Identification and genotoxicity evaluation of two carbamate impurities in rasagiline

Article abstract of DOI:10.1039/c6ra20810j

During the synthesis of a second-generation monoamine oxidase-B inhibitor rasagiline, two unknown impurities (impurity A and impurity B) were detected and isolated by preparative liquid chromatography. Based on mass spectroscopy and NMR, these two impurities were characterized as the by-products with a propargyl carbamate structure, whose generation was related to the carbon dioxide in the alkaline reaction solution. Because the carbamate structure has been highlighted as a class of potentially genotoxic impurities (GTIs), the genotoxicity of these two impurities was evaluated by the malformation test and the comet assay using zebrafish embryos. The results showed that the genotoxicity of impurity B was significant higher than that of rasagiline and other impurities. Thus, a HPLC-MS method was developed and validated for the determination of impurity B in rasagiline. The established method showed a good specificity, linearity, precision and accuracy. The detection limit of this method was 2.0?ppm with 0.1 mg ml^?1 rasagiline mesylate.

Full text of DOI:10.1039/c6ra20810j

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Identification and genotoxicity evaluation of two
carbamate impurities in rasagiline
Cite this: RSC Adv., 2016, 6, 106268
b
a
b
b
b
b
Yongqiang Sun,† Xinyu Zhang,† Yimin Yan, Yongrui Tu, Xiaohui Feng, Wei Jiang
ac
and Feng Zheng*
During the synthesis of a second-generation monoamine oxidase-B inhibitor rasagiline, two unknown
impurities (impurity A and impurity B) were detected and isolated by preparative liquid chromatography.
Based on mass spectroscopy and NMR, these two impurities were characterized as the by-products with
a propargyl carbamate structure, whose generation was related to the carbon dioxide in the alkaline
reaction solution. Because the carbamate structure has been highlighted as a class of potentially genotoxic
impurities (GTIs), the genotoxicity of these two impurities was evaluated by the malformation test and the
comet assay using zebrafish embryos. The results showed that the genotoxicity of impurity B was significant
higher than that of rasagiline and other impurities. Thus, a HPLC-MS method was developed and validated
for the determination of impurity B in rasagiline. The established method showed a good specificity, linearity,
Received 19th August 2016
Accepted 28th October 2016
DOI: 10.1039/c6ra20810j
ꢀ1
www.rsc.org/advances
precision and accuracy. The detection limit of this method was 2.0 ppm with 0.1 mg ml rasagiline mesylate.
as a class of structural alerts or potentially genotoxic impuri-
ties (PGIs).
Genotoxicity means damage to the DNA and accumulation of
1
. Introduction
1
1
Rasagiline, (R)-N-(prop-2-ynyl)-2,3-dihydro-1H-inden-1-amine,
is a second-generation selective and irreversible monoamine DNA damage in specic genes can lead to cancer, a process that
1
oxidase-B (MAO-B) inhibitor. It is able to ameliorate motor develops in the course of several years. Therefore, genotoxic
symptoms and prevent motor complications in Parkinson's impurities are garnering increased attention from regulatory
2
12,13
disease (PD). Unlike the rst-generation MAO-B inhibitor, agencies and the pharmaceutical industry in recent years.
The
rasagiline was not metabolized to L-methamphetamine deriv- control of these impurities is more strict than that of the common
atives in vivo, making rasagiline more effective and well impurities and their permitted limits in the long-term treatment
3
,4
14
tolerated. It gained UK- and EU-marketing authorization in drugs are usually in part per million (ppm) levels. Commonly
2
5
005 and US FDA approval in 2006, and gradually became used analytical methods for general impurity prole, i.e., HPLC-UV
a valuable therapeutic option for use in all stages of Parkin- and GC-FID, are not suitable for determination of such impuri-
15
son's disease. Separation and identication of impurities in ties. The higher cost methods, such as MS based methods, are
14–16
drugs is an important issue for drug development. A literature oen needed for trace analysis of genotoxic impurities.
