Journal of Pharmaceutical and Biomedical Analysis 99 (2014) 22–27

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Journal of Pharmaceutical and Biomedical Analysis

journal homepage: www.elsevier.com/locate/jpba

Isolation, identification and structure elucidation of two novel

process-related impurities of retigabine
Dengfeng Zhang, Xin Song, Jiangtao Su ∗
Department of Pharmacy, Food and Pharmaceutical Engineering College, Hubei University of Technology, Wuhan, Hubei 430068, China

a r t i c l e i n f o a b s t r a c t

Article history: Retigabine was the first neuronal potassium channel opener for the treatment of epilepsy. During the
Received 11 April 2014 manufacture of retigabine, two unknown impurities were present in laboratory batches in the range of
Received in revised form 12 June 2014 0.05–0.1% based upon HPLC analysis. These unknown impurities were obtained from the enriched reac-
Accepted 14 June 2014
tion mother liquor by column chromatography. The structure of these process-related impurities were
Available online 5 July 2014
elucidated using FT-IR, 1 H NMR, 13 C NMR, 2D NMR (HSQC, HMBC, NOESY) and MS spectral data. Based on
the complete spectral analysis and knowledge of the synthetic route of retigabine, these two new impu-
Keywords:
rities were designated as ethyl 4-fluorobenzyl(2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-yl)carbamate
Retigabine
Impurity
(impurity-II) and diethyl 5-((ethoxycarbonyl)(4-fluorobenzyl)amino)-2-oxo-1H-benzo[d]imidazole-
Isolation 1,3(2H)-dicarboxylate (impurity-III). Impurity identification, structure elucidation and the formation of
Identification impurities were also discussed.
Characterization © 2014 Elsevier B.V. All rights reserved.

1. Introduction mass analyzer (LC-ESI-Q-TOF-MS) [10]. That four process-related

Retigabine (D-23129, N-(2-amino-4-(4-fluorobenzlamino)-
phenyl) caramic acid ethyl ester) was discovered in a series of
desaza analogs of flupirtine. Retigabine (INN) or ezogabine (USAN),
is an anticonvulsant used as a treatment for partial epilepsies.
The drug was developed by Valeant Pharmaceuticals and Glaxo-
SmithKline. It was approved by the European Medicines Agency
under the trade name Trobalt on March 28, 2011, and by the
United States Food and Drug Administration (FDA), under the
trade name Potiga, on June 10, 2010. Retigabine works primarily
as a potassium channel opener – that is, by activating a certain
family of voltage-gated potassium channels in the brain [1–3].
Retigabine is a close structural analog of the centrally-acting
analgesic flupirtine (Katadolon® , D-9998; 2-amino-6-(4-fluoro-
benzylamino)-pyridin-3-yl-carbamic acid ethyl ester). The key
synthetic steps (Fig. 1) involved initial reduction of nitrobenzene
(compound 2) via catalytic hydrogenation followed by acylation
with ethyl chloroformate to give retigabine/ezogabine [4,5]. LC-UV
[6] and LC–MS/MS [7–9] methods were developed and reported for
the determination of retigabine and its N-acetyl or N-glucuronide
metabolites in biological samples. Four process-related impurities
were identified by liquid chromatography–tandem mass spectrom-
etry using electrospray ionization and quadrupole time-of-flight
∗ Corresponding author. Tel.: +86 13871056239.
E-mail address: jiangtsu@126.com (J. Su).
impurities were disclosed in samples of commercial retigabine
active pharmaceutical ingredient (API) which was prepared using
another synthetic route [10]. An experimentally validated HPLC
method was optimized in order to separate, selectively detect and
quantify dimer impurities of retigabine [11].
During the manufacture of retigabine, two unknown impuri-
ties were present in different batches in the range of 0.05–0.1%
based upon HPLC analysis. The synthetic route was wildly
reported and applied in laboratory sample preparation and
commercial production for its advantages of low-cost materi-
als, simple preparation process, and in good yield [4,5]. In our
study, we found that the key synthetic steps (the last two
steps of this route, Fig. 1) were directly related to the for-
mation of the two novel process-related impurities. These two
unknown impurities and three known impurities (process-related
impurities and degradation products: impurity-I, ring-closed
product, 5-(4-fluoro-benzylamino)-1,3-dihydro-benzimidazol-2-
one [12]; impurity-IV, dimer impurity, diethyl{4,4 -diamino-6,6 -
bis[(4-fluorobenzyl)amino]biphenyl-3,3 -diyl}biscarbamate [11];
impurity-V, diacylation product, diethyl-4-(4-fluorobenzylamino)-
l,2-phenylenedicarbamate [13]) still existed in the enriched
reaction mixture in the range of 0.1–10% respectively (Fig. 2b).
The stringent purity requirement [14] that all the individual
impurities, which are ≥0.1%, must be identified and character-
ized. This paper aims at the identification and characterization of
two prominent process-related impurities that are present at a
level of 0.05–0.1% in the bulk drug of retigabine. To the best of

