structure elucidation of ten autotaxin inhibitors and their … · and their metabolites using...
TRANSCRIPT
Submitted: 19/06/2014
MSc Chemistry
Analytical Sciences
“Structure elucidation of ten autotaxin inhibitors
and their metabolites using Liquid
Chromatography tandem Mass Spectrometry”
Master thesis
Georgios Psarros
Project Supervisor: Dr. H. Lingeman (Vrije Universiteit Amsterdam)
1
2
MSc Chemistry
Analytical Sciences
Master Thesis
Structural elucidation of ten autotaxin inhibitors using
Liquid Chromatography tandem Mass Spectrometry
by
Georgios Psarros
June 2014
Supervisor:
Prof Dr H. Lingeman
Daily Supervisor:
M. Mladic
Vrije Universiteit van Amsterdam
3
Preface
This thesis was prepared during the internship as a master student of the
Master of Analytical Sciences. The internship took place at the Department of
Biomolecular Analysis and Spectroscopy at VU University Amsterdam in the
period from February 2013 to October 2013.
I would like to thank Marija Mladic for her daily supervision and her
contribution at the experimental work, Dr. Jeroen Kool and Dr. Henk Lingeman
for their guidance during this project and Dr. Harald Albers for providing the
compounds.
4
Contents
Abstract................................................................................................................... 7
Introduction ............................................................................................................ 8
Anticancer drugs .................................................................................................. 8
Anticancer drugs directed at a drug target ........................................................... 9
Enzymes (as anti-cancer drug target) ................................................................. 11
Autotaxin ........................................................................................................... 12
Autotaxin inhibitors ........................................................................................... 14
Boronic acid-based autotaxin inhibitors ............................................................. 14
Metabolization of drugs ..................................................................................... 16
Experimental ......................................................................................................... 19
Apparatus .......................................................................................................... 19
Liquid Chromatography ................................................................................... 19
Mass Spectrometry ......................................................................................... 20
Methods............................................................................................................. 23
Metabolization with microsomes .................................................................... 23
Simulating Metabolism with Hydrogen Peroxide ............................................ 23
Results and Discussion .......................................................................................... 24
Proposed structure of Metabolites .................................................................... 28
Structural elucidation of metabolites using Mass Spectrometry ........................ 32
Fragmentation ................................................................................................ 32
5
Conclusion ............................................................................................................. 52
References ............................................................................................................ 54
Attachments.......................................................................................................... 56
6
List of Abbreviations
ACN Acetonitrile
ATX Autotaxin
CYPs Cytochrome P450 monooxygenases
ESI Electrospray ionization
GCPR G-Coupled Protein Receptors
GST Glutathione S-transferase
IC50 Half maximal inhibitory concentration
LPA Lysophosphatidic acid
LPC Lysophospholipids
MI Metabolic Incubation
NADPH Nicotinamide adenine dinucleotide phosphate-oxidase
NAT N-acetyl transferase
OE Oxidation Experiment
PST Phenol sulfotransferase
ST Sulfotransferase
TOF Time of Flight
UGT Glucuronosyl transferase
7
Abstract
This thesis describes the detection and the structure elucidation of ten potential
autotaxin inhibitors and their metabolites by the use of high performance liquid
chromatography (HPLC) tandem mass spectrometry (MS/MS). These compounds
were metabolized by incubation with pig liver microsomes and by oxidation
reactions with hydrogen peroxide (H2O2). The incubation mixtures then were
analyzed with HPLC-MS/MS. The principal metabolization pathway observed was
oxidative deboronation following by hydroxylation and in total 18 different
metabolites were identified. Useful in the structural investigation of the product
ions of the compounds and their metabolites were the accurate mass
measurements using a quadrupole-time of flight mass spectrometer.
8
Introduction
Anticancer drugs
What is Cancer?
Cancer is a term used for diseases in which abnormal cells divide without control
and are able to invade other tissues. Cancer cells can spread to other parts of the
body through the blood and lymph systems [1]. More than 100 different types of
cancer are known and they are named after the organ in which they start.
Anticancer drug development
Since cancer is the second leading cause of death in Europe and North America,
enormous resources are being invested in treatment, prevention and diagnosis of
this disease. For many pharmaceutical companies the key focus are the exploration
and the development of anticancer agents. Typical anticancer drug discovery and
development have focused on the cytotoxic agents and that was triggered by the
discovery of the toxic action of nitrogen mustards on cells of the haematopoietic
system 50 years ago [2].
Cytotoxic drugs
Cytotoxic drugs include any drug that inhibits or prevents the function of cells.
Cytotoxic drugs not only prohibit the rapid growth and division of cancer cells but
also affect the growth of other quick dividing cells in the body such as hair follicles
9
and the lining of the digestive system. As a consequence of the anticancer
treatment, many normal cells are damaged with the cancer cells [3].
Anticancer drugs directed at a drug target
In the last few decades drug development has been directed to a target-based drug
design focusing at the major drug targets categories, such as:
G Coupled Protein Receptors (44%)
Enzymes (29%)
Transporter proteins (15%)
ligands, structural and adhesion proteins, enzyme-interacting proteins and
other (12%) [4].
Figure 1: Major categories of drug targets.
Despite the fact that the traditional approach has achieved significant progress in
anticancer therapy, recent developments in molecular biology and the ability of
44%
29%
15%
3% 3% 3% 3%GPCR
Enzymes
Transporter proteins
Ligands
Enzyme-interacrion proteins
Structural and adhesion proteins
Other
10
understanding cancer at a molecular level have helped researchers to come up with
target-based drugs. These are agents that are designed to inhibit and/or modify a
selected molecular marker deemed important in cancer prognosis, growth, and/or
metastasis.
Several target-based compounds are developed in past years. Some examples are:
Imatinib mesylate (Gleevec1, Novartis) is a small-molecule compound that
inhibits a specific tyrosine kinase enzyme, the Bcr–Abl fusion oncoprotein. It
is used for gastrointestinal stromal tumor and chronic myeloid leukemia.
Gefitinib (Iressa1, AstraZeneca & Teva) is a small-molecule inhibitor of the
epidermal growth factor receptor’s (EGFR, or erbB1) tyrosine kinase domain.
It is used for non-small-cell lung cancer.
Bortezomib (Velcade1, Millenniums Pharmaceuticals) is the first of a new
class of agents called proteasome inhibitors and the first treatment in more
than a decade to be approved for patients with multiple myeloma.
