Characterization of major degradation products of an adenosine A 2A receptor antagonist under stressed conditions by LC-MS and FT tandem MS analysis

Article abstract of DOI:10.1002/jms.1695

Parkinson's disease (PD) is a very serious neurological disorder, and current methods of treatment fail to achieve long-term control. SCH 420814 is a potent, selective and orally active adenosine A_2A receptor antagonist discovered by Schering-Plough. Stability testing provides evidence of the quality of a bulk drug when exposed to the influence of environmental factors. Understanding the drug degradation profiles is critical to the safety and potency assessment of the drug candidate for clinical trials. As a result, identification of degradation products has taken an important role in drug development process. In this study, a rapid and sensitive method was developed for the structural determination of the degradation products of SCH 420814 formed under different forced conditions. The study utilizes a combination of liquid chromatography-tandem-mass spectrometry (LC-MS/MS) and Fourier Transform (FT) MS techniques to obtain complementary information for structure elucidation of the unknowns. This combination approach has significant impact on degradation product identification. A total of ten degradation products of SCH 420814 were characterized using the developed method. Copyright

Full text of DOI:10.1002/jms.1695

Research Article
Received: 17 July 2009
Accepted: 16 October 2009
Published online in Wiley Interscience: 12 November 2009
(www.interscience.com) DOI 10.1002/jms.1695
Characterization of major degradation
products of an adenosine A2A receptor
antagonist under stressed conditions by LC-MS
and FT tandem MS analysis
Li-Kang Zhang∗ and Birendra N. Pramanik
Parkinson’s disease (PD) is a very serious neurological disorder, and current methods of treatment fail to achieve long-term
control. SCH 420814 is a potent, selective and orally active adenosine A2A receptor antagonist discovered by Schering-Plough.
Stability testing provides evidence of the quality of a bulk drug when exposed to the influence of environmental factors.
Understanding the drug degradation profiles is critical to the safety and potency assessment of the drug candidate for clinical
trials. As a result, identification of degradation products has taken an important role in drug development process. In this study,
arapidandsensitivemethodwasdevelopedforthestructuraldeterminationofthedegradationproductsofSCH420814formed
under different forced conditions. The study utilizes a combination of liquid chromatography–tandem-mass spectrometry
(LC-MS/MS) and Fourier Transform (FT) MS techniques to obtain complementary information for structure elucidation of the
unknowns. This combination approach has significant impact on degradation product identification. A total of ten degradation
c
products of SCH 420814 were characterized using the developed method. Copyright ꢀ 2009 John Wiley & Sons, Ltd.
Keywords: antagonist; degradation; FT MS; MS/MS; structure; LC/MS/MS
Introduction
novel compounds to prepare drug candidates with improved
stability. It has been well documented that drug products will
undergo physicochemical degradation during manufacturing
processes and storage.[10] Understanding the drug degradation
profiles is also critical to the safety and potency assessment of
the drug candidate for clinical trials. In addition, stress testing
can help in the selection of more-stable drug substance salt
forms and drug formulations.[11] It is well accepted that the
drug degradation in formulations is highly complex and often
unpredictable process. The degradation products usually arise
from the ingredients used in dosage formulation and/or in
the process of formulation where temperature, humidity and
light may all play a part. The degradation products are able
to be generated from hydrolysis, oxidation, adduct formation,
dimerization, rearrangement and often the combination of these
processes. To accelerate drug development, various stress-testing
protocols had been designed to emulate stresses the compound
may experience during manufacturing and storage conditions.[9]
These methods expose drug candidates to forced degradation
conditions such as acid, base, heat, oxidation and exposure to
light.
SCH 420814 (2-(furan-2-yl)-7-(2-(4-(4-(2-methoxyethoxy)phenyl)
piperazin-1-yl)ethyl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c] pyr-
imidin-5-amine) is a novel chemical entity which is being
investigated for the treatment of Parkinson’s disease (PD), a very
serious neurological disorder.[1–3] The effects of adenosine are
mediated through at least four specific cell membrane receptors
classified as A1, A2A, A2B and A3. These receptors belong to the
G protein-coupled receptor class. It is reported that the A2A and
A2B receptors can couple to Gs/olf proteins that activate adenylate
cyclase and increase intracellular levels of cAMP. On the other
hand, the A1 and A3 receptors decrease cellular cAMP levels
through their coupling to Gi proteins, which inhibit adenylate
cyclase.[4] Adenosine A2A receptors modulate the release of GABA
in the striatum, which appears to regulate the activity of medium
spiny neurons. By reducing GABA output, A2A antagonism helps
restore normal function in the basal ganglia following dopamine
depletion.[5] Thus, A2A antagonism affords a possible treatment for
PD.[6–8] SCH 420814 is a potent, selective and orally active adeno-
sine A2A receptor antagonist discovered by Schering-Plough. It
exhibits high affinity for both human and rat A2A receptors, with
Ki values of 1.1 and 2.5 nM, respectively.[1] Moreover, SCH 420814
is more than 1000-fold selective for human A2A receptors over
A1, A2B and A3 receptors, with Ki values at human A1, A2B and A3
receptors of >1000, >1700 and >1000 nM, respectively.[1] The
compound is currently in the phase II clinical trials.
