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Q0. Q1. Q2. Q3. 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. HT ADME Sample History 2000-2003. 80000. 3500000. 70000. 60000. 3000000. 50000.

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500000

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  1. Q0 Q1 Q2 Q3 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 August 8-11, 2005 Bristol, Rhode Island

  2. HT ADME Sample History 2000-2003 80000 3500000 70000 60000 3000000 50000 2500000 40000 # Samples/week 2000000 # Samples Analyzed 30000 1500000 20000 1000000 10000 500000 0 0 2000 2001 2002 2003 Year August 8-11, 2005 Bristol, Rhode Island

  3. MALDI Potential For HT Quantitation Speed: potential for <1sec/sample Simple analyses: no pumps, solvents, etc Capacity: ~2mm sample size; no format constraints Universality: ??? August 8-11, 2005 Bristol, Rhode Island

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

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

  6. Q1 MS 4ng Carbamazepine 316.0 287.9 2.9e7 MH+ 237 172.2 115.9 270.4 145.7 190.0 147.1 378.9 317.4 304.4 106.1 335.2 244.2 Intensity, cps 89.1 122.0 298.4 191.0 237.1 250.2 290.3 416.4 264.1 162.0 256.3 88.2 194.1 272.3 117.9 313.2 234.0 223.0 102.0 156.1 336.2 251.1 278.2 245.2 361.3 376.9 207.0 69.9 299.4 305.3 266.2 130.9 76.8 175.1 413.1 441.0 477.2 197.0 363.3 400.9 64.8 94.8 328.2 232.2 394.9 282.2 92.0 459.2 114.7 185.0 465.1 449.0 384.1 351.2 181.0 150.1 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 m/z 5500 194.2 1.7e6 5000 4500 4000 3500 3000 Intensity, cps 2500 Intensity, cps 2000 192.1 1500 1000 237.3 500 0 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 m/z Time, min Carbamazepine MS/MS 237 25pg Carbamazepine SRM 237/194 August 8-11, 2005 Bristol, Rhode Island

  7. ~200msec 0.502 0.504 0.506 0.508 0.510 0.512 0.514 0.516 0.518 0.520 Time, min “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 ~280 Laser Shots August 8-11, 2005 Bristol, Rhode Island

  8. 18 16 14 12 energy per pulse at 355 nm (uJ) 10 8 6 4 2 0 0 500 1000 1500 2000 laser firing rate (Hz) Energy Per Pulse vs. Laser Firing Rate At 355 nm August 8-11, 2005 Bristol, Rhode Island

  9. 650 600 550 2.6e4 500 2.4e4 450 2.2e4 400 2.0e4 350 1.8e4 300 1.6e4 250 1.4e4 200 1.2e4 150 1.0e4 100 8000 50 6000 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 Time, min 4000 2000 0.0 2.404 2.406 2.408 2.410 2.412 2.414 2.416 2.418 2.420 2.422 2.424 Time, min Desorption Time Is Linear With Laser Rate “band focusing” effect Laser rate = 10 Hz Laser rate = 1400 Hz Area = 1597 Area = 1625 24 sec 240 shots 180 msec 252 shots August 8-11, 2005 Bristol, Rhode Island

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

  11. Cleanup biological samples with SPE Elute with 25uL MALDI matrix soln. Pipette directly onto MALDI target Sample Cleanup By Simple SPE With MALDI Matrix In Eluent • SPE removes suppressing interferences • Eluent contains matrix and int. std. • High co-crystallization uniformity • Rapid and easy to automate August 8-11, 2005 Bristol, Rhode Island

  12. Aqueous MP Organic MP Mass Spectrometer Waste SPE-Type Sample Cleanup Is Comparable To Contemporary HT Methodology Current HT Column-Switching LC/MS 3M/Tomtec SPExpress Card System August 8-11, 2005 Bristol, Rhode Island

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

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

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

  16. 4.4e4 0.0 0.2 0.3 0.1 0.4 0.6 0.7 0.8 0.5 0.9 Time, min 8.9e5 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 Time, min Example of Microsomal Incubate Timecourse 0.25uM Std. Analyte T=0 min T=5 min T=15 min T=30 min T=45 min Blank Internal Standard August 8-11, 2005 Bristol, Rhode Island

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

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

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

  20. Compound 1 Microsomal Timecourse Compound 2 Microsomal Timecourse Analyte Analyte Int. Std. Int. Std. Ratio Ratio 0 5 10 15 20 25 30 35 40 45 50 0 5 10 15 20 25 30 35 40 45 50 time (min) time (min) Internal Standard is Necessary for Quantitation August 8-11, 2005 Bristol, Rhode Island

  21. Verapamil Std. Curve Buspirone Std. Curve 20 8 y = 0.2461x - 0.0429 18 y = 0.0033x - 0.0106 2 = 0.9986 R 7 2 = 0.9788 R 16 6 14 5 12 Analyte/IS Analyte/IS 10 4 8 3 6 2 4 1 2 0 0 0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 0 5 10 15 20 25 30 Concentration (ng/ml) Concentration (uM) August 8-11, 2005 Bristol, Rhode Island

  22. y = 1.0228x + 1.8996 2 R = 0.9372 Human Microsome T1/2 ESI vs. MALDI 140.2 120.2 100.2 ESI T1/2 (min) 80.2 60.2 40.2 20.2 0.2 0.2 20.2 40.2 60.2 80.2 100.2 120.2 140.2 MALDI T1/2 (min) August 8-11, 2005 Bristol, Rhode Island

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

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

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

  26. Weekly Percent Compound Failure By ESI 14.0 12.0 10.0 % Failed Compounds 8.0 6.0 4.0 2.0 0.0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 Week # (2002) 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 August 8-11, 2005 Bristol, Rhode Island

  27. 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 Time, min 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 1.9 Time, min Sensitivity: Sample Consumption 0.25uM Std. MALDI MS T=0 min T=5 min T=15 min T=30 min T=45 min Blank ESI LC/MS August 8-11, 2005 Bristol, Rhode Island

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

  29. 0.77 0.78 0.79 0.80 0.81 0.82 0.83 0.84 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 Time, min Time, min 0.77 0.78 0.79 0.80 0.81 0.82 0.83 0.84 Time, min Speed: Effect On Precision Multiple Measurements vs. Single Measurement August 8-11, 2005 Bristol, Rhode Island

  30. Averaged vs. Single(n=8) Measurements 5000 T1/2 = 30.7 min C.V. = 7% 4% CV 4500 6% CV 4000 8% CV 3500 3000 area ratio (x10000) 6% CV 2500 10% CV 2000 1500 1000 500 0 0 5 10 15 20 25 30 35 40 45 50 time (min) August 8-11, 2005 Bristol, Rhode Island

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

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

  33. 8ng Quinidine 9.5e4 Capillary LC method and data collection by Dr. John Soglia 9.0e4 8.5e4 8.0e4 7.5e4 7.0e4 6.5e4 Intensity, cps 6.0e4 5.5e4 5.0e4 4.5e4 4.0e4 3.5e4 3.0e4 2.5e4 2.0e4 10ng Clozapine 1.5e4 1.0e4 5000.0 0.0 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 August 8-11, 2005 Bristol, Rhode Island

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

  35. Potential Uses For MALDI Analyses(small molecule measurements) Large numbers of samples (ADME screening) Rapid just-in-time or real-time analyses (spot checking, quick analyses, designing larger studies) Asynchronous LC-MS “write once, read many times” August 8-11, 2005 Bristol, Rhode Island

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

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

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

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