Speeding Up Drug Discovery with DART-based Mass Spectrometry Musselman Speeding Up Drug...
Transcript of Speeding Up Drug Discovery with DART-based Mass Spectrometry Musselman Speeding Up Drug...
Speeding Up Drug Discoverywith DART-based Mass
Spectrometry
Brian D. Musselman, IonSense, Inc.Land O Lakes Conference
September 2008
What is DART?
• DART stands for Direct Analysis in Real Time• Generates ions directly from gases, liquid, and solids by
using gas containing heated either metastable atoms ormolecules to transfer energy into the sample environment.
• Provides a non-contact surface sampling technique for usein mass spectrometry based analysis
• Samples are presented for analysis in open air atatmospheric pressure in this case by using a robot samplehander.
• Developed by J. Laramee and R. Cody at JEOL USA, Inc.
DART Schematic
• Gas flows throughDART
• Electrical dischargecreates a plasma
• Lenses removecharged particles
• Grid prevents ion-ionrecombination at exit,and other functions
• No exposed highvoltages
• Operated at ambientpressure in open air
Gas in
Needle electrode
Perforated disk electrodes
Gas heater
Grid electrode
Insulatorcap
Gas out
Gas in
Needle electrode
Perforated disk electrodes
Gas heater
Grid electrode
Insulatorcap
Gas in
Needle electrode
Perforated disk electrodes
Gas heater
Grid electrode
Insulatorcap
Gas out
Experimental Protocol
• Dip into the biological matrix and analyze
MSDARTSource
DART of UrineHigh Resolution Time-of-Flight MS
Pyridoxinecarboxylic acidHypoxanthine
Ranitidine+ClRanitidine
Ascorbic acid
Creatinine
Lactate
Pyruvate
Phenylacetyl glutamine
pGlu
Data courtesy of Dr. Cody, JEOL USA
Computer Analysis of SpectrumJEOL AccuTOF-MS
Data courtesy of Dr. Cody, JEOL USA
Blood droplet:Amino Acids (Positive Ions)
40 60 80 100 120 140 160 180 200
m/z
Urea
80 100 120 140 160
m/z
GlyAla
ProVal
Thr
SerPhe
Leu/IleI/L
PV
No sample prep,no cleanup, no
derivatization, noMS/MS
Data courtesy of Dr. Cody, JEOL USA
Gamma Hydroxybutyrate (GHB) in Urine(Deuterated I.S.)
R2 = 0.9991
0.00
100.00
200.00
300.00
400.00
500.00
600.00
700.00
800.00
900.00
0 100 200 300 400 500 600 700 800 900
ppm
[GH
B]/
[IS
]
Series1
Linear (Series1)
Data courtesy of Dr. Eshwar Jagedeo, FBI Academy
DART: Promazine in Urine
y = 0.0397x - 0.2301
R2
= 0.9952
-5
0
5
10
15
20
25
0 100 200 300 400 500 600
ppm
Are
a/A
rea
(IS
)
Promazine in UrineChlorpromazine internal standard
What makes DART different?
300 310 320 330 340 350
m/z
0
20
300 310 320 330 340 350
m/z
0
20
300 310 320 330 340 350
m/z
0
20
300 310 320 330 340 350
m/z
0
20
Saturated NaCl
DI Water
Saturated Phosphate Buffer
Raw Urine
Chlorpromazine[M+H]+
No alkali metalcation adducts
No multiplecharging
No apparentsuppression
[M+H]+
Initial Evaluation Instrument Configuration
In Vitro Intrinsic Clearance SamplesInitial Effort – May 07
Jing-Tao Wu, Shaoxia Yu, Millennium Pharma
• Experimental
– Incubate compounds in human liver S9. Afterquenching, split the samples for LC/MS andDART analysis
– Compare the intrinsic clearance values obtainedby each method for comparison
– Can a simple protocol be established?
DART for In Vitro SamplesX I C o f + M R M ( 2 p a ir s ) : 4 8 2 . 5 / 4 1 4 . 2 a m u f r o m S a m p l e 8 ( M L N 6 6 3 4 7 5 ) o f H u m a n _ S 9 . w i f f ( T u r b o S p r a y ) M a x . 5 8 4 0 . 0 c p s .
