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Journal of Pharmaceutical and Biomedical Analysis 168 (2019)
181–188
Contents lists available at ScienceDirect
Journal of Pharmaceutical and Biomedical Analysis
j o ur na l ho mepage: www.elsev ier .com/ locate / jpba
olution degradant of mirabegron extended release tablets
resultingrom a Strecker-like reaction between mirabegron, minute
amountsf hydrogen cyanide in acetonitrile, and formaldehyde in PEG
duringample preparation
insheng Lina, Tianpei Huanga, Mengxue Fenga, Dan Lia, Danna
Zhaob, Jichao Wanga,ianyang Jina, Wenquan Zhua, Min Lia,c,∗
Center of Excellence for Modern Analytical Technologies (CEMAT),
Zhejiang Huahai Pharmaceutical Co. Ltd., Xunqiao, Linhai, Zhejiang,
317204, PR ChinaQuality Control Department of Formulation, Zhejiang
Huahai Pharmaceutical Co. Ltd., Xunqiao, Linhai, Zhejiang, 317204,
PR ChinaHuahai US, Inc. 700 Atrium Drive, Somerset, NJ 08873,
USA
r t i c l e i n f o
rticle history:eceived 2 December 2018eceived in revised form 22
January 2019ccepted 26 January 2019vailable online 1 February
2019
eywords:irabegron
yanomethylationC–MSEGcetonitriletrecker-like reaction
a b s t r a c t
During the related substances testing of mirabegron extended
release tablets, an unknown peak wasobserved in HPLC chromatograms
in a level exceeding the identification threshold. By using a
strategythat combines LC–PDA/UV-MSn with mechanism-based stress
studies, the unknown peak was rapidlyidentified as cyanomethyl
mirabegron, a solution degradant that is caused by a Strecker-like
reactionbetween the API, formaldehyde (an impurity in PEG), and HCN
(an impurity in HPLC grade acetoni-trile). The mechanism of the
solution degradation chemistry was verified by stressing mirabegron
withformaldehyde and trimethylsilyl cyanide (TMSCN, a synthetic
reagent that generates HCN upon contactwith water), in which the
secondary amine group of mirabegron first reacts with formaldehyde
to formthe iminium ion intermediate; the latter then undergoes a
nucleophilic attack by cyanide to yield thecyanomethyl mirabegron.
The structure of the impurity was further confirmed through the
synthesisof the impurity and subsequent structure characterization
by 1D and 2D NMR. Due to the ubiquitouspresence of formaldehyde in
pharmaceutical excipients (e.g., PEG and polysorbate) and trace
amount of
HCN in HPLC grade acetonitrile, this type of solution
degradation would likely occur in sample prepara-tions of
pharmaceutical finished products containing APIs with primary and
secondary amine moieties.In a GMP environment, such an event may
trigger undesirable out-of-specification (OOS) investigations;the
results of this paper should help resolve such OOS investigations
or even prevent these events fromhappening in the first place.
© 2019 Elsevier B.V. All rights reserved.
. Introduction
Mirabegron, chemically known as
2-(2-aminothiazol-4-yl)N-[4-(2-{[(2R)-
2-hydroxy-2-phenylethyl]amino}ethyl)phenyl]cetamide, is the
first-in-class, potent and selective ˇ3-drenoceptor agonist for the
treatment of overactive bladder
OAB) [1,2]. In the process of our pharmaceutical developmentor
mirabegron extended release tablets, an unknown peak wasbserved at
a relative retention time (RRT) of 1.76 during analysis
∗ Corresponding author at: Center of Excellent for Modern
Analytical Technolo-ies (CEMAT), Zhejiang Huahai Pharmaceutical Co.
Ltd., Xunqiao, Linhai, Zhejiang,17204, PR China.
E-mail address: [email protected] (M. Li).
ttps://doi.org/10.1016/j.jpba.2019.01.045731-7085/© 2019
Elsevier B.V. All rights reserved.
of certain tablet samples by the related substances method.
Uponinitial investigation, it was found that the amount of this
unknownpeak could vary from 0.08% to 0.29% for the very same batch
of thetablets, dependent upon the sources of the HPLC grade
acetonitrile(used as part of the sample diluent and mobile phase).
Meanwhile,the same impurity was found to be below the detection
limit of0.02% in the corresponding API lot, when the latter sample
wasanalyzed by the same related substances method for the tablets.
