Isolation and structural characterization of degradation products of afatinib dimaleate by LC-Q-TOF/MS/MS and NMR: cytotoxicity evaluation of afatinib and isolated degradation products

Article abstract of DOI:10.1016/j.jpba.2019.01.004

Afatinib is an irreversible tyrosine kinase receptor inhibitor which was approved lately by USFDA for the treatment of metastatic non-small cell lung cancer (NSCLC). AFT was subjected to stress degradation studies under hydrolytic (acid, base and neutral), oxidative, thermal and photolytic conditions to investigate the inherent stability of the drug. The present study describes the simple and rapid HPLC method development for the selective separation of the AFT and its degradation products. The drug and degradation products were separated on Agilent Eclipse plus C18 (150 × 4.6 mm, 5 μ) column with ammonium acetate buffer (10 mM, pH 6.7) in gradient elution mode. The drug was found to be unstable in all the conditions studied. The developed chromatographic method was extended to tandem mass spectrometry (QTOF-MS) for the characterization of the degradation products. A total of 11 unknown degradation products were characterized using liquid chromatography quadrupole time-of-flight mass spectrometry (LC-Q-TOF/MS/MS). Two major degradation products (DP2 and DP3) were isolated using preparative HPLC and their structures were confirmed by conducting ¹H and ¹³C NMR experiments. The isolated DPs were evaluated for their anticancer potential using non small cell lung cancer cell line A549. The IC₅₀ values for AFT, DP2 and DP3 were found to be 15.02 ± 1.49, 25.00 ± 1.26 and 32.56 ± 0.11 respectively. The in silico toxicity studies were performed employing ProTox-II software for the assessment of toxicity potential of drug and its degradation products. Finally, the developed chromatographic method was validated as per the International Conference on Harmonization guideline Q2 (R1).

Full text of DOI:10.1016/j.jpba.2019.01.004

Journal of Pharmaceutical and Biomedical Analysis 166 (2019) 139–146
Contents lists available at ScienceDirect
Journal of Pharmaceutical and Biomedical Analysis
journal homepage: www.elsevier.com/locate/jpba
Isolation and structural characterization of degradation products of
afatinib dimaleate by LC-Q-TOF/MS/MS and NMR: cytotoxicity
evaluation of afatinib and isolated degradation products
Balasaheb B. Chavana, Sawant Vitthala, Roshan M. Borkarb, Ragampeta Srinivasa,b
,
M.V.N. Kumar Talluria,∗
a Department of Pharmaceutical Analysis, National Institute of Pharmaceutical Education & Research, IDPL R&D Campus, Balanagar, Hyderabad- 500 037.
India
b National Center for Mass Spectrometry, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500 607, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Afatinib is an irreversible tyrosine kinase receptor inhibitor which was approved lately by USFDA for
the treatment of metastatic non-small cell lung cancer (NSCLC). AFT was subjected to stress degradation
studies under hydrolytic (acid, base and neutral), oxidative, thermal and photolytic conditions to inves-
tigate the inherent stability of the drug. The present study describes the simple and rapid HPLC method
development for the selective separation of the AFT and its degradation products. The drug and degrada-
tion products were separated on Agilent Eclipse plus C18 (150 × 4.6 mm, 5 ␮) column with ammonium
acetate buffer (10 mM, pH 6.7) in gradient elution mode. The drug was found to be unstable in all the
conditions studied. The developed chromatographic method was extended to tandem mass spectrome-
try (QTOF-MS) for the characterization of the degradation products. A total of 11 unknown degradation
products were characterized using liquid chromatography quadrupole time-of-flight mass spectrome-
try (LC-Q-TOF/MS/MS). Two major degradation products (DP2 and DP3) were isolated using preparative
HPLC and their structures were confirmed by conducting 1H and 13C NMR experiments. The isolated DPs
were evaluated for their anticancer potential using non small cell lung cancer cell line A549. The IC50
values for AFT, DP2 and DP3 were found to be 15.02 1.49, 25.00 1.26 and 32.56 0.11 respectively.
The in silico toxicity studies were performed employing ProTox-II software for the assessment of toxic-
ity potential of drug and its degradation products. Finally, the developed chromatographic method was
validated as per the International Conference on Harmonization guideline Q2 (R1).
Received 24 August 2018
Received in revised form 1 January 2019
Accepted 5 January 2019
Available online 7 January 2019
Keywords:
Afatinib
Stress degradation
Degradation products
LC-Q-TOF/MS/MS
NMR
In silico toxicity
© 2019 Published by Elsevier B.V.
1. Introduction
and Drug Administration granted approval to AFT for a broadened
indication in first-line treatment of patients with metastatic non-
Afatinib diamaleate (AFT) ([N-[4-[(3-chloro-4-fluorophenyl)
small cell lung cancer (NSCLC) whose tumors have non-resistant
EGFR mutations as detected by an FDA-approved test. The most
common adverse reactions reported for AFT across clinical trials
are diarrhea, rash/acne dermatitis which varies with individuals
[3].
