1. Molecular imaging and metabolite profiling of tissues sections by FTICR-MS MALDI and LESA C. Logan MackayRichard Goodwin SIRCAMSDMPK School of ChemistryR&D University of EdinburghAstraZeneca EdinburghSödertälje UKSweden

2. AimMolecular imaging - unlabelled ProblemMetabolites missing SolutionLESA profiling MS Imaging - Small molecules Summary

3. Raclopride (a dopamine D2 receptor antagonist) C 15 H 20 Cl 2 N 2 O 3 [M+H]m/z 347.0924 Organs:Brain Liver KidneyWhole body Dose:7.52 mg/kg Time:1530 m.a.d Matrix:α-Cyano-4-hydroxycinnamic acid Application:solvent free dry matrix “Qualitative and quantitative MALDI imaging of the positron emission tomography ligands raclopride (a D2 dopamine antagonist) and SCH 23390 (a D1 dopamine antagonist) in rat brain tissue sections using a solvent-free dry matrix application method.” Anal. Chem. 15;83(24):9694-9701 Goodwin RJ, Mackay CL, Nilsson A, Harrison DJ, Farde L, Andren PE, Iverson SL (2011) Ultraflex eXtreme Ultraflex IISynapt G2 12T FT-ICR-MS LESA TOF/TOF TOF/TOF Liquid extraction surface analysis

4. Synapt MS/MS Synapt MS Raclopride 2 mg/kg 1 min post i.v MALDI MS imaging analysis of neurological positron emission tomography (PET) ligands: application to both established and developmental compounds Tue: 2.20pm

5. 1 minute after dose 1 mg/kg BrainLiverHeartStomachSpinal cord FT-ICR MS 350 µm Quantitative Whole Body Autoradiography (QWBA) 14 C MS imaging

6. 1 5 30 min Vehicle 100 % Raclopride 2 mg/kg 1 min post i.v 30 min post i.v Raclopride 2 mg/kg Quantitative Whole Body Autoradiography (QWBA) 10.9 ± 0.6 nmol/g 1.7 ± 0.1 nmol/g Relative abundance 15% after 30 Cortex 12.9 ± 0.3 nmol/g Cortex 5.4 ± 0.2 nmol/g Relative abundance 40% after 30 14 C

7. m/z 347 (Raclopride) 1 m.a.d.30 m.a.d. 2mg/kg100% (5.1 a.u)22.4% (1.1 a.u) 7.5mg/kg100% (16.1 a.u)21.1% (3.4 a.u) MS Imaging Conclusion 1.Linear response between dose and detection by MSI 2.In brain - correlation between MSI relative abundance and autoradiography abundance but not in kidney and liver 100 % Raclopride 2 mg/kg 10.9 ± 0.6 nmol/g 1.7 ± 0.1 nmol/g Relative abundance 15% after 30 1 min post i.v 30 min post i.v

8. Plasma 30 m.a.d. M4 M1M2M3 Raclopride M1 m/z 381 M2 m/z 523 M3 m/z 409 M4 m/z 333 Raclopride m/z 347 30 minute after dose 2 mg/kg Unable to detect any metabolites by MALDI MSI

9. Plasma 30 m.a.d. M4 M1M2M3 Raclopride M1 m/z 381 M2 m/z 523 M3 m/z 409 M4 m/z 333 Raclopride m/z 347 30 minute after dose 2 mg/kg Unable to detect any metabolites by MALDI MSI LESA Liquid Extraction Surface Analysis

10. 700 nL MeOH/H 2 O (0.1 formic) Quad isolation (m/z 400 ± 175)

11. 30 minute after dose Raclopride 2 mg/kg Contribution to QWBA abundance Theoretical Measured

12. m/z 381.0111m/z 381.0794 m/z 381.0518 Contribution to QWBA abundance Theoretical Measured Theoretical isotope distribution Measured isotope distribution

13. 30 minute after dose Raclopride 2 mg/kg Theoretical Measured

14. Theoretical isotope distribution Measured isotope distribution m/z 526.1246 m/z 526.1245 Theoretical Measured

15. 30 minute after dose Raclopride 2 mg/kg S.D 7% S.D 56% N = 7

16. QWBA30 minute after dose Raclopride 2 mg/kg Dissected kidney 30 minute after dose raclopride 2 mg/kg MSI m/z 347

17. Whole body Tissue specific Relative distribution Absolute Quant. Label available MALDI MS imaging ESI LESA profiling QWBA Time scale & Scope of analysis Study Summary Fast Cheap Effective

18. Acknowledgments University of Edinburgh (SIRCAMS) Pat Langridge-Smith- Oral - Tue 9:10 (Room 118-120) David Clarke- Oral – Wed 9:30 (Exhibit hall A) Andrew Dennison- Oral – Tue 3:30 (Ballroom CD) Uppsala University Per Andren Anna Nilsson- Oral – Tue 2:50 (Ballroom B) AstraZenecaAdvion Suzanne IversonMark Allen (UK) Goran EklundMark Baumert (UK) Daniel Borg