Inorder to find out the root cause for the occurrence and
associatedinconsistency of this unknown impurity, its structure
needed to beelucidated.
In general, structure elucidation of pharmaceutical impuritiesat
low levels (e.g., ∼0.1–0.2%) could be quite challenging due to
anumber of factors. One of such factors is the limited
availabilityof a properly purified impurity sample, particularly
from samples
https://doi.org/10.1016/j.jpba.2019.01.045http://www.sciencedirect.com/science/journal/07317085http://www.elsevier.com/locate/jpbahttp://crossmark.crossref.org/dialog/?doi=10.1016/j.jpba.2019.01.045&domain=pdfmailto:[email protected]://doi.org/10.1016/j.jpba.2019.01.045
-
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82 J. Lin, T. Huang, M. Feng, et al. / Journal of Pharma
f a finished drug product, for structure determination via 1D
andD NMR spectroscopy. Even in cases where the sample supply
wasbundant, isolation of impurities at such minute amounts coulde
very laborious and time-consuming. A strategy, developed athe
former laboratories of the corresponding author of this paper,as
been demonstrated to be capable of overcoming these chal-
enges and rapidly elucidating the structures of these impurities
atrace levels [3–8]. This strategy combines LC–PDA/UV-MSn
(partic-larly MSn molecular fingerprinting) with mechanism-based
stresstudies and in doing so, the structure of the impurity can
usuallye inferred, within a very short period of time (very often
in aater of a few days), with a very high confidence level. In
caseshere the initially inferred structure needs additional
assurance
y 1D and 2D NMR spectroscopy, an ample supply of the impu-ity
sample can generally be produced from the mechanism-basedtress
studies, followed by isolation of the impurity via prepara-ive or
semi-preparative chromatography. The key component ofhis strategy
is the design of relevant stress studies (a.k.a. forcedegradation)
based on the plausible formation mechanism of the
mpurity, which may be revealed by the initial LC–PDA/UV-MSn
esults. The mechanism-based stress studies typically can
affordhe target impurity in amounts much higher than those that
onean isolate from the samples of pharmaceutical finished productsn
which the impurity was originally observed.
In the present investigation of the RRT 1.76 impurity in
theolutions of mirabegron extended release tablets, this strategy
ofombining LC–PDA/UV-MSn with mechanism-based stress stud-es was
adopted, and consequently the unknown peak was rapidlydentified as
cyanomethyl mirabegron, a solution degradant that isaused by a
Strecker-like reaction [9] between the API, formalde-yde (an
impurity in PEG) [10–12], and HCN (an impurity inPLC grade
acetonitrile) [13,14]. The structure of the impurityas further
confirmed via synthesis of the impurity and subse-
uent structure characterization by 1D and 2D NMR spectroscopy.ue
to the ubiquitous presence of formaldehyde in pharmaceu-
ical excipients (e.g., PEG and polysorbate) and trace amount
ofCN in HPLC grade acetonitrile, such type of solution degrada-
ion due to the Strecker-like reaction would likely occur in
samplereparations of pharmaceutical finished products containing
pri-ary and secondary amine APIs in other pharmaceutical
testing
aboratories.
. Experimental
.1. Materials
Mirabegron API and its extended-release tablets were
manufac-ured by Zhejiang Huahai Pharmaceutical Co., Ltd. PEG 6000
was
product of Spectrum Chemical MFG Corp. (New Brunswick, NJ8901,
USA). Acetonitrile and ethanol were purchased from MerckGaA
(Pittsburgh, PA, USA), and acetonitrile was also purchased
rom Sigma-Aldrich Corp. (St. Louis, MO, USA). Other reagents
pro-ured from Sigma-Aldrich Corp included methanol, ammoniumcetate,
and ammonia solution. Sodium acetate was purchasedrom Fisher
Chemical Corp. Sodium hydroxide (extra pure, 50t% solution in
water) was purchased from Acros Organics Corp.cetic acid was
supplied by Hangzhou Chemical Reagent Co., Ltd
Hangzhou, China). Aqueous formaldehyde solution (37%) was
pro-uced by Shanghai Jiuyi Chemical Reagent Co., Ltd
(Shanghai,hina). Bromoacetonitrile was purchased from Shanghai
Ourchemhemical Reagent Co., Ltd (Shanghai, China) and
trimethylsilyl
yanide (TMSCN) was procured from Shanghai Aladdin Biochem-cal
Technology Co., Ltd (Shanghai, China). Pre-coated TLC plates
ith a layer of 0.25 mm silica gel (SIL G-25) were produced
byacherey-Nagel GmbH & Co. KG.