Stress stability studies of drug substances and drug products are
important part of drug development process and primarily used to
forecast drug stability. Stability studies can be useful in selection of
storage conditions, packaging materials and handling. In addition
these studies help to identify the degradation products, in turn aid
in establishing the degradation pathways and inherent stability of
the molecule. Thus, as per regulatory guidelines forced degrada-
tion studies and characterization of formed degradation products
(DPs) or impurities at or above 0.1% should be carried out in drug
substance [4,5].
amino]-7-[[(3S)-tetrahydro-3-furanyl]
4(dimethylamino)-2-butenamide])
oxy]-6-quinazolinyl]-
anilinoquinazoline
an
derivative is an irreversible tyrosine kinase receptor inhibitor
used in the therapy of metastatic non-small cell lung cancer
(NSCLC) which is the common type of lung cancer [1]. Afatinib
selectively and irreversibly binds and inhibits the epidermal
growth factor receptors and certain epidermal growth factor
receptor (EGFR) mutants which result in the inhibition of tumor
growth and angiogenesis in tumor cells [2]. Recently, the Food
∗
Corresponding author.
E-mail addresses: narendra.talluri@gmail.com, narendra@niperhyd.ac.in
(M.V.N.K. Talluri).
https://doi.org/10.1016/j.jpba.2019.01.004
0731-7085/© 2019 Published by Elsevier B.V.

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B.B. Chavan et al. / Journal of Pharmaceutical and Biomedical Analysis 166 (2019) 139–146
A thorough literature search revealed that, few reports are avail-
temperature control unit and photo diode array (PDA) detector.
Empower 3 software was used for the collection of data. The flow
rate of the mobile phase and injection volume were optimized at
1 mL min−1 and 5 ␮L, respectively. The column was equilibrated
with mobile phase for 10 min. prior to sample injection.
able in the literature about the liquid chromatographic tandem
mass spectrometric method for the determination of AFT in animals
[6] and humans [7]. Also, liquid chromatographic determination of
several tyrosine kinase inhibitors including AFT was reported using
diode array detection [8,9]. In addition, pharmacokinetic [10] and
metabolism studies [11] of the drug in humans has been reported.
Very few reports are present on determination of drug in bulk
and dosage forms [12]. To the best of our knowledge, there are
no reports which explain the stability indicating HPLC method
development and comprehensive forced degradation studies of
AFT using LC-Q-TOF/MS/MS and NMR. Over the last decade Liquid
chromatography-tandem mass spectrometry (LC–MS/MS) in com-
bination with high resolution and NMR has been widely utilized for
the identification and characterization of forced degradation prod-
ucts (DPs) [13–16]. The in vitro studies were carried out to check
the cytotoxicity of isolated DPs by MTT assay using A549 cell line.
The in silico toxicity studies is a useful tool for the prediction of
toxicities of the molecules. In the present study, the in silico toxic-
ity studies were carried out using ProTox-II software, to assess the
toxicological potential of the drug and DPs, which can be useful to
develop safer analogues.
LC–MS experiments were carried out on Agilent 1200 series
(Agilent technologies, USA) attached to quadruple-time of flight
(Q-TOF LC/MS 6540 series, Agilent Technologies, USA) where ESI
was used as ionization source in positive scan mode. Mass Hunter
Workstation software was used for data acquisition. Typical mass
operating source conditions were optimized as fragmentation volt-
age was 170 V, capillary voltage at 3500 V, capillary temperature
250 ◦C, skimmer voltage at 60 V. Nitrogen was used as the drying
(350 ◦C, 10 L min−1) and the nebulizing gas at pressure 45 psi.
The 1H and 13C NMR experiments were conducted using a
500 MHz NMR (AVANCE III HD 500, Bruker, Switzerland) spectrom-
eter. TMS and deuterated DMSO were used as an internal standard
and solvent for the NMR experiments, respectively. The chemical
shift values were reported on the ı scale in ppm.
2.3. Stress degradation study
Hence, the aim of present study was to investigate degrada-
tion behavior of the drug which includes: (i) to carry out stress
degradation studies (hydrolytic, oxidative, thermal and photolytic)
according to ICH prescribed guideline Q1A (R2) [4], (ii) develop-
ment of HPLC method for selective separation of drug and its DPs,
(iii) isolate the major DPs (DP2 and DP3) by preparative HPLC, (iv)
characterize the DPs using LC–MS/MS and NMR (v) in vitro toxic-
ity evaluation of DP2 and DP3 by MTT assay (vi) in silico toxicity
assessment of drug and its DPs using ProTox-II software and (vii)
validation of the developed HPLC method as per ICH guideline [17].
Stress degradation studies were carried out as per the ICH Q1 A
(R2) guideline. The degradation studies were carried out at 1000 ␮g
mL−1 concentration. The degradation samples were collected at
regular intervals to check the formation of DPs. Hydrolytic degra-
dation studies were carried out in acid, base and neutral conditions.
