From data to answers:
direct reporting of
ADME results
Rapid Resolution LC with TripleQuadrupole MS detection and
MassHunter software for
High Productivity ADME
ADME - From Data to Answers
Page 1
Month ##, 200X
Introduction
During the development of new drug substances any new potential drug compounds
and modifications of those are tested in certain assays to get information about their
physiological behavior. These in-vitro assays are meant to mimic crucial parts of the
human body. Typically the drug absorption to the body, its distribution over the body,
its metabolization and finally the excretion is tested. These experiments are covered
under the abbreviation ADME.
Absorption
As these tests happen during early stages of the drug discovery
process – in a early pre-clinical phase – the numbers of
compounds to test are high. Typically these assays are performed
in a microtiter-based format and with a certain degree of
automation.
Often, these assays are subcontracted by the big pharmaceutical
companies to CRO companies as these assays are highly
standardized.
Distribution
Metabolism
Excretion
ADME - From Data to Answers
Page 2
Month ##, 200X
Study - Overview
This study covered the high throughput analysis of 10 compounds in three typical assays performed in
early ADME profiling of new hits:
•
Human Liver Microsome (HLM) Stability to estimate the rate of metabolization
• Plasma Protein Binding (PPB) to estimate the distribution of the drug
• Caco-2-Permability (Caco-2) required as well to estimate the distribution
After performing the assays with each individual compound, several compounds have been pooled to
reduce the number of samples. All samples included internal standards. Quantization was done
according to the relative responses between target and ISTD. Additionally, every assay used certain
control substances.
ADME - From Data to Answers
Page 3
Month ##, 200X
Assays - Overview
6 Controls
10 Compounds
3 Assays
2 ISTDs
HLM
PPB
Caco-2
18 MRM Transitions & Optimizations
Pooling of samples
490 individual concentration
values plus blanks, in
Triplicates
> 1500 data points
14 MS methods
121 individual
samples
ADME - From Data to Answers
Page 4
Month ##, 200X
LC-MS/MS System - Overview
Agilent High throughput QQQ-RRLC-System
• 2 Binary Pumps SL
• CTC HTC-Pal autosampler with active wash station and cooled stack
• Thermostatted Column Compartment SL with 2ps/10pt valve for
Alternating Column Regeneration
• 2 Agilent Zorbax RRHT SB C18 2.1mm x 50 mm, 1.8µm columns
• 6410 Triple Quadrupole MS
• MassHunter Software for Acquisition, Qualitative and Quantitative
Data Analysis
ADME - From Data to Answers
Page 5
Month ##, 200X
Method - HPLC
Solvent A:
Solvent B:
Flow:
Temperature:
Water (10 mM NH4OAc, 0.1% HOAc)
Acetonitrile (0.1% HOAc)
1.1 ml/min
50 °C
%B
95
Eluent pump
System Overhead Time
min. 0.15min*
5
%B
95
Regeneration pump
5
Draw, Inject, Clean
HTC Pal Autosampler
ca. 0.5 min
0.3
0.5
0.7
0.85 1.00
time
*) System overhead time will be longer if method changes!