Based
survey reveals that two HPLC methods with different chiral on the bacterial reverse mutation assay (Ames test), some struc-
stationary phases were developed to separate the rasagiline turally alerting functional groups have been known to be involved
6
,7
11
from its analogues and its (S)-enantiomer, respectively.
in reactions with DNA. However, in vitro genotoxicity tests have
17
There are also several reports on the quantitative analysis of a high rate of false positive results with rodent carcinogenicity.
8
rasagiline mesylate in microspheres and separation of
Zebrash embryos have been proposed as an in vitro animal
process related impurities and degradation products of rasa- model which could bridge the gap between simple assays based
9
,10
giline mesylate by HPLC. During the synthesis of rasagiline on cell or tissue culture, and biological validation in whole
18
mesylate, we detected and identied two new carbamate animals such as rodents during the drug discovery. In recent
impurities by HPLC-MS and NMR. Concern about the presence years, the zebrash embryos method has been proven to be
of these two impurities was extremely crucial for the drug's a practicable and suitable avenue for detecting DNA damage
19,20
safety, because the carbamate structure has been highlighted induced by various genotoxicants.
The present study,
therefore, attempts to differentiate the genotoxic impurities
from the common impurities present in rasagiline mesylate
using zebrash embryos. Then a HPLC-MS method was devel-
oped and validated for the genotoxic impurity control. Our
a
Department of Pharmaceutical Analysis, China Pharmaceutical University, 24
Tongjiaxiang, Nanjing 210009, China. E-mail: cpu_analyst@126.com
b
Changzhou No.4 Pharmaceutical Co., Ltd, Changzhou 213004, China
c
Key Laboratory of Drug Quality Control and Pharmacovigilance, Ministry of investigations will help the safety of this active pharmaceutical
Education, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China
ingredients during its long-term clinical treatment.
†
These two authors contributed equally to this article.
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the detection of impurities. Chromatographic separations were
2
. Experimental
performed on an Agilent Poroshell 120 EC-C18 (50 ꢂ 4.6 mm,
2.1 Chemicals and reagents
2.7 mm) column. A mixture of acetonitrile–water with 0.1% for-
Rasagiline mesylate, the N-propargyl derivative, and (R)-1-ami- mic acid (1 : 1, v/v) was used as the mobile phase. The ow rate
ꢀ1
ꢁ
noindan were synthesised in Changzhou No. 4 Pharmaceutical was 0.3 ml min and the column temperature was kept at 30 C.
Co., Ltd. The purity of these compounds (>97.0%) were deter- The injection volume was 3 ml and the UV spectra were recorded
mined by respective HPLC methods. All reagents and solvents at 215 nm. The MS operating conditions were as follows: positive
2
used in the synthesis were obtained from Nanjing Chemical ionization mode, scan range from m/z 100 to 500, drying gas (N )
ꢀ
1
ꢁ
Reagent Co., Ltd (Nanjing, China). Puried water was obtained ow rate of 10 l min , drying gas temperature of 350 C,
by a Milli-Q system from Millipore (Bedford, MA, USA). Trie- nebulizer pressure of 40 psi, capillary voltage of 4000 V.
thylamine, acetic acid, formic acid, HPLC grade acetonitrile and
methanol were purchased from Merck (Darmstadt, Germany), 2.4 Preparative HPLC conditions for the isolation of
which were used for the preparation of mobile phases.
impurities
These two impurities were isolated using an Agilent 1200 series
preparative HPLC system which was equipped with an auto-
2.2 Synthesis of rasagiline crude product
The synthesis of rasagiline crude product followed the proce- mated fraction collector, a photodiode array detector and
dures reported by the ref. 21 and the synthesis scheme was a Chemstation soware. An Hypersil Prep. C18 (100 ꢂ 50 mm,
shown in Fig. 1. Briey, (R)-1-aminoindan (1 mol) was dissolved 10 mm) column was applied. Solvent A was water with 0.1%
by DMF into a round bottom ask with potassium carbonate (1 formic acid and solvent B was acetonitrile. The gradient
mol), followed by dropwise addition of propargyl methanesul- program [time (min)/% solvent B] was set as 0/20, 30/50 and 60/
ꢀ1
fonate ester (1 mol) in DMF. The reaction mixture was stirred at 50. The ow rate was set at 40 ml min and the injection
ꢁ
2
5–35 C for more than 20 h. Aer completion of the reaction, volume was 500 ml. The impurities containing fractions were
required amount of water was added. The aqueous layer was collected separately and were concentrated to powder using
extracted twice with dichloromethane. The combined Rotavapor under high vacuum.
