For Research Use Only. Not for use in diagnostic procedures Quantitative Analysis of Vit D Metabolites in Human Plasma using Exactive System Clinical Research Applications Group Thermo Fisher Scientific San Jose CA For Research Use Only. Not for use in diagnostic procedures

Presentation Overview Goals Introduction to Exactive Sample preparation and LC/MS method for 25 OH-Vit D Calibration curves, LOQ and results on QCs Blinded sample results comparison Appendix QQQ: Triple Quadrupole MS/MS system For Research Use Only. Not for use in diagnostic procedures

For Research Use Only. Not for use in diagnostic procedures Goal Results comparison between Exactive and QQQ method for quantitation of 25-OH-Vit D2 and 25-OH-Vit D3 in human plasma Exactive@Thermo - Off line sample prep and 2 min gradient QQQ@Service lab - Protein precipitation followed by Turboflow and 6min gradient Evaluate Exactive system for analysis of 1, 25 di hydroxy Vit D3 For Research Use Only. Not for use in diagnostic procedures 3

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

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)   What’s different about this Orbitrap compared to the one in the LTQ Orbitrap? Orbitrap itself is very similar but its mounting and wiring have been made more compact. How do you get faster scan speed? Why is it faster than the LTQ Orbitrap? We operate orbitrap mass analyzer at higher voltage which results in higher frequencies. In parallel, we have reduced overheads on ion transfer (as there is no LTQ) and thus we need less time for a spectrum and less time for inter-spectrum delay. What’s the ultimate resolution? 100,000 at m/z 200 and 1 sec acquisition. What’s the dynamic range At least similar to that in LTQ Orbitrap, Full MS mode. Pumping system? There is a novel split-flow pump which allows to keep vacuum chamber smaller and make it from aluminium. Final vacuum is similar to that in a standard LTQ Orbitrap. C Trap is this the same as the current LTQ Orbitrap Yes, C-trap and ion optics are the same though their mounting is different (from a flange) HCD Scan – how does this work and – what’s new? Ions get accelerated through the C-trap into a quadrupolar collision cell where they fragment and get stored. After that, voltages are ramped and ions are transferred back to the C-trap from which they are injected into the orbitrap. There is no mass selection so only a narrow mass range of ions fragment with optimal sequence coverage, with lower m/z fragmenting excessively and higher m/z fragmenting only weakly. Is the mass accuracy the same at the LTQ Orbitrap?  (based on work in Bremen, it was better than the LTQ Orbitrap) Actually, we have a better layout and components of central electrode power supply so we expect mass accuracy at least as good as on LTQ Orbitrap XL. For Research Use Only. Not for use in diagnostic procedures 5

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

For Research Use Only. Not for use in diagnostic procedures Ethinyl-Estradiol at Different Mass Resolutions Resolution: 10k, 30k, 50k, 100k Butyl-Phthalate, 279.15909 (ubiquitous background ion) 100 90 80 70 60 Ethinyl-Estradiol, 279.17434 50 Relative Abundance 40 30 20 10 279.12 279.14 279.16 279.18 279.20 m/z Note:Ethinyl Estradiol used just to demonstrate the power of resolution For Research Use Only. Not for use in diagnostic procedures

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 17 18 19 Time (min) 50 100 Relative Abundance 18.03 19.32 17.22 18.17 16.40 18.76 17.73 RP = 100,000 Phthalate Estradiol The isobaric phthalate background ion interferes with the Ethinyl-Estradiol ion. At resolution of 10,000 the steroid mass is off because the isobaric ions are not resolved. 17 18 19 Time (min) 50 100 Relative Abundance 16.68 17.81 18.07 18.94 17.59 19.04 RP = 10,000 Phthalate Estradiol Note:Ethnyl Estradiol used just to demonstrate the power of resolution For Research Use Only. Not for use in diagnostic procedures

For Research Use Only. Not for use in diagnostic procedures Sample Preparation and LC/MS Method for 25 OH Vit D Analysis For Research Use Only. Not for use in diagnostic procedures

Exactive Method – Sample Preparation Standards preparation 100 µL plasma + 200 mL IS 50 ng/mL [2H6]-25OH-D3 100 µg/mL 25-OH-D3 and 25-OH-D2 mix in Ethanol Serial dilutions down to 100 ng/mL Vortex Centrifuge 10 min @ 13200 RPM 10 µL std + 990 uL of 50% Ethanol Supernatant Calibration standards 1 to 200 ng/mL 50 µL inject Note: Stock IS in ACN; Standards, QC and samples were processed using identical procedure For Research Use Only. Not for use in diagnostic procedures