Rituximab (Rituxan1, Biogen Idec & Genentech) is a monoclonal antibody
used in the treatment of B-cell non-Hodgkin’s lymphoma and B-cell leukemia.
It binds the CD20 antigen on the CD20+ B-cells, causing their apoptosis.
Trastuzumab (Herceptin1, Genentech) is a monoclonal antibody that binds
the cell surface HER2/neu (erbB2) receptor and is used in the therapy of
erbB2+ breast cancer [2].
11
Enzymes (as anti-cancer drug target)
Enzymes catalyze multistep chemical reactions and achieve phenomenal rate
accelerations by matching protein and substrate chemical groups in the transition
state [5]. The compounds that prevent that kind of chemical interactions to happen
are called inhibitors and they are among the most dominant and effective drugs in
the market. The key to design potent inhibitors is to study the catalytic chemistry
of enzymes which makes them a special class of drug target.
All the drug targets except for nucleic acids, evoke biological functions through
ligand binding. However, enzymes are catalysts, which means forming and breaking
covalent chemical bonds since enzymes are more capable from their nature of
chemical transformations than ligand binding. This makes enzymes a different drug
target.
Enzyme inhibitors are about 30% of the drugs marketed, therefore it is important
to consider enzymes as a separate class of drug targets.
Figure 2: Classification of enzyme target class. *enzymes are further classified as soluble or
membrane-associated; the number in the brackets corresponds to the number of
membrane-associated enzymes in each class.
12
Autotaxin
Autotaxin, ATX, is a secreted ecto-enzyme responsible for lysophosphatidic acid
(LPA) production in blood. It is a member of the family of ecto-nucleotide
pyrophosphatase /phosphodiesterase (NPP1-7) and is also referred, as NPP2 [6]. It
is the only family member capable of producing LPA by hydrolysis of IC50 (figure 3).
Figure 3: Autotaxin (ATX) is responsible for hydrolyzing the lipid lysophosphatidylcholine (LPC)
into lysophosphatidic acid (LPA) and choline [7].
The active LPA stimulates migration, proliferation and survival of many cells by
acting on specific G protein-coupled receptors [7].
Autotaxin plays an important role in vascular development and is found
overexpressed in some human cancers. Studies have shown that the main product
of ATX, LPA, mediates chemotaxis and proliferation in melanoma cells [6].
13
Recent experiments suggest that ATX expression is one of the factors involved in
metastasis of melanoma cells (Figure 4).
Figure 4: Metastasis of melanoma cells. Autotoxin’s main product, LPA, mediates chemotaxis and
proliferation in melanoma cells.
Inhibition of ATX production blocks LPA-induced migration of melanoma cells. It has
been detected that melanoma metastatic specimens have increased ATX level, and
ATX expression in primary melanoma is higher than in melanoma in situ [8].
Therefore inhibition of LPA production by ATX is therapeutic interesting.
14
Autotaxin inhibitors
The approved melanoma therapy lacks significant efficiency, hence, new potent
ATX inhibitors are under investigation. LPA receptors are not attractive targets since
LPA acts on multiple receptors that show overlapping activities. The first published
ATX inhibitor is L-Histidine and since then two additional categories of inhibitors
have been described. The first category consists of analogs of bioactive lipids
including LPA, and the second category of non-lipid small molecule inhibitors [6].
Boronic acid-based autotaxin inhibitors
In the early 1970s, boronic acids were initially used as inhibitors of proteasome and
they were established as possible transition state analogs of serine proteases [9].
During the 1980s, peptides with boronic moiety attached to them showed efficient
inhibition to trypsin, chymotrypsin, α-lytic protease, pancreatic elastase, leukocyte
elastase, thrombin, and β-lactamases [10]. Since then, they have been studied for
potent use in various disease states and lately, peptidyl boronic acids were
indicated as potent proteasome inhibitors. Furthermore, high antitumor and anti-
inflammatory effectiveness in vitro and in animal models was observed [12,13]. In
2003, the drug VELCADE (bortezomib, figure 5) was used for the medication of
relapsed refractory multiple myeloma and became the first boronic acid available
in the market as a therapeutic agent [12].
15
Figure 5: Structure of Bortezomib.
Recently, Albers et al. described the discovery of a boronic acid-based ATX inhibitor
which helped to shorten the half-life of LPA (∼5 min) in vivo. Interestingly, the
introduction of a boronic acid group, designed that way to target the active site
threonine in autotoxin, showed a great increase of inhibitory efficacy with the most
drastic of the compound to inhibit ATX activity in a nanomolar range.
The idea of the boronic acid moiety introduction was encouraged by the already in
use therapeutic proteasome inhibitor bortezomib, which targets the N-terminal
threonine oxygen nucleophile in the proteasome through a boronic acid.
The finding that ATX can be targeted by boronic acids may aid the development of
ATX inhibitors for therapeutic use in ATX/LPA-dependent pathologies, including
chronic inflammation, tumor progression and fibrotic diseases.
16
Figure 6: Hypothesis on the binding of boronic acid with the T210 oxygen nucleophile in the ATX
active site [13].
Metabolization of drugs
Most medicines, except for some very polar substances that might be directly
excreted into the urine via the kidneys, are liposoluble and they are subjected to
metabolism. Subsequently, more polar species are generating, which can easily
avoid the reabsorption from the kidneys and excreted into the urine [14]. Drug
metabolism together with bile and renal excretion determine for how long the drug
will stay in patients’ body. Metabolism can lead to many unfortunate effects to the
parent compound – a toxic parent medicine can be detoxified or a nontoxic parent
compound can generate active metabolites by biotransformation [15]. Other
adverse consequences are:
change or loss of selectivity
deposition/accumulation of metabolites
bioactive metabolites with irreversible action
acquired ability to cross the blood-brain barrier
17
Drug Metabolism has various chemical pathways, but they are now categorized into
two phases.
Phase 1 oxidation. A polar group, usually hydroxyl moiety (-OH group), is added to
a non-polar molecule. This oxidation is mainly carried out by cytochrome P450
monooxygenases (CYPs).
Phase 2 conjugation. A very polar endogenous molecule such as glucose or sulfate
is added to the organic group which was possibly formed from phase 1 reaction,
usually at –OH site. Conjugation reactions may directly occur in drugs with the
proper functional groups [14]. Conjugation enzymes like glucuronosyl transferase
(UGT) and phenol sulfotransferase (PST) are usually present at phase 2 reactions.
