Quantitative Analysis of Vit D Metabolites in Human Plasma ... quantitation of 25-OH-Vit D2 and...
Transcript of Quantitative Analysis of Vit D Metabolites in Human Plasma ... quantitation of 25-OH-Vit D2 and...
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Clinical Research use only, Not for use in Diagnostics Procedure
Quantitative Analysis of Vit D Metabolites in
Human Plasma using Exactive System
Marta Kozak
Clinical Research Applications Group
Thermo Fisher Scientific San Jose CA
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2 Clinical Research use only, Not for use in Diagnostics Procedure
Calibrators and Controls
• Six calibrators at 2, 5 10, 25, 50 and100ng/mL in BSA
• Three QCs at 5ng/mL, 40 ng/mL and 80 ng/mL in BSA
• Cals and QC’s prepared by fortifying bovine serum albumin diluent with 200 ng/mL 25-hydryoxyvitamin D2 and D3 standard mix.
• Precipitating reagent was prepared by adding deuterated D6-25-
hydroxyvitamin D3 to acetonitrile for a final concentration of 75ng/mL.
• Pooled donor serum samples were crashed 2 to 1 with Acetonitrile and spiked with analytes for a final concentration of 20 ng/mL for 25-hydroxyvitamin D2
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3 Clinical Research use only, Not for use in Diagnostics Procedure
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
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4 Clinical Research use only, Not for use in Diagnostics Procedure
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
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5 Clinical Research use only, Not for use in Diagnostics Procedure
Exactive System Introduction
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6 Clinical Research use only, Not for use in Diagnostics Procedure
Exactive Benchtop LC-MS
• Resolution100,000 at 1 scan per second 10,000 at 10 scans per second
• Mass accuracySub ppm
•Dynamic range>10,000 within a spectrum
• Scan speedUp to 10 scans per second
• Mass rangem/z 50 - 4000
• Polarity switchingOne positive and one negative scan < 1 second (25K Resolution)
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7 Clinical Research use only, Not for use in Diagnostics Procedure
Orbitrap – Principle of Operation
{ })/ln(2/2
),(222
mm RrRrzk
zrU ⋅+−⋅=
z
φ
Hyper-logarithmic potential distribution: “ideal Kingdon trap”
r
12
2
−
=
R
Rmzωωϕ 2
2
−
=
R
Rmzr ωω
qm
kz
/=ω
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� Characteristic frequencies:• Frequency of rotation ωφ• Frequency of radial oscillations ω
r
• Frequency of axial oscillations ωz
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8 Clinical Research use only, Not for use in Diagnostics Procedure
Ethinyl-Estradiol at Different Mass Resolutions
Resolution: 10k, 30k, 50k, 100k
279.12 279.14 279.16 279.18 279.20
m/z
0
10
20
30
40
50
60
70
80
90
100
Re
lative A
bundance
Ethinyl-Estradiol, 279.17434
Butyl-Phthalate, 279.15909 (ubiquitous background ion)
Note:Ethinyl Estradiol used just to demonstrate the power of resolution
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9 Clinical Research use only, Not for use in Diagnostics Procedure
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 off because
the isobaric ions are
not resolved.