0 . 5 1 . 0 1 . 5 2 . 0 2 . 5 3 . 0 3 . 5 4 . 0 4 . 5 5 . 0T im e , m i n
0
5 0 0
1 0 0 0
1 5 0 0
2 0 0 0
2 5 0 0
3 0 0 0
3 5 0 0
4 0 0 0
4 5 0 0
5 0 0 0
5 5 0 0
5 8 4 04 . 1 7
3 . 7 6
3 . 3 8
2 . 9 6
2 . 1 9
1 . 7 3
2 . 5 7
1 . 3 2
0 . 9 3
0 . 1 9
0 . 5 7 3 . 4 20 . 5 4
X I C o f + M R M ( 2 p a i r s ) : 5 3 4 . 2 / 4 1 6 . 3 a m u f r o m S a m p l e 7 ( M L 6 6 2 8 8 3 ) o f H u m a n S 9 . w i f f ( T u r b o S p r a y ) M a x . 7 2 0 . 0 c p s .
0 . 5 1 . 0 1 . 5 2 . 0 2 . 5 3 . 0 3 . 5 4 . 0 4 . 5 5 . 0T im e , m i n
0
5 0
1 0 0
1 5 0
2 0 0
2 5 0
3 0 0
3 5 0
4 0 0
4 5 0
5 0 0
5 5 0
6 0 0
6 5 0
7 0 0
7 2 03 . 7 8
3 . 4 0
4 . 1 9
2 . 9 8
1 . 7 5
2 . 5 92 . 2 0
0 . 9 7
1 . 3 7
2 . 2 3
2 . 6 1
Compound 1
Compound 2
0 min3 min
7 min12 min
20 min
0 min3 min
7 min
12 min
20 min
DART for In Vitro Samples
Compound 2
0.000
0.500
1.000
1.500
2.000
2.500
0 5 10 15 20 25
Incubation Time
Compound 1
0.000
0.500
1.000
1.500
2.000
2.500
0 5 10 15 20 25
Incubation Time
DART for In Vitro SamplesComparison of LC/MS and DART data
2.080.61Compound 4
1.681.02Compound 3
3.663.17Compound 2
2.671.41Compound 1
CLint (L/hr/kg)
DART
CLint (L/hr/kg)
LC/MS
DART for PK Samples
• Experimental (Compound A)Rat PK study, 4 animals at 10 mg/kg PO
Plasma samples were extracted and analyzed by LC/MS
Plasma were re-aliquoted and mixed with high aqueousIS before subject to DART analysis
Comparison between DART and LC/MS results
Rat PK Study for Compound AX I C o f + M R M ( 2 p a ir s ) : 5 1 4 . 2 / 2 6 9 . 2 a m u f r o m S a m p l e 3 ( M L N 0 4 1 5 ) o f M L N 0 4 1 5 _ N F k B _ s a m p le . w if f ( T u r b o S p r a y ) M a x . 3 7 0 6 . 7 c p s .
0 . 5 1 . 0 1 . 5 2 . 0 2 . 5 3 . 0 3 . 5 4 . 0 4 . 5 5 . 0T im e , m i n
0
2 0 0
4 0 0
6 0 0
8 0 0
1 0 0 0
1 2 0 0
1 4 0 0
1 6 0 0
1 8 0 0
2 0 0 0
2 2 0 0
2 4 0 0
2 6 0 0
2 8 0 0
3 0 0 0
3 2 0 0
3 4 0 0
3 6 0 0
3 7 0 74 . 1 6
3 . 3 7
4 . 5 9
1 . 7 22 . 1 8 2 . 9 40 . 9 3 2 . 5 61 . 3 2
X I C o f + M R M ( 2 p a ir s ) : 5 1 4 . 2 / 2 6 9 . 2 a m u f r o m S a m p l e 7 ( M L N 0 4 1 5 ) o f M L N 0 4 1 5 _ N F k B _ s a m p le . w if f ( T u r b o S p r a y ) M a x . 9 3 3 3 . 3 c p s .