LC/MS has become the technique of choice in the degradation
product studies due to its inherently high level of sensitivity
and specificity. There have been a number of reports in the
∗
Correspondence to: Li-Kang Zhang, Chemical Research, Schering-Plough
Stress testing of a drug candidate is a critical component of both
drug discovery and drug development.[9] The identification of
drug degradation products in various dosage/formulations plays
animportantroleinthedrugdiscoveryprocess.Informationonthe
structures of degradation products can help chemists modify their
Research Institute, 2015 Galloping Hill Road, K-15-1/1945, Kenilworth, NJ
07033, USA. E-mail: li-kang.zhang@spcorp.com
Chemical Research, Schering-Plough Research Institute, 2015 Galloping Hill
Road, Kenilworth, NJ 07033, USA
c
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Copyright ꢀ 2009 John Wiley & Sons, Ltd.

Characterization of degradation products of A2A antagonist by LC-MS and FT tandem MS analysis
literature that applied LC-MS and LC-MS/MS for characterization
of degradation products.[12–28] A procedure for the rapid structure
elucidation of drug degradation products induced by acid, base
and heat was reported by Rourick et al. in 1996.[17] The same
group demonstrated that the similar strategy could be applied to
obtain the structural information of the degradation products of
paclitaxel(Taxol).[19] In2000,Wu[21] reviewedtheapplicationofLC-
MS in the analysis of drug degradation products in pharmaceutical
formulationsundervariousstressconditions(oxidation,hydrolysis,
dimerization and adduct formation with excipients). Feng et al.[14]
investigated the oxidative degradation products of an antifungal
agent,SCH56 592,bybothLC/MSandLC/NMRanalysis.Fourmajor
oxidative degradation products of SCH 56 592 were characterized,
and the oxidations were found to be occurred at the piperazine
ring in the center of the drug molecule. More recently, Li et al.[29]
reportedtheuseofmultiple-stagetandemMS(LC-MSn)technique,
in conjunction with mechanism-based stress studies, to identify
the drug degradation products in pharmaceutical development.
Exact mass measurements and elemental-composition assign-
ment are essential for the characterization of small molecules.[22]
Accurate mass measurement of the product ions, formed in an
MS/MS experiment, facilitates the structure elucidation of new or
unknown materials.[22] Hybrid quadrupole/time-of-flight MS had
been employed for accurate mass measurement for well over a
decade.[30–32] The mass measurement precision/accuracy can be
5 ppm with internal calibration methods. One limitation of the
instruments of this type was the narrow ion abundance range
over which accurate mass measurements could be made with a
high degree of certainty.[30–33] Currently, FT-ICR MS provides the
highest mass resolving power and mass accuracy among all the
mass spectrometric methods.[22] Using external calibration, FT-ICR
MSiscapableofachieving massmeasurementaccuraciesof1 ppm
or better.[22] Smith and co-workers recently reported that by using
a new trapped-ion cell with improved DC potential harmonic-
ity, they achieved under 0.05 ppm root-mean-square precision.[34]
This is impressive application that is not possible with any other
knownmassspectrometricequipment.AlthoughFTMShasrapidly
developed as one of the mosteffective techniques for the determi-
nation of unknowns, its application to drug degradation products
characterization is much less routine than for the drug metabo-
lites screening and drug impurities identification.[29,33,35–40] Bai
et al.[41] evaluated the application of MALDI-FT-ICR MS for the
identification of the degradation products of surfactant proteins.
They reported that owing to the high-resolving power, FT-ICR MS
was found to provide substantial advantages for the structural
identification of surfactant proteins and their degradants from
complex biological matrixes with high mass accuracy.[41]
measure the accurate mass and to establish the formula of SCH
420814 degradation products for structural assignments.
Experimental
Materials
SCH 420814, 2-(furan-2-yl)-7-(2-(4-(4-(2-methoxyethoxy)phenyl)
piperazin-1-yl)ethyl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c] pyr-
imidin-5-amine, was provided by the Chemical Development
Group at Schering-Plough Research Institute. The stock solution
of SCH 420814 was prepared by dissolving the compound in
acetonitrile/water (80 : 20, v : v) at the concentration of 1.0 mg/ml.
Triflouroacetic acid (99%) was purchased from Sigma-Aldrich
Chemical Co. (St Louis, MO, USA) and used without further
purification. Acetonitrile, deionized water and tetrahydrofuran
were also obtained from Aldrich Chemical Co. Hydro chloric acid
(2N), sodium hydroxide and hydrogen peroxide were obtained
from Fisher Scientific (Fair Lawn, NJ, USA).