dichloromethane layer was washed with acid water and the
aqueous layer was separated. Sodium hydroxide solution was 2.5 Nuclear magnetic resonance (NMR) spectrometer
added to the aqueous layer, then it was extracted twice with
NMR experiments were performed at ambient temperature (300
ethyl acetate. The combined ethyl acetate layer was dried and
K) on a BRUKER AV-500 spectrometer, and were operated at 500
distilled off under vacuum to get the title compound.
MHz. CDCl
TMS) was used as the internal standard. The H and
chemical shi values were reported on the d scale in ppm,
The rasagiline crude product was dissolved in acetonitrile– relative to TMS (d ¼ 0.00 ppm). For exchangeable proton
3
was used as the solvent and tetramethylsilane
1
13
(
C
2.3 Conditions for the detection of impurities
ꢀ
1
2
water (1 : 1, v/v) to form 1 mg ml solution. An Agilent 6240 LC- experiments, 10% D O was added to provide a lock signal. Two-
TOF mass spectrometer (Agilent Technologies, Santa Clara, dimensional spectra (HMBC, HMQC and COSY) were recorded
USA) with an electrospray ionization (ESI) interface was used for using standard impulse sequences provided by BRUKER.
2.6 Conditions for the determination of impurity B
Determination of impurity B was performed using Agilent 1200
SL series HPLC system interfaced with a quadrupole mass
spectrometer equipped with an electrospray source (Agilent
Technologies, CA, USA). The chromatographic condition was
almost the same as mentioned in Section 2.3 except for using
methanol–water with 0.1% acetic acid (80 : 20, v/v) as the
mobile phase. Selective ion monitoring (SIM) mode was applied
+
and the impurity B was monitored with its adduct ion [M + Na]
m/z 276.1. The other MS operating conditions were the same as
mentioned in Section 2.3.
2.7 Zebrash embryos assays
Adult AB line zebrash (Danio rerio) were obtained from a local
aquarium and maintained by standard culture techniques. Fish
ꢁ
were kept at temperature 28 ꢃ 0.5 C, pH 7.0 ꢃ 0.5, a constant
light cycle (14 h light/10 h dark) and fed a tropical sh
commercial ake food twice a day. Embryos were obtained from
19
Fig. 1 The synthesis scheme for rasagiline, impurity A and impurity B. healthy adult sh and cultured using a standard procedure.
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For the malformation test, each tested compound at
different concentrations was incubated with 50% epiboly
embryos for 48 h using water as the solvent. Standard culture
solution without any toxicant was used as a control. The
3
. Results and discussion
3.1 Detection and structural elucidation of impurity A and
impurity B
development status of zebrash embryos were observed with an The rasagiline crude product was analyzed by HPLC-UV for the
inverted microscope (IMT 2, Olympus, Tokyo, Japan) and detection of possible impurities. As shown in Fig. 2, there were
documented photographically at the specied time.
For the comet assay, 10 zebrash embryos aer 48 h incu- rasagiline peak. Table 1 shows the accurate molecular weight
four main impurity peaks in the chromatogram except the
ꢀ
1
bation with 2 mg ml impurity A or impurity B, were homoge- and formulas of rasagiline and each impurity in positive ioni-
nized in 1 ml ice-cold potassium phosphate buffer, pH 7.4. The zation mode. By comparison of the mass spectra and retention
ꢁ
homogenate was centrifuged for 10 min at 4 C and 200 ꢂ g. The time with those of authentic standard, the impurity peak at
pellet was resuspended in 1 ml phosphate buffer and again 2.5 min was identied as the 1-aminoindan and the impurity
ꢁ
centrifuged 10 min at 4 C and 180 ꢂ g. The pellet was resus- peak at 23.7 min was identied as the N-propargyl impurity.
pended in 100 ml phosphate buffer and mixed with 90 ml However, the mass spectral data of impurity A (9.0 min) and
low melting agarose. The comet assay followed the procedures impurity B (16.7 min) did not match with the formula of any
reported by the ref. 19. Comets were analysed by means of rasagiline related compound. Therefore, these two unknown
image analysis soware Comet Assay IV, version 4.3 (Perceptive impurities were isolated by preparative liquid chromatography
Instruments Ltd).
for the further structural elucidation.