17 18 19Time (min)
0
50
100
0
50
100
Rela
tive A
bu
nd
ance
16.68 17.81 18.07 18.9417.5919.04
RP = 10,000Phthalate
Estradiol
17 18 19Time (min)
0
50
100
0
50
100
Rela
tive A
bu
nd
ance
18.03 19.3217.22 18.1716.40 18.76
17.73 RP = 100,000Phthalate
Estradiol
Note:Ethnyl Estradiol used just to demonstrate the power of resolution
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10 Clinical Research use only, Not for use in Diagnostics Procedure
Exactive method – Sample preparation
Vortex
100 µL plasma + 200 µL IS 50 ng/mL [2H6]-25OH-D3
Centrifuge 10 min
@ 13200 RPM
Supernatant
50 µL inject
Sample preparation
Note: Stock IS in ACN; Standards, QC and samples were processed using identical procedure
10 µL std + 990 uL of
50% Ethanol
Serial dilutions down
to 100 ng/mL
100 µg/mL 25-OH-D3
and 25-OH-D2 mix in Ethanol
Calibration standards
1 to 200 ng/mL
Standards preparation
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11 Clinical Research use only, Not for use in Diagnostics Procedure
Data acceptance criteria
15200Cal Standard 7
15100Cal Standard 6
1550Cal Standard 5
1520Cal Standard 4
1510Cal Standard 3
152Cal Standard 2
201Cal Standard 1
15200QC 3
1550QC 2
205 for Vit-D2 and 20 for Vit-D3*QC 1*
Required Precision [%]Concentration [ng/mL]Samples
* QC1 spiked with 5 ng/mL of Vit-D3 and Vit-D2. Only total concentration was analyzed for QC1 sample.
Current generally acceptable LC/MS precision
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12 Clinical Research use only, Not for use in Diagnostics Procedure
25OH-Vit D2 and D3 – Exactive mass spectrum
D2
D2 - H2O
D2 - (2 x H2O)
D3 - H2O
D3 – (2 x H2O)
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13 Clinical Research use only, Not for use in Diagnostics Procedure
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
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14 Clinical Research use only, Not for use in Diagnostics Procedure
• 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
Exactive – LC/MS Method
Note: Details in appendix
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15 Clinical Research use only, Not for use in Diagnostics Procedure
Linearity and Accuracy – 25OH-Vit D3
% Difference
200 ng/mL
100 ng/mL
50 ng/mL
20 ng/mL
10 ng/mL
2 ng/mL
1 ng/mL
Standard
7.67.913.0
-1.7-2.32.9
-7.9-2.4-7.5
7.84.9-0.02
-6.5-9.6-10.5
0.81.52.2
Precision just below set criteria
Validation_3Validation_2Validation_1
Note: Calibration curve using neat standards
Vit D3 - Validation1
Y = 0.00576964+0.0027313*X R^2 = 0.9918 W: 1/X^2
0 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
Are
a R
atio
Vit D3 - Validation 2
Y = 0.0056991+0.00298291*X R^2 = 0.9954 W: 1/X^2
0 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
Are
a R
atio
Vit D3 - Validation 3
Y = 0.00571591+0.00317597*X R^2 = 0.9946 W: 1/X^2
0 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
Are
a R
atio
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16 Clinical Research use only, Not for use in Diagnostics Procedure
% Difference
200 ng/mL
100 ng/mL
50 ng/mL
20 ng/mL
10 ng/mL
2 ng/mL
1 ng/mL
Standard
2.18.29.7
0.3-5.30.8
-1.4-5.9-8.6
2.02.17.5
-2.9-4.4-4.3
-0.79.7-10.6
