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

August 8-11, 2005 Bristol, Rhode Island # Samples Analyzed Year HT ADME Sample History # Samples/week

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: ???

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

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

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

August 8-11, 2005 Bristol, Rhode Island 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

August 8-11, 2005 Bristol, Rhode Island Energy Per Pulse vs. Laser Firing Rate At 355 nm laser firing rate (Hz) energy per pulse at 355 nm (uJ)

August 8-11, 2005 Bristol, Rhode Island Time, min e4 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

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

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

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

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

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

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 Time, min 3.4e4

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

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

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

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

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

August 8-11, 2005 Bristol, Rhode Island Verapamil Std. Curve y = x R 2 = Concentration (ng/ml) Analyte/IS Buspirone Std. Curve y = x R 2 = Concentration (uM) Analyte/IS

August 8-11, 2005 Bristol, Rhode Island Human Microsome T 1/2 ESI vs. MALDI y = x R 2 = MALDI T 1/2 (min) ESI T 1/2 (min)

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

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

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

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/ % Failed ESI, Good MALDI: 4

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

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

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

August 8-11, 2005 Bristol, Rhode Island

Time, min e4 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

August 8-11, 2005 Bristol, Rhode Island

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”

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