2
The molecular mass of impurity A was 44 units (CO ) more
than that of rasagiline. Compared with rasagiline, impurity A
1
3
showed an additional C-NMR signal at 155.2 ppm corre-
sponded to a carbonyl carbon, suggesting that the additive
structure may be a carboxyl group or an ester group. However,
no exchangeable proton was observed in the D O exchange
2
experiment, indicating the absence of a carboxyl proton or
other reactive protons in the structure of impurity A. Inter-
1
1
estingly, the H– H COSY spectra exhibited clear correlations
between 5.05 ppm and 5.22 ppm (H ), which indicated
1
the presence of amide proton at 5.05 ppm in the structure
of impurity A. A reasonable expectation of this non-
exchangeable amide proton was that the formation of carba-
mate ester resulted in the low reaction activity of amide
proton. The other evidences for the existence of a carbamate
Fig. 2 Chromatogram of rasagiline and its impurities.
1
13
structure were that the H and C chemical shi values of
position 11, 12 and 13 of impurity A were higher than that of
rasagiline, and no correlated proton of the amide proton was
Table 1 Accurate molecular weight and formulas of rasagiline and
each impurity
+
a
Peak
[M + H] (m/z)
Error (ppm)
Formula
1
1
1
observed in the H– H COSY spectra except the H . Two-
dimensional spectra HMBC, HMQC and COSY, were used
for the assignments in the Fig. 3. Based on the above data, the
molecule formula of impurity A was conrmed as C H NO
2
2
5
9
1
2
.5 min
.6 min
.0 min
6.7 min
3.7 min
134.0968
172.1127
216.1026
254.1180
210.1282
ꢀ3.47
ꢀ3.17
ꢀ3.37
ꢀ1.76
ꢀ2.02
C
C
C
C
C
9 11
H N
12
13
16
15
H
H
H
H
13
N
13NO
15NO
2
1
3
13
2
(
Fig. 2).
The molecular mass of impurity B was 38 units (C H ) higher
15
N
3
2
a
Difference between the measured and calculated mass.
than that of impurity A. Therefore, the structure of impurity B
1
13
Fig. 3 H and C-NMR of rasagiline impurity A.
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1
Fig. 4 H-NMR of rasagiline impurity B.
3.3 Genotoxicity evaluation of impurity A and impurity B
At present it is extremely difficult to experimentally prove the
Scheme 1
existence of a threshold for the genotoxicity of a given impu-
27
rity. In this study, the malformation rates of impurity A and
impurity B with other impurities present in rasagiline were
compared to differentiate the genotoxic impurities from the
common impurities. Then the genotoxicity of impurity A and
impurity B was tested using the comet assay.
might contain another propargyl group when compared with
the structure of impurity A. In the H-NMR spectra of impurity B
1
(
(
Fig. 4), alkyne proton signals (2.15 ppm) and methylene proton
3.51 ppm, 4.01 ppm) were observed, while the amide proton
In the malformation test, the digital images of embryonic
malformations were used for the morphological assessment.
All impurity-exposed embryos exhibited several morphological
defects in a concentration-dependent manner and the ED50
values (Fig. 5), determined using the probit method with the R
was absent. This indicated that another propargyl group was
connected with the N atom. Based on the above spectral data,
impurity B was conrmed as C16H15NO (Fig. 2).
2
2
8
soware, were used to represent the concentration of each
3.2 The formation reason of impurity A and impurity B
The formation reason of the carbamate structure in impurity A
and impurity B was analyzed next. Previous reports have indi-
22,23
cated that carbon dioxide readily reacts with nucleophiles.
For example, urea is industrially produced by a reaction with
ammonia and secondary or primary amines react with CO to
2
give carbamic acids (Scheme 1).
Rasagiline is currently available by multiple synthetic
schemes and most of its synthetic routes encountered the same
24–26
intermediate (R)-1-aminoindan.
