A Single Quadrupole Compact Mass Spectrometer Enabling Early Stage Synthetic Optimization of Verubecestat (MK-8931)

Article abstract of DOI:10.1021/acs.oprd.9b00370

A low-cost compact mass spectrometer was effectively utilized for identifying and tracking process impurities in the early stage development of a multistep synthetic protocol used to prepare the verubecestat drug substance. Process optimizations were infl

Full text of DOI:10.1021/acs.oprd.9b00370

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Communication
A Single Quadrupole Compact Mass Spectrometer Enabling
Early Stage Synthetic Optimization of Verubecestat (MK-8931)
Daniel Zewge, Xiaodong Bu, Li Zhang, David A. Thaisrivongs, Yanke Xu, and Xiaoyi Gong
Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.9b00370 • Publication Date (Web): 19 Nov 2019
Downloaded from pubs.acs.org on November 20, 2019
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A Single Quadrupole Compact Mass Spectrometer Enabling Early Stage
Synthetic Optimization of Verubecestat (MK-8931)
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Daniel Zewge,*a Xiaodong Bu,*a Li Zhang,a David Thaisrivongs,b Yanke Xu,c and Xiaoyi Gonga
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b
aSmall Molecule Analytical Research and Development; Small Molecule Process Research and
c
Development; Chemical Engineering Research and Development. Merck & Co., Inc., P.O. Box
2000, Rahway, NJ 07065, USA
*Corresponding author, E-mail address: daniel_zewge@merck.com; xiaodong_bu@merck.com
ABSTRACT:
A low-cost compact mass spectrometer was effectively utilized for identifying and tracking
process impurities in the early stage development of a multi-step synthetic protocol used to prepare
the verubecestat drug substance. Process optimizations were influenced by the type, quantity, and
final fate of impurities generated during synthesis. Employing a compact mass spectrometer for
impurity identification and tracking served as a guide to redesign synthetic strategies, which
enabled the minimization or avoidance of undesired side products.
Keywords: Verubecestat; Compact mass spectrometer; Impurity identification; Process chemistry
development
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Process chemists’ efforts at designing and executing efficient and robust synthetic routes
to produce multi kilogram batches of active pharmaceutical ingredients (APIs) rely on accurate,
precise, and reliable analytical techniques.1,2 Process related impurities are tracked throughout
synthetic protocols and their fate is determined to avoid compromising drug safety and efficacy.
As the production of high-quality pharmaceuticals requires continued optimizations of reaction
conditions geared towards developing efficient synthetic protocols, these efforts must be coupled
with continuous generation of actionable analytical data to guide the optimization processes. While
traditional analytical support is focused around developing chromatographic methodologies that
identify products from starting material and intermediates, the characterization and identification
of side products and impurities via mass spectrometry (MS) is becoming more prevalent. As
process chemists face the difficult challenge of developing processes for the commercial
manufacturing of an API within a short period of time, rapid understanding of the nature of
undesired side products and low-level impurities during synthetic protocols is critical towards
understanding molecular interactions that guide the development of the commercial synthetic
route.
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Mass Spectrometry, therefore, is becoming a much more common instrument in the
pharmaceutical industry, playing a critical role in the structural elucidation of small and large
molecules.3 While its potential in assisting analytical, process, and medicinal chemists is
undeniable, it remains less abundant as a laboratory instrument than high-performance liquid
chromatography (HPLC) and gas chromatography (GC) due mainly to cost and space constraints.
In that regard, the availability of an affordable compact mass spectrometer (less than $50,000)4 as
a chromatographic detector to analyze and track small quantity side products while also analyzing
the yield and purity of crude reaction mixtures would be appealing to the synthetic chemist. The
recently introduced Waters ACQUITY QDa mass spectrometer, a quadrupole-based instrument,
can reach up to 1250 ± 0.1 m/z in both negative and positive modes, and can operate at flow rates
of up to of 1 mL/min, with full scan and SIM (single ion monitoring) modes at a scan rate of 10,000
m/z units*sec-1. Although the mass range and resolution (0.7 Da) are somewhat reduced relative
to conventional MS instruments, the compact mass spectrometer is sufficient for most of the early
stage analyses to support synthetic chemistry, where the sample is generally abundant and unit
mass resolution is often acceptable.