The metabolization enzymes are in principal in the liver, however metabolism may
occur in different sites such as in the brain, lungs or intestines. The Cytochromes
P450 (CYPs) are the most intensively studied enzymes due to the complicated
processes that they produce. The past years, scientists using the knowledge from
the study of CYPs were enabled to prognosticate the effects of drug metabolism in
vivo. A few in vitro liver models have been established during the past decades
which are: perfused liver, liver slices, primary hepatocytes, cytosol, S9 fractions,
supersomes, cell lines, transgenic cell lines and microsomes [16].
The most efficient in vitro model to study drug metabolism seems to be with the
use of microsomes which are subcellular fractions consisting of fragmented
endoplasmic reticulum to which ribosomes are attached [17]. The absence of
enzymes like N-acetyl transferase (NAT), sulfotransferase (ST), glutathione S-
18
transferase (GST) and cytosolic cofactors (Phase 2 enzymes) leads to an incomplete
production of metabolites, however due to the simplicity in use and the fact that
they are one the best characterized in vitro models for drug metabolism research
they are still extremely helpful [16].
In this project pig liver microsomes were used to metabolize the lead compounds
and mixtures of metabolites were produced therefore an analytical separation was
necessary for their study.
19
Experimental
Apparatus
Liquid Chromatography
An autosampler Shimadzu SIL-30AC was used. For the gradient LC separations (with
two Shimadzu LC-30AD parallel pumps), a Waters XBridge C18 column (4.6
x100mm, 5μm particles) with a guard column was used. A column oven Shimadzu
CTD-30A was used with a temperature of 40oC. The injection volume was 50 μl.
Mobile phase A consisted of H2O-ACN-formic acid (98%-2%-0.1%) and mobile phase
B of H2O-ACN-formic acid (2%-98%-0.1%). The following gradient (0.6mL/min flow)
was used: 3 min at 5% B → 10 min linear increase to 95% B →5 min at 95% B → 1
min linear decrease to 5% B→10 min at 5% B (figure). On-line UV measurements
were performed with a Shimadzu SPD-M20A prominence diode array detector. The
flow from the UV detector was directed into the mass spectrometer with a minimal
amount of PEEK tubing. Data analysis was performed with Postrun Analysis
software.
Figure 7: Graphic display of HPLC gradient program.
20
Mass Spectrometry
ULTIMA MICROMASS
In the early phase of the experiments a less accurate Mass Spectrometer was used.
The MS was a MICROMASS Q-TOF Ultima equipped with an electrospray ionization
source. Positive ion electrospray was performed with a capillary voltage of 4 kV.
Spectrum acquisition was performed between m/z 50 and 700 with 1 sec Scan time
and 0.1 sec Inter-Scan delay in MS mode and 0.5 sec Scan time and 0.1 sec Inter-
Scan delay in MS2 mode. The Source and Desolvation Temperature was 120 and
300 respectively. With the help of a divert valve, a solvent delay of 4 min in the
beginning and 4 min in the end of the run was used in order to prevent
contamination of the ESI source. Data analysis was performed with Mass Lynx
software.
BRUKER
A triple Quadrupole Time of Flight (Q-TOF) mass spectrometer from BRUKER
(Bremen-Germany) was used for the mass spectrometric detection. All experiments
were performed with electrospray ionization (ESI) in the positive ion mode. The
capillary voltage was 4.5 kV, the dry gas Temperature was set at 200oC and the Gas
flow at 10 l/min. Spectrum acquisition was performed with a scan range of 50-3000
m/z. Data depended MS2 analysis was performed with collision energy of 5eV. The
instrument was calibrated with recommended solutions (Sodium Formate). In order
to achieve higher mass accuracy, in the beginning of every measurement 1 min of
calibrant was measured giving the option to “correct” the masses afterwards if that
was necessary. To manage that an extra pump was introduced into the system and
21
a loop with the calibrant (figure). Data analysis was performed with Bruker
DataAnalysis software.
0-1 minute of the run
1-29 minute of the run
Figure 8: System scheme after calibration loop was installed.
22
Figure 9: Schematic representation of BRUKER micrOTOF compass 1.3.
23
Methods
Metabolization with microsomes
Pig liver microsomes were diluted 10 times in a reaction buffer consisted of 50 mM
KH2PO4- , 5mM MgCl2and 5 mM glucose-6-phosphate at pH 7,4 (adjusted with
NaOH). At 4 oC the solution was divided into Eppendorf tubes and compounds of
final concentration 100μM were added with 5 activity units/mL of glucose-6-
phosphate-dehydrogenase. The incubation was initiated at room temperature by
the addition of 10% (v/v) 30mM NADPH (which was used as a regenerator) and then
the tubes were transferred in a water bath at 37oC. At 30 and 60 minutes same
volume of 10mM NADPH was added and after 90 minutes the incubation was
stopped with 200% (v/v) ice-cold acetonitrile. The tubes were centrifuged at 10.000
rpm for 5 min at 4 oC and the supernatant was transferred to new tubes and
thereafter evaporated in a LABCONCO SpeedVac. The residues were re-dissolved in
mobile phase A and stored at -20oC [18].
Simulating Metabolism with Hydrogen Peroxide
Boiling compounds in hydrogen peroxide (H2O2) is a way to simulate metabolism
and since it is cheap and fast procedure high quantities of metabolites can be easily
produced. This method was tested for all the compounds, and in some cases
different metabolites were produced. Two series of samples were obtained, the
first one after 1 hour of boiling the parent compounds in H2O2 and the second after
7 hours. In that way, the comparison of the metabolite formation rate was enabled,
which is a crucial step in the study of metabolites.
(The experiments were performed at the Netherlands Cancer Institute (NKI) by
Harald Albers)
24
Results and Discussion
Aim of the thesis
The aim of this project was the study of 10 potential autotaxin inhibitors regarding
their metabolization pattern and the structural elucidation of their metabolites.
Also the metabolites produced by metabolic incubations with pig liver microsomes
in vitro were qualitatively compared with the ones produced with the oxidation of
the parent compounds with hydrogen peroxide.
Structure of compounds to be metabolized
We were provided with 10 different parent compounds which we can divide in 5
different groups regarding to their structure. Groups A-D contain a boron moiety in
their structure whereas group E does not.
Group A (compounds HA278/HA285)
Group B (compounds HA280/HA289)
25
Group C (compounds HA281/HA288)
Group D (compounds HA295/HA296)
Group E (compounds HA286/HA287)
26
Metabolization results of compounds HA278-HA296
After the metabolization of the parent compounds the usage of a chromatographic
separation technique was necessary since mixtures of parent compounds and
metabolites were formed. For the structural elucidation of the metabolites a Q-TOF
mass spectrometer was used coupled to a HPLC system. The experimental setup is
outlined in figure 10. Online UV measurements were performed with the flow
directed after the separation column into the UV detector and after that into the
MS detector with minimal amount of PEEK tubing.
