1. The world leader in serving science For Research Use Only. Not for use in diagnostic procedures Quantitative Analysis of 4 Immunosuppressant Drugs in Whole Blood Using Exactive System Clinical Research Applications Group Thermo Fisher Scientific San Jose CA

2. 2 For Research Use Only. Not for use in diagnostic procedures Presentation Overview  Goal  Introduction to Exactive  Sample Preparation and LC/MS Method for ISD Drugs  Linearity, LOQ and Results on QCs  Conclusions  Appendix ISD: Immunosuppressant Drugs

3. 3 For Research Use Only. Not for use in diagnostic procedures Goal  Evaluate Exactive for quantitation of Tacrolimus, Sirolimus, Everolimus and Cyclosporin A in whole blood

4. 4 For Research Use Only. Not for use in diagnostic procedures Exactive System Introduction

5. 5 For Research Use Only. Not for use in diagnostic procedures Exactive Benchtop LC-MS Resolution 100,000 at 1 scan per second 10,000 at 10 scans per second Mass accuracy Sub ppm Dynamic range >10,000 within a spectrum Scan speed Up to 10 scans per second Mass range m/z 50 - 4000 Polarity switching One positive and one negative scan < 1 second (25K Resolution)

6. 6 For Research Use Only. Not for use in diagnostic procedures Orbitrap – Principle of Operation z φ Hyper-logarithmic potential distribution: “ideal Kingdon trap” r Makarov A. Anal. Chem. 2000, 72, 1156-1162.  Characteristic frequencies: Frequency of rotation ω φ Frequency of radial oscillations ω r Frequency of axial oscillations ω z

7. 7 For Research Use Only. Not for use in diagnostic procedures Ethinyl-Estradiol at Different Mass Resolutions Resolution: 10k, 30k, 50k, 100k 279.12279.14279.16279.18279.20 m/z 0 10 20 30 40 50 60 70 80 90 100 Relative Abundance Ethinyl-Estradiol, 279.17434 Butyl-Phthalate, 279.15909 (ubiquitous background ion) Note:Ethinyl Estradiol used just to demonstrate the power of resolution

8. 8 For Research Use Only. Not for use in diagnostic procedures 100 ppb Hormone Sample (500 pg on-column) 100 ppb Hormone sample measured @ different resolution settings Ethinyl-Estradiol The isobaric phthalate background ion interferes with the Ethinyl-Estradiol ion. At resolution of 10,000 the steroid mass is shifted because the isobaric ions are not resolved. 171819 Time (min) 0 50 100 0 50 100 Relative Abundance 16.68 17.8118.0718.94 17.59 19.04 RP = 10,000 Phthalate Estradiol 171819 Time (min) 0 50 100 0 50 100 Relative Abundance 18.03 19.32 17.22 18.1716.40 18.76 17.73 RP = 100,000 Phthalate Estradiol Note:Ethinyl Estradiol used just to demonstrate the power of resolution

9. 9 For Research Use Only. Not for use in diagnostic procedures Sample Preparation and LC/MS Method for ISDs

10. 10 For Research Use Only. Not for use in diagnostic procedures Sample Preparation Vortex 50 uL blood + 150 uL of protein precipitation solution containing internal standards Centrifuge 10 min @ 13200 RPM Supernatant 50 µL inject Sample Preparation Shake for 30 min Note: Same sample prep procedure for standards, QCs and samples Internal Standards Add 1 volume of 0.2 M ZnSO4 2 volumes of 20ng/mL Ascomysin and 250 ng/mL of Cyclosporin D in Methanol Precipitation Solution Prep Acsomycin for Tacrolimus, Sirolimus and Everolimus Cyclosporin D for Cyclosporin A

11. 11 For Research Use Only. Not for use in diagnostic procedures Calibration Standards in Whole Blood Calibration Standards CalibratorTacrolimus (ng/mL)Sirolimus (ng/mL)Everolimus (ng/mL)Cyclosporin A (ng/mL) Cal 10.970.941.029.8 Cal 22.072.101.9526.4 Cal 35.115.215.1373.0 Cal 410.5710.0210.36208.8 Cal 528.2226.2828.17725.1 Cal 653.9249.9151.572067.2 QC Samples – Expected Concentration QC sampleTacrolimus (ng/mL)Sirolimus (mg/mL)Everolimus (ng/mL)Cyclosporin A (ng/mL) QC12.973.062.9331.0 QC213.6612.7413.58134.0 QC333.0630.6632.40386.8