0 . 5 1 . 0 1 . 5 2 . 0 2 . 5 3 . 0 3 . 5 4 . 0 4 . 5 5 . 0 5 . 5T im e , m i n
0
5 0 0
1 0 0 0
1 5 0 0
2 0 0 0
2 5 0 0
3 0 0 0
3 5 0 0
4 0 0 0
4 5 0 0
5 0 0 0
5 5 0 0
6 0 0 0
6 5 0 0
7 0 0 0
7 5 0 0
8 0 0 0
8 5 0 0
9 0 0 0
9 3 3 33 . 8 0
4 . 2 0
3 . 4 3
4 . 6 51 . 7 7
2 . 6 12 . 2 2
3 . 0 0
0 . 5 8
0 . 9 7
1 . 3 7
0 . 2 1
Predose24 hr
6 hr
4 hr
2 hr 1 hr
Rat PK Study for Compound A
0
0. 2
0. 4
0. 6
0. 8
1
1. 2
1. 4
0 1000 2000 3000 4000 5000
Concent r at i on ( ng/ mL)
height
ratio
16.746704000
17.428202400
-15.225.430
Bias%Calculated Concentration
(ng/mL)Norminal Concentration
(ng/mL)
QC performance
Conclusions 07’
• Evaluation for Bioanalysis indicated that matrixeffect were not overwhelming
• Applications for in vivo and in vitro ADMEstudies suggested that additional effort wasnecessary to achieve more optimal results
• Improvement in precision and sensitivity required
Jing-Tao Wu, Millennium PharmaAPA Boston 9/2006
Instrument Configuration – Circa 2008
Gas Ion Separator Developed 2008MS People are Plumbing People
DART DESORPTION PROFILE
Data courtesy of Dr. Shaoxia Yu, Takeda Pharmaceuticals
Data courtesy of Dr. Shaoxia Yu, Takeda Pharmaceuticals
Improved Reproducibility
N=9, CV=3.1%
XICof +MRM(4pairs): 309.1/281.0amufromSample2(test curve)of test curve.wiff (Photospray) Max.2.8e4cps.
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0Time, min
0.0
2000.0
4000.0
6000.0
8000.0
1.0e4
1.2e4
1.4e4
1.6e4
1.8e4
2.0e4
2.2e4
2.4e4
2.6e4
2.8e40.79
1.97
3.61
2.80
3.201.60
2.43
1.17
N=8 CV=27.7%
XICof +MRM(2pairs): 293.1/171.1amufromSample2(TuneSampleID) of Benzoylecgonine_std1.wiff (TurboSpray) Max. 5.6e6cps.
3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0Time, min
0.0
5.0e5
1.0e6
1.5e6
2.0e6
2.5e6
3.0e6
3.5e6
4.0e6
4.5e6
5.0e6
5.5e610.22
8.507.636.765.86 9.355.023.26
4.15
4.30 5.17
Now Before
Data courtesy of Dr. Shaoxia Yu, Takeda Pharmaceuticals
Improved Sensitivity
XICof +MRM(1 pair): 514.2/269.2amu fromSample 1(MLN0415) of std1_rat_PL_MLN0415.wiff (TurboSpray) Max. 1.5e6cps.
8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 12.0 12.5 13.0 13.5 14.0 14.5 15.0 15.5 16.0 16.5Time, min
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
5500
6000
6500
6680
Compound A
1ng/mL2.5ng/mL
10ng/mL
50ng/mLN=3
6680
W/GIST5000
W/out GIST
5000 ng/mLCompound A N=11
Data courtesy of Dr. Shaoxia Yu, Takeda Pharmaceuticals
Reevaluate for Bioanalysis
Dilution Effect of Verapamil
Signal reduction due to the dilution is canceled out by less matrix effect.This is particularly useful when sample is in low volume.
XICof +MRM(1 pair): 455.3/164.9 amu fromSample 3(ben std)of dilution3_Dec21_07.wiff (TurboSpray) Max. 2.0e5cps.
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5Time, min
0.0
1.0e4
2.0e4
3.0e4
4.0e4
5.0e4
6.0e4
7.0e4
8.0e4
9.0e4
1.0e5
1.1e5
1.2e5
1.3e5
1.4e5
1.5e5
1.6e5
1.7e5
1.8e5
1.9e5
2.0e52.37
1.52
0.64
3.24
4.12
4.97
2.521.680.79
Original plasma2x Dilution with H2O
Data courtesy of Dr. Shaoxia Yu, Takeda Pharmaceuticals
Dilution Effect of Verapamil
Further dilution is not beneficial for sensitivity
XICof +MRM(1pair): 455.3/164.9amufromSample1(benstd)of dilution2_Dec21_07.wiff (TurboSpray) Max. 2.5e5cps.