Data Acceptance Criteria Current generally acceptable LC/MS precision Samples Concentration [ng/mL] Required Precision [%] Cal Standard 1 1 20 Cal Standard 2 2 15 Cal Standard 3 10 Cal Standard 4 Cal Standard 5 50 Cal Standard 6 100 Cal Standard 7 200 QC 1* 5 for Vit-D2 and 20 for Vit-D3* QC 2 QC 3 * QC1 spiked with 5 ng/mL of Vit-D3 and Vit-D2. Only total concentration was analyzed for QC1 sample. For Research Use Only. Not for use in diagnostic procedures

25OH-Vit D2 and D3 – Exactive Mass Spectrum D2 - H2O D3 - H2O D3 – (2 x H2O) D2 - (2 x H2O) D2 For Research Use Only. Not for use in diagnostic procedures

Exactive – 3 Selected M/Z Analyte Monitored m/z 25-OH-D3 401.3411 (M+H) 383.3306 (M - H2O + H) 365.3200 (M - 2 x H2O) + H) 25-OH-D2 413.3411 (M+H) 395.3305 (M - H2O + H) 377.3199 (M - 2 x H2O) + H [2H6] 25-OH-D3 (Internal Standard) 407.3787 (M+H) 389.3679 (M - H2O + H) 371.3576 (M - 2 x H2O) + H m/z with minimum interference For Research Use Only. Not for use in diagnostic procedures 13

For Research Use Only. Not for use in diagnostic procedures Exactive – LC/MS Method LC method Thermo Hypersil aQGold 50 x 2.1 mm, 5um (1, 25 Vit D3) Thermo Hypersil aQGold 50 x 2.1 mm, 5µm (25 OH Vit D) Mobile phase A: water containing 0.1% formic acid Mobile phase B: methanol containing 0.1% formic acid Column Temp: 50 Deg C Exactive MS method HESI source Full scan MS acquisition @ 50,000 Resolution External mass calibration Extract Ion Chromatogram for molecule of interest (m/z)@ better than 5ppm mass accuracy Confirm the identity using mass accuracy and RT Quantitate using peak area in the chromatogram Note: Details in appendix For Research Use Only. Not for use in diagnostic procedures

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

Linearity and Accuracy – 25OH-Vit D3 Vit D3 - Validation 1 Y = 0.00576964+0.0027313*X R^2 = 0.9918 W: 1/X^2 20 40 60 80 100 120 140 160 180 200 220 ng/mL 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 0.65 Area Ratio Vit D3 - Validation 2 Y = 0.0056991+0.00298291*X R^2 = 0.9954 W: 1/X^2 20 40 60 80 100 120 140 160 180 200 220 ng/mL 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 0.65 0.70 Area Ratio Vit D3 - Validation 3 Y = 0.00571591+0.00317597*X R^2 = 0.9946 W: 1/X^2 20 40 60 80 100 120 140 160 180 200 220 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 0.65 0.70 0.75 Area Ratio % Difference Standard Validation_1 Validation_2 Validation_3 1 ng/mL Precision just below set criteria 2 ng/mL 2.2 1.5 0.8 10 ng/mL -10.5 -9.6 -6.5 20 ng/mL -0.02 4.9 7.8 50 ng/mL -7.5 -2.4 -7.9 100 ng/mL 2.9 -2.3 -1.7 200 ng/mL 13.0 7.9 7.6 Note: Calibration curve using neat standards For Research Use Only. Not for use in diagnostic procedures

Linearity and Accuracy – 25OH-Vit D2 Vit D2 – Validation 1 Y = 0.00432057+0.0119399*X R^2 = 0.9919 W: 1/X^2 20 40 60 80 100 120 140 160 180 200 220 ng/mL 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 2.8 Area Ratio Vit D2 – Validation 2 Y = 0.0010089+0.0121633*X R^2 = 0.9943 W: 1/X^2 20 40 60 80 100 120 140 160 180 200 220 ng/mL 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 2.8 Area Ratio Vit D2 – Validation 3 Y = -0.000622327+0.0121577*X R^2 = 0.9996 W: 1/X^2 20 40 60 80 100 120 140 160 180 200 220 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 Area Ratio % Difference Standard Validation_1 Validation_2 Validation_3 1 ng/mL 5.4 -4.4 0.6 2 ng/mL -10.6 9.7 -0.7 10 ng/mL -4.3 -2.9 20 ng/mL 7.5 2.1 2.0 50 ng/mL -8.6 -5.9 -1.4 100 ng/mL 0.8 -5.3 0.3 200 ng/mL 8.2 Note: Calibration curve using neat standards For Research Use Only. Not for use in diagnostic procedures