al and Biomedical Analysis 168 (2019) 181–188
2.2. HPLC analyses
2.2.1. Sample solution preparationTen extended release tablets
of mirabegron, either 25 mg or
50 mg, were randomly selected and then placed into a 200 mL
vol-umetric flask, followed by the addition of 60 mL of the first
samplediluent (MeOH/CH3CN = 1:1, v/v). The flask was vigorously
shakento fully disintegrate the tablets and 60 mL of the second
samplediluent (H2O/CH3CN = 4:1, v/v) was added. The sample
prepara-tion mixture was then sonicated for a minimum of 20 min
withthe temperature of the sonication bath set at 30◦, during
whichprocess the mixture was occasionally shaken. After sonication,
themixture was diluted to volume with the second sample
diluent.Finally, the sample preparation mixture was filtered
through a0.22 �m nylon membrane filter and the first 2 mL filtrate
was dis-carded.
2.2.2. HPLC conditionsA Thermo Scientific Dionex Ultimate 3000
HPLC system,
equipped with a Waters Xterra MS C18 column (150 mm × 4.6 mm,3.5
�m) and a PDA/UV detector, was used for conducting therelated
substances method. The mobile phase system consistedof A (water
with 15 mM ammonium acetate) and B (acetonitrile),with the gradient
varied according to the following program: 0 min(10% B), 2 min (10%
B), 30 min (50% B), 30.1 min (10% B), and35 min (10% B). The
analyses were performed at a flow rate of1.0 mL/min and a column
temperature of 40◦. UV spectra werecollected from the PDA/UV
detector with a wavelength range of190–400 nm.
2.3. LC–PDA/UV–MSn analysis
An Agilent HPLC instrument (1260 series, Agilent Technolo-gies,
USA) equipped with a single quadrupole mass spectrometer(6120
series, Agilent Technologies) was used for the initial
LC–MSanalysis. Another Agilent HPLC instrument (1260 series,
AgilentTechnologies, USA) interfaced to a quadrupole time-of-flight
(Q-TOF) mass spectrometer (6545 series, Agilent Technologies,
USA)was used for comprehensive LC-PDA/UV-MSn (n = 1, 2) analyses
ofsamples. The chromatographic conditions of the LC–MS methodwere
the same as those of the related substances method. UV spec-tra
were collected from 190 nm to 400 nm by the PDA detectors.The Q-TOF
mass spectrometer was operated at positive ESI modewith the
following source parameters: gas flow 6 L/min, nebulizerpressure 60
psi, source temperature 320◦, sheath gas temperature350◦, sheath
gas flow 12 L/min and capillary voltage 3.5 kV. Reser-pine and
purine were used as the reference compounds for accuratemass
measurement. The mass acquisition range was 100–1700 Daand for the
MS2 analyses, the collision energy was set at 10, 30, and45 eV,
respectively.
2.4. Stress studies, preparation, and purification of the RRT
1.76impurity
For the mechanism-based stress study to generate
thecyanomethylated impurity, a mixture of mirabegron (10 mg,0.025
mmol), 37% formaldehyde (50 �L, 1.8 mmol) and TMSCN(50 �L, 0.4
mmol) in methanol (1.0 mL) was shaken at 30 ◦C for 3 h.Aliquots of
the reaction solution were analyzed by the HPLC andLC–MSn methods
as described in Sections 2.2 and 2.3, respectively.The targeted
impurity, cyanomethyl mirabegron, was obtained in∼49% yield. The
same stress study was also performed in acetoni-
trile, and the yield of cyanomethyl mirabegron increased to
67%.