ADME - From Data to Answers
Page 6
Month ##, 200X
Chromatographic Performance
MRM chromatogram of three pooled samples and added ISTD:
PWHH = 0.016 min
ISTD
0.8 min
ADME - From Data to Answers
Page 7
Month ##, 200X
Method - MS
General:
Time Filter:
DeltaEMV:
Source:
Ion Source:
ESI
DGasHeater:
250°C
DGasFlow:
11 L/min
Nebulizer Pressure: 50 psi
VCap: 4000 V
MRM-Settings:
MS1Res:
MS2Res:
Dwell:
Prec.Ion:
Prod.Ion:
Fragmentor:
CE:
Off
800
Unit (Warfarine, Digoxine, Diclofenac Flurbiprofen: Wide)
Wide
individually set to have ca. 15 data point/sec,
i.e. dwell-time in the range of 5-30 ms depending on number of transitions
see following tables
see following tables
see following tables
see following tables
ADME - From Data to Answers
Page 8
Month ##, 200X
Method - MS
Pola
Precursor Ion
rity
Product Ion
pos
798.4
651.4
Nefazodon
pos
470.2
274.1
15
160
35
Buspirone
pos
386.3
122
15
180
25
Nadolol
pos
310.2
74.1
15
140
25
HLM-Methods
Compound
Method_1
Digoxin
Dwell time
[ms]
15
Fragmentor
[V]
CE
[V]
120
10
Method_1neg
Flurbiprofen
neg
199.1
199.1
50
100
0
time seg. 0.5min
Nadolol
pos
310.2
74.1
50
140
25
Method_2
Diclofenac
pos
296
215
10
90
6
Tramadol
pos
264.2
58
10
100
15
Atenolol
pos
267.2
145
10
140
25
Labetolol
pos
329.2
162
10
120
25
Nadolol
pos
310.2
74.1
10
140
25
Nimodipin
pos
343.1
301
20
120
20
Propranolol
pos
260.1
183
20
120
25
Nadolol
pos
310.2
74.1
20
140
25
Verapramil
pos
455.1
165.1
30
140
25
Nadolol
pos
310.2
74.1
30
140
25
Dextrometorphan
pos
272.2
170.9
30
140
25
Nadolol
pos
310.2
74.1
30
140
25
Method_3
Method_4
Method_5
Control Substance
ISTD
ADME - From Data to Answers
Page 9
Month ##, 200X
Method - MS
Pola
Precursor Ion
rity
Product Ion
Dwell time
[ms]
Fragmentor
[V]
CE
[V]
PPB Methods
Compound
Method_9
Diclofenac
pos
296
215
5
90
6
Digoxin
pos
798.4
651.4
5
120
10
Tramadol
pos
264.2
58
5
100
15
Nimodipin
pos
343.1
301
5
120
20
Propranolol
pos
260.1
183
5
120
25
Nefazodon
pos
470.2
274.1
5
160
35
Warfarin
pos
309.2
162.9
5
140
25
Metoprolol
pos
268.2
74.1
5
140
25
Method_9neg
time segmented
Flurbiprofen
neg
199.1
199.1
50
100
0
Metoprolol
pos
268.2
74.1
50
140
25
Method_10
Atenolol
pos
267.2
145
10
140
25
Buspirone
pos
386.3
122
10
180
25
Labetolol
pos
329.2
162
10
120
25
Warfarin
pos
309.2
162.9
10
140
25
Metoprolol
pos
268.2
74.1
10
140
25
ADME - From Data to Answers
Page 10
Month ##, 200X
Method - MS
Pola
Precursor Ion
rity
Product Ion
Dwell time
[ms]
Fragmentor
[V]
CE
[V]
CaCO2 Methods
Compound
Method_6
Tramadol
pos
264.2
58
13
100
15
Propranolol
pos
260.1
183
13
120
25
Atenolol
pos
267.2
145
13
140
25
Metoprolol
pos
268.2
74.1
13
140
25
Method_6neg
time seg. 0.5min
Flurbiprofen
neg
199.1
199.1
50
100
0
Metoprolol
pos
268.2
74.1
50
140
25
Method_7
Diclofenac
pos
296
215
10
90
10
Digoxin
pos
798.4
651.4
10
120
10
Nimodipin
pos
343.1
301
10
120
20
Nefazodon
pos
470.2
274.1
10
160
35
Metoprolol
pos
268.2
74.1
10
140
25
Labetolol
pos
329.2
162
20
120
25
Propranolol
pos
260.1
183
20
120
25
Metoprolol
pos
268.2
74.1
20
140
25
Buspirone
pos
386.3
122
13
180
25
Atenolol
pos
267.2
145
13
140
25
Talinolol
pos
364.3
100.1
13
140
25
Metoprolol
pos
268.2
74.1
13
140
25
Method_11
Method_8
ADME - From Data to Answers
Page 11
Month ##, 200X
Software Overview - Data analysis in Quant Analysis
All Information at one glance!
• MRM extraction of targets and ISTD
• Integration
• Check of signal height
• Chromatographic issues
Toggle between compounds
Results for first target
Results for ISTD
Sample list
Toggle between
compounds and
samples
Target
Chromatogram
Spectrum
ISTD
ADME - From Data to Answers
Page 12
Month ##, 200X
Software – MassHunter Quantitative analysis
MRM information
for Quantifier,
Qualifier, ISTD
information, etc.
directly pulled from
acquisiton method!