http://dx.doi.org/10.1016/j.jpba.2014.06.023
0731-7085/© 2014 Elsevier B.V. All rights reserved.
 D. Zhang et al. / Journal of Pharmaceutical and Biomedical Analysis 99 (2014) 22–27 23

Fig. 1. The key synthetic steps of retigabine.

Fig. 2. HPLC chromatogram of (a) retigabine bulk drug, (b) reaction mixture and (c) impurities mixture.
24 D. Zhang et al. / Journal of Pharmaceutical and Biomedical Analysis 99 (2014) 22–27
our knowledge, impurity-II, impurity-III and structure elucidation
of impurity-I are first reported in this paper. Hence, this study
will provide useful reference information for a large number of
organic chemists from pharmaceutical companies and drug reg-
ulatory authorities.
2. Experimental
2.1. Samples, chemicals and reagents
The samples of different batches of retigabine bulk drug (No.
20140201, 1.21 kg; No. 20140211, 1.33 kg; No. 20140221, 1.30 kg)
was prepared in our laboratory referring to literature [4,5]. Enriched
reaction mixture was collected for isolation of impurity-II (HPLC
purity: 98.8%) and impurity-III (HPLC purity: 99.2%). Impurity-
I (HPLC purity: 98.7%), impurity-IV (HPLC purity: 99.0%) and
impurity-V (HPLC purity: 98.4%) were synthesized by efficient syn-
thetic method [11,13,15] with high purity.
HPLC grade acetonitrile was purchased from Thermo Fisher Sci-
entific Inc. (Waltham, MA, USA). TFA (trifluoroacetic acid, HPLC
grade) was purchased from Sinopharm Chemical Regent Co., Ltd.
(Shanghai, China). Water used for the preparation of mobile phase
was purified using Millipore Milli-Q plus (Milford, MA, USA) purifi-
cation system. Solvent for NMR, DMSO-d6 , (Isotech, 99.9 Atom %D,
with 0.05% TMS, v/v) was purchased from Sigma–Aldrich Trading
Co., Ltd. (Shanghai, China).
2.1.1. Column chromatography
Column chromatography was carried out with silica gel 60H
from Qingdao Haiyang Chemical Co., Ltd., China. The separation
was carried out using petroleum ether and ethyl acetate gradi-
ents from (100:1, v/v) to (20:1, v/v). All fractions were analyzed by
HPLC method described in Section 2.2. The major impurity contain-
ing fractions was collected and solvent was removed by reduced
pressure to obtain the impurity-II and impurity-III as white solid.
2.2. High performance liquid chromatography (analytical)
A Waters Model Alliance 2695 Separation module (Waters Cor-
poration, Milford, MA, USA) equipped with a Waters 2489 photo
diode array UV detector (Waters Corporation, Milford, MA, USA)
was used. Chromatographic separations were carried out on a
reversed-phase Agilent-C18, 100 mm × 4.6 mm, and 2.7 ␮m parti-
cle size column (Agilent Technologies, Santa Clara, CA, USA). The
gradient elution employed solutions A and B as mobile phase com-
ponents. The solvent A was 0.1% trifluoroacetic acid (v/v) in water,
while solvent B was 0.1% trifluoroacetic acid (v/v) in acetonitrile.
The gradient program was set as follows: time/% of solvent B:
0/25, 0.5/25, 7.5/75, 9.5/75, 12.0/25, 15.0/25. The mobile phase was
pumped at 0.8 ml/min and the column was thermostated at a tem-
perature of 40 ◦ C. UV detector was carried out at 250 ␮m. The data
was recorded using Empower pro data handing system (Waters cor-
poration, Milford, MA, USA). This HPLC method was able to detect
these impurities which ranged from 0.05 to 0.1% in the presence of
parent compound (Fig. 2a).
2.3. NMR spectroscopy
The 1 H and two dimensional (2D) NMR (HSQC, HMBC, NOESY)
measurements were performed on Varian Mercury plus 400 MHz
NMR instrument at 25 ◦ C in DMSO-d6 . 13 C NMR experiments were
performed on a Varian Mercury 100 MHz instrument, model 2000