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Herein, we demonstrate the use of QDa compact mass spectrometer in the identification
and tracking of impurities during early stage process optimizations of the synthesis of a beta
secretase inhibitor, verubecestat (MK-8931). Three case studies were selected to illustrate how
rapid data generation assisted in the immediate redesign and optimization of reaction protocols.
The presence of a quaternary chiral center in 10 makes synthesis of structurally distinct
novel thiadiazine core in verubecestat challenging.5 Depicted in Scheme 1, a five-step protocol
was developed by the process chemistry team which involves installation of a chiral auxiliary on
5-bromo-2-fluoroacetophenone 1 to generate intermediate 3. Early stage synthesis of ketimine 3
indicated that 1 is not inert to basic conditions and could generate polymeric impurities.6 Synthesis
of para-methoxybenzyl protected N-methylmethanesulfonamide 4 is performed via reductive
amination of 4-methoxybenzaldehyde followed by sulfonylation of the resulting secondary amine
with methanesulfonyl chloride. Lithiation of 4 followed by nucleophilic addition to 3 resulted in
the formation of 5 (not isolated), which was subsequently subjected to a copper catalyzed CN
coupling with 5-fluoropicolinamide 6 generating key intermediates 7a and 7b (scheme 1).
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O
S
O
S
O
Ti(OEt)4
EtOAc
O
O
O
S
N
F
t-Bu
t-Bu
NH
n-HexLi
THF
O
O
Br
S
Me
Me
NPMB
Me
Br
Br
S
H2N
t-Bu
Me
NPMB
Me
Step 1
Me
F
F
Step 2
1
2
4
3
5
O
F
CuI, trans-N,N'-dimethyl-
cyclohexane-1,2-diamine
F
H2N
S
O
O
F
H
N
t-Bu
NH
TFA
O
O
S
H
NH2
N
NPMB
Me
N
S
N
N
Me
NPMB
toluene
K2CO3, toluene, H2O
O
Me
F
O
O
F
Me
Step 3
Step 2 continued
6
7a
7b
NH3·Br
NH2
F
Me
F
F
H2N HN
S
Me
Me
O
;
K2CO3
H
N
N
S
N
N
S
BrCN
IPAc, MeCN
Step 4
H
H
N
N
O
N
N
O
O
N
N
Me
F
IPAc, n-heptane
O
O
O
Me
F
Me
F
O
O
Step 5
8
10
verubecestat (
)
9
Scheme 1: Early stage five step synthetic protocol developed to prepare verubecestat (10)
Case study 1: Copper catalyzed coupling of 5 and 6 was investigated using various ligands
including dimethylethane-1,2-diamine (L1) and cyclohexane-1,2-dimethylamine (L2). During
process development, we were puzzled by the observation of a 10 to 15% drop in the yield when
using L1 compared to L2, because UPLC-UV analysis showed a similar profile for crude samples
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from both ligands with no new impurities observed. Subsequently, UPLC with compact MS
detector was utilized to investigate the challenging issue. Further examination of the MS data
revealed that there was a suspicious MS peak co-eluting with the toluene solvent peak (Figure 1)
with molecular mass corresponding to a derivative of the new ligand, compound 26. The sample
was then subjected to overnight freeze drying to remove the solvent and re-injected to confirm the
generation of up to ~3% of ligand coupled impurity 26 compared to ≤ 0.02% of 27 (impurity
generated from coupling of L2 to 7a). This resulted in quick selection of L2 as the best option.
Use of compact MS detector to discover the new impurity co-eluting with the toluene solvent peak
was the key to understanding the yield issue and the subsequent identification and determination
of the impurity generation pathway. Identification of 26 provided an insight into reactivity of the
ligand towards the fluoropyridyl side chain and assisted in the final decision to further develop the
process with the less reactive and optimal ligand, L2.7
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Toluene
UV 210nm
ESI (+) TIC
.00
2.00
4.00
6.00
8.00
10.00
12.00
14.00
677.6
O
S
Me
N
t-Bu
NH
O
O
H
N
S
NH
Me
N
NPMB
Me
Me
F
O
26
C32H45FN6O5S2
Exact Mass: 676.2877
00.00
400.00
600.00
m/z
800.00
1000.00
Figure 1: Crude reaction mixture injected on UPLC/QDa (method A) to evaluate the unintended
side reaction generating 26.