Figure 10: Experimental setup
27
Using a high resolution Bruker Mass Spectrometer we could confirm that masses
within a range of +/- 5ppm (or +/-3 mDa) from the calculated mass correspond to
the right fragments.
After a series of analyses we manage to identify in total 18 different metabolites
from 10 parent compounds with 2 different metabolization techniques (metabolic
incubation and oxidation with H2O2). The proposed structures of the metabolites
for all ten compounds and the table with the experimental conditions are given
below.
Table 1: Experimental conditions during metabolite identification.
28
Proposed structure of Metabolites
Compound HA278
Compound HA280
29
Compound HA281
Compound HA285
Compound HA286
30
Compound HA287
Compound HA288
31
Compound HA289
Compound HA295
Compound HA296
32
Structural elucidation of metabolites using Mass Spectrometry
The molecular structure of the metabolization products originating from 10
different autotaxin inhibitors was elucidated using a High resolution Q-TOF
instrument.
Fragmentation
For practical reasons in the next paragraphs we will show in detail the
fragmentation of one only compound per group. All the needed information can be
found in the end of this chapter in table 20.
It would be useful, before starting with fragmentation, to give some information
about the isotopic abundances of our compounds’ characteristic elements.
Isotope Atomic Mass (ma/u) Natural abundance (atom %)
10B 10.0129 19.9
11B 11.0093 80.1
19F 18.9984 100
35Cl 34.9688 75.78
37Cl 36.9659 24.22
Table 2: Isotopes of Boron, Fluorine and Chlorine.
33
Group A
HA278
Parent
Figure 11: MS and MS/MS spectra of parent compound.
On the right: proposed fragmentation of HA278.
Fragment Formula Exact mass Measured mass Mass error (mDa)
F1 C7H6BO3+ 149.0405 149.0407 -0.2
F2 C6H8BO3+ 139.0561 139.053 3.1
F3 C6H6BO2+ 121.0455 121.0458 -0.3
F4 C9H5F6+ 227.0290 227.0272 1.8
F5 C20H19F6N2O+ 417.1396 417.1349 4.7
Table 3: Mass accuracy measurements of HA278 parent fragment ions.
79.0188
103.9558
195.0911218.9578
264.9584
318.0691 350.0621 425.2246
505.1369
146.0061
385.0986
+MS, 11.9min #704
0.0
0.2
0.4
0.6
0.8
4x10
Intens.
100 150 200 250 300 350 400 450 500 m/z
149.0407
227.0272
505.1366
+MS2(505.1366), 15.364eV, 11.8min #701
0.0
0.5
1.0
1.5
4x10
Intens.
100 150 200 250 300 350 400 450 500 m/z
Full MS
MS2 505
34
Metabolite M1
Figure 12: MS and MS/MS spectra of M1.
On the right: proposed structure and fragmentation of M1.
Fragment Formula Exact mass Measured mass Mass error (mDa)
F1 C14H15F6N2O2+ 357.1032 357.1025 0.7
F2 C9H5F6+ 227.0290 227.0272 1.8
F3 C7H5BO2+ 121.0284 121.0284 0
F4 C6H9N2O3+ 157.0608 157.0603 0.5
Table 4: Mass accuracy measurements of HA278 M1 fragment ions
144.9806 194.1162
223.0455 256.2490296.2500 352.2461
477.1214
103.9551
141.9238
+MS, 12.1-12.2min #(718-722)
0
250
500
750
1000
1250
1500
Intens.
100 150 200 250 300 350 400 450 m/z
121.0281
357.1022
477.1266
+MS2(477.1266), 15eV, 12.1min #719
0
1000
2000
3000
4000
5000
6000
Intens.
100 150 200 250 300 350 400 450 m/z
Full MS
MS2 477
35
Metabolite M2
Figure 13: MS and MS/MS spectra of M2.
On the right: proposed structure and fragmentation of M2.
Fragment Formula Exact mass Measured mass Mass error (mDa)
F1 C7H5F6+ 105.0355 105.0344 1.1
F2 C9H5F6+ 227.0290 227.0312 -2.2
Table 5: Mass accuracy measurements of HA278 M2 fragment ions.
The fragments that indicate a change in the structure of the parent compound are
found at the A part of the molecule. Specifically we can see that the main fragment
(m/z 149.0407) of the parent compound corresponds to the part A. At MS/MS
spectra of the metabolites the m/z difference compared to the parent compound
is Δ -28.0126 and -44.0063 for each metabolite respectively. These differences
indicate loss of boron and one hydroxyl group (BOH) for the M1 and loss of boron
and both hydroxyl groups [B(OH)2] for the M2. Taking the above into consideration
the proposed structures of M1 and M2 are verified.
105.0344
227.0312
461.1307
+MS2(461.1307), 15eV, 13.2min #782
0
200
400
600
800
1000
Intens.
100 150 200 250 300 350 400 450 500 m/z
103.9555
144.9824
195.0891
210.1069 255.2323281.1755
352.2451
368.2408
398.2118 425.2170
461.1313
483.1106
104.0389
+MS, 13.2-13.2min #(781-785)
0
500
1000
1500
2000
2500
Intens.
100 150 200 250 300 350 400 450 m/z
Full MS
MS2 461
36
Figure 14: MS/MS spectra of parent compound, M1 and M2.
On the right: structure of autotaxin inhibitor HA278.
149.0407
227.0272
505.1366
+MS2(505.1366), 15.364eV, 11.8min #701
0.0
0.5
1.0
1.5
4x10
Intens.
100 150 200 250 300 350 400 450 500 m/z
105.0344
227.0312
461.1307
+MS2(461.1307), 15eV, 13.2min #782
0
200
400
600
800
1000
Intens.
100 150 200 250 300 350 400 450 500 m/z
MS2 461
121.0281
357.1022
477.1266
+MS2(477.1266), 15eV, 12.1min #719
0
1000
2000
3000
4000
5000
6000
Intens.
100 150 200 250 300 350 400 450 m/z
MS2 477
MS2 505
37
Group B
HA289
Parent
Figure 15: MS and MS/MS spectra of parent compound.
On the right: proposed fragmentation of HA289.
