1. Plasma Free Metanephrines Analysis using LC-MS/MS with Porous Graphitic Carbon Column Xiang He (Kevin) and Marta Kozak Thermo Fisher Scientific

2. 2 Presentation Overview Method summary Sample preparation protocol Linearity study Ion suppression Patient sample analysis Epinephrine separation Accuracy and precision study Carry-over study Conclusions Appendix

3. 3 Method Summary SPE based single step sample preparation HPLC using Hypercarb fast and stable graphite carbon column TSQ Vantage MS system Method validation using charcoal stripped serum and human plasma LOQ of 10 pg/mL Linearity 3.90- 500 and 7.81 -1000 to 7.81 pg/mL for MN and NMN, respectively Detailed validation using mixing study Run time 10 minutes

4. 4 Sample Preparation Precondition with 0.1% PFHA Load 500ul plasma Wash with 0.1% PFHA Elute with 60% Acetonitrile HyperSep C18 Dry under N 2 Recon with 200ul 2% Formic acid Inject 40ul into LC-MS/MS Note: SPE details in appendix

5. 5 Linearity Study Design Spiked charcoal striped serum MN = 500 pg/mL NMN =1000 pg/mL A serial 2-fold dilution with charcoal stripped serum Eight levels of linearity samples MN concentrations = 500 to 3.90 pg/mL NMN = 1000 to 7.81 pg/mL All levels analyzed in triplicate with one set of calibrators (also 8 levels). The calibration curve constructed by plotting the analyte/IS area ratio vs. analyte concentration Note: SPE details in appendix

6. 6 Linearity Results Deming regression. Excellent Linearity in the measured range

7. 7 Metanephrines – Quantitation Performance LOQ = 7.8 pg/mL Dynamic range = 3.9 – 500 pg/mL MN at 3.9 pg/mL MN d3 at 400 pg/mLCal curve 3.9 – 500 pg/mL

8. 8 Nor Metanephrines – Quantitation Performance LOQ = 15.6 pg/mL Dynamic range = 15.6 – 1000 pg/mL NMN at 15.6 pg/mL NMN d3 at 400 pg/mLCal curve 7.8 – 1000 pg/mL

9. 9 Ion suppression Post column infusion of MN-d3 and NMN-d3 at 100 pg/mL No ion suppression with pooled plasma Note: Details on ion suppression study using mixed samples in appendix

10. 10 20 12 29 25 31 15 Chromatograms- Patient Samples 104 68 63 85 93 46 Good S/N for all samples Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Patient 6 MN (pg/mL) NMN (pg/mL)

11. 11 IS signal stability Stable IS signal in all samples Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Patient 6

12. 12 Isobaric Epinephrine Separation NMN-d3 EPI-d3 No Epinephrine interference

13. 13 Accuracy and Precision Study Design CSS spiked with MN at 25 pg/mL Note: SPE details in appendix CSS spiked with MN at 250 pg/mL CSS spiked with NMN at 50 pg/mL CSS spiked with NMN at 500 pg/mL Five measurements in a batch Repeat over three days Inter and Intra Assay Accuracy and Precision Workflow

14. 14 Accuracy and Precision Results Inter assay CSS Intra assay Plasma Inter and Intra Assay Accuracy ~ 100% Precision better than 10%

15. 15 Accuracy and Precision Study Design Inject CSS spiked with MN at 500 and NMN at 1000 pg/mL Inject CSS spiked with MN at 7.8 and NMN at 15.6 pg/mL Inject CSS spiked with MN at 500 and NMN at 1000 pg/mL Inject CSS spiked with MN at 7.8 and NMN at 15.6 pg/mL Measure experimental value (Low 1) Measure experimental value (Low 2) Inject CSS spiked with MN at 500 and NMN at 1000 pg/mL Inject CSS spiked with MN at 7.8 and NMN at 15.6 pg/mL Measure experimental value (Low 3)

16. 16 Carry-over Study Results Excellent Accuracy and Precision in three measurements

17. 17 Linearity test Linearity: Charcoal striped serum was spiked with MN and NMN to achieve a final concentration of 500 pg/mL and 1000 pg/mL, respectively. A serial 2-fold dilution with charcoal stripped serum was performed to make 8 levels of linearity samples with concentration ranges of 500 to 3.90 pg/mL and 1000 to 7.81 pg/mL for MN and NMN respectively. Linearity samples were analyzed in triplicates along with one set of calibrators (also 8 levels). The calibration curve was constructed by plotting the analyte:IS area ratio vs. analyte concentration.

18. 18 Conclusion Easy sample prep: SPE, dry, shoot. No matrix effect for MN. Insignificant ion suppression for NMN, corrected by I.S. Hypercarb LC: fast equilibrium, fast run time, full resolution from EPI, great peak shape, stable performance. LOW, Intra- and inter-assay, LOQ, dynamic range, carry over, interference: all validated with single transition and TIC (two SRMs). Overall, a very robust, fast and sensitive method for Pmets quantitation. It is expected that this method will work on Ultra with similar LOQs.

19. 19 Linearity (serial dilution in CSS) 2/3/2011 LOQ Deming regression.

20. 20 Appendix

21. 21 LC-MS/MS setup LC column: Hypercarb 50 × 2.1 mm, 5  m, 70  C

22. 22 LC-MS/MS setup TSQ Vantage EMR

23. 23 Ion Suppression Test Post-column infusion: a constant flow (5 µL/min) of MN-d3 or NMN-d3 (both at 100 ng/mL) was infused post-column into the mobile phase using a T junction while ion-pairing SPE extracted pooled human plasma samples without internal standards and mobile phase buffer (blank) were injected. SRMs of both MN-d3 and NMN-d3 were monitored for the entire LC gradient. The obtained SRM chromatograms were visually inspected for obvious ion suppression or enhancement.

24. 24 Mixing study test Purpose: To decide which blank matrix to use to prepare calibrators To examine matrix effect from real patient plasma samples Also to check sample prep consistency Design: Spiked charcoal stripped serum samples Spiked with 100 and 200 pg/mL of MN and NMN 4 replicate SPE sample preps Measure concentration and take average Six patient plasma samples P1, P2,…P6 Mixed samples, M1, M2, …M6 (half of patient sample + half of spiked charcoal stripped serum sample Process using SPE and measure concentration Calibration curve prepared in charcoal stripped serum Ca1, Cal 2…Cal8 Passing criteria for mixing test: 0.8 <= 2*M/(A+P) <= 1.2

25. 25 Mixing Study Design

26. 26 Mixing results

27. 27 Ion Suppression – CSS vs pooled plasma Minimum ion suppression

28. 28 Things evolve