at 25 ◦ C in DMSO-d6 . The chemical shift values were reported on
the ı scale in ppm, relative to TMS (ı = 0.00 ppm) and DMSO-d6
(ı = 39.5 ppm) as internal standard, respectively. Assignments were
further confirmed by running two-dimensional chemical shift cor-
relation experiments.
2.4. FT-IR spectroscopy
FT-IR spectra for impurity-I, impurity-II and impurity-III were
recorded in the solid state as KBr dispersion using a Shimadzu
Corporation Affinity-1 FT-IR spectrophotometer.
2.5. Mass spectroscopy
Mass spectra were recorded on a triple quadruple mass spec-
trometer PE Sciex model API 3000. Detection of ions were
performed in electrospray ionization, positive and negative ion
mode.
3. Results and discussion
3.1. Detection of impurities
Retigabine API sample prepared by known synthetic route was
analyzed using the typical HPLC method as described in Section 2.2,
revealing the presence of two new impurities (RT 5.79 min, RRT
1.33; RT 9.29 min, RRT 2.13). These two new impurities (isolated
from enriched reaction mother liquor) and three known impuri-
ties were spiked with retigabine and RRTs were compared. Three
known impurities were marked as impurity-I (RT 1.93 min, RRT
0.44), impurity-IV (RT 6.69 min, RRT 1.54), and impurity-V (RT
6.23 min, RRT 1.43). HPLC chromatogram of (a) retigabine bulk
drug, (b) reaction mixture, and (c) impurities mixture were shown
in Fig. 2.
3.2. Structural elucidation of impurities
Impurity-I was synthesized referring to the previous work [15]
and impurity-II and impurity-III were independently isolated by a
designed reaction (described in Section 2.2).
The 1 H and 13 C NMR spectrum data of impurities are shown in
Table 1. The structural elucidation of retigabine has been described
in detail in literature [11]. MS spectra and plausible fragmentation
pathways for the impurities are shown in Fig. 3. The key NOESY and
HMBC correlations of impurity-II are shown in Fig. 4.
The chemical structures of impurities I–III and numbering
scheme for NMR are shown in Table 1. The IR and MS data of
impurity-I, impurity-II and impurity-III are shown in Table 2.
3.2.1. Impurity-I
ESI mass spectrum of impurity-I displayed protonated molecule
peak at m/z 258 [M+H]+ in positive ion mode, indicating the mass of
this impurity to be 257 which is 46 amu less than that of retigabine.
The MS spectra and plausible fragmentation of impurity-I were
shown in Fig. 3. In the 1 H NMR spectrum of this impurity, signals
corresponding to CH3 (1.20 ppm, t, 3H) and CH2 (4.05 ppm, q, 2H)
protons of retigabine were disappeared and signals corresponding
CH3 (15.11 ppm) and CH2 (46.35 ppm) carbon were as well disap-
peared in the 13 C NMR spectrum. Besides, the number of reactive
hydrogen protons in impurity-I is just one less then that of retiga-
bine. In FT-IR spectrum of impurity-I exhibited one characteristic
absorption band at 1678 cm−1 indicating the presence of a carbonyl
group in impurity-I. Retigabine was not stable, which might lose
a molecule of ethanol to format impurity-I. Based on the above
spectral data the molecule formula of impurity-I was confirmed as
C14 H12 FN3 O and the corresponding structure was characterized as
5-((4-fluorobenzyl)amino)-1H-benzo[d]imidazol-2(3H)-one.
 D. Zhang et al. / Journal of Pharmaceutical and Biomedical Analysis 99 (2014) 22–27 25

Table 1
1
H and 13 C NMR data of impurity-I, impurity-II and impurity-III.

Position ı (ppm) multiplicity 13

C ı (ppm), J (Hz)a Position ı (ppm) multiplicity 13
C ı (ppm), J (Hz)a Position ı (ppm) multiplicity 13
C ı (ppm), J (Hz)a

