1. 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 Q0Q1Q2Q3

2. August 8-11, 2005 Bristol, Rhode Island 0 500000 1000000 1500000 2000000 2500000 3000000 3500000 # Samples Analyzed 2000200120022003 Year HT ADME Sample History 2000-2003 0 10000 20000 30000 40000 50000 60000 70000 80000 # Samples/week

3. August 8-11, 2005 Bristol, Rhode Island 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: ???

4. August 8-11, 2005 Bristol, Rhode Island Small Molecule MALDI Quantitation Has 4 Requirements 1.MS/MS 2.High repetition (firing) rate laser 3.Sample Cleanup 4.Internal Standard

5. August 8-11, 2005 Bristol, Rhode Island MS/MS Is Critical For Distinguishing Signal

6. August 8-11, 2005 Bristol, Rhode Island 25pg Carbamazepine SRM 237/194Carbamazepine MS/MS 237

7. August 8-11, 2005 Bristol, Rhode Island 0.5020.5040.5060.5080.5100.5120.5140.5160.5180.520 Time, min ~280 Laser 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

8. August 8-11, 2005 Bristol, Rhode Island Energy Per Pulse vs. Laser Firing Rate At 355 nm 0 2 4 6 8 10 12 14 16 18 0500100015002000 laser firing rate (Hz) energy per pulse at 355 nm (uJ)

9. August 8-11, 2005 Bristol, Rhode Island 2.4042.4062.4082.4102.4122.4142.4162.4182.4202.4222.424 Time, min 0.0 2000 4000 6000 8000 1.0e4 1.2e4 1.4e4 1.6e4 1.8e4 2.0e4 2.2e4 2.4e4 2.6e4 Laser rate = 10 HzLaser rate = 1400 Hz 180 msec 252 shots 24 sec 240 shots Desorption Time Is Linear With Laser Rate “band focusing” effect Area = 1597 Area = 1625

10. August 8-11, 2005 Bristol, Rhode Island “Good” crystals from clean samples “Bad” crystals from raw samples Raster Laser Direct Sampling Of Biological Matrices Does Not Produce Useful Crystals

11. August 8-11, 2005 Bristol, Rhode Island 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

12. August 8-11, 2005 Bristol, Rhode Island Aqueous MP Organic MP Waste Mass Spectrometer Current HT Column-Switching LC/MS3M/Tomtec SPExpress Card System SPE-Type Sample Cleanup Is Comparable To Contemporary HT Methodology

13. August 8-11, 2005 Bristol, Rhode Island Data Collected By Rastering Laser Across Samples

14. August 8-11, 2005 Bristol, Rhode Island 0.700.710.720.730.740.750.760.770.780.790.800.810.820.830.84 Time, min 3.4e4 ~1.5 mm

15. August 8-11, 2005 Bristol, Rhode Island “Peak” Integration for Quantitation Each “peak” consists of ~10 separate measurements Each measurement is an average of ~280 laser shots 0.700.710.720.730.740.750.760.770.780.790.800.810.820.830.84 Time, min 3.4e4

16. August 8-11, 2005 Bristol, Rhode Island 0.1 0.20.3 0.4 0.5 0.60.70.8 0.9 Time, min 0.0 4.4e4 Internal Standard Analyte Example of Microsomal Incubate Timecourse Blank T=45 min T=30 min T=15 min T=5 min T=0 min 0.25uM Std.

17. August 8-11, 2005 Bristol, Rhode Island Internal Standard Means Never Having To Say You’re Sloppy

18. August 8-11, 2005 Bristol, Rhode Island 0.200.220.240.260.280.300.320.340.360.380.400.42 Time, min 3.3e4

19. August 8-11, 2005 Bristol, Rhode Island 0.700.710.720.730.740.750.760.770.780.790.800.810.820.830.84 Time, min 3.4e4 ~1.5 mm

20. August 8-11, 2005 Bristol, Rhode Island Internal Standard is Necessary for Quantitation 05101520253035404550 time (min) Analyte Int. Std. Ratio Compound 1 Microsomal Timecourse 05101520253035404550 time (min) Analyte Int. Std. Ratio Compound 2 Microsomal Timecourse

21. August 8-11, 2005 Bristol, Rhode Island Verapamil Std. Curve y = 0.0033x - 0.0106 R 2 = 0.9788 0 2 4 6 8 10 12 14 16 18 20 0500100015002000250030003500400045005000 Concentration (ng/ml) Analyte/IS Buspirone Std. Curve y = 0.2461x - 0.0429 R 2 = 0.9986 0 1 2 3 4 5 6 7 8 051015202530 Concentration (uM) Analyte/IS

22. August 8-11, 2005 Bristol, Rhode Island Human Microsome T 1/2 ESI vs. MALDI y = 1.0228x + 1.8996 R 2 = 0.9372 0.2 20.2 40.2 60.2 80.2 100.2 120.2 140.2 0.220.240.260.280.2100.2120.2140.2 MALDI T 1/2 (min) ESI T 1/2 (min)

23. August 8-11, 2005 Bristol, Rhode Island Other Biological Samples Analyzed 1.Hepatocyte Metabolic Stability 2.Caco-2 Absorption 3.MDR Pgp Transport 4.Serum - standard curves

24. August 8-11, 2005 Bristol, Rhode Island The Inevitable Comparison: MALDI vs. ESI For Small Molecule Analysis Universality Coverage Sensitivity Speed

25. August 8-11, 2005 Bristol, Rhode Island 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

26. August 8-11, 2005 Bristol, Rhode Island 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

27. August 8-11, 2005 Bristol, Rhode Island 0.10.20.30.40.50.60.70.80.91.01.11.21.31.41.51.61.71.81.9 Time, min 0.10.20.3 0.40.50.60.70.80.9 Time, min MALDI MS ESI LC/MS Sensitivity: Sample Consumption Blank T=45 min T=30 min T=15 min T=5 min T=0 min 0.25uM Std.

28. August 8-11, 2005 Bristol, Rhode Island MALDI MS ESI LC/MS Ionization/Ion Transfer Efficiency 25 uL injected Area = 217,100 25 nL consumed Area = 26,970 2.2nL MALDI: 100-fold increase in efficiency over ESI

29. August 8-11, 2005 Bristol, Rhode Island 0.770.780.790.800.810.820.830.84 Time, min 0.770.780.790.800.810.820.830.84 Time, min 0.10.20.3 0.40.50.60.70.80.9 Time, min Speed: Effect On Precision Multiple Measurements vs. Single Measurement

30. August 8-11, 2005 Bristol, Rhode Island

33. 0.20.40.60.81.01.21.41.61.82.02.22.42.6 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 Intensity, cps

34. August 8-11, 2005 Bristol, Rhode Island

35. 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”

36. August 8-11, 2005 Bristol, Rhode Island 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

37. August 8-11, 2005 Bristol, Rhode Island 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

38. August 8-11, 2005 Bristol, Rhode Island Acknowledgements Mark Cole John Janiszewski Jay Corr Peter Kovarik Tom Covey Nora Wallace Sabrina Zhao John Soglia