Fragment Formula Exact mass Measured mass Mass error (mDa)
F1 C12H15BNO3+ 232.1140 232.1142 -0.2
F2 C7H6BO3+ 149.0405 149.0407 -0.2
F3 C6H6BO2+ 121.0455 121.0458 -0.3
F4 C8H13N2O2+ 169.0972 169.0971 0.1
F5 C7H5Cl2+ 158.9763 158.9757 0.6
Table 6: Mass accuracy measurements of HA289 parent fragment ions.
520.1570
186.2221 520.6913103.9553
+MS, 9.4-9.5min #(555-563)
0
1
2
3
4
4x10
Intens.
50 100 150 200 250 300 350 400 450 500 m/z
149.0407
169.0971
232.1142
370.1097
520.1565
+MS2(520.9081), 16.4575eV, 9.4min #560
0.0
0.2
0.4
0.6
0.8
5x10
Intens.
50 100 150 200 250 300 350 400 450 500 m/z
Full MS
MS2 520
38
Metabolite M1
Figure 16: MS and MS/MS spectra of M1.
On the right: proposed structure and fragmentation of M1.
Fragment Formula Exact mass Measured mass Mass error (mDa)
F1 C12H14NO2+ 204.1019 204.1012 0.7
F2 C7H5O2+ 121.0284 121.0277 0.7
F3 C8H13N2O2+ 169.0972 169.0964 0.8
F4 C17H25Cl2N3O2+ 372.1240 372.1224 1.6
F5 C7H5Cl2+ 158.9763 158.9739 2.4
Table 7: Mass accuracy measurements of HA289 M1 fragment ions.
290.1858
492.1452
493.1481
334.1750159.9789
+MS, 9.6-9.6min #(567-571)
0
1
2
3
4x10
Intens.
100 150 200 250 300 350 400 450 500 m/z
Full MS
MS2 492
121.0277
204.1012
372.1224
492.1449
+MS2(492.8918), 15eV, 9.6min #568
0
2
4
6
8
4x10
Intens.
100 150 200 250 300 350 400 450 500 m/z
39
Metabolite M2
Oxidation Experiment
Figure 17: MS and MS/MS spectra of M2.
On the right: proposed structure and fragmentation of M2.
Fragment Formula Exact mass Measured mass Mass error (mDa)
F1 C7H5O2+ 121.0284 121.0275 0.9
F2 C12H14NO2+ 204.1019 204.1013 0.6
F3 C7H5Cl2+ 158.9763 158.9758 0.5
Table 8: Mass accuracy measurements of HA289 M2 fragment ions.
136.0587 185.9413 218.9547
508.1394
509.1428
255.9204105.9553
+MS, 9.8min #(579)
0
2000
4000
6000
8000
Intens.
100 150 200 250 300 350 400 450 500 m/z
121.0275
204.1013
508.1438
+MS2(509.1415), 15.6277eV, 9.7min #576
0
2
4
6
4x10
Intens.
100 150 200 250 300 350 400 450 500 m/z
Full MS
MS2 508
40
Metabolic Incubation
Figure 18: MS and MS/MS spectra of M2.
On the right: proposed structure and fragmentation of M2.
Metabolite M2 with m/z 508 is present with two different oxidation positions. With
metabolic incubation, the oxidation took place in A-ring whereas in oxidation
experiment in C-ring. The fragments that indicate this difference are 121 and 204 in
figure 17 while in figure 18 these fragments are found with a difference of +16
which indicates the addition of a hydroxyl group. However, the exact position in the
ring is unclear.
Fragment Formula Exact mass Measured mass Mass error (mDa)
F1 C7H5O3+ 137.0233 137.0241 -0.8
F2 C12H14NO3+ 220.0968 220.0971 -0.3
F3 C17H24Cl2N3O2+ 372.1240 372.1289 -4.9*
Table 9: Mass accuracy measurements of HA289 M2 fragment ions.
*even though the error is higher than expected, this is the proposed structure.
123.0549
155.1103
195.0901
252.1601
327.0742
508.1403
212.1650
509.1422229.1431
+MS, 9.4min #(560)
0
250
500
750
1000
1250
Intens.
50 100 150 200 250 300 350 400 450 500 m/z
137.0241
220.1022372.1289
508.1398
+MS2(509.1534), 15.6288eV, 9.5min #562
0
200
400
600
800
1000
Intens.
100 150 200 250 300 350 400 450 500 m/z
Full MS
MS2 508
41
Metabolite M3
Figure 19: MS and MS/MS spectra of M3.
On the right: proposed structure and fragmentation of M3.
Fragment Formula Exact mass Measured mass Mass error (mDa)
F1 C7H5O+ 105.0335 105.0330 0.5
F2 C12H14NO+ 188.1070 188.1078 -0.8
Table 10: Mass accuracy measurements of HA289 M3 fragment ions.
112.0276
155.1083
170.1148
195.0918
226.1804
246.0187
266.1753
295.2291
335.2249
353.2338
392.1742
414.3021
476.1544
432.3088
477.1551
450.3237
+MS, 10.1min #600
0
500
1000
1500
Intens.
100 150 200 250 300 350 400 450 m/z
105.0330
188.1078
476.1519
+MS2(477.1539), 15eV, 10.1min #602
0
100
200
300
400
500
Intens.
100 150 200 250 300 350 400 450 m/z
Full MS
MS2 476
42
Group C
HA288
Parent
Figure 20: MS and MS/MS spectra of parent compound.
On the right: proposed fragmentation of HA288.
Fragment Formula Exact mass Measured mass Mass error (mDa)
F1 C15H15F6N2O2+ 369.1032 369.1031 0.1
F2 C9H5F6N + 227.0290 227.0291 -0.1
F3 C12H10F6N + 282.0712 282.0718 -0.6
F4 C14H15F6N2+ 325.1134 325.1130 0.4
Table 11: Mass accuracy measurements of HA288 parent fragment ions.
369.1031
+MS2(533.1688), 17.3255eV, 10.5min #622
0
1
2
3
4
5
65x10
Intens.
100 150 200 250 300 350 400 450 500 m/z
369.1026
533.1677
149.9523103.9549
+MS, 10.4-10.5min #(617-625)
0.0
0.2
0.4
0.6
0.8
1.0
5x10
Intens.
50 100 150 200 250 300 350 400 450 500 m/z
43
Metabolite M1
Figure 21: MS and MS/MS spectra of M1.
On the right: proposed structure and fragmentation of M1.