Alongside oxidative, thermal and photolytic stress degradation
studies were performed. The drug was found to be degraded under
hydrolytic, oxidative, photolytic and thermal condition. The acidic,
basic and neutral degradation was executed by reflux heating of the
AFT in 1 N HCl, 0.02 N NaOH and water at 80 ◦C, respectively. The
oxidation of the drug was investigated in 30% of H2O2 for 3 days.
Photolytic forced degradation studies were performed to investi-
gate the effect of UV and fluorescence light on the drug using a
photostability chamber (Newtronic life care sciences equipment
Pvt. Ltd. India) equipped with an illumination bank made of light
source as described in the ICH guideline Q1B [18]. The thermal
degradation studies were performed in a hot air oven (Oswarld sci-
entific Pvt. Ltd. India, model no. 00GS S-90) where the drug was
kept in the petri plate as a thin layer. The photostability studies
were conducted in solid as well as in liquid form in photostabil-
ity chamber by exposing total dose of 200 W h m-2 of UV light and
1.2 × 106 lux h of florescence light at 40 ◦C and 75%RH. Alongside set
of the control samples were stored in dark at the same temperature
to serve as control.
2. Experimental
2.1. Chemicals and reagents
AFT was obtained as a gratis sample from Sun Pharmaceuti-
cal Industries Ltd. Vadodara, Gujarat, India. LC–MS CHROMASOLV®
grade acetonitrile (ACN) were procured from Sigma- Aldrich
(Bangalore, India). Analytical reagent grade Ammonium acetate,
ammonium formate, formic acid, hydrochloric acid (HCl), sodium
hydroxide (NaOH) and acetic acid were purchased from SD Fine
Chemicals (Mumbai, India). Methanol (MeOH) and acetonitrile
(ACN) were procured from Sigma-Aldrich (Bangalore, India). AR
grade hydrogen peroxide (H2O2) was purchased from Merck
(Mumbai, India). HPLC grade water was used for the preparation of
all the samples and buffer which was obtained from Evoqua Inte-
grated Ultra Care TW GP Water purification system (United States).
Tetramethylsilane (TMS) and DMSO were purchased from Sigma-
Aldrich (Bangalore, India) and Cambridge Isotope Laboratories, Inc.
Andover (MA, USA), respectively. The (3-(4,5-Dimethylthiazol-2-
Yl)-2,5-Diphenyltetrazolium Bromide (MTT) reagent was obtained
from Hi Media (Hyderabad, India). Non small cell lung cancer cell
line A549 line was procured from NCCS (Pune, India).
2.4. Sample preparation
Stock solution of AFT (1000 ␮g mL−1) was prepared by dissolv-
ing the drug in diluent (water: ACN 50:50 v/v). The stock solution
was further diluted for the preparation working standard (200 ␮g
mL−1). Acidic and basic samples were neutralized with appropriate
strengths of base and acid respectively before injecting the sample.
All the degradation samples were filtered through 0.22 ␮m nylon
membrane syringe filter before subjected to HPLC and LC–MS/MS
analysis and analyzed at 200 ␮g mL−1 concentrations.
2.2. LC separation of AFT and DPs
2.5. Isolation of DPs by preparative HPLC
Chromatographic separation of AFT and DPs were achieved on
Agilent Eclipse plus C18 (150 × 4.6 mm, 5 ␮) column with 10 mM
ammonium acetate (pH 6.7) as solvent A and ACN as solvent B using
the following linear gradient: 0 min 10%B; 5 min 50%B, 7 min 80%B,
10 min 80%B, 11 min 10%B and 12 min 10% B using a Waters 2695
series HPLC system (M.A, USA) equipped with auto sampler, quater-
nary gradient pump, in-line degasser, column compartment with
The developed chromatographic method was transferred from
analytical HPLC to preparative HPLC for isolation of the DPs. The
preparative isolation of the major degradation products was car-
ried out on a preparative HPLC from Waters (Milford, MA, USA)
equipped with 515 HPLC pumps and 2489 UV–vis detectors. The
separation and isolation of the DPs (DP2 and DP3) was carried out

B.B. Chavan et al. / Journal of Pharmaceutical and Biomedical Analysis 166 (2019) 139–146
141
(Table S1). The precision of the method was studied as repeatabil-
ity and inter day precision. Repeatability is expressed over short
interval of time and also termed as intraday precision. The repeata-
bility of the drug was determined at 100% of the test concentration
(200 ␮g mL−1) using six determinations and assay value obtained
was expressed in terms of % RSD. The % RSD value was found to
be less than 1%, indicating repeatability of the method. The inter
day precision expresses inter day variation on different days and
evaluated for three consecutive days at the assay concentration
(200 ␮g mL−1). The results of the study were expressed as % RSD
values and at each day RSD values were below 2%, indicate preci-
sion of the method (Table S2). The robustness of the method was
determined by deliberate change in certain method parameters like
pH of mobile phase (6.7 0.2), flow rate (1.0 0.2 mL min−1), col-
umn temperature (25 5 ◦C). No significant change was found in
the assay value of the AFT which indicates the robustness of the
method.
on a Waters X bridge C18 column (250 × 19 mm × 5 ␮m, 130 Å).