Lower Points in Calibration Curve – Good Specificity LOQ D3 -1ng/mL D3 -2ng/mL D3 -10ng/mL %RSD>20% LOQ D2 -10ng/mL D2 -1ng/mL D2 -2ng/mL Note:Extracted Ion Chromatogram (XIC)@accuracy of 5 ppm For Research Use Only. Not for use in diagnostic procedures 18

QC Results - 25OH-Vit D3 Inter and Intra Assay Excellent accuracy, precision and recovery QC1 (22 ng/mL) Replicate Val 1 Val 2 Val 3 1 19.6 21.5 20.3 2 21.3 19.9 20.1 3 19.0 20.9 4 22.1 22.6 20.4 5 23.1 19.3 Mean 20.7 21.6 20.0 %RSD 6.5 5.8 2.2 %Rec ND QC2 (50 ng/mL) Replicate Val 1 Val 2 Val 3 1 47.4 50.2 48.2 2 53.1 49.7 54.9 3 55.8 47.7 4 53.5 53.9 49.1 5 49.6 54.7 47.3 Mean 51.9 52.7 49.4 %RSD 6.4 4.7 6.3 %Rec 104 105 98.9 QC3 (200 ng/mL) Replicate Val 1 Val 2 Val 3 1 209 215 204 2 224 3 218 212 201 4 200 5 220 213 207 Mean 205 %RSD 3.8 2.3 1.5 %Rsc 106 108 102 For Research Use Only. Not for use in diagnostic procedures

QC Results - 25OH-Vit D2 Inter and Intra Assay Excellent accuracy, precision and recovery QC1 (5 ng/mL) Replicate Val1 Val 2 Val 3 1 5.0 4.4 4.3 2 4.8 4.1 4.7 3 4.2 5.1 3.9 4 5.3 5 4.9 Mean 4.6 SD 0.37 0.40 0.28 %Rec 96.9 93.0 87.4 QC2 (50 ng/mL) Replicate Val 1 Val 2 Val 3 1 47.3 45.6 49.7 2 51.1 48.5 48.1 3 48.9 51.6 4 54.2 45.0 49.1 5 52.2 54.0 48.0 Mean 50.4 48.4 49.3 %RSD 6.0 7.3 3.0 %Rec 101 96.8 98.6 QC3 (200 ng/mL) Replicate Val 1 Val 2 Val 3 1 189 197 230 2 191 209 3 186 184 4 177 188 199 5 187 190 Mean 193 202 %RSD 3.4 5.0 8.9 %Rec 93.9 96.7 101 For Research Use Only. Not for use in diagnostic procedures

For Research Use Only. Not for use in diagnostic procedures Blinded Sample Results Comparison For Research Use Only. Not for use in diagnostic procedures

Blinded Sample - Results Comparison Seven blinded samples from service lab analyzed Comparison with service labs 6 min TurboFlowTM QQQ method 5 replicate analysis on Exactive to asses precision For Research Use Only. Not for use in diagnostic procedures

Blinded Samples – Results Summary Good agreement between Exactive and service lab QQQ results Service lab ng/mL Note: External service lab used Access QQQ For Research Use Only. Not for use in diagnostic procedures

Chromatograms - Blinded Sample A Exactive 39.6 ng/mL Vitamin D3 Access 33.5 ng/mL Note: 5 injections on Exactive and 3 injections on Access For Research Use Only. Not for use in diagnostic procedures

Chromatograms - Blinded Sample A Exactive <LOQ Vitamin D2 Access <LOQ See appendix for other 6 samples Note: 5 injections on Exactive and 3 injections on Access For Research Use Only. Not for use in diagnostic procedures

For Research Use Only. Not for use in diagnostic procedures Appendix 25 OH Vit D Analysis For Research Use Only. Not for use in diagnostic procedures

Reproducibility - Blinded Sample B Exactive 34.0 ng/mL Vitamin D3 Access 34.8 ng/mL Note: 5 injections on Exactive and 4 injections on Access For Research Use Only. Not for use in diagnostic procedures

Reproducibility - Blinded Sample C Exactive 14.6 ng/mL Vitamin D3 Access 16.6 ng/mL Note: 5 injections on Exactive and 4 injections on Access For Research Use Only. Not for use in diagnostic procedures