Method
set-up
tasks
Method set-up in two minutes
(Additional
columns available
for all available
tasks)
Sample Information
(TIC of all compounds)
Compound Information
(Target, ISTD;
Quantifier,Qualifier)
ADME - From Data to Answers
Page 13
Month ##, 200X
Software Overview – The Power of Excel-based reporting
The data-flow in MassHunter
Results File
MassHunter
Quantitative
Analysis
Raw Data
Excel™ Template
Excel
worksheet and
print-out with
assay-results
ADME - From Data to Answers
Page 14
Month ##, 200X
Software Overview – The Power of Excel-based reporting
Create once a template in Excel:
Custom calculations on the data
after data import
Data import area during processing
All available data in the
data-file, as a XML-map
Just drag and drop!
ADME - From Data to Answers
Page 15
Month ##, 200X
Software Overview – The Power of Excel-based reporting
Results after automatic processing – example HLM-assay:
Microsomal Stability Results
Labetalol
Target Compound
k-Value
k=
by Michael Frank
Half life time
t 1/2 =
-0.00988
Stability
70.1901666
70.2 min
The compound half life
time directly on the
MassHunter report
Summary
SLOPE(y-range,
x-range)
time
av. Ln(RR)
[min]
stdev (RR)
av. RT
[%RSD]
[min]
stdev (RT)
Answers, not just data – directly
from your LCMS software!!!
[%RSD]
0
1.1233
3.71%
0.4941
1.27%
5
1.1452
9.06%
0.4939
1.19%
15
0.9980
0.96%
0.4944
1.02%
30
0.9246
1.54%
0.4940
1.05%
45
0.6795
7.25%
0.4930
1.28%
=a/b [as percent]
Raw Data
Response
131417
ISTD Resp.
41029
Timepoint2
0
Ret. Time
Rel. Resp. Ratio Stdev(RR)
3.2030
3.2030 0.4898
3.0382
3.0382 0.5013
119633
39377
0
108957
36517
0
2.9837
2.9837
0
AVERAGE(range)
3.0750
0.1142 0.4941
0.4911
STDEV(range)
Ret. Time
Rel. Resp. Ratio Stdev(RR)
2.7426
2.7426 0.4901
2.6990
2.6990 0.4999
Response
101295
ISTD Resp.
36933
Timepoint2
15
93517
34649
15
85528
31717
15
2.6966
2.6966
15
2.7127
0.0259 0.4944
Response
ISTD Resp.
Timepoint2
81574
32889
30
2.4803
85500
33863
30
2.5249
79013
30894
30
30
Rel. Resp. Ratio Stdev(RR)
0.4932
Ret. Time
Data File
0.4898 HLM_0min_M2_1.d
0.5013 HLM_0min_M2_2.d
Stdev(RT)
0.0063
Data File
0.4901 HLM_15min_M2_1.d
0.4999 HLM_15min_M2_2.d
Stdev(RT)
0.4932 HLM_15min_M2_3.d
0.0050
Stdev(RT)
Data File
0.4903 HLM_30min_M2_1.d
0.4999 HLM_30min_M2_2.d
2.5576
0.4903
2.5249 0.4999
2.5576 0.4918
2.5209
0.0388 0.4940
0.0052
Response
ISTD Resp.
Timepoint2
70683
34711
45
2.0363
69698
33620
45
2.0731
2.4803
Rel. Resp. Ratio Stdev(RR)
Ret. Time
0.4908
2.0731 0.5001
2.0363
Excel-features applied here:
0.4911 HLM_0min_M2_3.d
0.4918 HLM_30min_M2_3.d
Stdev(RT)
Æ Formulas (e.g. linear regression
analysis of a data set, simple
formula like average, stdev,etc.)
Æ Conditioned formatting
Æ Formatting
Data File
0.4908 HLM_45min_M2_1.d
0.5001 HLM_45min_M2_2.d
ADME - From Data to Answers
Page 16
Month ##, 200X
Software Overview – The Power of Excel-based reporting
Pooled compounds on separate worksheets, of course:
Page brakes and sheet brakes freely definable
ADME - From Data to Answers
Page 17
Month ##, 200X
Software Overview – The Power of Excel-based reporting
…and of course nice print-outs:
ADME - From Data to Answers
Page 18
Month ##, 200X
ADME-Assay Examples
ADME - From Data to Answers
Page 19
Month ##, 200X
HLM Stability Assay
Question
What is the metabolic half life time
of my compound?