The mobile phase used was solvent-A ammonium acetate (pH 6.7;
10 mM) with solvent-B ACN as organic solvent in gradient elution
mode set as 0/10, 6/50, 9/80, 13/80, 15/10, 16/10, 18/10 (Time
min/% solvent B). The flow rate and injection volume was 10 mL
min−1 and 1 mL, respectively. The wavelength for identification of
DPs was fixed at 258 nm. The DP2 and DP3 were isolated for struc-
tural confirmation by NMR. The drug (1000 mg mL−1) was allowed
to degrade in 0.5 N NaOH and 3 N HCl solution for 4 h to get a
higher percentage of the DP2 and DP3, respectively. The degraded
solutions of acidic and basic degradation were neutralized with
the appropriate concentrations of NaOH and HCl, respectively. The
fraction of DPs were collected by multiple injections into prepara-
tive HPLC and pooled together. Pooled fractions were concentrated
at 35 ◦C using a rotary evaporator to remove ACN and further
lyophilized to get the solid compound. The isolated DPs were found
to be pure with chromatographic purity >98%. The solid form of the
DPs was dissolved in DMSO and submitted for NMR analysis.
3.2. Stability indicating method of AFT
2.6. In vitro cell viability assay
AFT was studied and found to be susceptible under all stud-
ied forced degradation conditions (hydrolytic, oxidative, photolytic
and thermal) with optimized conditions reported in Table S3. A
total of 11 DPs were formed under the various stress conditions
and were separated chromatographically (Fig. 1) and were charac-
terized by ESI/QTOF MS/MS, with the proposed structures shown
in Scheme 1 and 2. Extracted UV spectra of drug and DPs are shown
in Fig. S1 and Fig. S2.
The effect of AFT and its DPs (DP2 and DP3) on viability of non
small cell lung cancer cell line A549 was evaluated using MTT assay.
Briefly, 5 × 103 cells per well were seeded in 96-well plate. After
24 h, cells were treated with AFT, DP2 and DP3 at suitable con-
centrations in respective wells (highest concentration 40␮M). Post
treatment (48 h), media was aspirated and 100 ␮L of MTT solu-
tion was added at a final concentration of 0.5 mg mL−1 followed by
incubation for 4 h. Then, MTT solution was discarded and 200 ␮L of
DMSO was added to dissolve the formazan crystals and incubated
for 30 min. Absorbance was measured at 570 nm with a multi-mode
spectrophotometer (Molecular devices, USA). Experiments were
performed in triplicates and data was expressed as percentage cell
viability versus concentration of the compound by taking control
cells as 100% viable cells.
3.2.1. Hydrolytic degradation
Acid hydrolysis was performed using 1 N HCl (4 h at 80 ◦C) where
optimum degradation of the AFT was found (DP1, DP2 and DP3)
(Fig. 1b). Excess degradation was observed when AFT was refluxed
with 1 N NaOH. At 0.02 N NaOH for 4 h at 80 ◦C, DP2, DP3 and DP4
were formed (Fig. 1c). For neutral degradation study, the drug was
refluxed for 24 h at 80 ◦C in diluent. The drug was degraded to form
four DPs (DP1, DP2, DP3 and DP5) (Fig. 1d).
3. Results and discussion
3.1. Validation of the HPLC method
3.2.2. Oxidative degradation
The oxidative degradation study results in the formation of DP1
and DP6 in 30% H2O2 after 3 days (Fig. 1e).
The developed HPLC method for AFT was validated according
to the ICH recommended guidelines Q2 (R1). The specificity of the
method was evaluated by the purity angle and purity threshold
values of the peaks by PDA detector. The peak purity assessment
shows that purity angle of AFT was found to be less than the purity
threshold in presence of all the DPs suggests the purity of the peak.
The limit of detection (LOD) and limit of quantification (LOQ) indi-
cates the concentration of the drug where signal to noise (S/N) ratio
would be 3 and 10, respectively. The LOD and LOQ for AFT were
found to be 3 and 7 ␮g mL−1. The linearity of the developed method
was determined by plotting calibration curve using standard solu-
tion of the drug at eight different concentrations (triplicate) in the
range of 10–350 ␮g mL−1. Standard calibration curve were plot-
ted by taking average peak area of each concentration. The data
was subjected to statistical analysis using linear regression model.