The fragmentation of the metabolite M1 was not god enough so fragments of the
part of the molecule that changed were not observed. However, comparing the
mass difference Δm/z= - 28 with previous compounds (metabolite M1 of HA278 and
HA289) we concluded that this loss derives from the deboronation of the parent
compound following with hydroxylation in the same ring.
Fragment Formula Exact mass Measured mass Mass error (mDa)
F1 C15H15F6N2O2+ 369.1032 369.1017 1.4
Table 12: Mass accuracy measurements of HA288 M1 fragment ions.
103.9543
185.9438 218.9577 253.9233 369.1043 403.0732
505.1552
144.9798154.0024
+MS, 10.6-10.7min #(628-636)
0
1000
2000
3000
4000
5000
6000
Intens.
100 150 200 250 300 350 400 450 500 m/z
369.1017
505.1551
+MS2(505.1551), 15.3807eV, 10.7min #633
0.0
0.5
1.0
1.5
2.0
2.5
4x10
Intens.
100 150 200 250 300 350 400 450 500 m/z
44
Metabolite M2
Figure 22: MS and MS/MS spectra of M2.
On the right: proposed structure and fragmentation of M2.
Fragment Formula Exact mass Measured mass Mass error (mDa)
F1 C13H15F6N2+ 313.1134 313.1126 0.8
F2 C9H5F6+ 227.0290 227.0279 1.1
F3 C4H9N2+ 85.0760 85.0733 2.7
F4 C5H9N2O+ 113.0709 113.0710 -0.1
F5 C11H10F6N+ 270.0712 270.0688 2.4
F6 C5H9N2O2+ 129.0659 129.0659 0
Table 13: Mass accuracy measurements of HA288 M2 fragment ions.
357.1025
103.9541 185.9488
+MS, 9.8-9.9min #(584-588)
0
2
4
6
4x10
Intens.
100 150 200 250 300 350 m/z
113.0710
227.0279
313.1126357.1023
+MS2(357.1023), 15eV, 9.9min #589
0
2
4
6
4x10
Intens.
100 150 200 250 300 350 m/z
45
Metabolite M3
Figure 23: MS and MS/MS spectra of M3.
On the right: proposed structure and fragmentation of M3.
Fragment Formula Exact mass Measured mass Mass error (mDa)
F1 C15H15F6N2O2+ 369.1032 369.1014 1.8
F2 C5H10NO2+ 116.0706 116.0710 -0.4
F3 C6H10NO2+ 128.0706 128.0713 -0.7
F4 C16H17F6N2O2+ 383.1189 383.1178 1.1
Table 14: Mass accuracy measurements of HA288 M2 fragment ions.
89.0677
103.9541149.9512 185.9415 218.9559
314.0584
445.1199
419.0582386.0484
+MS, 10.3-10.4min #(613-617)
0
1000
2000
3000
4000
Intens.
100 150 200 250 300 350 400 450 m/z
116.0679
369.1014
445.1174
+MS2(445.1174), 15eV, 10.4min #618
0.0
0.5
1.0
1.5
4x10
Intens.
100 150 200 250 300 350 400 450 m/z
46
Group D
HA295
Parent
Figure 24: MS and MS/MS spectra of parent compound.
On the right: proposed fragmentation of HA295.
Fragment Formula Exact mass Measured mass Mass error (mDa)
F1 C8H6BO3+ 161.0405 161.0401 0.4
F2 C7H8BO3+ 151.0561 115.0546 1.5
F3 C7H6BO2+ 133.0455 133.0451 0.4
F4 C5H9N2O+ 113.0709 113.0716 -0.7
Table 15: Mass accuracy measurements of HA295 parent fragment ions.
103.9553
144.9815
449.0843
450.0859
244.2614
+MS, 11.7-11.8min #(694-702)
0.0
0.5
1.0
1.5
4x10
Intens.
100 150 200 250 300 350 400 450 m/z
161.0401
275.0023 319.0076449.0813
+MS2(450.0873), 15eV, 11.8min #699
0
1
2
3
4
4x10
Intens.
150 200 250 300 350 400 450 m/z
Full MS
MS2 449
47
Metabolite M1
Figure 25: MS and MS/MS spectra of M1.
On the right: proposed structure and fragmentation of M1.
Fragment Formula Exact mass Measured mass Mass error (mDa)
F1 C8H7O3+ 151.0390 151.0389 0.1
F2 C8H5O2+ 133.0284 133.0285 -0.1
F3 C6H9N2O3+ 157.0608 157.0594 1.4
F4 C7H5Cl2+ 158.9763 158.9747 1.6
Table 16: Mass accuracy measurements of HA295 M1 fragment ions.
112.0288151.0381
195.0912228.1963 250.1784
343.2947421.0746
439.0839
313.2395295.2252
440.0888
+MS, 11.0-11.2min #(655-667)
0.00
0.25
0.50
0.75
1.00
1.25
4x10
Intens.
100 150 200 250 300 350 400 450 m/z
113.0708
133.0285
151.0389
219.1140 291.0034
+MS2(440.0807), 15eV, 11.1min #660
0
1
2
3
4
5
6
4x10
Intens.
100 150 200 250 300 350 400 m/z
Full MS
MS2 439
48
Group E
HA286
Parent
Figure 26: MS and MS/MS spectra of parent compound.
On the right: proposed fragmentation of HA286.
Fragment Formula Exact mass Measured mass Mass error (mDa)
F1 C7H5Cl2+ 158.9763 158.9739 2.4
F2 C13H15Cl2N2O2+ 301.0505 301.0491 1.4
Table 17: Mass accuracy measurements of HA286 parent fragment ions.
103.9545158.9752 266.1717
478.0913
479.0952
226.1793
301.0505
+MS, 9.9-10.0min #(590-594)
0
2000
4000
6000
8000
Intens.
100 150 200 250 300 350 400 450 m/z
158.9739
301.0491
+MS2(479.1041), 15eV, 10.0min #595
0
1
2
3
4x10
Intens.
150 200 250 300 350 400 450 m/z
Full MS
MS2 478
49
Metabolite M1
Figure 27: MS and MS/MS spectra of M1.
On the right: proposed structure and fragmentation of M1.
Fragment Formula Exact mass Measured mass Mass error (mDa)
F1 C5H9N2O+ 113.0709 113.0721 -1.2 F2 C9H10Cl2N+ 202.0185 202.0184 0.1 F3 C5H9N2O2
+ 129.0659 129.0652 0.7 F4 C11H15Cl2N2
+ 245.0607 245.0600 0.7 F5 C7H5Cl2+ 158.9763 158.9763 0 F6 C7H4Cl+ 122.9996 123.0013 -1.7 F7 C4H9N2
+ 85.0760 85.0743 1.7
Table 18: Mass accuracy measurements of HA286 M1 fragment ions.
