An Integrated Approach for Metabolic Stability and Detailed Metabolite Profiling from a Single Injection on an

LTQ Linear Ion Trap Robyn A. Rourick, Hesham Ghobarah and John P. Walsh; Kalypsys, Inc., San Diego, CA 92121

OVERVIEW CROSS-VALIDATION RESULTS: API4000 vs. LTQ

Assignment Tools: Structure Drawing & Fragmentation
• Early evaluation of drug metabolism has placed new pressures on the 120%
Buspirone 120%
Imipramine

techniques used to acquire and accurately interpret lead optimization data. 100%

80%
100%

80%
Combined Modifications List Used

% Remaining
% Remaining
60% 60% N

Parent m/z 421 R1

• Highly automated methods featuring fast chromatographic separations along
40% 40%

N
20% 20%

with mass spectrometry analysis provide powerful methods for quantitative 0%

0 5 10 15
Time (min)

API4000 LT Q
20 25 30
0%
0 5 10 15
Time (min)

API4000 LT Q
20 25 30

N
analysis. 120%
Lidocaine 120%
Methoxyverapamil
m/z 421 + 16
R1
N
N

R1
N NH
100%

O
100%

R2
O
• Quadrupole mass spectrometers have clearly been benchmarked as the
80% 80%

% Remaining

% Remaining
60%
60%

m/z 421 - 2
quantitative standard while ion traps have been more widely utilized for
40%
40%

20%
20%

qualitative structural studies. 0%

0 5 10 15
Time (min)

API4000 LT Q
20 25 30
0%
0 5 10 15
Time (min)

API4000 LT Q
20 25 30

Propranolol Verapamil

• The LTQ linear ion trap provided the sensitivity and flexibility to successfully
120% 120%

100% 100%

provide qualitative and quantitative results of microsomal incubates. 80% 80%

Figure 6: Database Manager: Assign Parent Fragments

% Remaining

% Remaining
60% 60%

40% 40%

• Recent software advances prove indispensable when mining the qualitative

20% 20%

Database Manager: Library Search Utilities

0% 0%
0 5 10 15 20 25 30 0 5 10 15 20 25 30
Time (min) Time (min)

data available from data dependent experiments. Figure 3: Metabolite ID 2.0 - EICs Resulting from Modification Step RLM Metabolites
API4000 LT Q API4000 LT Q

Figure 1: Rat Liver Microsomal Incubations with Reference Standards

30mpk Rat Plasma

KLYP 1 KLYP 2
MASS FRONTIER 4.0 – MODULE HIGHLIGHTS
INTRODUCTION 120%

100%
120%

100%

80% 80%

Segmented linear traps with radial ejection and dual detection systems now
60%

40%
60%

40%
• Chromatogram Processor
20% 20% component detection for automated extraction of individual spectra
enable improved detection efficiency and sensitivity. These benefits, coupled with 0%
0 5 10 15 20 25 30 35
0%
0 5 10 15 20 25 30 35
or MSn trees from complex data sets
fast scan times, produce better peak shapes affording improved quantitation over
API4000 LTQ API4000 LTQ

a wider linear dynamic range. We have established the LTQ as the front-line KLYP 3 KLYP 4
• Fragments & Mechanisms
120% 120% an expert system for the automated generation of fragmentation &
platform for metabolic stability and profiling after a rigorous cross validation 100%

80%
100%

80%
rearrangement mechanisms – simulation of MSn experiments
against an API 4000 using reference and proprietary compounds. Data 60% 60%

• Structure Editor
40% 40%

dependent acquisition of a single incubation is acquired to simultaneously supply

20% 20%

full featured structure drawing tool

0% 0%
0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35

quantitative and qualitative results. Intelligent assessment of the stability data API4000 LTQ API4000 LTQ

involves identifying compounds which display significant metabolic liabilities (i.e. • Database Manager
KLYP 5 KLYP 6
organizing & processing mass spectra, chemical structures & Figure 7: Build Proprietary In-house Libraries
< 30% at 30min). Interpretation of the data dependent results provides insights 140%

120%
120%

100%
libraries
100%

into the metabolic fate of in-house medicinal chemistry collections. 80%

80%

60%
Database Manager: In Vivo/In Vitro Comparison
60%
40%
40%

20% 20%

0% 0%
0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35

METHODS
API4000 LTQ API4000 LTQ

RT: 0.00 - 7.00 SM: 5G

0.36 NL: 2.47E6

Figure 2: Rat Liver Microsomal Incubations with Kalypsys Compounds

100 0.68 TIC F: ITMS + c
ESI Full ms [
90
150.00-1000.00]
MS
Difference
• 2µM Concentration
80