ADME - From Data to Answers
Page 20
Month ##, 200X
HLM Stability Assay - Details
Microsomes
Phosphate-Buffer
Microsomes
Testcompound
Phosphate-Buffer
3
1
2
Add NADPH
1’
Matrix Control
Control
(no NADPH)
Aliquots
Calculation of
half life time t1/2
Stop by addition of MeOH at different time points
4
LC-MS/MS Analysis
Work-up
0min
5min
15min
30min 45min
Matrix
Contr
5
ADME - From Data to Answers
Page 21
Month ##, 200X
HLM Stability Assay - Details
Protocol Summary
Final test compound concentration:
Final DMSO concentration:
3 µM
0.25%
ISTD:
Final ISTD concentration:
Nadolol
0.18 µM
Microsomes:
Microsomes final concentration:
Human, pooled male and female
0.5 mg/mL
Phosphate-Buffer:
Incubation temperature:
Final incubation volume:
Final Volume:
0.1 M, pH=7.4
37 °C
25 µL
75 µL
Reactions are stopped by the addition of 50 µL methanol containing
internal standard at the appropriate time points followed by a 1:1 dilution by
water.
Incubation plates are centrifuged at 2,500rpm for 20min at 4°C to
precipitate the protein.
ADME - From Data to Answers
Page 22
Month ##, 200X
HLM stability assay – Results Summary
half life
[min]
CLint
[mL/(min*kg)]
CLint
[L/(h*kg)]
Atenolol
363.0
3.44
0.21
Buspirone
10.2
122.32
7.34
Diclofenac
18.2
68.55
4.11
∞
0.00
0.00
Labetalol
70.2
17.77
1.07
Nefazodone
7.6
164.17
9.85
Nimodipine
4.6
271.23
16.27
Propranolol
69.2
18.03
1.08
Tramadol
392.0
3.18
0.19
Compound
Digoxine (stable)
Microsome concentration: 0.5 mg/mL; 45 mg microsomes/g liver; 20 g liver/kg body
Formula: CLint= ln(2) / t1/2 * (1/cmicrosomes * 45 mg/g * 20 g/kg)
ADME - From Data to Answers
Page 23
Month ##, 200X
Caco-2 Assay
Question
What is the permeability of my
compound in the gut?
ADME - From Data to Answers
Page 24
Month ##, 200X
Caco-2 Assay - Details
1
Add Test-compound
to apical compartment
Grow Caco-2 cell to a
confluent layer (20 days)
Test for confluency!
HBSS-Buffer
2
3
Wait for equilibration
Test-compound
travels through cell layer
into the basolateral
compartment
Sample from both
compartments
LC-MS/MS Analysis
1’
Dilution series of test
compound in buffer
for calibration curve
Calculation of
apparent
Permeability Papp
4
ADME - From Data to Answers
Page 25
Month ##, 200X
Caco-2 Assay - Details
Caco-2 Assays Plates:
Taken from the Millipore Protocol Note - PC1060EN00: “MultiScreen® Caco-2 --Optimization of Caco-2 cell growth and
differentiation for drug transport assay studies using a 96-well assay system”
Cross-sectional EM of Differentiated Caco-2 Cells:
Taken from the Dissertation of Stephan A. Motz, Universität des Saarlands,
Saarbrücken, 2007
ADME - From Data to Answers
Page 26
Month ##, 200X
Caco-2 Assay - Details
Protocol Summary
Final test compound concentration:
Final DMSO concentration:
10 µM
1.0%
ISTD:
Final ISTD concentration:
Metoprolol
0.2 µM
Caco-2 cells:
obtained from the ATCC ( passage numbers 40-60), seeded on to
Millipore Multiscreen Caco-2 plates at 1 x 105 cells/cm2,
cultured for 20 days in DMEM, media is changed every two or three
days. On day 21 the permeability study is performed
Buffer:
Hanks Balanced Salt Solution (HBSS), pH 7.4 buffer with 25 mM
HEPES and 4.45 mM glucose at 37°C
Cell Integrity test:
By Lucifer yellow method
Incubation temperature:
Incubation time:
37 °C
120 min
After incubation samples from the Donor and Receiver plates are taken and 30 µL methanol containing
internal standard are added to all samples, giving a final volume of 100µL.