129.0659
158.9765 245.0610
289.0509
195.0902
290.0548
112.0292
+MS, 9.1-9.3min #(543-551)
0
1
2
3
4
4x10
Intens.
100 120 140 160 180 200 220 240 260 280 m/z
158.9763
245.0600
+MS2(289.0495), 15eV, 9.2min #548
0.00
0.25
0.50
0.75
1.00
1.25
1.50
5x10
Intens.
100 120 140 160 180 200 220 240 260 280 m/z
Full MS
MS2 289
50
Table 19: Structural elucidation of the compounds HA285, HA280, HA281, HA296, HA287 and their metabolites.
Compound m/z (mass error) Fragments (mass error) Proposed Structure
HA285 437.0837 (0.7mDa) 158.9763 (0.7mDa)
149.0405 (0.7mDa)
-
M1 409.0716 (0.8mDa) 158.9763 (-0.5mDa),
121.0284 (0.1mDa),
93.0335 (-1.3mDa)
289.0505 (-2.1mDa)
Parent - BOH
HA280 588.2099 (1.7mDa)
149.0405 (0.4mDa)
232.1140 (0.2mDa)
438.1611 (-2.8mDa)
M1 560.1979 (1.4mDa) 121.0284 (-0.1mDa)
204.1019 (-0.7mDa)
357.1032 (-1mDa)
440.1767 (-0.8mDa)
227.0290 (1.1mDa)
Parent - BOH
M2 576.1928 (0.8mDa) 121.0284 (1.1mDa)
204.1019 (0 mDa)
227.0290(0.4mDa)
Parent – BOH,
+OH
HA281 465.1150 (1.4mDa) 301.0505 (1.6mDa)
158.9763 (0mDa)
245.0607 (0.9mDa)
M1 437.1029 (0.2mDa) 301.0505(-0.1mDa) 158.9761 (0.2mDa)
Parent - BOH
M2 289.0505 (-0.1mDa) 245.0607 (0.4mDa) 158.9765 (-0.2mDa)
N- dealkylation of
parent compound
(-C9H9BO3)
M3* 377.0666 (0mDa) 301.0477 (2.8mDa) 158.9763 (-1.2mDa) 128.0706 (0.8mDa) 116.0706 (0.2mDa)
M2 + C3H4O3
51
HA296 517.1364 (-1.5mDa) 161.0405 (0.5mDa)
277.0290 (0.1mDa)
133.0455 (3.1mDa)
M1 507.1349 (-0.4mDa) 151.0390 (1.7mDA)
227.0290 (2.2mDa)
357.1032 (1.7mDa)
157.0608 (0.9mDa)
133.0284 (2.3mDa)
Parent – BOH,
+OH
HA287 546.1458 (0mDa) 369.1032 (0.3mDa)
227.0290 (-2.9mDa)
M1 357.1032 (-0.9mDa) 227.0290 (-0.9mDa)
313.1134 (-0.6mDa)
129.0659 (0.9mDa)
85.0760 (0.4mDa)
113.0709 (-0.3mDa)
270.0712 (-2.9mDa)
N- dealkylation of
parent compound
(-C10H7NO3)
*M3 is visible only at oxidation experiment
52
Conclusion
Metabolic stability of ten ATX inhibitors was successfully assessed using HPLC-
MS/MS analysis of metabolic mixtures generated from the parent compounds. Two
different approaches were used to generate metabolic mixtures of ATX inhibitors.
The incubation with pig liver microsomes was first used to simulate the in vivo
metabolism of the compounds and estimate the relative stability of the compounds.
Next, oxidation experiment using hydrogen-peroxide was used to test the ability of
alternative way of producing the metabolites of investigated compounds.
Metabolic mixtures generated in both experiments were then subjected to the
HPLC-MS/MS analysis. The mixtures were easily separated on a C18 column and
accurate MS and MS/MS measurements allowed full or partial structure elucidation
of the formed metabolites.
For the compounds with the boron moiety, the primary route of metabolism
observed in metabolic incubation was the deboronation of the parent compound
followed by hydroxylation in the same position (M1). Metabolites with double
hydroxylation after the deboronation were also formed for two of the compounds
and simple deboronation was also observed in some cases but with low
concentration. N-dealkylation of the parent compounds was also present.
For the two compounds without the boron moiety in their structure only one
metabolite was observed, which was formed after N-dealkylation of the parent
compound. Therefore, these structures were marked to be relatively stable.
The same main metabolites were produced in the oxidation experiment with the
hydrogen-peroxide, which demonstrated that this technique could be used for the
53
study of metabolic stability of these and similar compounds. Furthermore, it could
be used to generate large amounts of the metabolites for eventual pharmacological
characterization, and toxicology and pharmacokinetic studies.
54
References
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[11] J. Adams, M. Behnke, S. Chen, A. A. Cruickshank, L. R. Dick, L. Grenier, J. M. Klunder, Y.-T. Ma, L. Plamondon, and R. L. Stein, “Potent and selective inhibitors of the proteasome: Dipeptidyl boronic acids,” Bioorg. Med. Chem. Lett., vol. 8, no. 4, pp. 333–338, Feb. 1998.
[12] V. Uttamsingh, C. Lu, G. Miwa, and L.-S. Gan, “Relative contributions of the five major human cytochromes P450, 1A2, 2C9, 2C19, 2D6, and 3A4, to the hepatic metabolism of the proteasome inhibitor bortezomib.,” Drug Metab. Dispos., vol. 33, no. 11, pp. 1723–8, Nov. 2005.
[13] H. M. H. G. Albers, L. a van Meeteren, D. a Egan, E. W. van Tilburg, W. H. Moolenaar, and H. Ovaa, “Discovery and optimization of boronic acid based inhibitors of autotaxin.,” J. Med. Chem., vol. 53, no. 13, pp. 4958–67, Jul. 2010.
[14] M. S. Lennard and A. V. Stachulski, “Drug Metabolism: The Body’s Defense against Chemical Attack,” J. Chem. Educ., vol. 77, no. 3, p. 349, Mar. 2000.
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[16] S. Asha and M. Vidyavathi, “Role of human liver microsomes in in vitro metabolism of drugs-a review.,” Appl. Biochem. Biotechnol., vol. 160, no. 6, pp. 1699–722, Mar. 2010.