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 7.0 7.5 8.0Time, min
0.0
1.0e4
2.0e4
3.0e4
4.0e4
5.0e4
6.0e4
7.0e4
8.0e4
9.0e4
1.0e5
1.1e5
1.2e5
1.3e5
1.4e5
1.5e5
1.6e5
1.7e5
1.8e5
1.9e5
2.0e5
2.1e5
2.2e5
2.3e5
2.4e5
2.5e5
1.73
2.60
0.84
6.946.03
7.79
3.46 5.234.33
1.00
2.751.88
Original plasma
3x dilution with H2O
3x dilution with0.1% FA/H2O
Matrix Effects in rat plasma
87.3%8.0%122.7%56.9%17.2%1:1 (plasma:H2O)
43.2%5.4%68.4%37.1%6.9%Plasma
CompoundE
PraparacaineAlprozolamVerapamilMethyacina
Matrix effect is reflected by the analyte response in matrix over that in neat solvent
Compound A with Internal StandardDirect Ionization of Rat Plasma
666n
4.42.818.0%Bias
16.04.222.5%CV
16700257011.8Mean
11400271010.16
19000266012.35
17300247011.14
169002570~16.63
1790025508.882
17500243012.11
High(16000)
Mid(2500 )
Low(10.0 )
QC concentration(ng/mL)
Compound A with IS in rat Plasma
R2 = 0.9958
-50
0
50
100
150
200
250
0 5000 10000 15000 20000 25000
222222212n
6.012.08.2-1.5-6.7-6.0-6.6-13.02.0%Bias
21200112005410197093347093.417.45.10Mean
20200110004740168092149878.717.45.002
222001130060702250945441108*29.35.201
200001000050002000100050010020.05.00STD Concentration
with IS (ng/mL)
Compound A in rat Plasma No IS
R2 = 0.9936
-100000
0
100000
200000
300000
400000
500000
600000
700000
800000
0 5000 10000 15000 20000 25000
666n
3.82.018.0%Bias
13.34.514.5%CV
16600255011.8Mean
13000257012.96
17300247011.75
18900267012.54
153002590~14.03
1850023709.332
16300265010.41
High(16000 )
Mid(2500 )
Low(10.0)
QC concentration(ng/mL)
Compound A without IS in Rat Plasma
222222222n
8.013.00.0-3.0-13.1-6.4-2.26.0-1.2%Bias
21600113005000194086946897.821.24.94Mean
20000109004960198085444991.516.14.672
23200116005030189088448610426.25.211
200001000050002000100050010020.05.00STD
Concentration No IS(ng/mL)
Indomethacin in Rat Plasma
standatd Curve of Indomethacin in rat plasma
R2 = 0.9907
0
10000
20000
30000
40000
50000
60000
70000
80000
0 2000 4000 6000 8000 10000 12000
33333333n
8.21.81.410.38.011.424.211.5%CV
13.08.82.0-3.1-9.8-10.4-1.61.0Mean %Bias
113005440255096945122449.210.1Mean
1240053602560105042720652.010.93
108005420258085743425359.410.62
108005550251099949321236.18.751
100005000250010005002505010.0STD Concentration (ng/mL)
Terfenadine in Rat Plasma
666n
-10.09.6-5.9%bias
10.14.74.3%CV
1440027409.41Mean
1530029209.076
1560025408.905
1430027409.194
1530027109.723
1400028309.812
1170026809.781
High
(16000)
Mid
(2500)
Low
(10.0)
QCConcentration
(ng/mL)Terfennadine in rat Plasma
R2 = 0.9977
0
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
0 5000 10000 15000 20000 25000
2222222222n
3.57.05.66.53.0-4.4-8.7-8.0-7.64.0%Bias
207001070052802130103047891.318.44.622.08Mean
198001100052801990100049889.719.04.512.012
215001040052802270105045792.817.74.722.151
200001000050002000100050010020.05.002.00STD concentration
(ng/mL)
XICof +MRM(1pair): 455.3/164.9amufromSample1(verapmil) of A&P_carryover.wiff (TurboSpray) Max. 4.9e6cps.
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38Time, min
0.0
2.0e5
4.0e5
6.0e5
8.0e5
1.0e6
1.2e6
1.4e6
1.6e6
1.8e6
2.0e6
2.2e6
2.4e6
2.6e6
2.8e6
3.0e6
3.2e6
3.4e6
3.6e6
3.8e6
4.0e6
4.2e6
4.4e6
4.6e6
4.8e64.9e6
15.82
16.60
32.6034.1431.08
34.90
14.29
12.75
28.8130.3326.51
11.25
9.71
8.16
7.39
STD
blank
High QC
XICof +MRM(1 pair): 455.3/164.9 amu fromSample1(verapmil) ofA&P_carryover.wiff (Turbo Spray) Max. 4.9e6cps.