Sample preparation
For all of the solution stability studies, the stability samples were
prepared by mixing a stock solution of SCH 420814 (1 mg/ml) with
various media (30 : 70; v : v). Acid and alkaline stress studies were
performed in 0.1 N HCl and 0.1 N NaOH at room temperature
for 5 days, respectively. The study in oxidation condition was
carried out in 1% H2O2 for 24 h. The photo stability study was
carried out in a quartz cylindrical cell with Teflon stopper (6445A
Photostability Chambers, CARON, Marietta, OH, USA). The SCH
420814 solution (acetonitrile/H2O; 50/50; v/v) was exposed to
UV/VIS in a temperature-controlled (25 ◦C) light chamber. The
total UVA and CWF exposure were equal to 208 W h/m2 and
1.3 million lux-h, respectively. To assess the physical stability
of the compound, SCH 420814 was combined with a common
tablet excipient. The active/excipient capsule was stored on an
open dish (RH4) for 1 month (Lab-Line and Barnstead/Thermolyne
EC14075 small stability chamber, Thermo Scientific, Waltham,
MA, USA). The HPLC sample solution was prepared by diluting
the active/excipient mixture with water/acetonitrile (50 : 50, v : v).
Following forced decomposition, all samples are stored at −20 ◦C
to minimize further degradation and allow for future use.
LC-MS, LC-MS/MS, FT MS and LC-FT MS analyses
LC/ESI-MS and LC/ESI-MS/MS analyses were performed on a PE-
Sciex QStar Pulsar Q-TOF mass spectrometer (Applied Biosystems,
Foster City, CA, USA) coupled to a Shimazu HPLC system,
operated in the positive-ion mode. The HPLC system consisted
The aims of this research were to perform stress studies on
SCH 420814 in order to evaluate its inherent stability, and to
develop rapid online LC-MS(/MS) and ultra-high resolution FT
MS methods to characterize the degradation products. Although
the main focus of the study was chemical degradation of SCH
420814 in solutions, the assessment of the physical stability of the
stressed tablets by RH4 condition (open dish, 40 ◦C/75% relative
humidity, 1 month) was also exhibited. The research reported here
utilized a combination of LC-MS(/MS) and FT MS techniques to
obtain complementary information for structural elucidation of
the unknowns. Although the LC-MS offered the molecular weight
of the degradation product, the LC-MS/MS provided the detailed
structural elucidation information. We will also demonstrated that
the ultra-high resolving power of the FT MS was a useful tool to
of solvent delivery module (LC-10ATVP), auto injector (SIL-10AVP
)
and UV-visible dual-wavelength detector (SPD-10AVP) (Shimadzu
Corporation, Tokyo, Japan). All HPLC separations were performed
at ambient temperature. The samples were analyzed on a reverse-
phase C18 column (YMC ODS-A, 250 mm × 4.6 mm, I.D., 5 µm)
using A (water/tetrahydrofuran/trifluoroacetic acid; 90 : 10 : 0.1,
v : v : v) and B (acetonitrile/water/tetrahydrofuran/trifluoroacetic
acid; 50 : 40 : 10 : 0.1, v : v : v : v) as mobile phases at a flow rate of
1.0 ml/min. The injection volume was 30 µl and UV detection was
set at 262 nm. The mobile phase gradient started at 0% B, and
linearly increased to 100% B over 40 min. The tandem MS analysis
of all the compounds was performed on the same Q-TOF MS
instrument in the MS-MS mode, with Turbo-Ion-Spray ionization.
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J. Mass. Spectrom. 2010, 45, 146–156
Copyright ꢀ 2009 John Wiley & Sons, Ltd.
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L.-K. Zhang and B. N. Pramanik
The collision energy was 35 eV for all the analytes and standards.
Thetime-of-flightwasoperatedwithapulsingfrequencyof10 kHz.
Calibration was achieved using CsI at m/z 133, Verapamil at m/z
455 and a peptide (ALILTLVS) at m/z 829. The resolution of the
time-of-flight was 10 000, full width half maximum (FWHM) at m/z
455. The quadrupole mass analyzer was set to unit resolution.
High-resolution MS spectrum were acquired on a Bruker
Daltonics APEX IV 70e FT mass spectrometer (Bruker Daltonic,
Billerica, MA, USA) equipped an Apollo electrospray ionization
source. A source temperature of 120 ◦C was used for all
experiments. The samples were infused at a flow rate of 1 µl/min
into the electrospray source, to which a voltage of 3.8 kV was
applied. Ions were stored in the source region in a hexapole
guide for 2 s and pulsed into the detection cell through a series
of electrostatic lenses. Ions were finally trapped in the cell using
either gas-assisted dynamic trapping (Xe pulses, upper pressure
around 10−7 mbar) with front and back trapping voltages of 3.0
and 3.5 V for trapping, reduced to 0.5 and 0.6 V for detection or
static trapping. Mass spectra were acquired from m/z 120 to 2000
with 1024 K data points, and peaks were automatically labeled
using the XMASS 7.08 software (Bruker Daltonik GmbH, Bremen,
Germany). The external calibration was achieved using Verapamil
at m/z 455.2904, a peptide (ALILTLVS) at m/z 829.5393 as well as
the product ion of ALILTLVS at m/z 724.4967 (ALILTLV), 625.4283
(ALILTL) and 512.3443 (ALILT). The resolution of the time-of-flight
was 150 000, FWHM at m/z 455.2904. For sustained off resonance
irradiation collision-induced dissociation (SORI-CID) experiment,
the ion to fragment was isolated by radio frequency (rf) ejection of
all unwanted ions using both low-voltage single rf pulses at their
resonance frequencies and a chirp excitation covering the region
of interest. The fragmentation was performed using excitation
duration of 400 µs at a frequency 500 Hz higher than the cyclotron
frequency of the ion of interest. Xenon was used as the collision
gas and introduced several times through a pulsed valve to get a
pressure in the cell up to 10−7 mbar. The excitation/detection of
all fragment ions was performed after a 3 s pumping delay.
