500000

August 8-11, 2005 Bristol, Rhode Island

Characterization and Performance of MALDI on a Triple Quadrupole Mass Spectrometer for Analysis and Quantification of Small Molecules

Jason S. Gobey, Mark J. Cole, John S. Janiszewski

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HT ADME Sample History 2000-2003

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MALDI Potential For HT Quantitation

1. Speed: potential for <1sec/sample

2. Simple analyses: no pumps, solvents, etc

3. Capacity: ~2mm sample size; no format constraints

4. Universality: ???


Small Molecule MALDI Quantitation Has 4 Requirements

1. MS/MS

2. High repetition (firing) rate laser

3. Sample Cleanup

4. Internal Standard


MS/MS Is Critical For Distinguishing Signal

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300m/z

0.0

1.0e6

2.0e6

3.0e6

4.0e6

5.0e6

6.0e6

7.0e6

8.0e6

9.0e6

1.0e7

1.1e7

1.2e7

1.3e7

1.4e7

1.5e7

Inte

nsity, cp

s

378.8288.1189.8

172.9143.6

335.2171.5

294.2

156.1

316.3122.0

90.9107.1

360.8188.0130.9 278.1

524.1 568.0270.1238.0

115.9 304.2362.9

202.1 323.3220.9 438.8423.077.2 522.1 643.8 665.9141.1 512.1309.2 855.1757.1 887.0 962.9 1054.1

Q1 MS scan of -cyano matrix (“blank”)

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300m/z

0.0

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5.0e6

6.0e6

7.0e6

8.0e6

9.0e6

1.0e7

1.1e7

1.2e7

1.3e7

1.4e7

1.5e7

Inte

nsity, cp

s

378.8288.1189.8

172.9143.6

335.2171.5

294.2

156.1

316.3122.0

90.9107.1

360.8188.0130.9 278.1

524.1 568.0270.1238.0

115.9 304.2362.9

202.1 323.3220.9 438.8423.077.2 522.1 643.8 665.9141.1 512.1309.2 855.1757.1 887.0 962.9 1054.1

Q1 MS scan of -cyano matrix (“blank”)


Q1 MS 4ng Carbamazepine

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500m/z

2.9e7

Inten

sity, cps

316.0287.9

172.2115.9

270.4145.7 190.0

147.1 378.9317.4

304.4106.1 335.2244.289.1 122.0 298.4191.0 237.1 250.2

290.3 416.4264.1162.0 256.388.2 194.1 272.3117.9 313.2234.0223.0102.0 336.2156.1 251.1 278.2245.2 361.3 376.9207.069.9 299.4305.3266.2130.976.8 175.1 413.1 441.0 477.2197.0 363.3 400.994.8 328.264.8 232.2 394.9282.292.0 459.2114.7 185.0 465.1449.0384.1351.2181.0150.1

MH+ 237

40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260m/z

1.7e6

Inten

sity, cps

194.2

192.1

237.3

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0Time, min

0

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Intensity, cps

25pg Carbamazepine SRM 237/194Carbamazepine MS/MS 237


0.502 0.504 0.506 0.508 0.510 0.512 0.514 0.516 0.518 0.520

Time, min

~280Laser Shots

~200msec

“Peaks” Produced By Laser Drilling Through Sample

Laser:• JDSU Nanolaser• Solid state diode-

pumped• 355nm wavelength• 500psec pulse width• ~16uJ/pulse• 1,400Hz pulse rate


Energy Per Pulse vs. Laser Firing Rate At 355 nm

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laser firing rate (Hz)

energ

y p

er

puls

e a

t 3

55

nm

(uJ)


0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8Time, min

0

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100

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2.4042.4062.4082.4102.412 2.4142.4162.418 2.420 2.4222.424Time, min

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1.0e4

1.2e4

1.4e4

1.6e4

1.8e4

2.0e4

2.2e4

2.4e4

2.6e4

Laser rate = 10 Hz Laser rate = 1400 Hz

180 msec252 shots

24 sec240 shots

Desorption Time Is Linear With Laser Rate“band focusing” effect

Area = 1597 Area = 1625


“Good” crystals from clean samples

“Bad” crystals from raw samples

Raster Laser

Raster Laser

Direct Sampling Of Biological Matrices Does Not Produce Useful Crystals


Sample Cleanup By Simple SPE With MALDI Matrix In Eluent

Elute with 25uL MALDI matrix soln.

Cleanup biological samples with SPE

Pipette directly onto MALDI target

• SPE removes suppressing interferences• Eluent contains matrix and int. std.• High co-crystallization uniformity• Rapid and easy to automate


Aqueous MP

Organic MP

Waste

MassSpectrometer

Current HT Column-Switching LC/MS 3M/Tomtec SPExpress Card System

SPE-Type Sample Cleanup Is ComparableTo Contemporary HT Methodology


Data Collected By Rastering Laser Across Samples


0.70 0.71 0.72 0.73 0.74 0.75 0.76 0.77 0.78 0.79 0.80 0.81 0.82 0.83 0.84

Time, min

3.4e4

~1.5 mm~1.5 mm


“Peak” Integration for Quantitation

Each “peak” consists of ~10 separate measurementsEach measurement is an average of ~280 laser shots

0.70 0.71 0.72 0.73 0.74 0.75 0.76 0.77 0.78 0.79 0.80 0.81 0.82 0.83 0.84

Time, min

3.4e4


0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9Time, min

0.0

4.4e4

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9Time, min

0.0

8.9e5Internal Standard

Analyte

Example of Microsomal Incubate Timecourse

Blank

T=45 min

T=30 minT=15 min

T=5 min

T=0 min

0.25uM Std.