Because TMSCN is highly toxic, we chose an alternative
syntheticroute to prepare a larger quantity of cyanomethyl
mirabegron forNMR structure confirmation. In this alternate route,
bromoacetoni-
-
J. Lin, T. Huang, M. Feng, et al. / Journal of Pharmaceutical
and Biomedical Analysis 168 (2019) 181–188 183
F e mira solut2 m the
ttwbptoodmv
2
bdNAaesd
ig. 1. (a) The original HPLC chromatogram at 254 nm of the
sample solution from tht 22.918 min. (b) The UV chromatogram at 240
nm of the same mirabegron tablet4.177 min. (c) High resolution MS
spectrum of the RRT 1.76 unknown impurity fro
rile was reacted with mirabegron in the presence of
triethylamine:o a solution of mirabegron (0.5 g, 1.26 mmol) in
methanol (7 mL)as added bromoacetonitrile (0.105 mL, 1.50 mmol),
followed
y the addition of triethylamine (0.383 mL, 2.77 mmol) in
oneortion. The mixture was allowed to stir overnight at
roomemperature and approximately 1/3 of the mixture was loadednto
three TLC plates for isolation. The target compound wasbtained (89
mg, 53% yield) with a solvent system
containingichloromethane/methanol (10:1, v/v). The yield of
cyanomethylirabegron decreased to 31% (HPLC yield) when the
reaction sol-
ent was changed from methanol to acetonitrile.
.5. 1D and 2D NMR determination
Approximately 15 mg of cyanomethyl mirabegron, preparedy the
reaction between mirabegron and bromoacetonitrile asescribed in
Section 2.4, was dissolved in 1 mL of CDCl3. 1H, 13CMR and 2D NMR
spectra of the compound were acquired on agilent 400 MHz
spectrometer at 25◦. 1H and 13C resonances were
ssigned and confirmed by the results from the following 2D
NMRxperiments: gCOSY, gHSQC, and gHMBC. The corresponding NMRpectra
of mirabegron were recorded similarly, except that DMSO-6 was used
as the solvent.
abegron 50 mg extended release tablets; the RRT 1.76 unknown
peak was observedion as in (a) from the initial LC-PDA/UV-MS
analysis; the target impurity eluted at
same mirabegron tablet solution as in (a).
2.6. Different sources of HPLC grade acetonitrile and
methanolversus the occurrence of the RRT 1.76 impurity; trace
amount ofHCN in HPLC grade acetonitrile
We have learned from our past experience that residual HCNmay
exist in HPLC grade acetonitrile and the level seemed to
varydependent upon the sources or vendors of the solvent [13]. On
theother hand, HPLC grade methanol does not seem to contain
HCN.Hence, HPLC grade acetonitrile and methanol from different
ven-dors were used in the preparation of the sample diluent and
mobilephase to examine the impact of different solvents or
different ven-dors on the occurrence of the RRT 1.76 impurity.
2.7. Ion chromatographic (IC) analysis
A Thermo Scientific ICS-5000+ DP IC system equipped with
anelectrochemical detector was used for analyzing residual HCN
indifferent solvents. A Dionex IonPacTM AS7 ion exchange column(150
mm × 4 mm) was used for the analysis and a Dionex Ion-PacTM AG7
column (50 mm × 4 mm) was used as guard column.The mobile phase
consisted of A (water with 0.5 M sodium acetate)
and B (water with 0.1 M sodium hydroxide). The analyses were
per-formed at a flow rate of 1.0 mL/min and a column temperature
of30◦. Electrochemical spectra were collected from the
electrochem-ical detector (mode: Integrated amperometric; waveform:
silver,
-
184 J. Lin, T. Huang, M. Feng, et al. / Journal of
Pharmaceutical and Biomedical Analysis 168 (2019) 181–188
Fig. 2. LC-ESI-MS/MS spectra of (a) [M+H]+ of the RRT 1.76
impurity (m/z 436) and (b) [M+H]+ of mirabegron (m/z 397) at the
collision energy of 30 eV. The experimentsw ed in( obileg rom 2
AA
3
3
irtwtarshczdrtioff4tm
ere performed on the Agilent LC-QTOF MS system with MS
parameters describ150 mm × 4.6 mm, 5 �m) maintained at 40 ◦C and
the elution was affected with mradient at flow rate of 1.0 mL/min:
10% B from 0 to 2 min and increasing to 50% B f
g-AgCl RE, S-2, CN-, I-) equipped with working electrode
andg/AgCl reference electrode.