Then (R)-1-aminoindan was
used to react with different propargyl ester under various alka-
line conditions, which provided a suitable environment for the
2
existence of an amount of CO . Therefore, the side reaction
resulting in the formation of impurity A and impurity B (Fig. 1)
might occur at these conditions.
Fig. 6 The percentage of DNA in the tail (% tail DNA) after 48 h
ꢀ1
ꢀ1
incubation with 0.5% water (control), 2 mg ml impurity A or 2 mg ml
impurity B (n ¼ 3).
Fig. 5 The embryonic malformation rate after 48 h incubation with rasagiline or its impurities at different concentration (n ¼ 3).
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Table 2 The 50% of embryonic malformation concentration (ED50) of rasagiline and its impurities
Compound
Rasagiline
280
(R)-1-Aminoindan
N-Propargyl impurity
Impurity A
33
Impurity B
2
ꢀ1
EC50 (mg ml
)
167
31
compound that produces 50% malformation rate. As shown in 3.4 Development and validation of HPLC-MS method for the
Table 2, the ED50 value of impurity B was lowest among the determination of impurity B
tested compound. The ED50 value of impurity A was higher
Different ions were assessed for quantitation, including
molecular ion [M + H] and adduct ions [M + Na] , [M + K] . In
addition, different mobile phase such as acetonitrile and
than those of rasagiline and (R)-1-aminoindan, but equal with
that of N-propargyl impurity. In the comet assay, the
percentage of DNA in the tail (% tail DNA) was used to repre-
+
+
+
methanol have been tested. Quantitation of impurity B was
1
9
sent the extent of DNA strand breaks. As shown in Fig. 6, the
tail DNA was signicant higher in the group that was
+

nally achieved by monitoring with adduct ion [M + Na] using
%
methanol–water with 0.1% formic acid (80 : 20, v/v) as the
mobile phase. Then the analytical performance of the proposed
method was investigated with respect to specicity, linearity,
precision, accuracy, limits of detection (LODs) and limits of
quantitation (LOQs).
ꢀ
1
incubated with 2 mg ml impurity B than in the control group.
But there was no signicant difference in the % tail DNA
between the control and the group that was incubated with
ꢀ
1
2
mg ml impurity A.
The malformation test and the comet assay indicated the
As shown in Fig. 7, the retention time of impurity B was
highest DNA damage ability of impurity B among these impu-
rities. Therefore, a MS based method was needed to differen-
tiate impurity B from the other impurities present in rasagiline.
3.23 min and blank solvent (mobile phase) had no interference
with it. Different standard concentrations of impurity B at
ꢀ1
0
.20–20 ng ml were prepared in mobile phase to evaluate the
Fig. 7 Chromatograms of blank solvent (a) and impurity B (b).
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method linearity. Standard curve was constructed by plotting
the peak area of impurity B (Y) against the impurity B concen-
tration (X). The linearity correlation equation obtained was Y ¼
3 A. H. Schapira, Monoamine oxidase B inhibitors for the
treatment of Parkinson's disease: a review of symptomatic
and potential disease: a review of symptomatic and
potential disease-modifying effects, CNS Drugs, 2011, 12,
1067–1071.
2
47.8 ꢂ X ꢀ 25.9, r ¼ 0.998.
The limit of detection (LOD) was obtained based on a signal-
to-noise ratio (S/N) of 3, and the limit of quantication (LOQ)
was obtained based on a S/N of 10. The determined LOD value
4 Z. A. Abassi, O. Binah and M. B. Youdim, Cardiovascular
activity of rasagiline, a selective and potent inhibitor of
mitochondrial monoamine oxidase B: comparison with
selegiline, Br. J. Pharmacol., 2004, 143, 371–378.
5 S. E. Lakhan, From a Parkinson's disease expert: rasagiline
and the future of therapy, Mol. Neurodegener., 2007, 2, 13–15.
ꢀ
1
ꢀ1
was 0.07 ng ml
respectively.
and the LOQ value was 0.20 ng ml ,
ꢀ
1
ꢀ1
The 0.20 ng ml and 20 ng ml impurity B solutions were
separately prepared and injected for six times to evaluate the
injection precision and % RSD values for peak responses of
impurity B were separately calculated and it was found to be
well within acceptance criteria of not more than 2.15%.