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The coupling reaction with optimized conditions was efficient, affording intermediate 7a
in 85% yield. While the process resulted in approximately 12% of the partially de-protected
impurity 7b, once identified via MS analysis the partially de-protected impurity was considered as
part of the product, as de-protection will eventually remove the sulfinamide chiral auxiliary
exposing the free amine (Scheme 1). The rapid identification of this impurity enabled the process
chemists to redirect their optimization efforts.
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The use of copper (I) iodide in the process (coupling of 5 and 6) had resulted in an iodo
derivative 15 (due to incomplete conversion of a presumed reactive intermediate to coupling
products 7a) and des-bromo impurity 11. However, the use of catalytic amounts of copper (I)
iodide eliminated the iodo derivative and minimized the des-bromo impurity. Subjecting 11 to
subsequent de-protection and cyanation resulted in the ring closed product 13. Identification of the
ring closed impurity (Scheme 2) was very useful for impurity tracking purposes. Other side
products 28 and 29 (Scheme 5) resulting from coupling of 1 with 6 and nucleophilic addition of 4
to 1 were also detected and tracked in early optimizations.
O
O
O
CuI, trans-N,N'-dimethyl-
cyclohexane-1,2-diamine
S
S
F
S
t-Bu
NH
F
O
O
t-Bu
NH
O
O
t-Bu
NH
O
O
H
I
S
Br
NH2
S
N
S
NPMB
Me
N
NPMB
Me
N
NPMB
Me
K2CO3, toluene, H2O
Me
F
Me
F
Me
F
O
O
Step 2
15
6
7a
14
Iodo intermediate
O
S
NH3·Br
Me
Me
O
t-Bu
NH
N
N
S
H2N HN
S
O
O
TFA
BrCN
H
H
S
H
O
NPMB
Me
toluene
IPAc, MeCN
O
O
Me
F
Me
F
Me
F
Step 3
Step 4
13
Des-bromo impurity
12
Des-bromo impurity
11
Des-bromo impurity
Scheme 2: De-halogenated/halogen exchanged impurities detected in early process development
Case study 2: Dual de-protection of sulfinamide chiral auxiliary and para-methoxybenzyl groups
in compounds 7a and 7b was done using trifluoroacetic acid (TFA). While the de-protection step
is very efficient, giving a 96% isolated yield of de-protected 8 (Scheme 1), the presence of small
amounts of water (0.5 wt.%) in TFA resulted in generation of up to 1.12% of hydrolyzed product
16 (Figure 2) which eventually converts to the API analog 17 (Scheme 3). Based on these
observations, a specification was set to control the water in TFA to <0.1 wt%.
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NH
F
Me
F
Me
O
HN
OH
O
N
H
BrCN
wet TFA
H
N
S O
7a 7b
+
N
S
N
N
Me
IPAc, MeCN
Step 4
O
Me
O
O
O
F
F
16
17
9
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Scheme 3: Tracking fate of hydroxylated impurity 16.
Despite these corrective measures, a more effective de-protection reagent, methanesulfonic
acid (MSA), was later selected to remove the protecting groups and gain better control of the
impurity. De-protection reaction using MSA in acetic acid reduced the hydroxy impurity 16 to ND
levels (<0.03%). The valuable information obtained from UPLC/compact MS detector analysis
was useful in helping to optimize and implement a synthetic step thus avoiding formation of 16 as
well as its derivative 17.
1.40
1.20
Desired product
UV 210nm
1.00
0.80
0.60
hydroxyImpurity (~1.12%)
0.40
0.20
0.00
0.00
2.00
4.00
6.00
8.00
Minutes
10.00
12.00
14.00
F
Me
386.17
OH
HN
S
H
N
O
N
O
Me
F
O
275.13
16
C16H17F2N3O4S
Exact Mass: 385.0908
355.22
331.42
270.53
301.27
221.94
377.57
200.00
250.00
300.00
350.00
400.00
m/z
Figure 2: Crude reaction mixture injected on UPLC/QDa (method A) to track hydrolysis impurity
16.