0.6-4.45.4
Validation_3Validation_2Validation_1
Vit D2 – Validation 1
Y = 0.00432057+0.0119399*X R^2 = 0.9919 W: 1/X^2
0 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
Are
a R
atio
Vit D2 – Validation 2
Y = 0.0010089+0.0121633*X R^2 = 0.9943 W: 1/X^2
0 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
Are
a R
atio
Vit D2 – Validation 3
Y = -0.000622327+0.0121577*X R^2 = 0.9996 W: 1/X^2
0 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
Are
a R
atio
Note: Calibration curve using neat standards
Linearity and Accuracy – 25OH-Vit D2
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17 Clinical Research use only, Not for use in Diagnostics Procedure
Lower points in calibration curve – Good ppecificity
1ng_std - m/z= 365.32-365.32 SM: 5 RT: 0.75 - 1.50 NL: 4.36E4F: {0,0} + p APCI Full ms [ 350.00-420.00]
0.8 0.9 1.0 1.1 1.2 1.3 1.4
Time (min)
0
5
10
15
20
25
30
35
40
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50
55
60
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70
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100
Re
lati
ve
In
ten
sit
y
RT: 1.12
1.20
1.470.970.77 0.87
Note:Extracted Ion Chromatogram (XIC)@accuracy of 5 ppm
2ng_std - m/z= 365.32-365.32 SM: 5 RT: 0.76 - 1.51 NL: 5.39E4F: {0,0} + p APCI Full ms [ 350.00-420.00]
0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5
Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
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95
100
Re
lati
ve
In
ten
sit
y
RT: 1.13
1.321.441.250.78 0.960.880.83
10ng_std - m/z= 365.32-365.32 SM: 5 RT: 0.76 - 1.51 NL: 1.49E5F: {0,0} + p APCI Full ms [ 350.00-420.00]
0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5
Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
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95
100
Re
lati
ve
In
ten
sit
y
RT: 1.13
1.25 1.411.310.81 1.480.980.89 1.03
1ng_std - m/z= 395.33-395.33 SM: 5 RT: 0.79 - 1.54 NL: 7.47E4F: {0,0} + p APCI Full ms [ 350.00-420.00]
0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5
Time (min)
0
5
10
15
20
25
30
35
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Re
lati
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In
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y
RT: 1.16
1.29
1.000.86 0.92
2ng_std - m/z= 395.33-395.33 SM: 5 RT: 0.78 - 1.53 NL: 1.37E5F: {0,0} + p APCI Full ms [ 350.00-420.00]
0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5
Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
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95
100
Re
lativ
e In
ten
sit
y
RT: 1.16
1.411.31 1.351.050.91 0.990.83
10ng_std - m/z= 395.33-395.33 SM: 5 RT: 0.80 - 1.55 NL: 5.80E5F: {0,0} + p APCI Full ms [ 350.00-420.00]
0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5
Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
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100
Re
lati
ve In
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RT: 1.17
1.370.990.86
D3 -1ng/mL D3 -10ng/mLD3 -2ng/mL
D2 -1ng/mLD2 -10ng/mL
D2 -2ng/mLLOQ
LOQ
%RSD>20%
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18 Clinical Research use only, Not for use in Diagnostics Procedure