ADME - From Data to Answers
Page 27
Month ##, 200X
Caco-2 Assay - Results
Data analysis with MassHunter Quantitative Analysis, reporting similar to the HLM assays by
averaging across triplicates of two individual assays wells and automatic calculation of the Papp
value in the Excel-Report.
Example:
Nefazodone
Target Chromatogram of receiver well
Calibration curve
y=6.7495E-4 x +0.0086, no
outliers, R2=0.9955
ISTD Chromatogram of receiver well
ADME - From Data to Answers
Page 28
Month ##, 200X
Caco-2 Assay - Results
Compound
Papp [10-6 cm/s]
Recovery [%]
Flurbiprofen
53
68
Diclofenac
62.7
71
Digoxine
1.82
69
Tramadol
49.7
92
Nimodipine
60.4
48
Propranolol
53.6
70
Nefazodon
59.2
32
Atenolol
0.612
93
Buspirone
52.1
79
Labetolol
15.5
84
Talinolol
0.485
84
Initial Concentration of Donor: 10000nM, Volume Receiver:0.21mL, Incubation time 7200 s, cell area 0.11cm2,
Formula: Papp=creceiver/cdonor,initial * Vrec/(time * Area)
ADME - From Data to Answers
Page 29
Month ##, 200X
PPB Assay
Question
What is the fraction
unbound of my compound in
plasma?
ADME - From Data to Answers
Page 30
Month ##, 200X
PPB Assay - Details
Test-compound
2
Phosphate-Buffer
Plasma
Compartments
separated by a semipermeable membrane
Test compound
bound to
plasma proteins
1
Wait for equilibration
3
Work-up
LC-MS/MS Analysis
1’
Work-up
Dilution series of test
compound in buffer
and in plasma for
calibration curve
4
Calculation of fraction
unbound fu
Measurement of concentration
• Protein free final
• Protein free initial
• Protein containing final
• Protein containing initial
ADME - From Data to Answers
Page 31
Month ##, 200X
PPB Assay - Details
Protocol Summary
Final test compound concentration:
Final DMSO concentration:
5 µM
0.5%
ISTD:
Final ISTD concentration:
Metoprolol
0.18 µM
Plasma:
Human, 50% (v/v) in phosphate
buffer
Phosphate-Buffer:
0.1 M, pH=7.4
Method:
Equilibrium dialysis
Incubation temperature:
Incubation time:
Final Volume:
37 °C
overnight
130 µL
After incubation 26 µL methanol containing internal standard are added to
all samples.
Incubation plates are centrifuged at 2,500rpm for 20min at 4°C to
precipitate the protein.
ADME - From Data to Answers
Page 32
Month ##, 200X
PPB Assay - Results
Fraction
unbound
(50% plasma)
Fraction
unbound
(100% plasma)
Recovery
[%]
Atenolol
0.912
0.838
96
Buspirone
0.572
0.401
94
Digoxine
0.792
0.655
54
Labetalol
0.524
0.355
93
Nefazodone
0.063
0.033
71
Nimodipine
0.074
0.038
68
Propranolol
0.539
0.369
89
Tramadol
0.939
0.884
87
Compound
ADME - From Data to Answers
Page 33
Month ##, 200X
Summary
The Agilent 6410 TripleQuad LC-MS/MS System together with the 1200 Series
Rapid Resolution LC system and MassHunter software offers a very high
productivity by:
•
Parallel column operation to reduce the overall cycle-time.
•
Easy and straight forward software operation.
•
Direct reporting of typical ADME values and not only individual concentration
values by fully customizable Excel reports.
•
Direct generation of Excel files for any further processing, documentation etc.
(no csv-file export/import or file conversion actions).
ADME - From Data to Answers
Page 34
Month ##, 200X
Thank you!!!
ADME - From Data to Answers
Page 35
Month ##, 200X
Appendix
ADME - From Data to Answers
Page 36
Month ##, 200X
Dwell Time Evaluation
200 pg Alprazolam 100, 20, 5 ms dwell times
2.8 ·103
Dwell
100 ms
20 ms
5 ms
Area
14860
13605
13202
% RSD
0.61
0.74
2.25
2.8 ·103
3.0·103
5 ms dwell
ADME - From Data to Answers
Page 37
Month ##, 200X