21.4 21.6 21.8 22.0 22.2 22.4 22.6 22.8 23.0 23.2 23.4 23.6 23.8 24.0 24.2 24.4 24.6 24.8 25.0 25.2 25.4 25.6 25.8 26.0 26.2Time, min
0
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
2400
2600
2800
3000
3200
3400
3600
3800
3985
Low QC
Mid QC
Verapamil in Rat Plasma
Verapamil in Rat Plasma
Verapmil std curve in rat plasma
R2 = 0.9972
0
1000000
2000000
3000000
4000000
5000000
6000000
7000000
0 5000 10000 15000 20000 25000666n
-15.0-6.0-10.8%bias
3.75.212.9%CV
1360023508.92Mean
1360023009.136
1280025207.725
1320024908.294
1390022307.953
13600227010.72
1420023009.711
High(16000)
Mid(2500)
Low(10.0)
QC Concentration(ng/mL)
2222222212n
-16.0-11.1-7.09.00.04.69.07.520.0-5.0%Bias
16800889046502180100052310921.56.001.90Mean
16400844046902190101052311121.9*7.302.042
1710093304600216099352310721.16.001.761
200001000050002000100050010020.05.002.00STDconcentrat
ion(ng/mL)
Applications for in Vivo and in
Vitro ADME Studies
1. Mouse PK Study for Compound A (plasmasamples)
• 7 groups,3 animals at 25 mg/kg PO
a) Plasma samples were extracted and analyzed byLC/MS/MS
b) Plasma samples were directly subjected toDART analysis
Mouse PK StudyMeasured Plasma Concentrations By LC/MS/MS & DART
BQL1.39C21 24hr
BQLBQLC20 24hr
BQLBQLC19 24hr
BQLBQLC18 16hr
BQL2.00C17 16hr
BQLBQLC16 16hr
-2.231303270C15 8hr
5.321111900C14 8hr
-4.328513110C13 8hr
2.847664510C12 4hr
2.736723480C11 4hr
-4.726742940C10 4hr
2.930732900C9 2hr
10.623401890C8 2hr
11.744303500C7 2hr
9.730112480C6 1 hr
16.458464200C5 1 hr
10.242973500C4 1 hr
11.829182300C3 30min
5.844123930C2 30min
2.241703990C1 30min
% MPDDART
Concentration (nM)LC/MS /MS
Concentration (nM)Sample ID
Applications for in Vivo and in VitroADME Studies
2. Mouse PK Study for Compound B (wholeblood samples)
• 16 groups,3 animals at 60 mg/kg SC
a) Whole blood samples were extracted andanalyzed by LC/MS/MS
b) Whole blood samples were directlysubjected to DART analysis
Mouse PK StudyMeasured Whole Blood Concentrations Between LC/MS/MS & DART
1.53418733200HH3
22.42829044600HH2
-3.73085333200HH1
1.02429223800GG3
-3.62011321600GG2
19.13287622300GG1
5.91743415500FF3
-6.11876321200FF2
-1.81985820600FF1
BQLBQLEE3
BQLBQLEE2
BQLBQLEE1
-8.82993935700DD3
-11.51913124100CC2
5.42372421300CC1
3.62383622200BB3
3.81716215900BB2
9.32327119300BB1
BQLBQLAA3
BQLBQLAA2
BQLBQLAA1
%MPDDART (nM)LC/MS/MS
Concentration (nM)Sample ID
-1.43383934800PP1
-1.31763918100OO2
8.91985216600OO1
3.41712616000NN3
-4.91460916100NN2
8.81360611400NN1
BQLBQLMM3
BQLBQLMM2
BQLBQLMM1
9.03809231800LL3
-6.72772231700LL2
3.42397822400LL1
-5.31816820200KK3
8.02220818900KK2
-3.81640917700KK1
-5.41302714500JJ3
-0.11467714700JJ2
10.51592712900JJ1
BQLBQLII3
BQLBQLII2
BQLBQLII1
%MPDDART (nM)LC/MS/MS
Concentration(nM)Sample ID
Applications for in Vivo and in VitroADME Studies
3. Intrinsic Clearance Study for In vitro Samples
0.920.91Mouse MicrosomesCompound D
<0.16<0.16Rat S9Compound C
<0.19<0.19Human S9Compound C
Hepatic ExtractionRatio
DART
Hepatic ExtractionRatio
LC/MS/MS
Matrix
Other Speedy Methods
Rapid Fire LC/MS (BioTrove)UPLC/MS
MALDI / QTOFDESI / DAPSI
Laser Diode Thermal DesorptionLDTD MS
Conclusions
• The reproducibility, Sensitivity and Practicabilityof DART has been significantly improved withnew instrumentation.
• DART was found to meet the general requirementfor bioanalysis without sample preparation.
• Compared to results generated with LC/MS/MS,DART has produced comparable results for PKand in vitro ADME studies.
• Speed is on its way, one way or another
Acknowledgements
Shaoxia Yu, Jing-Tao Wu, Gordon Justin, ChuangLu, Ji Zhang, Cicely Berg, and members of theTakeda bioanalytical group for their help in thiswork.