The linear regression equation and correlation coefficient (r2) were
y = 11352x + 54,857 and 0.999, respectively. The data shows good
linearity. Accuracy of the developed HPLC method was determined
by standard addition method. Standard drug (AFT) at three dif-
ferent concentration levels (80, 100 and 120%) were added into
the sample solutions and analyzed in triplicate (n = 3). The per-
centage recovery of the drug was calculated by comparison of the
peak area values of test samples with standard drug solutions. The
percentage recovery rang and relative standard deviation (RSD) val-
ues were found to be 99.58–100.48% and within 1%, respectively
3.2.3. Thermal and photolytic degradation
AFT was found to be degraded after 2 days at 80 ◦C and showed
formation of DP2, DP5 and DP7 (Fig. 1f). Photolytic degradation
of AFT was observed in solid state with four DPs (DP2, DP8, DP9
and DP10) and liquid state with three DPs (DP2, DP9 and DP11)
(Fig. 1g-j).
3.3. LC–ESI-Q-TOF/MS/MS and NMR studies of AFT dimaleate and
its DPs
Structural elucidation of all the DPs formed under various stress
conditions was carried out with the help of LC/ESI-Q-TOF-MS/MS
experiments. The most probable structures for the DPs were pro-
posed based on the MS/MS fragmentation pattern of the drug and
DPs. The elemental composition of the AFT and protonated DPs
along with product ions formed are provided in Table 1. The ele-
mental composition of all the product ions of drug and DPs along
with the mass error has been provided in the Table S4. The MS/MS
fragmentation patterns for the drug and degradation products are
given in Fig. S3-S5. Further, major DPs (DP2 and DP3) were isolated
for the structural confirmation by NMR studies.

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B.B. Chavan et al. / Journal of Pharmaceutical and Biomedical Analysis 166 (2019) 139–146
Fig. 1. The overlay of HPLC chromatogram of (a) AFT (b) acidic (c) basic (d) neutral (e) oxidation (f) thermal (g) UV solid (h) UV liquid (i) fluorescence solid (j) fluorescence
liquid conditions.
Table 1
Elemental composition of afatinib (AFT) and its degradation products (DP1-DP11).
AFT and
its DPs
Molecular
Formula
Calculated
m/z
Observed
m/z
Error
(ppm)
MS/MS fragment ions
+
AFT
C24H26ClFN5O3
C18H23N4O4
486.1703
359.1714
459.1230
375.1019
504.1808
602.1812
484.1746
441.1124
475.1179
486.1703
403.0968
457.1073
486.1700
359.1710
459.1220
375.1011
504.1801
602.1811
484.1749
441.1124
475.1184
486.1701
403.0969
457.1077
−0.62
−1.11
−2.18
−2.13
−1.39
−0.17
0.62
0.00
1.05
−0.41
−0.25
0.88
441, 429, 416, 398, 375, 371, 359, 343, 331, 317, 305, 288, 226, 213, 112, 94, 84, 71, 58
314, 289, 244, 216, 178, 112, 94, 84, 71
441, 389, 371, 343, 305, 71
305, 288, 277, 269, 261, 250, 160, 135, 108, 71
486, 459, 441, 389, 375, 371, 305, 130, 112, 94,84
558, 532, 514, 487, 421, 377, 112, 94, 84, 71
439, 414, 396, 369, 341, 329, 303, 112, 94, 84
371, 343, 304, 288, 71
+
DP1
DP2
DP3
DP4
DP5
DP6
DP7
DP8
DP9
DP10
DP11
+
+
+
+
C22H21ClFN4O4
C18H17ClFN4O2
C24H28ClFN5O4
C28H30ClFN5O7
+
C24H27ClN5O4
+
+
+
+
+
C22H19ClFN4O3
C22H21ClFN4O5
C24H26ClFN5O3
C19H17ClFN4O3
C22H19ClFN4O4
405, 387, 375, 359, 341, 305, 71
441, 429, 416, 398, 375, 371, 359, 343, 331, 317, 305, 288, 226, 213, 112, 94, 84, 71, 58
333, 315, 305, 160
387, 369, 359, 341, 272, 242, 71
3.3.1. MS/MS and NMR of AFT dimaleate and DP9
ions at m/z 416 and m/z 359 with the loss of C4H6O from m/z 486
and m/z 429, respectively confirms the presence of tetrahydrofuran
moiety in the drug and DP9. The formation of prominent fragment
ion at m/z 112 indicates the presence of dimethylamino but-2-enal
moiety. Further, structure specific fragment ion at m/z 441 formed
from AFT/DP9 indicates the presence of terminal dimethylamine
group (Scheme S1). The formation of product ion at m/z 441 can
be explained by ‘H’ migration from the methylene group to the ter-
minal nitrogen followed by dimethylamine loss. The formation of
characteristic fragment ions similar to the drug confirms that DP9
to be Z-isomer of AFT. The probable mechanism for the formation of
The MS/MS data of [M+H]+ ions of AFT and DP9 with an ele-
+
mental composition of C24H26ClFN5O3 and accurate mass m/z
486.1701 displays similar fragmentation pattern. The DP9 was
observed in photolytic conditions and believed to be the Z-isomer
of the drug. The ESI-MS/MS spectrum of [M+H]+ ion of proto-
nated AFT (Fig. S3a) (retention time (tR) = 7.4 min.) and DP9 (Fig.