in positive mode. The ESI source parameters were optimized to
reduce the background signals and to minimize fragmentation
reactions. The molecular weight of SCH 420814 (Rt 18.5 min)
was confirmed to be 503 Da by LC/MS analysis (spectrum not
presented). The elemental composition of SCH 420814 was
established by high-resolution FT MS (MH+: C25H30N9O3). The
calculated exact mass and measured mass was 504.2472 and
504.2474 amu, respectively. The mass measurement error of
−0.5 ppm was achieved using external calibration. According
to the LC-UV and LC-MS data, there was no impurity detected in
this batch of SCH 420814.
An initial step in elucidating degradant structures of SCH
420814 is to understand the fragmentation pattern of the
drug substance. Extensive tandem-mass spectrometry analysis
of the fragmentation pattern of SCH 420814 provides a basis
for assessing structural assignment for the degradation products.
The ability of FT MS to provide an exact mass measurement for
each of the ions produced in tandem MS assists greatly in the
investigation of the fragmentation mechanism of SCH 420814.
Accurate mass characterization of SCH 420814 (m/z 504) was
performed on a Bruker FT MS instrument using SORI-CID. Collision-
induced decomposition (CID) of the ESI-produced MH+ precursor
generates ions at m/z 311, 263, 234 and 206 (Fig. 1). The most
abundant product ion (m/z 263.1754) observed in the spectrum
is formed by a neutral loss of 2-(furan-2-yl)-7H-pyrazolo[4,3-
e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine (Fig. 1). It could further
decompose to an ion of m/z 234.1364 by a loss of ethyl radical
in the gas phase (Fig. 1). Two complementary fragment ions of
SCH 420814 are also observed at m/z 206.1175 and 311.1363
(Fig. 1). The mass measurement errors of these fragment ions are
determined to be −0.02, 0.22, 0.51 and 0.53 ppm, respectively
(Fig. 1). SCH 420814 was also submitted to LC/MS/MS analysis on a
hybrid Qq-TOF instrument. The product-ion spectrum generated
from the Q-TOF instrument is dominated by the same fragments
as in the FT SORI-CID spectrum (m/z 311, 268, 263, 234 and
206) (product-ion spectrum not shown). The relative intensities of
these ions recorded on the two instruments are also very similar.
LC/ESI-FT MS was performed on the same instrument coupled
to an Agilent 1100 series liquid chromatograph (Agilent G1312A
binary pump, G1313A Auto sample injector, G1316A Column oven
and G1314A VWD UV detector; Agilent Technologies, Wilmington,
DE,USA).Thebase-stressedSCH420814wasanalyzedbyLC-FTMS.
All HPLC separations were performed at 25 ◦C. The samples were
analyzed on a reverse-phase C18 column (YMC ODS-A, 250 mm ×
4.6 mm, I.D., 5 µm) using 0.1% formic acid and acetonitrile as
a mobile phase at a flow rate over the column of 500 µl/min
in the split-flow LC systems. The LC systems were controlled
through HyStar version 3.3 (Bruker), and the FT MS was controlled
by apexControl 2.0. The injection volume was 10 µl. The mobile
phase gradient started at 40% acetonitrile in 0.1% formic acid, and
linearly increased to 80% acetonitrile over 6 min, and then linearly
increased to 90% in 4 min. The FT MS conditions were the same as
those described above for the standards. DataAnalysis 3.3 (Bruker)
was applied to process the FT MS data.
Degradation behaviors of SCH 420814
The forced degradation samples of SCH 420814 (acidic, basic,
oxidative and photolytic) were analyzed by LC-MS(/MS) to identify
the degradation products. The LC-MS total ion chromatograms
(TIC) were obtained for SCH 420814 at each stress conditions
(Fig. 2). A total of ten degradation products of SCH 420814 were
characterized using extensive tandem MS analysis.
Studies under acidic conditions
After 5 days exposure to the acidic media at room temperature,
less than 10% degradation was observed according to the LC/UV
data. Three degradation products (A, B and C) were detected
in the acid-stressed sample (Fig. 2(A)), and the relative retention
times (RRT) of the three components were found to be 0.61, 0.81
and 0.87, respectively (Fig. 2(A)). According to the LC/MS data, the
molecular weight of A, B and C were determined to be 353, 489
and 477 Da, respectively.
Results and Discussion
Mass accuracy is critical in establishing compound identity.