6 K.
M. Tak, E. J. Park and M. H. Hyun, Liquid
chromatographic resolution of racemic rasagiline and its
analogues on a chiral stationary phase based on (+)-(18-
crown-6)-2,3,11,12-tetracarboxylic acid, J. Sep. Sci., 2013, 36,
3682–3687.
7 P. S. Reddy, K. S. Babu and N. Kumar, A validated normal
phase LC method for enantiomeric separation of rasagiline
mesylate and its (S)-enantiomer on cellulose derivative-
based chiral stationary phase, Chirality, 2013, 25, 324–327.
8 M. Fern ´a ndez, E. Barcia and S. Negro, Development and
validation of a reverse phase liquid chromatography
method for the quantication of rasagiline mesylate in
biodegradable PLGA microspheres, J. Pharm. Biomed. Anal.,
ꢀ
1
Solution stability was studied by injecting the 0.20 ng ml
ꢀ
1
and 20 ng ml solutions at T ¼ 0 h, 30 min, 1 h, 2 h, 4 h and 8 h
and no signicant difference in the area of impurity B solutions
were observed within the 8 h (less than 2.03%).
Accuracy of the method was demonstrated though spiked
recovery experiments. Authentic impurity B was spiked into
ꢀ
1
0
(
.1 mg ml rasagiline mesylate in triplicates at three levels
2 ppm, 20 ppm and 200 ppm). The average recovery was 88.74%
with RSD of 5.68% (n ¼ 9).
2
009, 49, 1185–1191.
4
. Conclusion
9
P. G. Shelke and A. V. Chandewar, Study of stress
degradation behavior of rasagiline mesylate under
hydrolytic conditions by high performance liquid
chromatography, Drug Res., 2014, 64, 182–185.
Two carbamate impurities were detected and isolated during the
synthesis of rasagiline, and their identity was established by the
mass spectra and NMR. These two impurities may come from the
intermediate (R)-1-aminoindan reacting with the carbon dioxide 10 P. S. Reddy, K. Sudhakar Babu and N. Kumar, Development
in the alkaline reaction solution. Because the carbamate struc-
ture has been highlighted as a class of potentially genotoxic
impurities (GTIs), the genotoxicity of these two impurities was
evaluated by the malformation test and the comet assay using
zebrash embryos. The results showed that the genotoxicity of
and validation of a stability-indicating RP-HPLC method for
the simultaneous estimation of process related impurities
and degradation products of rasagiline mesylate in
pharmaceutical formulation, J. Chromatogr. Sci., 2013, 51,
242–249.
impurity B was signicant higher than those of rasagiline and 11 L. M u¨ ller, R. J. Mauthe, C. M. Riley, et al., A rationale for
other impurities. Thus, a HPLC-MS method was developed and
validated to determine impurity B in rasagiline mesylate. This
study will help the safety of this active pharmaceutical ingredi-
ents during its long-term clinical treatment.
determining, testing, and controlling specic impurities in
pharmaceuticals that possess potential for genotoxicity,
Regul. Toxicol. Pharmacol., 2006, 44, 198–211.
12 US FDA, Guidance for Industry: Genotoxic and Carcinogenic
Impurities in Drug Substances and Products, Recommended
Approaches, 2008.
3 ICH, M7: Assessment and Control of DNA Reactive (mutagenic)
Impurities in Pharmaceuticals to Limit Potential Carcinogenic
Risk, 2013.
Acknowledgements
1
This is a Project Funded by the Priority Academic Program
Development of Jiangsu Higher Education Institutions and the
Open Project Program of MOE Laboratory of Drug Quality 14 D. Q. Liu, M. Sun and A. S. Kord, Recent advances in trace
Control and Pharmacovigilance (No. DQCP2015MS04).
analysis of pharmaceutical genotoxic impurities, J. Pharm.
Biomed. Anal., 2010, 51, 999–1014.
1
5 N. V. V. S. S. Raman, A. V. S. S. Prasad and K. R. Reddy,
Strategies for the identication, control and determination
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