S5b) (tR = 7.0 min.) with m/z 486 displays characteristic high abun-
dant fragment ion at m/z 371 and m/z 305 formed by the loss of
tetrahydrofuran (from m/z 441) and (E)-4-(dimethylamino) but-
2-enal (from m/z 416), respectively. Further, formation of product

B.B. Chavan et al. / Journal of Pharmaceutical and Biomedical Analysis 166 (2019) 139–146
143
Scheme 1. Probable mechanism for the formation of DP8-DP11.
Scheme 2. Probable mechanism for the formation of DP1-DP7.

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B.B. Chavan et al. / Journal of Pharmaceutical and Biomedical Analysis 166 (2019) 139–146
Table 2
1H and 13C NMR assignments for AFT dimaleate and its DPs.
Position
Afatinib dimaleate
DP2
DP3
␦
(ppm), Multiplicity
␦
(ppm)
␦
–
(ppm), Multiplicity
␦
–
29.2
174.13
83.88
162.06
–
(ppm)
␦
–
–
–
–
–
(ppm), Multiplicity
␦
–
–
–
–
–
–
(ppm)
H
C
H
C
H
C
1/3
4
5
6
7
9
2.83 (s, 6 H)
3.84 (m, 2 H)
6.81 (m, 1 H)
6.81 (m, 1 H)
42.70
57.43
136.99
132.19
162.90
–
2.39 (m, 3 H)
–
6.33 (m, 1 H)
–
–
10.02 (s, 1 H)
–
5.38 (s, 2 H)
10
11
12
13
–
133.72
108.01
117.36
157.52
–
–
137.10
109.34
123.96
157.39
–
–
139.36
101.56
110.96
155.51
–
8.59 (s, 1 H)
–
–
8.39 (s, 1 H)
–
–
7.80 (m, 1 H)
–
–
14
9.77 (s, 1 H)
9.89 (s, 1 H)
9.39 (s, 1 H)
15
16
17
19
20
21
24
26
27
28
30
31
33
34
35
–
116.98
124.42
127.75
119.26
123.24
152.96
154.15
148.30
109.21
154.90
79.48
72.48
67.07
32.08
–
–
116.97
125.71
127.22
119.23
122.76
152.78
154.71
151.49
109.44
155.37
78.76
72.66
66.97
32.93
–
–
116.87
122.97
138.02
119.10
121.96
150.87
152.20
145.08
107.87
154.15
78.53
72.62
67.03
32.97
–
7.28 (s, 1 H)
–
7.31 (d, 1 H)
–
7.05 (s, 1 H)
–
7.40 (d, 1 H)
7.38 (d, 1 H)
7.78 (m, 1 H)
7.45 (t, 1 H)
–
8.96 (s, 1 H)
–
7.84 (m, 1 H)
7.42 (t, 1 H)
–
8.60 (s, 1 H)
–
–
8.37 (s, 1 H)
–
8.19 (dd, 1 H)
–
5.24 (m, 1 H)
3.90 (m, 2 H)
3.90 (m, 2 H)
2.23 (m, 2 H)
8.11 (dd, 1 H)
–
8.19 (dd, 1 H)
–
5.32 (m, 1 H)
3.99 (m, 2 H)
3.99 (m, 2 H)
2.25 (m, 2 H)
–
5.30 (m, 1 H)
3.82 (m, 2 H)
3.82 (m, 2 H)
2.28 (m, 2 H)
3.98 (m, 1 H)
–
–
–
–
36/39 (dimaleate)
37/38(dimaleate)
–
167.44
133.87
–
–
–
–
6.15 (s, 4 H)
–
s = singlet; d = doublet; t = triplet; m = multiplet; dd = doublet of doublets.
DP9 under photolytic conditions is shown in Scheme 1 [19]. The 1
H
(Fig. S8) and carbon (Fig. S9) NMR spectrum of DP2 proved that
DP2 was devoid of dimethylamine moiety (Table 2). Further, the
presence of 1H NMR signal of DP2 for the proton at H35 and absence
of 1H NMR signal for the proton at H5 when compared with the
drug, confirmed that the DP2 was formed by the hydroxylation at
position 5. Additionally, the 13C spectrum of DP2 shows downfield
and upfield shift for the carbon at C5 and C6, respectively which
demonstrates the hydroxylation at position 5. Based on all these
data the structure for the DP2 was identified as shown in Scheme S3.
and 13C NMR spectra of AFT dimaleate were given in Fig. S6 and S7,
respectively (supplementary information). The NMR signals that
are structure specific for the AFT dimaleate includes the presence
of (E)-4-(dimethylamino)but-2-enamide (atom no. 1, 3, 4, 5, 6, 7
and 9), tetrahydrofuran (atom no. 30, 31, 33 and 34) and dimaleate
(atom no. 36, 37, 38 and 39) signals (Table 2). The 1H and 13C NMR
signals of these groups were utilized for the identification of the
major DPs (DP2 and DP3).