Accurate mass measurements (i.e. those less than 2 ppm) can aid
in the characterization of the degradation products by placing
constraints on elemental formulas of the unknowns. In order to
obtain the accurate masses of the three components, the acid-
stressed SCH 420814 sample was submitted to high-resolution
SCH 420814 structure characterization by LC-MS, LC-MS/MS,
and FT MS analyses
ToassessthepurityofSCH420814,aMS-friendlyHPLCmethodwas
developed as described in the experimental section. The synthetic
compound was analyzed by both LC/ESI-MS and LC/ESI-MS/MS
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Characterization of degradation products of A2A antagonist by LC-MS and FT tandem MS analysis
Figure 1. Proposed fragmentation pathways and ESI-FT MS product-ion spectrum of SCH 420814.
FT MS analysis. The elemental compositions of A, B and C
were established to be C16H20N9O (MH+: Cal. 354.1785, found
354.1787; error 0.47 ppm), C24H28N9O3 (MH+: Cal. 490.2309, found
490.2312; error 0.48 ppm) and C23H28N9O (MH+: Cal. 478.2315,
found 478.2318; error 0.60 ppm), respectively.
LC-MS/MS experiments were carried out on the molecular ions
of the degradation products in order to obtain their fragmentation
patterns for structural analysis. The tandem MS data suggest that
compound A is a hydrolysis product of SCH 420814 (Scheme 1).
Upon CID, the MH+ ions of compound A (m/z 354) decompose
to give a product-ion spectrum of ions of m/z 311, 268, 242
and 113 (Fig. 3(A)). The most abundant fragment ion peak in
the spectrum (m/z 113) corresponds to a 1-ethylidenepiperazin-
1-ium ion (Scheme 1). The product ion at m/z 311 is generated
by a neutral loss of ethenamine (MW 43) from A. The further
loss of ethenamine from the ion at m/z 311 forms the ion at
m/z 268 (Scheme 1). Comparing with the formula of SCH 420814
(MH+: C25H30N9O3), the component B (MH+: C24H28N9O3) is a
decomposition product via the net loss of a CH2 group (Scheme 2).
Majorproductionsfromtheprecursorion(m/z 490)weredetected
atm/z 311, 268, 249, 220, 192and84asshowninFig. 3(B). Themost
intense peak (m/z 249) in the spectrum corresponds to loss of the
terminal methyl group from SCH 420814 (Scheme 1). The product
ions of m/z 220 and 192 originate from m/z 249 as proposed
in Scheme 1. The structure of degradant B was also confirmed
with a chemically synthesized standard using LC-UV, LC-MS and
LC-MS/MS techniques. The retention time and the product-ion
spectrum of the degradation product are identical to those of
the standard. Compound C is another degradant detected in the
acid-stressed SCH 420814. High-resolution FT MS data confirm
the difference between the m/z 504.2474 (SCH 420814) and m/z
478.2318 (compound C) is 26.0156 amu or equivalent to a C2H2
group. According to the LC/MS/MS data, product ions from the
molecular ion (m/z 478) were detected at m/z 311, 285, 268, 242,
237 and 194, as shown in Fig. 3(C). The abundant fragment ions
of m/z 311 and 268 are also observed in product-ion spectrum
Figure 2. LC-UV chromatograms of the stressed SCH 420814 by 0.1 N HCl
for 5 days (A); 0.1 N NaOH for 5 days (B); 1% H2O2 for 1 day (C); as well as
UV/VIS in a temperature-controlled (25 ◦C) light chamber (D).
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L.-K. Zhang and B. N. Pramanik
+H+
NH
+H+
NH
N
N
2 N
2 N
NH
2 N
NH
N
2 N
N
N
O
O
N
N
O
N
N
N
N
HN
N
O
N
N
N
N
HN
N
N
N
HN
HN
N
N
A
m/z 311
m/z 268
m/z 242
m/z 113
m/z 354
NH
NH
2 N
2 N
HO
N
N
N
O
N
NH
O
2 N
N
N
O
N
N
N
N
N
N
O
HN
N
HO
N
N
N
O
m/z 311
m/z 268
m/z 249
N
N
N
B
HO
HO
m/z 490
O
O
N
N
N
m/z 220
m/z 192
+H+
NH
2 N
NH
NH
2
2 N
N
N
N
O
N
N
+H+
O
O
N
N
N
N
N
N
NH2
N
N
N
N
HN
N
N
N
H2N
N
O
N
m/z 311
m/z 285
m/z 268
MeO
N
O
N
NHHN
+H+
NH
N
2 N
MeO
MeO
H
N
C
N
O
O
O
N
NHHN
m/z 478
HN
N
m/z 242
m/z 237
m/z 194
Scheme 1. Proposed fragmentation mechanism for compounds A, B and C.
of SCH 420814, indicating that the part of the molecule is intact
(Scheme 1). The major product ion observed at m/z 237 is a crucial
evidence for the breakdown of the center piperazine ring, as
interpreted in Scheme 1. Moreover, the product ions of m/z 285,
242 and 194 are well consistent with the proposed fragmentation
mechanism of the compound C in Scheme 1.
measurement errors were determined to be −2.2 ppm (MH+
found: 522.2560) and −3.5 ppm (MH+ found: 522.2554) for the
internal and external calibrations, respectively. A structure was
proposed for the compound D (Scheme 2) and verified by tandem
MS analysis (Fig. 4). Loss of NH3 from the precursor ion (m/z 522)
leads to an abundant product ion at m/z 505 (Fig. 4, Scheme 2).