. Results and discussion
.1. LC–PDA/UV–MSn (n = 1, 2) analysis
The unknown peak was originally observed at 22.918 min dur-ng
the analysis of certain samples of the mirabegron extendedelease
tablets (Fig. 1a) and its relative retention time (RRT) versushat
of mirabegron (13.008 min) was found to be 1.76. This peakas seen
at 24.177 min in the initial LC–PDA/UV–MS analysis of
he mirabegron tablets sample solution (Fig. 1b). For
consistencynd simplicity, this unknown peak is referred to as RRT
1.76 impu-ity or species in this paper. The UV spectrum of the RRT
1.76pecies is essentially identical to that of mirabegron with
bothaving a UV absorption peak at 250 nm, indicating that the
twoompounds have the same chromophore, i.e., the 2-amino thia-ole
moiety. The accurate MS spectrum of the RRT 1.76 speciesisplayed
three m/z values at 409.1689, 436.1804, and 494.2333,espectively
(Fig. 1c). Upon careful examination, it was determinedhat the m/z
436.1804 species is the protonated ion of RRT 1.76mpurity, while
m/z 494.2333 is the gas phase acetonitrile adductf the ammoniated
ion of RRT 1.76 impurity, and m/z 409.1689 is aragment of the ions
of RRT 1.76 impurity resulting from in-source
ragmentation. The m/z value of the protonated RRT 1.76
impurity,36.1804, matches a formula of C23H25N5O2S with an error of
lesshan 1.6 ppm. By comparing with the formula of the
protonated
irabegron, C21H24N4O2S, the RRT 1.76 impurity has an
additional
Experimental section. The system was equipped with a Zorbax SB
C18 column phases A (0.1% formic acid in water) and B
(acetonitrile) according to the follow
to 30 min.
C2HN component. The MS/MS spectra of both the protonated RRT1.76
species (m/z 436) and mirabegron (m/z 397) showed severalcommon
fragment ions at m/z 379, m/z 260, m/z 239, m/z 146, m/z120, and
m/z 113 at the collision energy of 30 eV (Fig. 2a & b),
sug-gesting that the RRT 1.76 species may have the same core
structureas mirabegron.
Based upon the fact that the RRT 1.76 unknown species hasextra
C2HN component over the molecular formula of mirabegronbut
essentially the same UV absorption spectrum as mirabegron,we
surmised that the unknown species could result from
theStrecker-like reaction (during the sample preparation step)
inwhich the secondary amine moiety of mirabegron first
condensedwith formaldehyde to form the iminium intermediate,
followed bythe attack of HCN (Scheme 1). This hypothesis seemed to
be reason-able as the two components of the reaction, formaldehyde
and HCN,can be present in the excipients (PEG) [10–12] of the
mirabegrontablets and sample diluent (acetonitrile) [13,14],
respectively. Sucha cyanomethylation on the secondary amine group
of mirabegrondoes not generate any new UV chromophore, which is
consistentwith the observation that the UV spectra of the RRT 1.76
unknownspecies and mirabegron are essentially the same. With that
pre-sumed structure in mind, we started to examine the
fragmentationpatterns in the MS2 spectrum of the RRT 1.76 impurity
versusthose of mirabegron (the latter was previously studied by
Kalariya[15]), in order to uncover the fragmentation pathway
indicative ofthe cyanomethylated amine moiety. The vast majority of
the frag-
ments are the same between the RRT 1.76 impurity and
mirabegron(Fig. 2), which is not unexpected considering that the
two structuresare very similar. On the other hand, the fragment
ions at m/z 409 andm/z 391 are unique only to the cyanomethylated
mirabegron. More
-
J. Lin, T. Huang, M. Feng, et al. / Journal of Pharmaceutical
and Biomedical Analysis 168 (2019) 181–188 185
Scheme 1. Proposed formation pathway of cynaomethyl mirabegron
in mirabegron extended release tablets sample solution due to the
Strecker-like reaction betweenmirabegron, trace amount of
formaldehyde in PEG, and trace amount of HCN in acetonitrile.
Scheme 2. Proposed fragmentation pathway of the protonated RRT
1.76 impurity observed in certain sample solutions of mirabegron
extended release tablets.