Internal Standard Means Never Having To Say You’re Sloppy


0.20 0.22 0.24 0.26 0.28 0.30 0.32 0.34 0.36 0.38 0.40 0.42

Time, min

3.3e4


0.70 0.71 0.72 0.73 0.74 0.75 0.76 0.77 0.78 0.79 0.80 0.81 0.82 0.83 0.84

Time, min

3.4e4

~1.5 mm~1.5 mm


Internal Standard is Necessary for Quantitation

0 5 10 15 20 25 30 35 40 45 50

time (min)

AnalyteInt. Std.Ratio

Compound 1 Microsomal Timecourse

0 5 10 15 20 25 30 35 40 45 50

time (min)

AnalyteInt. Std.Ratio

Compound 2 Microsomal Timecourse


Verapamil Std. Curve

y = 0.0033x - 0.0106R2 = 0.9788

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Concentration (ng/ml)

Analy

te/I

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Buspirone Std. Curve

y = 0.2461x - 0.0429R 2 = 0.9986

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Concentration (uM)

Analy

te/I

S


Human Microsome T1/2 ESI vs. MALDI

y = 1.0228x + 1.8996

R2 = 0.9372

0.2

20.2

40.2

60.2

80.2

100.2

120.2

140.2

0.2 20.2 40.2 60.2 80.2 100.2 120.2 140.2

MALDI T1/2 (min)

ESI T

1/2

(min

)


Other Biological Samples Analyzed

1. Hepatocyte Metabolic Stability

2. Caco-2 Absorption

3. MDR Pgp Transport

4. Serum - standard curves


The Inevitable Comparison:

MALDI vs. ESI For Small Molecule Analysis

• Universality

• Coverage

• Sensitivity

• Speed


MALDI vs ESI conditions

Generic system for high throughput LC/ESI/MS/MS• Standard API3000• Standardized mobile phases• Standardized columns• Use only a few template MS/MS conditions; define polarity

MRM and choose a collision energy (little optimization)

Generic system for high throughput MALDI/MS/MS• Modified API3000 with MALDI source -CHC used exclusively as MALDI matrix• Simple generic SPE cleanup used• Template MS/MS method taken from the ESI studies


Universality: Ionization Success

Experiment:

•208 compounds from compound collection•Representative of total chemical space•25 Std. Curves used to correlate 50nM signals with detection limits•MALDI success: S/N 5 and estimated detection limit <50nM

Failed by MALDI: 33/208 16%

Failed by ESI: 14/208 6.7%

Failed ESI, Good MALDI: 4

Weekly Percent Compound Failure By ESI

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Week # (2002)

% F

aile

d C

om

pound

s


0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9Time, min

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9Time, min

MALDI MS

ESI LC/MS

Sensitivity: Sample Consumption

Blank

T=45 min

T=30 minT=15 min

T=5 min

T=0 min

0.25uM Std.


MALDI MS

ESI LC/MS

Ionization/Ion Transfer Efficiency

25 uL injectedArea = 217,100

25 nL consumedArea = 26,970

2.2nL

MALDI: 100-fold increase in efficiency over ESI


0.77 0.78 0.79 0.80 0.81 0.82 0.83 0.84Time, min

0.77 0.78 0.79 0.80 0.81 0.82 0.83 0.84Time, min

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9Time, min

Speed: Effect On Precision

Multiple Measurementsvs.

Single Measurement


Averaged vs. Single(n=8) Measurements

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0 5 10 15 20 25 30 35 40 45 50

time (min)

are

a r

ati

o (

x1

00

00

)

4% CV

6% CV

8% CV

6% CV

10% CV

T1/2 = 30.7 min

C.V. = 7%


0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6

Time, min

0.0

5000.0

1.0e4

1.5e4

2.0e4

2.5e4

3.0e4

3.5e4

4.0e4

4.5e4

5.0e4

5.5e4

6.0e4

6.5e4

7.0e4

7.5e4

8.0e4

8.5e4

9.0e4

9.5e4

8ng Quinidine

10ng Clozapine

Capillary LC method and data collection by Dr. John Soglia

Inte

nsity,

cps


MALDI / MS/ MS LC/ ESI / MS/ MS

I on-suppression YES YES

Sample prep SPE ACN precipitation

Suffi cient Sensitivity f or HT ADME YES YES

Coverage GOOD EXCELLENT

Simple plumbing YES NO

Space constraints NO YES

Solvent use <50µL/ sample >1 mL/ sample

Current speed ~8 samples/ minute 3-4 samples/ minute

Potential speed 6-8 samples/ minute or

360 samples/ hr

1 sample/second or

3600 samples/hr


Potential Uses For MALDI Analyses(small molecule measurements)

1. Large numbers of samples (ADME screening)

2. Rapid just-in-time or real-time analyses (spot checking, quick analyses, designing larger studies)

3. Asynchronous LC-MS “write once, read many times”


Summary

• The instrument was capable of generating calibration curves for a variety of compounds. Suitable linearity and dynamic range was obtained to support typical ADME assays

• ADME data obtained using this prototype instrument was compared with data obtained using ESI/MS/MS, revealing a good correlation

• A calibration curve for verapamil in human serum was linear from 5-1000 ng/ml. Lower detection limits were not possible, due to severe ion-suppression

• Overall, the instrument shows promise as a tool for ADME screening, with possibilities for increased speed, perhaps plate-reader like speed


The future…

• “Autosampler” – not trivial, 3600 samples/hr• Faster motors• Software • Cheaper sample cleanup or more tolerant MALDI

methodology• Disposable plates/plates with sorbent• Negative ionization not addressed• Integration strategy to match analysis speed

with complementary use of ESI


Acknowledgements

• Mark Cole• John Janiszewski• Jay Corr• Peter Kovarik• Tom Covey• Nora Wallace• Sabrina Zhao• John Soglia

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