70
Relative Abundance

60

50
Active record in
40

30 2.33 spreadsheet

• 10µL injection CROSS-VALIDATION RESULTS: API4000 vs. LTQ

20

0.98 1.31 1.94

10 1.61 2.56 2.80 2.99 3.52 3.66 4.28 5.81 5.99 6.23 6.70
4.36 4.89 5.50
0
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5
Time (min)

RT: 0.00 - 7.00 SM: 5G

• Strong correlation shown between LTQ data dependent mode versus

RT: 2.33 NL: 4.60E5
100
m/z= 455.31-455.60

• Supernatant from precipitated sample

F: ITMS + c ESI Full
90
ms [ 150.00-1000.00]
MS
80

API4000 MRM mode for metabolic stability monitoring.

70
Relative Abundance

60

50

40

• Data-dependent acquisition 30

20

Selected Component
10

0
0.0
0.24 0.60

0.5
1.09

1.0
1.33

1.5
1.82 2.18

2.0 2.5 3.0

3.45

3.5
Time (min)
4.0
4.20 4.50

4.5
4.87

5.0
5.41

5.5
5.81 6.11 6.26

6.0
6.43

6.5
• Kalypsys compound correlations closely mimic results afforded from KLYP526 - 14

• 1x50mm 3µm column for separation of metabolites reference standards.

KLYP526 + 16

• Quantitation of [M+H]+ for stability determination INTEGRATION OF SOFTWARE APPLICATIONS Figure 8: Tree or Spectrum Search
Verapamil Timepoint (min) %Remaining Average Std. Dev. Ver apamil

102104_MetIDStability_61 0 95.6

Data Dependent experiment performed for

102104_MetIDStability_62 0 103.2 100.0 3.9 120

102104_MetIDStability_63 0 101.2 100

102104_MetIDStability_64 5 44.4
102104_MetIDStability_65
102104_MetIDStability_66
5
5
47.5
49.0
47.0 2.3
80

60
Metabolic Stability & ID Figure 4: Chromatogram Processor - MS1 Component Detection CONCLUSIONS
102104_MetIDStability_67 15 20.2 40

102104_MetIDStability_68 15 19.7 17.1 4.9

• Both metabolic stability data and qualitative metabolic profiling are achievable from a single microsomal incubate
20
102104_MetIDStability_69 15 11.5 SPECTRAL TREES
102104_MetIDStability_70 30 4.8 0
0 5 10 15 20 25 30 - Levels
<30% remaining at T=30
30 0.8
injection on an LTQ ion-trap.
102104_MetIDStability_71 6.4 5.7
102104_MetIDStability_72 30 5.8
T im e ( m in )
- Node Connectors
- Nodes
- Node items
CROSS-VALIDATION EXPERIMENTAL: API4000 vs. LTQ • Background subtraction Selected Component
• This integrated structure elucidation approach with Metabolite ID and Mass Frontier demonstrates practical analytical
merit by relieving the heavy reliance on experts to propose metabolites.
• Search for known and unknown modifications
API 4000 LTQ
HPLC Conditions • Identify isotope clusters • Software packages provide faster insight to lead optimization efforts of metabolic “soft spots”.
Injection Volume 5µL 10µL
Column 2x30mm C18, 4µm Polar-RP 1x50mm C18, 3µm Atlantis
• User interrogation of combined modifications &
control • Mass Frontier allows for the efficient management of the data resulting from data dependent acquisition.
Flow Rate 1000µL/min 200µL/min
Mobile Phase A:B Water:Acetonitrile (Formic Acid) Water:Acetonitrile (Formic Acid)
Runtime 2.7min 7min • Future work benefits from the ability to create and maintain searchable proprietary mass spectral & chromatographic
Time %B Time %B libraries.
0.00 0.0 0.00 10.0
• ID location of metabolism Selected Node Spectrum
1.00 100.0 0.10 10.0
Gradient • More accurate fragmentation predictions facilitates the interpretation process and the heavy reliance on the expert.
1.90 100.0 5.00 90.0 • Predictive fragmentation with
2.00 0.0 6.00 90.0 mechanisms Selected Component Tree
2.70 0.0 6.10 10.0
• Ability to store information as trees & ACKNOWLEDGEMENTS
MS Conditions search Figure 5: Chromatogram Processor: MS2 m/z 407 Component Detection • Stewart Noble
Mode MRM Full Scan Data Dependent
Ionization ESI+ ESI+ • Component detection • Eric Hemenway
Temperature 500°C 375°C • Brenda Kesler
• Database searching • Robert Mistrik
Spray Voltage 5.5kV 4kV