12. 12 For Research Use Only. Not for use in diagnostic procedures  LC method 10 x 2.1 mm id packed with 5 µm, C18 guard column A: water containing 10 mM NH4FA and 0.1% formic acid B: methanol containing 10 mM NH4FA and 0.1% formic acid C: ACN/IPA/Acetone = 45/45/10 v/v/v Column Temp: 80 Deg C  Exactive MS method APCI source Full scan MS acquisition 800-1250 amu @ 50,000 Resolution External mass calibration Extract chromatogram for drugs of interest (m/z)@ better than 5ppm mass accuracy Confirm the identity using mass accuracy and RT Quantitate using peak area in the chromatogram Exactive – LC/MS Method Note: See appendix for details

13. 13 For Research Use Only. Not for use in diagnostic procedures Linearity, LOQ and Results on QCs

14. 14 For Research Use Only. Not for use in diagnostic procedures Linearity and Accuracy Standard% Difference 0.97 ng/mL1.04 2.07 ng/mL0.34 5.11 ng/mL-6.41 10.57 ng/mL-1.62 28.22 ng/mL2.77 53.92 ng/mL3.88 Standard% Difference 0.944.83 2.1-12.57 5.213.31 10.021.88 26.28-1.61 49.914.16 Excellent Linearity and Accuracy Tacrolimus Sirolimus

15. 15 For Research Use Only. Not for use in diagnostic procedures Excellent Linearity and Accuracy Everolimus Cyclosporin A Standard% Difference 1.0217.85 1.95-39.05 5.1312.53 10.36-0.81 28.17-1.06 51.5710.62 Standard% Difference 9.8-3.47 26.48.81 73.01.65 208.80.93 725.1-3.24 2067.2-4.69 Cyclosporin A Y = 0.0011175+0.00238964*X R^2 = 0.9968 W: 1/X^2 0200400600800100012001400160018002000 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 Area Ratio Note: The 1.95 ng/mL Evrolimus standard was excluded Linearity and Accuracy

16. 16 For Research Use Only. Not for use in diagnostic procedures Chromatograms@ Low and High Concentration Note:Chromatograms reconstructed with m/z accuracy of 5 ppm 0.97 ng/mL 53.9 ng/mL 0.94 ng/mL 49.9 ng/mL 1.02 ng/mL 51.6ng/mL 9.8 ng/mL 2067 ng/mL TacrolimusSirolimusEverolimus Cyclosporin A Excellent Specificity and Peak Shapes

17. 17 For Research Use Only. Not for use in diagnostic procedures QCs – Performance Statistics Excellent Accuracy and Precision Everolimus Cyclosporin A Tacrolimus Sirolimus

18. 18 For Research Use Only. Not for use in diagnostic procedures Conclusion  Exactive system operated in full scan MS @ 50,000 resolution Has high specificity for analysis of drugs in whole blood Has required linearity and LOQ Is capable of accurate quantitation of immunosuppressant drugs for research

19. 19 For Research Use Only. Not for use in diagnostic procedures Appendix

20. 20 For Research Use Only. Not for use in diagnostic procedures Ionization source parameters Exactive Mass Spectrometer IonizationAPCI, positive, Ion Max™ source, Discharge current 4.0  A Vaporizer temperature (deg C)300 Sheath gas (arb) 15 Ion Sweep Gas Pressure:0.0 Aux gas (arb) 17 Capillary temperature (deg C)275

21. 21 For Research Use Only. Not for use in diagnostic procedures Exactive: Instrument Method Polarity: Positive Microscans: 1 Resolution: High (50 000) Target: Balanced Max Inject time: 250 Scan range: 800-1250 Divert valve: switch events 3 0-0.6 min – to waste 0.6-1.1 – to detector 1.1-2.0 – to waste

22. 22 For Research Use Only. Not for use in diagnostic procedures LC method  LC Conditions CTC autosampler and Accela pump Mobile Phase A: water containing 10 mM NH4FA and 0.1% formic acid B: methanol containing 10 mM NH4FA and 0.1% formic acid C: ACN/IPA/Acetone = 45/45/10 v/v/v Column 10 x 2.1 mm id packed with 5 µm, C18 guard column Column temperature: 80 deg C Injection volume: 50 µL Gradient Time (min)%A%B%CFlow rate (  L/min) 070300800 0.2570300800 0.501000800 1.0901000800 1.1001001000 1.5001001000 1.5170300800 2.070300800

23. 23 For Research Use Only. Not for use in diagnostic procedures Autosampler method