m/z 358.0832)
In Fig. 11, the LC-MS/MS line spectral data represented DPD-VII
having the m/z 358.1128 as [M+H] + which was observed due to the
elimination of pyrimidine and thiazole ring forming two primary
amines. The possible fragmentation order of DPD-VII is presented in
Fig. 19. The fragment with m/z 328.1146 was observed due to the loss
of diamine. The other molecular ion having m/z 327.1059 was built
from DPD-VII by the loss of sulfanone oxygen and an amine group.
The new ion with m/z 321.1035 was formed from DPD-VII by elim￾ination of difluorine and displacement of amine group with methyl.
From m/z 327.1059 another fragment was formed having m/z
312.0664 by loss of primary amine. From fragments with m/z
321.1035 elimination of methyl and hydrogenation of double bond
resulted in the molecular ion having m/z 309.1154. Then the reduc￾tion gave the other molecular ion owing m/z 307.0996. Further
elimination of methyl group, formed the fragment with m/z
295.1343. Again displacement of amine with a methyl group and
reduction to ethene gave the fragment owing m/z 292.0763. Another
fragment was formed from m/z 327.1059 by loss of difluorine atom
and ethyl group resulting in a molecular ion having m/z 277.0534.
The fragment with m/z 271.0439 formed from DPD-VII by elimina￾tion of fluorine atoms, sulfanone oxygen atoms also with the sub￾stitution of amine with a methyl group. From fragments with m/z
277.0534 loss of ethenamine and fluorine atom resulted in molecular
ion owing m/z 218.0304. The loss of difluorobenzene and another
fluorine atom from fragment 328.1146 gave a small fragment having
m/z 212.0608. With the above information, the structure of DPD-VII
could be characterized as N-(3-(1,3-diaminoprop-1-en-1-yl)-2-
fluorophenyl)-2,6-difluorobenzene sulfonamide ion.
3.4.8. DPD-VIII (m/z 518.0927)
The LC MS/MS line spectral data in Fig. 12 represented the mass
of DPD-VIII to be m/z 518.0926 as [M+H]+
, which was built by the
cyclization of the pyrimidine ring in dabrafenib. The fragmentation
order of DPD-VIII is presented in Fig. 20. The double charged DPD￾VIII formed the dimer ion having m/z 1035.1772. The molecular ion
owing m/z 341.1085 was formed by the elimination of difluoro
substituted benzene ring and substituted amine on pyrimidine ring.
The other molecular ion with m/z 314.0972 formed by the removal of
the isopropyl group and difluoro substituted benzene from dabra￾fenib. Further elimination of amine gave the fragment with m/z
285.0447. With the above information, the structure of DPD-VIII
Fig. 17. The probable ESI/MS/MS fragmentation order of protonated DPD-V.
P. Grover, M. Bhardwaj and L. Mehta Journal of Pharmaceutical and Biomedical Analysis 206 (2021) 114351
Fig. 18. The probable ESI/MS/MS fragmentation order of protonated DPD-VI.
P. Grover, M. Bhardwaj and L. Mehta Journal of Pharmaceutical and Biomedical Analysis 206 (2021) 114351
Fig. 19. The probable ESI/MS/MS fragmentation order of protonated DPD-VII.
P. Grover, M. Bhardwaj and L. Mehta Journal of Pharmaceutical and Biomedical Analysis 206 (2021) 114351
could be characterized asN-(5-amino-2-(tert-butyl)-11-fluorobenzo
[f]thiazolo[4,5-h]quinazolin-10-yl)-2,6-di￾fluorobenzenesulfonamide ion.
3.5. Degradation pathway of Dabrafenib
The most plausible degradation pathway of Dabrafenib under￾lined eight degradation products as shown in Fig. 21. Eight major
degradation products were obtained from DBR under forced de￾gradation study. The possible formation of DPs can be explained as
DPD-I was formed under photolytic degradation. Activation by light
results in the loss of di-fluorine atoms with the elimination of the
pyrimidine ring and also the opening of the thiazole ring resulting in
m/z 307.0989. With the action of peroxide, N-Oxide formation takes
place at thiazole nitrogen which gave DPD-II with m/z 536.1040.
Oxidative degradation then led to the removal of difluoro atoms and
methyl from DBR resulting in DPD-III with m/z 470.0816. Further N￾oxide formation took place at thiazole and pyrimidine nitrogen
under peroxide degradation which followed the formation of DPD-IV
having m/z 552.0990. Peroxide also caused the C-C bond formation
by cyclization of pyrimidine ring with a benzene ring and elim￾inating fluorine atom and amine moiety forming DPD-V with m/z
471.0659. Photolysis caused the addition of pyrimidine ring with
benzene ring resulting in cyclization and also the loss of fluorine
atom which gave DPD-VI having m/z 500.1013. Under oxidative de￾gradation, thiazole and pyrimidine ring elimination takes place with
the formation of two primary amines which ended in the formation
of DPD-VII with m/z 358.1128. Photolytic degradation also caused
cyclization with C-C bond formation between pyrimidine and ben￾zene ring that gave DPD-VIII having m/z 518.0926.
Fig. 20. The probable ESI/MS/MS fragmentation order of protonated DPD-VIII.
P. Grover, M. Bhardwaj and L. Mehta Journal of Pharmaceutical and Biomedical Analysis 206 (2021) 114351
4. Conclusion
Forced degradation (Oxidative, thermal, photolytic and hydro￾lytic) study of dabrafenib was performed as per the recommendation
of ICH guideline Q1 (R2). This study established the intrinsic stability
of drug substances. Dabrafenib was found to be highly sensitive in
presence of oxidative and photolytic degradation conditions
whereas it remained stable under hydrolytic (neutral, basic and
acidic) as well as thermal degradation conditions.
Eight new degradation products were obtained when the drug
substance was exposed to different stressed conditions. All these
degradation products were novel and never reported earlier. Out of
eight degradation products, five were obtained due to oxidative
degradation conditions and three major degradation products were
obtained due to photolytic exposure. All these degradation products
(DPD-I to DPD-VIII) were characterized with the help of the so￾phisticated technique of LC-MS/MS and accurate mass measurement
in ESI positive ionization mode. The current study explores the es￾tablishment of the degradation pathway of drug substance and
fragmentation pathway of drug substance as well as its DPs which
were never reported in any literature. The present study may also
use for the estimation of dabrafenib in bulk drug, formulation and
stability studies.
CRediT authorship contribution statement
Parul Grover: Conceptualization, Methodology, Writing − ori￾ginal draft preparation, Investigation, Validation. Monika Bhardwaj:
Resources, Supervision, Writing − original draft, Writing − review &
editing. Lovekesh Mehta: Data curation, Validation, Visualization,
Investigation.
Declaration of Competing Interest
The authors declare that they have no known competing fi￾nancial interests or personal relationships that could have appeared
to influence the work reported in this paper.
Fig. 21. Probable degradation pathway of dabrafenib.
P. Grover, M. Bhardwaj and L. Mehta Journal of Pharmaceutical and Biomedical Analysis 206 (2021) 114351
Acknowledgments
We are highly grateful to the Director Dr. (Col.) A. Garg and Joint
Director, Dr. Manoj Goel, KIET Group of Institutions and Dr. K.
Nagarajan, Principal, KIET School of Pharmacy, Ghaziabad for their
motivation, all-round support and arranging research facilities. We
are also very much thankful to MSN Laboratory, Hyderabad for
proving us with pure drug as a gift sample to carry out the research.
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