QC Results - 25OH-Vit D3 inter and intra assay
2.25.86.5%RSD
%Rec
Mean
5
4
3
2
1
Replicate
QC1 (22 ng/mL)
19.323.121.5
20.021.620.7
NDNDND
20.422.622.1
19.920.919.0
20.119.921.3
20.321.519.6
Val 3Val 2Val 1
%Rec
%RSD
Mean
5
4
3
2
1
Replicate
QC2 (50 ng/mL)
49.452.751.9
47.354.749.6
49.153.953.5
47.754.955.8
Val 3Val 2Val 1
98.9105104
6.34.76.4
54.949.753.1
48.250.247.4
%Rsc
%RSD
Mean
5
4
3
2
1
Replicate
QC3 (200 ng/mL)
205215212
207213220
204212200
201212218
Val 3Val 2Val 1
102108106
1.52.33.8
209224215
204215209
Excellent accuracy, precision and recovery
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19 Clinical Research use only, Not for use in Diagnostics Procedure
%Rec
SD
Mean
5
4
3
2
1
Replicate
QC1 (5 ng/mL)
4.44.94.9
4.44.64.8
0.280.400.37
87.493.096.9
4.44.85.3
3.95.14.2
4.74.14.8
4.34.45.0
Val 3Val 2Val1
%Rec
%RSD
Mean
5
4
3
2
1
Replicate
QC2 (50 ng/mL)
49.348.450.4
48.054.052.2
49.145.054.2
51.648.947.3
Val 3Val 2Val 1
98.696.8101
3.07.36.0
48.148.551.1
49.745.647.3
%Rec
%RSD
Mean
5
4
3
2
1
Replicate
QC3 (200 ng/mL)
202193188
190187191
199188177
184186191
Val 3Val 2Val 1
10196.793.9
8.95.03.4
209209191
230197189
Excellent accuracy, precision and recovery
QC Results - 25OH-Vit D2 inter and intra assay
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20 Clinical Research use only, Not for use in Diagnostics Procedure
Blinded Sample Results Comparison
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21 Clinical Research use only, Not for use in Diagnostics Procedure
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
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22 Clinical Research use only, Not for use in Diagnostics Procedure
Blinded samples – Results summary
Good agreement betweenExactive and service lab QQQ results
Note: External service lab used Access QQQ
Vitamin D2Vitamin D3
2.4
2.0
2.3
<1.9
7.2
2.1
<1.9
Clinical Lab (ng/mL)
0.6
-1.2
-12.4
3.5
13.7
2.3
-15.4
% Diff
ND<134.034.2G
ND<125.825.5F
ND<136.332.3E
ND<120.419.7D
67.44.316.614.6C
ND<134.834.0B
ND<133.539.6A
% DiffExactive(ng/mL)
Clinical Lab (ng/mL)
Exactive(ng/mL)
Sample#
Vitamin D2Vitamin D3
2.4
2.0
2.3
<1.9
7.2
2.1
<1.9
Clinical Lab (ng/mL)
0.6
-1.2
-12.4
3.5
13.7
2.3
-15.4
% Diff
ND<134.034.2G
ND<125.825.5F
ND<136.332.3E
ND<120.419.7D
67.44.316.614.6C
ND<134.834.0B
ND<133.539.6A
% DiffExactive(ng/mL)
Clinical Lab (ng/mL)
Exactive(ng/mL)
Sample#
Service lab
ng/mL
Service lab
ng/mL
![Page 23: Quantitative Analysis of Vit D Metabolites in Human Plasma ... quantitation of 25-OH-Vit D2 and 25-OH-Vit D3 in human plasma • Exactive@Thermo - Off line sample prep and 2 min gradient](https://reader034.fdocuments.in/reader034/viewer/2022050718/5e16fd3a28cbb144061fb54d/html5/thumbnails/23.jpg)
23 Clinical Research use only, Not for use in Diagnostics Procedure
Chromatograms - Blinded sample A
c:\xcalibur\...\rawfiles\sample_a5 7/13/2009 4:47:50 PM
RT: 0.00 - 2.02 SM: 5B
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0
Time (min)
0
50
100
0
50
100
0
50
100
Re
lati
ve
Ab
un
da
nc
e
0
50
100
0
50
100
RT: 1.14AA: 2580930
RT: 0.09AA: 10925
RT: 0.37AA: 21678 0.93 1.01 1.991.44 1.491.38 1.57 1.680.78 1.83 1.870.21 0.49 0.720.650.61