Further elimination of CO from this ion results a low abundant
ion at m/z 477 (Scheme 2). The most intense fragment ions of
m/z 263 and 234 are the same as those observed in the product-
ion spectrum of SCH 420814, indicating the hydroxylation does
not occur on this part of the molecule (Scheme 2). The product
ion of m/z 206 originates from m/z 263 and 234 as shown in
Scheme 2. The product ion of m/z 84 is the protonated 1,2,3,6-
tetrahydropyrazine. A characteristic product ion of m/z 312 fully
agrees with the proposed structure of D (Scheme 2).
Studies under basic conditions
There was only one degradation product, D (RRT 0.79, m/z 522),
observedintheLC/MSchromatogramofthebasic-stressedsample
(Fig. 2(B)). The molecular weight difference of 18 amu between
this compound and SCH 420814 (503 Da) suggests that it might
be a hydrolysis product of SCH 420814. In order to generate the
formula of compound D, the basic-stressed sample was submitted
to high-resolution FT ESI-MS analysis. However, no ionization was
observed for this unknown when it was direct infused into the
FT-ICR/MS operating in positive mode. This effect is probably due
to the very basic pH of the stressed sample. To overcome the
problem, the degradation product was analyzed by LC-FT MS,
and the generated data was processed by using DataAnalysis 3.3
software (Bruker). The formula of degradant D was confirmed to
be C25H32N9O4 (MH+: Cal. 522.2571) by applying both internal and
external calibrations. The external calibration approach made use
of Verapamil and a peptide (ALILTLVS) as calibrants. The internal
calibration was performed manually at single point correction
mode by using SCH 420814 as calibrant. The accurate mass
Studies under oxidative conditions
Almost 80% of the drug degraded upon exposure to 1% H2O2 for
24 h (Fig. 2(C)). On the basis of the LC/MS(/MS) data of the stressed
sample, three unknowns were characterized as compound E
(RRT 0.71, MW 535), F (RRT 0.76, MW 535) and G (RRT 0.88,
MW 519) (Schemes 3 and 4). According to the LC-UV and LC-MS
data, the relative amounts of the degradation products to that
of SCH 420814 after the H2O2 stress was found to be F > SCH
420814 > E > G. Comparing with the molecular weight 503
Da of SCH 420814, the unknown G, which has an additional 16
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J. Mass. Spectrom. 2010, 45, 146–156

Characterization of degradation products of A2A antagonist by LC-MS and FT tandem MS analysis
Figure 3. MS/MS product-ion spectra of the protonated molecular ions at m/z 354 (A), 490 (B) and 478 (C) for components A, B and C in Fig. 2A,
respectively.
N
N
N
O
N
+H+
O
H2N
N
H2N
N
O
O
NH2
N
N
MeO
N
N
O
-CO
H2N
N
O
O
N
N
N
N
N
N
m/z 477
MeO
O
N
N
N
m/z 312
m/z 505
m/z 263
D
MeO
MeO
MeO
m/z 522
O
N
N
O
O
N
N
N
m/z 234
m/z 206
Scheme 2. Proposed fragmentation mechanism for compound D.
mass units, may be the oxidized product via the addition of an
oxygen atoms. Similarly, unknown E and F may be the di-oxidation
product of SCH 420814. The stressed sample was analyzed by FT
MS, and the elemental composition of E, F and G were confirmed
to be C25H30N9O5 (MH+: Cal. 536.2364, found 536.2361; error
−0.63 ppm), C25H30N9O5 (MH+: Cal. 536.2364, found 536.2361;
error −0.63 ppm) and C25H30N9O4 (MH+: Cal. 520.2415, found
520.2413; error −0.43 ppm), respectively.
The informative tandem MS data reveal the structural infor-
mation pertaining to the two di-oxidation isomers (Fig. 5). The
mass fragmentation pattern for E is quite different from that for
F. Collisional activation of the precursor ion of the E results in a
number of product ions, with m/z 323, 313, 268, 252, 232, 206 and
193, as shown in Fig. 5(A). On the other hand, the collision-induced
dissociation of F yields major product ions of m/z 339, 311, 297,
268, 232 and 194 (Fig. 5(B)). Upon CID, unknown E and F forms
c
J. Mass. Spectrom. 2010, 45, 146–156
Copyright ꢀ 2009 John Wiley & Sons, Ltd.
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L.-K. Zhang and B. N. Pramanik
Figure 4. MS/MS product-ion spectrum of compound D (m/z 505) generated by alkaline stressed conditions.