Case study #3: In the late stage cyclization of the penultimate intermediate 8 with cyanogen
bromide (CNBr), a new impurity 19, with (MW = M-14) was observed (Figure 3). Subsequent
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pseudo-MS/MS experiments (in-source ion fragmentation) confirmed that an N-demethylation had
occurred. As N-demethylation commonly occurs under oxidative conditions, this impurity was
initially considered unlikely (no oxidation conditions involved in the protocol). However, the
experimental data was further supported by available literature which indicated the possibility of
N-demethylation (Von Braun reaction) in the presence of cyanogen bromide. As excess cyanogen
bromide (1.2 eq) was found to be the culprit the use of 0.98 to 1.0 equivalents of CNBr did
eventually minimize/eliminate the generation of the Von Braun product 19.
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0.25
0.20
0.15
Desired product
0.10
Demethylation
Impurity
0.05
0.00
2.00
4.00
6.00
8.00
10.00
12.00
14.00
Minutes
396.2
257.1
Pseudo Fragmentation @ 35V
202.1
200.00
300.00
400.00
500.00
600.00
700.00
150.00
200.00
250.00
m/z
300.00
350.00
m/z
Me
F
H
S
O
O
Fragment 1
C9H11FO2
Exact Mass: 202.0464
S
NH2
NH2
Excess
BrCN
F
Me
F
H
N
N
S
N
N
S
H
N
H
N
O
O
N
N
NH2
O
Me
F
O
Me
F
O
O
N
NH
S
10
19
O
O
Me
F
C16H15F2N5O3
Exact Mass: 395.0864
S
Fragment 2
C10H12FN3O2
S
Exact Mass: 257.0634
Figure 3: Crude reaction mixture with excess cyanogen bromide used for identification of
demethylation impurity 19 via UPLC/QDa (method B). Pseudo MS/MS experiments at 35V show
further fragmentation of demethylated ring.
Besides resulting in unintended demethylation, the use of excess CNBr also generated the
over cyanation product 20 (Figure 4a). While the addition of a nitrile functional group on one of
the active nitrogens was determined using UPLC/compact MS analysis data (pseudo MS/MS
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experiments), an LR-HSQMBC NMR experiment involving long range C-H coupling was
required to determine the exact position of cyanation.8 Early LC/MS experiments have also
identified over-cyanation on the pyridyl side chain 18.9
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8
9
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17
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19
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CN
NH2
HN
Excess
BrCN
F
Me
O
F
Me
O
N
N
N
N
S
H
H
N
S
N
N
N
O
Me
F
O
Me
F
O
O
10
20
Scheme 4: Excess cyanogen bromide intentionally charged to assess the effect of excess reagent
on ring closed product
In addition to side products that may be expected during chemical reactions, the possibility
of generating impurities via simple contamination of reaction vessels was examined. Indeed,
contamination of the reaction vessel with MeOH was found to be responsible for generating a
methoxy derivative (Figure 5B) via an SNAr reaction. Rapid identification of the methoxy
derivative 30 was critical in addressing and avoiding contamination issues, prompting elimination
of methanol as cleaning solvent for reaction vessels.10
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CN
435.1
HN
A)
F
Me
O
0.050
0.040
0.030
0.020
0.010
N
N
S
O
H
N-Cyanation
impurity
457.1
N
N
Me
F
O
Desired product
10
20
600.00
300.00
400.00
500.00
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m/z
5.50
6.00
6.50
7.00
7.50
8.00
8.50
9.00
Minutes
422.2
1.00
0.80
0.60
0.40
0.20
0.00
B)
NH2
Methoxy
impurity
MeO
Me
O
N
N
H
N
S
N
O
Me
O
F
30
C18H20FN5O4S
Exact Mass: 421.1220
350.00 400.00 450.00 500.00
0.00
2.00
4.00
6.00
8.00
10.00
12.00
14.00
Minutes
Figure 4: Crude reaction mixture injected on UPLC/QDa (method A) to identify/track (a)
overcyanation side product 20 (b) Impurity 30 resulting from contamination with MeOH via SNAr
reaction.
