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.