1 121.26 1 129.20 1 123.58

2 131.10 2 128.61 2 126.40
3 6.19, s, 1H 94.36 3 6.66, s, 1H 108.39 3 7.76, s, 1H 113.40
4 144.07 4 134.75 4 138.33
5 6.22, d, 1H 109.29 5 6.70, d, 1H 120.04 5 7.10, d, 1H 124.59
6 6.63, d, 1H 105.80 6 6.85, d, 1H 108.74 6 7.62, d, 1H 114.41
7 4.20, d, 2H 47.09 7 4.71, s, 2H 61.60 7 4.84, s, 2H 53.20
8 137.18 8 135.10 8 134.39
9, 13 7.38, dd, 2H 129.43 (8) 9, 13 7.25, dd, 2H 130.10 (8) 9, 13 7.26, dd, 2H 129.98 (8)
10, 12 7.17, dd, 2H 115.48 (21) 10, 12 7.15, dd, 2H 115.68 (21) 10, 12 7.16, dd, 2H 115.79 (21)
11 162.69 (240) 11 162.97 (240) 11 163.03 (240)
14 155.91 14 155.84 14 149.83
15 4.10, q, 2H 53.57 15 4.14, q, 2H 64.08
16 1.15, t, 3H 14.95 16 1.13, t, 3H 14.89
17 155.95 17, 20 146.42
a 10.19, s, 1H a 10.64, s, 1H 18, 21 4.36-4.44, m, 4H 64.08
b 10.06, s, 1H b 10.60, s, 1H 19, 22 1.24-1.37, m, 6H 14.40
c 5.87, br, 1H 23 155.49

s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; dd, doublet of doublet; br, broad singlet.
a 13
C–19 F coupling constants.

3.2.2. Impurity-II
ESI mass spectrum of impurity-II displayed protonated molecule
peak at m/z 330 [M+H]+ in positive ion mode, indicating the mass
of this impurity to be 329 which is 26 amu more than that of reti-
gabine. The MS spectra and plausible fragmentation of impurity-II
were shown in Fig. 3. In comparison with impurity-I (m/z = 257)
and its proposed process of formation (Fig. 5), the impurity-I could
excessively be acylated by one equivalent ethyl chloroformate to
product the m/z 329. FT-IR spectrum of impurity-II exhibited two
strong characteristic absorption band at 1702 and 1697 cm−1 indi-
cating the presence of two carbonyl group in this impurity.
One-dimensional 1 H NMR and 13 C NMR analyses reveal that
the impurity-II has two reactive hydrogen protons (10.60 ppm, s,
1H and 10.64 ppm, s, 1H in 1 H NMR) and two carbonyl carbon
signals were observed at 155.84 and 155.95 ppm independently
(Table 1). We performed Nuclear Overhauser Effect Spectroscopy
(NOESY) and Heteronuclear Multibond Coherence Spectroscopy
(HMBC) experiments to determine the configuration of this impu-
rity. The HMBC spectrum of impurity-II shows clearly correlations
between C14 (115.84 ppm, carbonyl carbon) and H7 (4.71 ppm, s,
2H, methylene protons) and between C14 and H15 (4.10 ppm, q,
2H, methylene protons). Of the different functional group isomers,
the only one that has strong Heteronuclear Multibond Coher-
ence Spectroscopy (HMBC) correlations is between C14 and H7
(Fig. 4). This proved the Hc position reactive hydrogen of impurity-
I was acylated by ethyl chloroformate. Furthermore, the existed
reactive hydrogen protons (Ha and Hb) showed clearly NOE cor-
relations between H6 (6.85 ppm, d, 1H) and H3 (6.66 ppm, s, 1H)

Table 2
IR and MS data of impurity-I, impurity-II and impurity-III.

Compound IR (cm−1 ) MS

Impurity-I 3115 (N H stretching), 3010 (aromatic C H stretching), 2902, 2831 (aliphatic C H stretching), 1755 (C O 258.05 [M+H], 259.07,
stretching amide I), 1607, 1456 (aromatic C C stretching) 1514 (N H bending amide II), 1234 1182 150.02, 149.00, 108.95
(asymmetric C O and C F stretching), 1030 (symmetric C O stretching), 810, 783, 763 (aromatic C H
bending)

Impurity-II 3196 (N H stretching), 2981 (aliphatic C H stretching), 1702, 1697 (C O stretching amide I), 1605 (aromatic 330.05 [M+H], 331.13,
C C stretching), 1508 (N H bending amide II), 1220, 1157 (asymmetric C O and C F stretching), 1050 274.21, 149.04
(symmetric C O stretching), 826, 756, 710 (aromatic C H bending)

Impurity-III 2980, 2943 (aliphatic C H stretching), 1803, 1746, 1703 (C O stretching amide I), 1607 (aromatic C C 474.16 [M+H], 475.18,
stretching), 1512, 1498 (N H bending amide II), 1261 (aromatic C N stretching) 1236, 1223 (asymmetric 402.03, 149.00, 108.95
C O and C F stretching), 1030 (symmetric C O stretching), 839, 765, 741 (aromatic C H bending)
26 D. Zhang et al. / Journal of Pharmaceutical and Biomedical Analysis 99 (2014) 22–27

Fig. 4. The key NOESY (a) and HMBC (b) correlations of impurity-II.