-
1 ceutical and Biomedical Analysis 168 (2019) 181–188
immr
3t
fri(mtstaswwttMi
trrmtosmrsmTegopb1
3
1iacdb(ptg2CF5Hs
Table 11H and 13C NMR spectroscopic data of mirabegron (DMSO-d6)
and the RRT 1.76impurity of mirabegron (CDCl3; ı in ppm).
positionmirabegron1 RRT 1.76 impurity1
H shifts C shifts H shifts C shifts
1 N.A.2 144.7 N.A. 141.22 7.29 125.9 7.31 (1H, o3) 125.93 7.27
127.9 7.32 (1H, o) 128.64 7.19 126.8 7.27 (1H, m) 128.05 7.27 127.9
7.32 (1H, o) 128.66 7.29 125.9 7.31 (1H, o) 125.97 4.57 71.5 4.65
(1H, dd, J = 9.4, 2.5 Hz) 70.28a
2.61 57.62.62 (1H, t, J = 12.0 Hz)
62.58b 2.82 (1H, o)9a
2.72 50.82.85 (1H, o)
55.59b 2.93(1H, m)10 2.62 35.4 2.75 (1H, dd, J = 16.0, 4.0 Hz)
33.411 N.A. 135.1 N.A. 134.712 7.08 128.8 7.11 (1H, d, J = 8.0 Hz)
129.113 7.46 119.0 7.45 (1H, d, J = 8.0 Hz) 120.414 N.A. 137.2 N.A.
136.715 7.46 119.0 7.45 (1H, d, J = 8.0 Hz) 120.416 7.08 128.8 7.11
(1H, d, J = 8.0 Hz) 129.117 N.A. 167.8 N.A. 167.718 3.43 39.8 3.57
(1H, s) 40.119 N.A. 145.9 N.A. 145.120 6.27 102.6 6.30 (1H, s)
105.721 N.A. 168.2 N.A. 168.722a N.A. N.A. 3.59 (1H, d, J = 16.0
Hz)
42.522b N.A. N.A. 3.72 (1H, d, J = 16.0 Hz)23 N.A. N.A. N.A.
115.1-NH 9.99 N.A. N.A. N.A.-NH2 6.89 N.A. 5.26 (2H, br s4) N.A.-OH
—— N.A. 9.03 (1H, s) N.A.
Notes: 1: The numbering of the carbon skeleton in the RRT 1.76
impurity in thesample solution‘n from the mirabegron extended
release tablets is shown as follows.The numbering for mirabegron
follows the same rule. 2: Abbreviations: N.A., notapplicable; o,
overlap signals; br s, broad singlet signal.
86 J. Lin, T. Huang, M. Feng, et al. / Journal of Pharma
nterestingly, the accurate mass of m/z 132.0805 (Fig. 2a,
Inset)atches a formula of C9H10N+, suggesting the m/z 132 fragment
isost likely deriving from m/z 391 and still retaining the
methylene
esidue on the secondary amine moiety (Scheme 2).
.2. Mechanism-based stress study, preparation and isolation ofhe
RRT 1.76 impurity
From the above LC-PDA/UV-MSn results, we had surmised
theormation mechanism and likely structure of the RRT 1.76
impu-ity. In order to verify the mechanism, a stress study was
designedn which mirabegron was reacted with formaldehyde and TMSCNa
cyanide donor) [16] as outlined in Section 2.4. In the UV chro-
atogram of the mirabegron drug substance sample solution afterhe
addition of TMSCN and aqueous formaldehyde (hence the
stressolution), a peak at 22.690 min was generated in ∼67% yield
afterhe stress solution was allowed to stand at 30◦ for 3 h (Fig.
3). The UVbsorption spectrum and MS2 fingerprint of this
stress-generatedpecies at 22.690 min matched those of the target
impurity thatas originally observed at a relative retention time
(RRT) of 1.76hen the original sample solution of mirabegron
extended release
ablets was analyzed by the related substances method (for the
lat-er method, refer to Section 2.2.2; the comparison of the UV
and
S2 spectra between the stress-generated species and the
targetmpurity is not shown).
The above stress study provided further evidence to supporthe
hypothesis that solution degradation due to the
Strecker-likeeaction is responsible for the formation of the RRT
1.76 impu-ity (Scheme 1). Nevertheless, this forced degradation
reactionay not be suitable for a somewhat larger scale preparation
of
he impurity for NMR structure confirmation, due to the toxicityf
TMSCN (which gives off HCN upon contact with water). In aeparate
study, Vivier et al. [17] reported that a secondary amineoiety can
easily react with bromoacetonitrile to afford the cor-
esponding cyanomethyl derivative. Thus, we decided to use
thisynthetic approach to prepare a quantity of the
cyanomethylatedirabegron that is sufficient for 1D and 2D NMR
determination.
he cyanomethylated mirabegron impurity thus prepared
displaysssentially identical retention time, UV spectrum, and MS2
fin-erprint as compared to those of the RRT 1.76 unknown
speciesbserved in the original mirabegron extended release tablets
sam-le solution. Hence, we concluded that the RRT 1.76 impurity
hadeen generated and we moved forward to verify its structure viaD
and 2D NMR spectroscopy.