3.3.2.3. DP3 (m/z 375). ESI/MS/MS spectrum (Fig. S3d) of DP3
showed the characteristic loss of tetrahydrofuran (m/z 70) which
leads to formation of structure specific fragment ion at m/z 305.
The product ions observed are reported in Scheme S1. The forma-
tion of DP3 in hydrolytic conditions can be explained by hydrolytic
cleavage of amide bond [20] as shown in Scheme 2. The struc-
ture of the DP3 was further established by accurate comparison
of NMR signals of AFT and DP3. The NMR study of DP3 showed
absence of peaks which correspond to (E)-4-(dimethylamino)but-
2-enal in both 1H (Fig. S10) and 13C NMR (Fig. S11) spectrum
compared to drug (Table 2). All these data indicated the structure of
DP3 to be N4-(3-chloro-4-fluorophenyl)-7-((tetrahydrofuran-3-yl)
oxy) quinazoline-4,6-diamine.
3.3.2. MS/MS and NMR of degradation products
3.3.2.1. DP1 (m/z 359). The ESI/MS/MS spectrum of DP1 (Fig. S3b)
displays structure indicative product ions at m/z 314 and m/z 244
and absence of drug specific fragment ions (m/z 441, m/z 371 and
m/z 305). Also, formation of fragment ions at m/z 112 and m/z 71
indicate that (E)-4-(dimethylamino) but-2-enal and tetrahydrofu-
ran moieties are intact. This clearly suggests the structure of DP1
(Scheme S2). The proposed mechanism for the formation of DP1-
DP7 was shown in Scheme 2.
3.3.2.2. DP2 (m/z 459). ESI/MS/MS spectrum of major hydrolytic
degradation product DP2 (Fig. S3c) showed the presence of product
ion at m/z 305 (as in AFT) and the absence of m/z 112 indicated
modification of the side chain (Scheme S3). To confirm the proposed
structure of the DP2, NMR (1H and 13C) studies were carried out. The
absence of NMR signals for the dimethylamine group in both proton
3.3.2.4. DP4 (m/z 504). The ESI/MS/MS spectrum of DP4 (Fig. S4a)
showed abundant precursor ion peak at m/z 504.1801. Further it

B.B. Chavan et al. / Journal of Pharmaceutical and Biomedical Analysis 166 (2019) 139–146
145
shows loss of water molecule (DP4 to m/z 486 and m/z 130 to m/z
112) and fragment ion at m/z 130 which suggests hydroxylation
of the AFT at (E)-4-(dimethylamino)-1-oxobut-2-en-1-ylium side
chain. (Scheme S3).
3.3.2.5. DP5 (m/z 602). The ESI/MS/MS spectrum of DP5 (Fig. S4b)
showed formation of distinctive product ions including the loss of
tetrahydrofuran from DP5, the loss of dimethylamine and C6H9NO
from m/z 532 and formation of m/z 112 which were in correlation
to the product ions formed in the MS/MS of the AFT. All this data
demonstrate that DP5 was an adduct of maleic acid with the drug
(Scheme S4).
Fig. 2. Cytotoxicity studies of AFT, DP2 and DP3 on non small cell lung cancer cell
line A549.
3.3.2.6. DP6 (m/z 484). The molecular formula of the oxidative DP6
(C24H27ClN5O4+) indicated an addition of oxygen with the loss of
fluorine atom as compared to drug. The ESI/MS/MS spectrum of
DP6 (Fig. S4c) showed product ions that correlate to product ions
observed in the MS/MS of the AFT. The formation of the ion at m/z
112 indicates that (E)-4-(dimethylamino) but-2-enal moiety was
retained (Scheme S5).
icity, organ toxicity (hepatotoxicity), toxicological endpoints (such
as mutagenicity, carcinotoxicity, cytotoxicity and immunotoxicity),
toxicological pathways and toxicity targets were incorporated. Dif-
ferent levels of toxicity target helps to provide insight into the most
likely possible molecular mechanism behind such toxic response.
The predicted outcomes for each target will show by the confi-
dence score. The score below 70% (0.7) is omitted whereas above
it where considered as active to the input molecule [21]. The pre-
dicted toxicity of different target for AFTand its DPs are shown in
the Table S5 (A–B). The predicted toxic dose (LD50) for all the DPs
are in the range of 500–1500 mg kg−1 and falls under toxicity class
IV, toxic. The toxicological endpoint, immunotoxicity, for DP2 to
DP11 is predicted to be active with confidence score >90% whereas
confidence score for hepatotoxicity, carcinogenicity, mutagenicity,
cytotoxicity and toxicological pathways were <70%. AFT and its
DPs showed no binding probability for both toxicological pathways
(AhR, AR, AR-LBD, ER, ER-LBD, aromatase, PPAR-Gamma, nrf2/ARE,
HSE, MMP, p53, ATAD5) and toxicological targets (Adenosine recep-
tor A2a, Beta-2 adrenergic receptor, Androgen receptor, Amine
oxidase A, Corticotropin-releasing factor receptor 1, D(3) dopamine
receptor, Estrogen receptor, Estrogen receptor beta, Glucocorti-
coid receptor,Histamine H1 receptor, Nuclear receptor subfamily
1 group, Kappa-type opioid receptor, Mu-type opioid receptor,
cAMP-specific 3, Prostaglandin G/H synthase 1, Nuclear receptor
subfamily 3 group C member 3. Thus ProTox-II predicts that there
is a possibility that the AFTand its DPs (DP2 and DP11) can be active
for immunotoxicity and thereby resulting in severe toxic effect.