Fig. 3. MS spectra and plausible fragmentation pathways for the impurities.
respectively (Fig. 4). From the above spectral data the molecular
formula of impurity-II was confirmed as C17 H16 FN3 O3 and corre-
sponding structure was characterized as ethyl 4-fluorobenzyl(2-
oxo-2,3-dihydro-1H-benzo[d]imidazol-5-yl)carbamate.
3.2.3. Impurity-III
ESI mass spectrum of impurity-III showed protonated molecule
peak at 474 [M+H]+ in positive ion mode, indicating the mass of
this impurity to be 473 which is 170 amu more than that of reti-
gabine. The MS spectra and plausible fragmentation of impurity-III
were shown in Fig. 3. In comparison with impurity-I (m/z = 257)
and its proposed process of formation (Fig. 5), the impurity-I could
 D. Zhang et al. / Journal of Pharmaceutical and Biomedical Analysis 99 (2014) 22–27 27

H
N
O
N N
H O
F O
O O
NH O
O O O
H HN N O
NH N N
O F + O
N N F
N NH2 H O N
H H
F N O
F impurity-I
O
O O
O impurity-II impurity-III
N
O
N N
H H
F

Fig. 5. Formation of impurities.

excessively be acylated by three equivalent ethyl chloroformate to
product the m/z 473.
One-dimensional 1 H NMR and 13 C NMR analyses reveal that
the impurity-III has no reactive hydrogen proton which indicat-
ing the corresponding reactive hydrogen protons (Ha, Hb and Hc)
of impurity-I were all acylated by ethyl chloroformate to prod-
uct impurity-III. FT-IR spectrum of impurity-III exhibited three
strong characteristic carbonyl group absorption band at 1803, 1746,
1703 cm−1 . In comparison to impurity-II, additional two methy-
lene group (4.36–4.44, m, 4H) and two methyl group (1.24–1.37,
m, 6H) were found in 1 H NMR spectra and the corresponding car-
bon signals were at 64.08 ppm and 14.40 ppm. Based on the above
spectral data the molecule formula of impurity-III was confirmed as
C23 H24 FN3 O7 and the corresponding structure was characterized
as diethyl 5-((ethoxycarbonyl)(4-fluorobenzyl)amino)-2-oxo-1H-
benzo[d]imidazole-1,3(2H)-dicarboxylate.
3.3. Formation of impurities
Retigabine is a close structural analog of the centrally-acting
analgesic flupirtine and they have the similar synthetic route [4,5].
Comparing to the previous impurity profile study of flupirtine [15],
there was a big difference in formation of process-related impu-
rities (Fig. 5). Compounds IIa and IIb (Fig. 5) were proposed to be
potential process-related impurities of retigabine in literature [16].
However, none of them but another functional group positional
isomer (impurity-II) was disclosed in the retigabine bulk drug.
The key synthetic steps (Fig. 1) involved initial reduction of 2-
amino-4-[(4-fluorobenzyl)amino]-1-nitrobenzene (compound 2)
via catalytic hydrogenation followed by acylation with ethyl chlo-
roformate to product retigabine, which might lose a molecule of
ethanol to product impurity-I. At the same time, the rest of other
reactive hydrogens could be further acylated by some amount of
unreacted ethyl choroformate and undesired impurities (impurity-
II and impurity-III) were formed.
4. Conclusion
The two new process-related impurities of retigabine
were found using typical HPLC-UV method and identified by
means of MS, 1 H, 13 C, 2D NMR (HSQC, HMBC and NOESY)
and FT-IR techniques as ethyl 4-fluorobenzyl(2-oxo-2,3-
dihydro-1H-benzo[d]imidazol-5-yl)carbamate (impurity-II) and
diethyl 5-((ethoxycarbonyl)(4-fluorobenzyl)amino)-2-oxo-1H-
benzo[d]imidazole-1,3(2H)-dicarboxylate (impurity-III). Besides,
structural elucidation of impurity-I was also first reported in this
paper.
Acknowledgment
The authors wish to thank the Shanghai Institute of Pharmaceu-
tical Industry for structure analysis services.
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