.3. 1D and 2D NMR characterization of the RRT 1.76 impurity
All the 1H NMR and 13C NMR data of mirabegron and the RRT.76
impurity are summarized in Table 1. The 1H and 13C chem-
cal shifts of mirabegron were reported by Rajendiran et al
[18],nd the NMR results of the in-house mirabegron API (Table 1)
areonsistent with those reported in the literature mentioned
above,espite the fact that different solvents were used. The NMR
signalsetween mirabegron and the RRT 1.76 impurity are very
similarTable 1), except for the H-22, C-22, and C-23 signals which
are onlyresent in the RRT 1.76 impurity. Based on their chemical
shifts,hese three sets of signals are very likely due to the
cyanomethylroup. Indeed, the HMBC analysis revealed the correlation
of H-2a/H-22b (�H 3.59, 1H, d, J = 16.0 Hz/�H 3.72, 1H, d, J = 16.0
Hz) with-22 (�C 42.5, t) and C-23 (�C 115.1, s), respectively (Fig.
4 HMBC).
urthermore, the HMBC correlations of H-22a/H-22b with C-9
(ıC5.5, t), C-8 (ıC 62.5, t), and C-23 (ıC 115.1, s) are also
present. TheseMBC results confirm that the cyanomethyl group is
linked to the
econdary amine moiety of mirabegron.
3.4. Different sources of acetonitrile and methanol used
ascomponent for sample diluent and mobile phase: correlation
withthe formation and/or level of the RRT 1.76 impurity
In the course of the investigation, we noticed that the levelof
the RRT 1.76 impurity appeared to correlate with the sourcesof
acetonitrile (they are referred to as Vendor 1 and Vendor
2,respectively). With the acetonitrile sourcing from Vendor 1 as
thecomponent for sample diluent and mobile phase, we found thatup
to 0.12% of the RRT 1.76 impurity was formed. On the con-trary,
when the acetonitrile sourcing from Vendor 2 was used, only0.04% of
this impurity was observed from the very same batchof mirabegron
extended release tablets samples. Furthermore, wealso found that
the content of the RRT 1.76 impurity was belowthe detection limit
(∼0.02%) in the mirabegron API sample solutioneven when Vendor 1
acetonitrile was used as diluent.
When the sample diluent component acetonitrile was replacedby
methanol during the sample preparation, the RRT 1.76 specieswas
essentially absent. It is also worth noting that replacing
ace-tonitrile in the mobile phase with methanol had no
meaningfulimpact on the formation of the RRT 1.76 species,
indicating thatthis impurity should be formed mainly during the
step of the sam-
ple solution preparation, in which all the three components of
theStrecker-like reaction, i.e., mirabegron, formaldehyde, and HCN,
arepresent.
-
J. Lin, T. Huang, M. Feng, et al. / Journal of Pharmaceutical
and Biomedical Analysis 168 (2019) 181–188 187
Fig. 3. UV chromatogram at 240 nm of the stress mirabegron
solution: a mirabegron drug substance solution in acetonitrile was
added TMSCN and 37% formaldehyde aqueoussolution, and the resulting
solution was allowed to stand at room temperature for 3 h. The HPLC
yield of the RRT 1.76 impurity (22.690 min) was ∼67%.
elation
aet0
3is
t
Fig. 4. The key gHMBC corr
We also measured the quantities of residual HCN in HPLC
gradecetonitrile from the two vendors using ion chromatography
withlectrochemical detection. The amounts of HCN in HPLC grade
ace-onitrile from Vendor 1 and 2 were observed at 0.65 �g/mL and.22
�g/mL, respectively.