Reproducibility - Blinded Sample D Exactive 19.7 ng/mL Vitamin D3 Access 20.4 ng/mL Note: 5 injections on Exactive and 4 injections on Access For Research Use Only. Not for use in diagnostic procedures

Reproducibility - Blinded Sample E Exactive 32.3 ng/mL Vitamin D3 Access 36.3 ng/mL Note: 5 injections on Exactive and 4 injections on Access For Research Use Only. Not for use in diagnostic procedures

Reproducibility - Blinded Sample F Exactive 25.5 ng/mL Vitamin D3 Access 25.8 ng/mL Note: 5 injections on Exactive and 4 injections on Access For Research Use Only. Not for use in diagnostic procedures

Reproducibility - Blinded Sample G Exactive 34.2 ng/mL Vitamin D3 Access 34.0 ng/mL Note: 5 injections on Exactive and 4 injections on Access For Research Use Only. Not for use in diagnostic procedures

Reproducibility - Blinded Sample B Exactive <LOQ Vitamin D2 Access <LOQ Note: 5 injections on Exactive and 4 injections on Access For Research Use Only. Not for use in diagnostic procedures

Reproducibility - Blinded Sample C Exactive <LOQ Vitamin D2 Access <LOQ Note: 5 injections on Exactive and 4 injections on Access For Research Use Only. Not for use in diagnostic procedures

Reproducibility - Blinded Sample D Exactive <LOQ Vitamin D2 Access <LOQ Note: 5 injections on Exactive and 4 injections on Access For Research Use Only. Not for use in diagnostic procedures

Reproducibility - Blinded Sample E Exactive <LOQ Access <LOQ Vitamin D2 Note: 5 injections on Exactive and 4 injections on Access For Research Use Only. Not for use in diagnostic procedures

Reproducibility - Blinded Sample F Exactive <LOQ Vitamin D2 Access <LOQ Note: 5 injections on Exactive and 4 injections on Access For Research Use Only. Not for use in diagnostic procedures

Reproducibility - Blinded Sample G Exactive <LOQ Vitamin D2 Access <LOQ Note: 5 injections on Exactive and 4 injections on Access For Research Use Only. Not for use in diagnostic procedures

For Research Use Only. Not for use in diagnostic procedures Reagents 25-OH-D3 was purchased from Cerilliant, 25-OH-D2 was purchased from Sigma. Both compounds were stored in -20 deg.C. [2H6]-25-OH-D3 (Internal Standard) was purchased from Medical Isotopes and it was stored in -70 deg.C. For Research Use Only. Not for use in diagnostic procedures 39

Method validation Confirm method accuracy Total quantity in QC sample Endogenous quantity in blank plasma measured using IS alone Actual spiked amount in QC sample minus = 25-OH-D3 = 16.5 ng/mL 25-OH-D2 = 0.8 ng/mL Intra assay variability - 5 replicates of each QC sample Inter assay variability - 5 replicates of each QC sample in 3 different batches For Research Use Only. Not for use in diagnostic procedures

25-OH-Vit D - Ionization source parameters Exactive Mass Spectrometer Ionization APCI, positive, Ion Max™ source, Discharge current 5.0 Vaporizer temperature (deg C) 390 Sheath gas 15 Ion Sweep Gas Pressure: 0.0 Aux gas 5 Capillary temperature (deg C) 275 For Research Use Only. Not for use in diagnostic procedures 41

25-OH-Vit D - Exactive Method Polarity: Positive Microscans: 1 Resolution: Ultra High (100 000) Target: Balanced Max Inject time: 250 Scan range: 350-420 Divert valve: switch events 4 1-0.95 min – to waste 0.95-1.4 – to detector 1.4-1.9 – to waste 1.9-2.0 – to detector For Research Use Only. Not for use in diagnostic procedures 42

For Research Use Only. Not for use in diagnostic procedures 25-OH-Vit D - LC method LC Conditions Surveyor autosampler and pump Mobile Phase A: water containing 0.1% formic acid B: methanol containing 0.1% formic acid Column 50 x 2.1 mm id packed with 3 µm, C18-aQ stationary phase Injection volume: 50 µL Flow rate: 700 µL/min Gradient Time (min) % A %B 0 20 80 0.1 20 80 0.2 5 95 1.4 5 95 1.5 20 80 2.0 20 80 For Research Use Only. Not for use in diagnostic procedures 43

25-OH-Vit D - Autosampler method For Research Use Only. Not for use in diagnostic procedures