1. 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

2. 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

3. 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
- Off line sample prep and 2 min gradient
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

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

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
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

6. Orbitrap – Principle of Operationz 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,
For Research Use Only. Not for use in diagnostic procedures

7. For Research Use Only. Not for use in diagnostic procedures
Ethinyl-Estradiol at Different Mass Resolutions
Resolution: 10k, 30k, 50k, 100k
Butyl-Phthalate, (ubiquitous background ion)
100 90 80 70 60
Ethinyl-Estradiol, 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

8. For Research Use Only. Not for use in diagnostic procedures
100 ppb Hormone Sample (500 pg on-column)
100 ppb Hormone sample 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

9. 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

10. Exactive Method – Sample Preparation
Standards preparation
100 µL plasma 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
@ RPM
10 µL std 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

11. 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

12. 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

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

14. 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 50,000 Resolution
External mass calibration
Extract Ion Chromatogram for molecule of interest 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

15. 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

16. Linearity and Accuracy – 25OH-Vit D3
Vit D3 - Validation 1
Y = *X R^2 = 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 = *X R^2 = 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 = *X R^2 = 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

17. Linearity and Accuracy – 25OH-Vit D2
Vit D2 – Validation 1
Y = *X R^2 = 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 = *X R^2 = 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 = *X R^2 = 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

18. 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 of 5 ppm
For Research Use Only. Not for use in diagnostic procedures 18

19. 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

20. 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

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

22. 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

23. 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

24. 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

25. 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

26. 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

27. 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

28. 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

29. 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

30. 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

31. 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

32. 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

33. 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

34. 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

35. 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

36. 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

37. 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

38. 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

39. 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

40. 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 different batches
For Research Use Only. Not for use in diagnostic procedures

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

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

43. 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
For Research Use Only. Not for use in diagnostic procedures 43

44. 25-OH-Vit D - Autosampler method
For Research Use Only. Not for use in diagnostic procedures