Figure 5. Comparison of the MS/MS spectra of SCH 420814 degradation products E (A) and F (B) generated by oxidative conditions.
a common fragment ion of m/z 268, which was also observed in
the product-ion spectrum of SCH 420814. This indicates this part
of SCH 420814 is intact (Scheme 3), and the di-oxidation could
occur only on the piperazine ring. For both E and F, the breaking
of the carbon-nitrogen bond, followed by consecutive loss of two
waters yielded the most abundant ion at m/z 232 (Scheme 3). For
compound E, the piperazine-ring cleavages give rise to two major
product ions of m/z 313 and 252 (Scheme 3). Further fragmen-
tation of the ion at m/z 252 generates product ion at m/z 193
via loss of ethene-1,2-diol radical (Scheme 3). Two characteristic
product ions of m/z 323 and 206 are also consistent with the
proposed structure of E as shown in Scheme 3. For compound
F, the through piperazine-ring cleavages leads to product ions of
m/z 339, 311, 297 and 194 (Fig. 5(B)), and their interpretation are
shown in Scheme 3. These distinctive fragment ions of compound
F are not seen in the product-ion spectrum of E (Fig. 5). Thus, the
study of the MS/MS fragmentation pathway of the two degrada-
tion products allows the rapid and direct differentiation of the two
proposed structures (E and F).
The mono-oxidation product G was also identified as a minor
degradant of the H2O2-stressed SCH 420814 (Fig. 2(C)). The char-
acteristic fragment ions of degradation product G are observed at
m/z 323, 311, 297, 279, 262, 249, 242, 234, 207 and 193 (spectrum
not shown), and a fragmentation pathway is proposed in
Scheme 4. The product-ion spectrum of G clearly indicates the hy-
droxyl group is locating on the piperazine ring; however, the exact
oxidation position cannot be elucidated solely by mass spectrom-
etry (Scheme 4). The most of the fragmentation is occurred at the
piperazine components of the molecule (i.e. m/z 323, 311, 207 and
193inScheme 4). Twocomplementaryfragmentionsofthedegra-
dation product were detected at m/z 279 and 242 (Scheme 4).
Recall that the fragment ion of m/z 263 is the most abundant
one observed in the product-ion spectrum of SCH 420814 (Fig. 1).
We believe the ion of m/z 279 is formed in an analogous manner
and agrees well with our proposed structure of G (Scheme 4). The
hydroxyl group, however, can locate on either the 2- or 3-position
of the piperazine ring (Scheme 4), and the two isomers cannot
be easily distinguished by the LC-MS/MS analysis. For further
c
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J. Mass. Spectrom. 2010, 45, 146–156

Characterization of degradation products of A2A antagonist by LC-MS and FT tandem MS analysis
NH2
MeO
MeO
HO OH
N
N
N
N
O
O
N
N
O
N
H+
N
NH2
N
m/z 268
m/z 252
NH2
m/z 193
N
N
O
N
MeO
HO OH
N
NH2
N
O
N
N
N
N
MeO
N
N
N
MeO
N
O
O
N
N
OH
O
HN
E
O
N
N
N
N
N
N
N
H
N
m/z 536
m/z 206
m/z 323
m/z 232
m/z 313
NH2
NH2
NH2
N
N
NH2
N
N
N
N
H+
N
O
N
N
O
N
N
N
N
O
NH2
N
N
O
N
N
N
N
N
N
N
HN
N
N
N
O
N
N
N
N
MeO
HO
N
H
N
OH
N
O
N
N
m/z 268
m/z 311
m/z 339
H+
m/z 297
OH
MeO
F
MeO
O
N
NH
m/z 536
O
NH
m/z 232
m/z 194
Scheme 3. Proposed fragmentation mechanism for compounds E and F.
+H+
MeO
NH2
N
NH2
HO
N3 2 N
MeO
N
N
N
O
N
N
N
O
O
O
N
N
N
H
N
N
N
HN
or
H+
N
NH2
N
m/z 297
m/z 242
+H+
m/z 262
MeO
OH
N3 2 N
N
N
O
N
MeO
HO
N
O
N
MeO
N
O
N
MeO
N
N
O
O
N
NH
OH
N
N
m/z 249
m/z 279
m/z 234
G
NH2
NH2
N
N
MeO
m/z 520
MeO
N
N
N
N
O
O
N
N
O
NH
O
N
N
N
N
N
N
HN
m/z 323
m/z 311
m/z 207
m/z 193
Scheme 4. Proposed fragmentation mechanism for compound G.
structural confirmations, the MS and NMR analysis of the synthetic
degradation products and/or isolated samples are required.
On the basis of our results, we conclude that the forced
oxidations are occurred at the piperazine ring in the center of
SCH 420814.
LC/MS/MS studies. Five major degradation products (A, C, H, I
and J) were detected as shown in Fig. 6. The relative amounts of
the degradants to that of SCH 420814 after the RH4 stress was
found to be SCH 420814 ꢁ H ∼ J > A ∼ I ∼ C (Fig. 6). The LC-MS
analysis confirmed the presence of decomposition products, A
(RRT 0.61, MW 353) and C (RRT 0.87, MW 477), in the stressed
sample. These two compounds had been previously identified in
the acid-stressed sample. The unknown, H (MW 284, RRT 0.56),
was determined to be a decomposition product of SCH 420814.
The proposed structure (H, Fig. 6) was confirmed by tandem MS
analysis. The collision-induced dissociation of the m/z 285 ion
gives product ions of m/z 268 ([MH+ –NH3], 242, 214, 187, 161
and 134 (product-ion spectrum not shown). The fragmentation
pattern of the product-ion spectrum was found to be consistent
with the proposed structures (H). The most abundant fragment
ion (m/z 242) was generated by neutral loss of ethenamine
from the molecular ion of compound H. The other product
ions are generated by the fragmentation of the 7H-pyrazolo[4,3-
e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine triaromatic ring.