O
O
NH3·Br
O
S
F
Me
O
S
S
H2NHN
S
F
Me
F
6, CuI,Ligand
H
N
N
t-Bu
4, n-HexLi
t-Bu
NH
N
N
S
2, Ti(OEt)4
t-Bu
NH
O
O
TFA
BrCN
O
O
Br
H
N
H
O
Me
Br
Br
S
N
O
N
S
Me
NPMB
Me
K2CO3, toluene, H2O
Step 2 continued
N
Me
F
O
N
NPMB
Me
THF
Step 2
EtOAc
Step 1
toluene
Step 3
IPAc, MeCN
Step 4
O
O
Me
F
Me
F
Me
F
F
O
O
F
1
3
8
9
5
7a
O
O
O
S
S
Me
Me
NPMB
Me
34
N
t-Bu
H
NH
O
O
S
O
O O
O
4
N
S
S
F
Me
O
NH
Me
N
NPMB
Me
Me
N
OH HN
S
Me
F
O
O
H
N
O
Me
OMe
S
N
O
26
Me
Cl
O
Me
F
Case study #1
33
MeO
NH2
S
O
S
16
N
HN
F
Case study #2
N
NH
Me
N
t-Bu
NH
H
N
O
O
S
OMe
H
OMe
O
N
N
23
N
NPMB
Me
O
Me
F
NH
Me
F
O
O
19
OMe
27
H
32
MeO
HO
Case study #3
O
O
OMe
OMe
H2
N
O
O
S
t-Bu
NH
Me
O
S
O
OMe
Br
S
NPMB
Me
21
I
Me
F
NH2
NPMB
Me
MeO
Me
31
N
N
S
F
O
36
H
15
N
O
Br
N
F
O
Me
F
O
O
Br
H
N
O
18
N
Me
S
t-Bu
NH
O
35
O
F
O
F
Br
O
Br
S
NPMB
Me
28
Me
F
CN
HN
O
F
F
11
OH
HO
O
O
Me
O
F
Br
Br
N
N
S
Br
S
H
NPMB
Me
N
Br
Br
Me
F
N
Me
Me
O
Me
F
F
F
O
29
24
F
F
20
25
Scheme 5: Impurity map for a five-step total synthesis of verubecestat. All impurities were
identified and tracked using UPLC/QDa.11
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In summary, this paper highlights impurity tracking case studies used in early process
optimizations of verubecestat. The study demonstrates the value of a compact mass spectrometer
to generate useful critical analytical data with significant contributions towards developing robust
synthetic protocols to prepare pharmaceuticals. Diligent impurity tracking and mapping (Scheme
5) during reaction optimizations was a crucial part of successful process optimizations affording
the API with improved overall yield (~50%) and high purity “≥ 99.8%”. The efficiency and
robustness of a compact mass spectrometer lend itself to the analysis and rapid generation of
valuable information from crude reaction mixtures. Knowledge of where impurities are generated
and determination of the fate of side products saves time and minimizes the risk of compromising
the quality of the final API due to small amount of impurities that may not be identified via fit-for-
purpose HPLC methodologies.
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These studies have illustrated the benefit of a readily available LC-MS on every bench
providing researchers in the chemical sciences with powerful, personalized analytical support
tools. The revolution in low cost, miniaturized LC-MS promises to move beyond the traditional
process research laboratory into new domains, while improving and accelerating the design and
optimization of commercial routes for drug substances.
EXPERIMENTAL SECTION:
Chemicals and Reagents: All chemicals including ammonium formate (99%) and acetontrile
were purchased from Sigma Aldrich. All other samples are from Merck & Co., Inc., Kenilworth,
NJ, USA.
Preparation of samples and Buffers: All samples were dissolved in acetonitrile. A stock solution
of ammonium formate (200 mM) was prepared by dissolving 12.61g of ammonium formate in
water in 1L volumetric flask. Stock solution (10 mL) is diluted to 1L to prepare 2 mM ammonium
formate solution. Required buffer pH conditions were attained by adjusting pH using (ammonium
hydroxide, pH ~8.6) and (formic acid) for pH ~3.5.
Instrument: Waters ACQUITY QDa mass spectrometer is a compact quadrupole-based
instrument. It can reach up to 1250 ± 0.1 m/z in both negative and positive modes and can operate
at flow rates up to 1 mL/min, with full scan and SIM modes at a scan rate of 10,000 m/z units*sec-1.
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The instrument features include automated calibration, which virtually eliminates the need for the
sample-specific or user based tuning typical of traditional mass spectrometers. and disposable
sample cones and probes. The module is connected to standard Waters ACQUITY H-class UPLC
chromatography system and is controlled via Empower software. The version of the system used
in the studies also includes a floor pump with lower vacuum for improved sensitivity.