3.3.2.7. DP7 (m/z 441). The degradation product DP7 was formed
by the loss of dimethylamine from AFT. The MS/MS spectrum of
DP7 (Fig. S4d) showed the formation of characteristic product ions
as observed in MS/MS spectrum of AFT (Scheme S1).
3.3.2.8. DP8 (m/z 475). The molecular formula of DP8
(C22H21ClFN4O5+) indicates an addition of two oxygen atoms
with the loss of dimethylamine moiety as compared to the drug.
The presence of fragment ion at m/z 375 and absence of charac-
teristic fragment ion of m/z 112 in the MS/MS spectrum (Fig. S5a)
of DP8 provide clear evidence of hydroxylation of the drug at the
side chain [(E)-4-(dimethylamino) but-2-enal] (Scheme S6). The
probable mechanism for the formation of DP8-DP11 was shown in
Scheme 1.
3.3.2.9. DP10 (m/z 403). The ESI/MS/MS spectrum of DP10 (Fig.
S5c) displayed characteristic loss of tetrahydrofuran (m/z 403 to
m/z 333), followed by water loss and CO loss from m/z 333. The loss
of CO (m/z 333 to m/z 305) suggests the presence of aldehyde group
in the structure of DP10 and the absence of product ion at m/z 112
indicates that DP10 was formed by the loss of N,N-dimethylprop-
2-en-1-amine side chain (Scheme S7).
3.3.2.10. DP11 (m/z 457). The ESI/MS/MS spectrum of DP11
(C22H19ClFN4O4+) (Fig. S5d) showed presence of structure indica-
tive product ions and the absence of m/z 112 which clearly suggests
that DP11 could be formed by the loss of dimethylamine followed
by oxidation (Scheme S6).
4. Conclusion
A selective stability indicating HPLC method was developed to
study the degradation behavior of AFT under hydrolytic, oxida-
tive, thermal and photolytic conditions. Stress degradation studies
were carried out by examining the factors suggested by ICH guide-
lines. The drug was found to be degraded in all above studied
conditions and results in formation of 11 new hitherto degrada-
tion products. All the DPs were identified and characterized using
LC-Q-TOF/MS/MS along with molecular formula generation. Struc-
tural confirmation was provided for the major DPs (DP2 and DP3)
using NMR experiments (1H and 13C). The mechanistic explana-
tion was provided for the formation of each DP. The MTT assay was
performed to evaluate the cytotoxicity of the isolated DPs using
A549 cell line. Additionally, in silico toxicity studies were performed
with ProTox-II tool where toxicological endpoint, immunotoxicity
of DP2 and DP11 were predicted with high probability score. The
developed HPLC method was validated in terms of specificity, lin-
earity, accuracy, precision and robustness. The developed HPLC and
LC–MS/MS method can be useful for quality control analysis and
detection and characterization of degradation impurities.
3.4. In vitro cell viability assay
The DPs (DP2 and DP3) of AFT were evaluated for their cytotoxi-
city on non small cell lung cancer cell line A549. AFT showed 88.8%
inhibition of cell growth, DP2 exerted cytotoxicity by inhibiting
78.3% of the cell growth and DP3 was found to inhibit cell growth
by 62.7% in A549 cells at highest screened concentration of 40␮M.
IC50 values of AFT, DP2 and DP3 were found to be 15.02 1.49,
25.00 1.26 and 32.56 0.11 respectively on A549 cells (Fig. 2)
which demonstrates decrease in cytotoxic activity of DPs on A549
cell line as compared to the AFT.
3.5. In silico toxicity studies
The potential toxicity of AFT and its stress DPs were predicted by
ProTox-II tool. Different levels of toxicity endpoints such as oral tox-

146
B.B. Chavan et al. / Journal of Pharmaceutical and Biomedical Analysis 166 (2019) 139–146
[8] M. Fouad, M. Helvenstein, B. Blankert, Ultra high performance liquid
Acknowledgements
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The authors thank Dr. Shashi Bala Singh, Director, NIPER-
Hyderabad, India for facilities and encouragement. Authors BC and
SV are grateful to Sun Pharmaceutical Industries Ltd. Vadodara,
India for the gift sample of the Afatinib dimaleate. The authors are
also thankful to the Department of Pharmaceuticals, Ministry of
Chemicals and Fertilizer, New Delhi, India, for providing a Research
Fellowship.
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Appendix A. Supplementary data
Supplementary material related to this article can be found,
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