.5. Solution degradation due to the Strecker-like reaction and
itsmplication toward drug molecules containing primary and
econdary amine moiety
All the evidence presented above clearly shows that duringhe
sample preparation of mirabegron extended release tablets,
s of the RRT 1.76 impurity.
mirabegron undergoes solution degradation due to the
Strecker-like reaction in which its secondary amine moiety was
modifiedthrough cyanomethylation. One of the key components of
theStrecker-like reaction, formaldehyde, is apparently from PEG,
akey excipient of the drug product. It is well known that PEG
canundergo oxidative degradation to form various impurities
includ-ing formaldehyde [10]. On the other hand, HCN is an impurity
inacetonitrile and its content in HPLC grade acetonitrile can
vary
among different vendors [13,14].
Due to the ubiquitous presence of formaldehyde in
pharmaceu-tical excipients (e.g., PEG and polysorbate) and trace
amount of HCNin HPLC grade acetonitrile, this type of solution
degradation would
-
1 ceutic
lpptlsgwtwrf
4
cwossshnmfistPaicGshf
A
mt
R
[
[
[
[
[
[
[
[
(2011) 5705–5710.
88 J. Lin, T. Huang, M. Feng, et al. / Journal of Pharma
ikely occur in sample preparations of pharmaceutical
finishedroducts containing primary and secondary amine APIs in
otherharmaceutical testing laboratories. It is somewhat surprising
thathere seems to have no previous report on this type of
cyanomethy-ated drug degradant, except for a forced oxidative
degradationtudy of drug molecules that contains primary and
secondary amineroups. In the latter study, the cyanomethylated drug
degradantsere reported as the major artifactual degradants
resulting from
he reaction between the drug molecules, formaldehyde, and
HCN,ith the last two components generated as byproducts from
the
adical initiator, 2,2’-azobis(2-methylpropionitrile) (AIBN),
usedor the free radical–mediated oxidative stress [9].
. Conclusions
A solution degradation product observed in HPLC analysis
ofertain sample solutions of mirabegron extended release tabletsas
rapidly identified as cyanomethyl mirabegron via the strategy
f using LC-PDA/UV-MSn in conjunction with mechanism-basedtress
studies. The solution degradation product has been demon-trated to
result from the Strecker-like reaction in which theecondary amine
group of mirabegron first reacts with formalde-yde to form the
iminium intermediate; the latter then undergoes aucleophilic attack
by hydrogen cyanide to yield the cyanomethylirabegron. The
structure of the impurity has been further con-
rmed through the synthesis of the impurity and
subsequenttructure characterization by 1D and 2D NMR. Due to the
ubiqui-ous presence of formaldehyde in pharmaceutical excipients
(e.g.,EG and polysorbate) and trace amount of HCN in HPLC
gradecetonitrile, this type of solution degradation would likely
occurn sample preparations of other pharmaceutical finished
productsontaining APIs with primary and secondary amine moieties.
In aMP environment, such an event may trigger undesirable
out-of-pecification (OOS) investigations; the results of this paper
shouldelp resolve such OOS investigations or even prevent these
events
rom happening in the first place.
cknowledgment
We gratefully acknowledge Quality Control Department of
For-ulation of Zhejiang Huahai Pharmaceutical Co. Ltd for their
echnical support during the course of this investigation.
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EAS Abstracts.pdfhttp://www.eas.org/askeas/2012 EAS
Abstracts.pdfhttp://www.eas.org/askeas/2012 EAS
Abstracts.pdfhttp://www.eas.org/askeas/2012 EAS
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Solution degradant of mirabegron extended release tablets
resulting from a Strecker-like reaction between mirabegron,
minu...1 Introduction2 Experimental2.1 Materials2.2 HPLC
analyses2.2.1 Sample solution preparation2.2.2 HPLC conditions
2.3 LC–PDA/UV–MSn analysis2.4 Stress studies, preparation, and
purification of the RRT 1.76 impurity2.5 1D and 2D NMR
determination2.6 Different sources of HPLC grade acetonitrile and
methanol versus the occurrence of the RRT 1.76 impurity; trace
amount...2.7 Ion chromatographic (IC) analysis
3 Results and discussion3.1 LC–PDA/UV–MSn (n = 1, 2) analysis3.2
Mechanism-based stress study, preparation and isolation of the RRT
1.76 impurity3.3 1D and 2D NMR characterization of the RRT 1.76
impurity3.4 Different sources of acetonitrile and methanol used as
component for sample diluent and mobile phase: correlation
with...3.5 Solution degradation due to the Strecker-like reaction
and its implication toward drug molecules containing primary
an...
4 ConclusionsAcknowledgmentReferences
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