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[18] J. Reinen, J. Kool, and N. P. E. Vermeulen, “Reversed-phase liquid chromatography coupled on-line to estrogen receptor bioaffinity detection based on fluorescence polarization.,” Anal. Bioanal. Chem., vol. 390, no. 8, pp. 1987–98, Apr. 2008.
56
Attachments
HA280-parent MS and MS/MS
HA280-Metabolite1 MS and MS/MS
588.2072
227.0284
+MS, 10.1min #597
0
2
4
6
4x10
Intens.
100 200 300 400 500 600 m/z
149.0401
232.1138
438.1583
588.2086
+MS2(588.2086), 21.1795eV, 10.0min #594
0
2
4
6
8
4x10
Intens.
150 200 250 300 350 400 450 500 550 m/z
560.1960
227.0279 334.1734
+MS, 10.2min #604
0.0
0.5
1.0
1.5
5x10
Intens.
100 200 300 400 500 600 m/z
57
HA280-metabolite2 MS and MS/MS
121.0273
204.1008
440.1758
560.1964
+MS2(560.5099), 19.2314eV, 10.2min #605
0.0
0.2
0.4
0.6
0.8
1.0
5x10
Intens.
100 200 300 400 500 m/z
227.0286
576.1931
576.7081112.0268 288.6017
+MS, 10.3min #(612)
0
1
2
3
4
5
6
4x10
Intens.
100 200 300 400 500 m/z
121.0273
204.1009
576.1920
+MS2(576.1920), 20.3517eV, 10.3min #609
0.0
0.5
1.0
1.5
5x10
Intens.
100 150 200 250 300 350 400 450 500 550 m/z
58
HA281-parent MS and MS/MS
HA281-metabolite1 MS and MS/MS
158.9763
301.0489
+MS2(466.1152), 15eV, 10.0min #591
0
2
4
6
4x10
Intens.
150 200 250 300 350 400 450 m/z
103.9555149.9518
465.1132
466.1145
302.0518
226.1799
+MS, 9.9-10.0min #(590-594)
0.0
0.2
0.4
0.6
0.8
1.0
4x10
Intens.
100 150 200 250 300 350 400 450 m/z
103.9552144.9813 195.0930
226.1800301.0512
437.1019
438.1059
302.0495112.0275
+MS, 10.1-10.1min #(598-602)
0
2000
4000
6000
Intens.
100 150 200 250 300 350 400 m/z
59
HA281-metabolite2 MS and MS/MS
158.9761
301.0508
+MS2(437.8954), 10.1min #(599)
0
1
2
3
4
5
4x10
Intens.
50 100 150 200 250 300 350 400 m/z
158.9750
245.0604
+MS2(289.8899), 15eV, 9.2min #547
0.0
0.5
1.0
1.5
2.0
4x10
Intens.
75 100 125 150 175 200 225 250 275 m/z
129.0668 195.0904
289.0499
290.0534
246.0648
158.9744
+MS, 9.2min #(545)
0.00
0.25
0.50
0.75
1.00
1.25
4x10
Intens.
100 125 150 175 200 225 250 275 m/z
60
HA281-metabolite3 MS and MS/MS
HA285-parent MS and MS/MS
123.0551 149.9516 195.0877218.9570
377.0675
378.0711
141.9586
103.9549
+MS, 9.7-9.8min #(574-582)
0
500
1000
1500
2000
Intens.
100 150 200 250 300 350 m/z
116.0704
128.0698 158.9775200.0811
301.0477
361.0519
377.0671
+MS2(378.0724), 15eV, 9.8min #579
0
1000
2000
3000
Intens.
100 150 200 250 300 350 m/z
121.0655144.9805 173.1542
218.9561267.1228
369.1667
387.1805
404.2057
437.0822
249.2058
105.0699
+MS, 11.4-11.6min #(677-689)
0
2000
4000
6000
8000
Intens.
100 150 200 250 300 350 400 m/z
61
HA285-metabolite1 MS and MS/MS
149.0398
262.9995439.0849
+MS2(437.0772), 15eV, 11.4min #678
0
1000
2000
3000
Intens.
150 200 250 300 350 400 450 m/z
103.9568 195.0891
409.0731
409.8189121.0948
+MS, 11.8min #(701)
0
1
2
3
4
4x10
Intens.
100 150 200 250 300 350 400 m/z
121.0285
158.9760
289.0541
+MS2(409.0749), 15eV, 11.8min #698
0.0
0.5
1.0
1.5
2.0
2.5
3.0
4x10
Intens.
100 150 200 250 300 350 400 m/z
62
HA287-parent MS and MS/MS
HA287-metabolite1 MS and MS/MS
103.9559185.9404 218.9568 369.1056
546.1455
253.9241149.9525
+MS, 10.6min #(627)
0.0
0.2
0.4
0.6
0.8
1.0
4x10
Intens.
50 100 150 200 250 300 350 400 450 500 550 m/z
369.1029
+MS2(546.1448), 18.2488eV, 10.6min #628
0.0
0.5
1.0
1.5
2.0
2.5
4x10
Intens.
50 100 150 200 250 300 350 400 450 500 m/z
112.0285195.0919
357.1045
236.1576 259.1005
+MS, 9.8-9.9min #(582-590)
0
1
2
3
4
4x10
Intens.
50 100 150 200 250 300 350 m/z
63
HA296-parent MS and MS/MS
113.0708
227.0297
313.1135 357.1043
+MS2(357.1043), 15eV, 9.9min #587
0
2
4
6
4x10
Intens.
100 150 200 250 300 350 m/z
103.9560
144.9823
517.1379
245.1058
+MS, 12.1-12.2min #(718-722)
0.0
0.5
1.0
1.5
2.0
4x10
Intens.
100 150 200 250 300 350 400 450 500 m/z
161.0400
227.0289
517.1384
+MS2(517.1384), 16.2022eV, 12.1min #719
0
1
2
3
4
5
4x10
Intens.
100 150 200 250 300 350 400 450 500 550 m/z
64
HA296-metabolite1 MS and MS/MS
112.0283151.0378 195.0907 249.2062
507.1368
387.1806
409.1631
425.1381
+MS, 11.5-11.7min #(681-693)
0.0
0.5
1.0
1.5
2.0
4x10
Intens.
150 200 250 300 350 400 450 500 m/z
133.0281
151.0385
247.1080 357.1046
489.1258507.1361
+MS2(507.1361), 15.5161eV, 11.5min #686
0
1
2
3
4x10
Intens.
150 200 250 300 350 400 450 500 m/z