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UPLC/MS method: Water acquity UPLC BEH C18 1.7 um, 100 x 2.1mm column, Temp = 40
oC, flow rate = 0.6 mL/min, UV = 210 nm, Time = 15 min, post time = 3 min. Mobile Phase: A =
2 mM Ammonium Formate (pH ~8.6), Mobile Phase B= ACN. Method A: Gradient Time = 0
min, %B = 5, Time = 3 min, %B = 5, Time = 13 min, %B = 95, Time = 15 min, %B = 95, Time =
15.1 min, %B = 5. Sample concentration = 0.5 mg/mL, injection volume = 2µL. Method B:
Gradient Time = 0 min, %B = 3, Time = 3 min, %B = 3, Time = 13 min, %B = 95, Time = 15 min,
%B = 95, Time = 15.1 min, %B = 5. Sample concentration = 0.5 mg/mL, injection volume = 2µL.
Acknowledgements
The authors acknowledge Dr. Steve Miller, Dr. Carmela Molinaro, Dr. Azzeddine Lekhal, Mrs.
Sarah Pulicare and Mr. Ashish Shah for helpful discussions, Dr. Huaming Sheng for HRMS
support and Mr. Ryan Cohen for NMR support.
References
1 Thaisrivongs, D. A.; Miller, S. P.; Molinaro, C.; Chen, Q.; Song, Z. J.; Tan, L.; Chen, L.;Chen, W.; Lekhal, A.; Pilicare,
S. K.; Xu, Y. Org. Lett., 2016, 18, 5780-5783.
2 (a) Isolation and characterization of low-level impurities is a time-consuming process requiring resources from
several parts of the organization. As a result, obtaining useful analytical data on crude reaction mixtures would be
very useful in allowing process chemists to make rapid decisions towards optimization of reaction protocols. (b)
Bari, S. B.; Kadam, B. R.; Jaiswal, Y. S.; Shirkhedkar, A. A. Eurasian J. Anal.l Chem., 2007, 2, 32-53. (C) Grodowska, K.;
Parczewski, K. Acta Poloniae Pharmaceutica-Drug Research. 2010, 67, 3-12.
3 (a) Chen, G.; Paramanik, B, N.; Liu, Y-H.; Mirza, U. A. Journal of Mass Spectrometry, 2007, 42, 279-287 (b)
Hamilton, S. E.; Mattrey, F.; Bu, X.; Murray, D.; McCullough, B.; Welch, C. J. Org. Process. Res. Dev, 2014, 18, 103-
108.
4 (a) Bu, X.; Regalado, E. L.; Hamilton, S. E.; Welch, C.J. The emergence of low-cost compact mass spectrometry
detectors for chromatographic analysis. Trends in Anal Chem. 2016, 82, 22-34. (b) Snyder, D. T.; Pulliam, C. J.;
Ouyang, Z.; Cooks, R. G. Miniature and Fieldable Mass Spectrometers: Recent Advances. Anal Chem. 2016, 88, 2 –
29.
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5 Christoffers, J.; Baro, A. Stereoselective Construction of Quaternary Stereocenters. Adv.Synth. Catal. 2005, 347,
1473-1482.
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6 Minor polymerization of bromo fluoroacetophenone 1 (Scheme 1) was observed during early chiral auxiliary
installation process development generating proposed impurity structures 24 and 25 (Scheme 5).
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7 Phillips, E. M.; Reibarkh, M.; Limanto, J.; Kieu, M.; Lekhal, A.; Zewge, D. Improved Process for a Copper-Catalyzed
C−N Coupling in the Synthesis of Verubecestat. J. Org. Process. Res. Dev, 2019, 23, 1674 -1678.
8 Williamson, R. T.; Buevich, A. V.; Martin, G. E.; Parella, T. J. Org. Chem, 2014, 79, 3887–3894.
9 Overcyanation on fluoropyridyl side chain in 10 was explained using pseudo MS/MS data using UPLC/QDa.
NH2
F
F
F
Me
O
N
N
S
H
N
NC
NC
N
N
N
O
O
O
Me
F
O
C7H2FN2O
C6H3FNO
18
Exact Mass: 149.0146
Exact Mass: 149.0146
Impurity
C18H16F2N6O3S
Exact Mass: 434.0973
Fragments detected by LC/MS
10 Contamination of API with methanol in reaction vessels and generation of impurity 30 was avoided by replacing
methanol with acetone.
11 Additional impurities were identified using UPLC/QDa and will be discussed in future communications.
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