Studies under photolytic conditions
The exposure of SCH 420814 to light (UV/VIS) did not result in
any significant degradation (Fig. 2D). Two components eluted at
10.4 and 16.0 min in the LC chromatogram was characterized as
A (RRT 0.61, MW 353) and G (RRT 0.88, MW 520), respectively. The
compound A had been identified as a degradation product of
SCH 420814 under acid stress condition (Scheme 1), whereas the
compound G is an oxidation product of SCH 420814 (Scheme 4).
Degradation behaviors of the stressed SCH 420814 tablets
The identification of degradant formed in stressed tablets of
SCH 420814 (open dish, RH4 for 1 month) was achieved by
c
J. Mass. Spectrom. 2010, 45, 146–156
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L.-K. Zhang and B. N. Pramanik
Figure 6. LC-MS chromatogram of the sample extracted from a SCH 420814/excipient capsule which had been stored on an open dish (RH4) for 1 month.
NH2
O
N
N
N
O
O
N
N
NH
N
N
m/z 268
m/z 221
NH2
N
N
N
O
N
O
N
+H+
O
N
N
N
NH2
N
I
O
N
N
m/z 488
O
N
O
N
N
N
N
N
N
m/z 430
m/z 247
Scheme 5. Proposed fragmentation mechanism for compound I.
The LC/MS analysis also confirmed the presence of a unique
degradation product, I (MW 487, RRT 0.77), in the stressed tablet.
The LC-MS/MS product-ion spectrum for product I contains a
fragment ion of m/z 268, indicating this part of SCH 420814 is
intact (Fig. 7, Scheme 5). An ion detected at m/z 221 is found to
be the complementary fragment ion of m/z 268 (Scheme 5). In
addition, the presence of the fragment ion at m/z 430 corresponds
to the loss of the diethanol from I (Scheme 5). On the basis of
the tandem MS data, we proposed structure I for this degradation
product as shown in Scheme 5. The observation of an abundant
fragment ion at m/z 247 also agrees well with the structure
assignment (Fig. 7, Scheme 5).
A mechanism was proposed for the formation of I by heat
stress in the solid state (40 ◦C/75% relative humidity, 1 month)
(Scheme 6). In the proposed mechanism, a dienone intermediate
(Scheme 6, I-1) could be formed directly from SCH 420814. The
dienone intermediate (I-1) would then undergo an intermolecular
ring-closure reaction to form I-2, followed by rapid methane loss
to yield compound I (Scheme 6).
Figure 7. MS/MS product-ion spectrum of the protonated molecular ion
at m/z 505 for the component I in Fig. 6.
The molecular weight of the unknown J (RRT 0.87) was
determined to be 517 Da by LC/MS analysis (Fig. 6). The molecular
weight of compound J is 14 Da higher than that of SCH 420814,
which suggests one oxygen atom is added with the removal of two
c
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J. Mass. Spectrom. 2010, 45, 146–156

Characterization of degradation products of A2A antagonist by LC-MS and FT tandem MS analysis
NH2
N
NH2
N
N
N
O
N
OMe
N
N
O
N
N
MeO
N
N
N
N
O
N
N
N
O
I-1
SCH 420814
NH2
NH2
N
N
N
N
N
N
O
N
N
O
O
-CH4
N
O H
O
N
N
N
N
N
O
N
N
I
I-2
Scheme 6. Proposed mechanism for the formation of I by RH4 in the solid state.
+H+
NH2
NH
N
N
2 N
NH
N
2 N
O
N
N
N
N
N
O
O
N
H
N
N
N
N
HN
N
N
HN
N
H+
m/z 311
m/z 297
m/z 242
NH2
N
N
N
O
N
MeO
O
N
O
N
N
MeO
N
O
N
-CO
-CO
-NH3
O
N
m/z 490
MeO
m/z 473
m/z 249
m/z 277
J
m/z 518
MeO
O
NH2
O
N
m/z 206
m/z 194
Scheme 7. Proposed fragmentation mechanism for compound J.
hydrogenatomsonSCH420814.TheMS/MSproduct-ionspectrum
shows major fragment ions at m/z 490, 473, 311, 297, 277, 242,
249, 206 and 194 (spectrum not shown). The consecutive losses
of CO and NH3 from the parent molecular ion (m/z 518) yield
the product ions at m/z 490 and 473, respectively (Scheme 7).
The observation of CID product ions at m/z 311, 297 and 242
clearly indicates the oxidation of SCH 420814 occurred on the
piperazine ring (Scheme 7). The rest of the major fragment ions
are also consistent with the proposed structure of the degradation
product as shown in Scheme 7.
drug tablets to RH4 for 1 month resulted in generation of five
degradation products also in small quantities.
Acknowledgements
The authors thank Schering-Plough Research Institute for financial
support. They also thank Drs Malcolm MacCoss and John Piwinski
for their support of this project.
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