RT: 1.14MA: 2040088
1.350.93 1.300.90 1.05 1.911.45 1.540.140.08 1.780.64 0.72 1.720.23 0.36 0.50
RT: 1.15MA: 2178738
1.33 1.370.94 1.950.89 1.480.410.14 1.671.590.35 1.75 1.790.820.22 0.630.45 0.58 0.690.03
RT: 1.15MA: 2205493
0.91 1.361.291.03 1.951.911.51 1.681.63 1.730.05 0.490.20 0.760.27 0.330.09 0.43 0.680.59
RT: 1.14MA: 1964455
0.90 1.05 1.33 1.900.86 1.49 1.821.65 1.690.08 0.14 0.19 0.35 0.55 0.770.59 1.600.48
NL: 5.21E5
m/z= 365.3182-365.3218 F: FTMS {0,0} + p APCI Full ms [350.00-420.00] MS ICIS Sample_A1
NL: 4.45E5
m/z= 365.3182-365.3218 F: FTMS {0,0} + p APCI Full ms [350.00-420.00] MS sample_a2
NL: 4.57E5
m/z= 365.3182-365.3218 F: FTMS {0,0} + p APCI Full ms [350.00-420.00] MS sample_a3
NL: 4.72E5
m/z= 365.3182-365.3218 F: FTMS {0,0} + p APCI Full ms [350.00-420.00] MS sample_a4
NL: 4.33E5
m/z= 365.3182-365.3218 F: FTMS {0,0} + p APCI Full ms [350.00-420.00] MS sample_a5
Exactive39.6 ng/mL
c:\xcalibur\...\rawfiles\sample_a_003 7/14/2009 3:31:06 PM
RT: 0.00 - 2.00 SM: 5B
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0
Time (min)
0
20
40
60
80
100R
ela
tiv
e A
bu
nd
an
ce
0
20
40
60
80
100
Re
lati
ve
Ab
un
da
nc
e
0
20
40
60
80
100
Re
lati
ve
Ab
un
da
nc
e
RT: 0.84MA: 1076719
0.961.050.74
1.19 1.28 1.46 1.741.67 1.860.09 0.24 0.47 0.540.37
RT: 0.84MA: 1093612
0.970.751.07
1.13 1.18 1.45 1.75 1.801.640.17 1.930.11 0.26 0.54 0.630.38
RT: 0.84MA: 844399
0.970.74 1.071.14 1.19 1.46 1.570.21 1.841.770.39 1.960.11 0.47 0.55
NL: 3.87E5
TIC F: + c APCI SRM ms2 383.280 [257.103-257.153, 365.075-365.125] MS Sample_A_001
NL: 3.70E5
TIC F: + c APCI SRM ms2 383.280 [257.103-257.153, 365.075-365.125] MS sample_a_002
NL: 2.84E5
TIC F: + c APCI SRM ms2 383.280 [257.103-257.153, 365.075-365.125] MS sample_a_003
Access33.5 ng/mL
Vitamin D3
Note: 5 injections on Exactive and 3 injections on Access
![Page 24: Quantitative Analysis of Vit D Metabolites in Human Plasma ... quantitation of 25-OH-Vit D2 and 25-OH-Vit D3 in human plasma • Exactive@Thermo - Off line sample prep and 2 min gradient](https://reader034.fdocuments.in/reader034/viewer/2022050718/5e16fd3a28cbb144061fb54d/html5/thumbnails/24.jpg)
24 Clinical Research use only, Not for use in Diagnostics Procedure
Chromatograms - Blinded sample A
Vitamin D2
c:\xcalibur\...\rawfiles\sample_a5 7/13/2009 4:47:50 PM
RT: 0.00 - 2.02 SM: 5B
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0
Time (min)
0
50
100
0
50
100
0
50
100
Re
lati
ve
Ab
un
da
nc
e
0
50
100
0
50
1001.25
0.90
1.46 1.52 1.671.421.82 1.89
0.03 0.34 0.670.230.15
1.19
1.151.98
1.94
1.631.46 1.841.760.12 0.680.33 0.430.26 0.56
1.17
0.901.290.87
1.48 1.52 1.671.42 1.76 1.890.550.08 0.710.64 0.760.320.28 0.49
1.98
0.930.88 1.161.94
1.131.551.52 1.63 1.891.760.590.13 0.19 0.750.30 0.490.43
1.97
1.920.87
1.63 1.831.570.52 0.71 0.740.24 0.590.280.180.13
NL: 5.32E3
m/z= 395.3285-395.3325 F: FTMS {0,0} + p APCI Full ms [350.00-420.00] MS Sample_A1
NL: 2.14E4
m/z= 395.3285-395.3325 F: FTMS {0,0} + p APCI Full ms [350.00-420.00] MS sample_a2
NL: 3.29E4
m/z= 395.3285-395.3325 F: FTMS {0,0} + p APCI Full ms [350.00-420.00] MS sample_a3
NL: 3.53E4
m/z= 395.3285-395.3325 F: FTMS {0,0} + p APCI Full ms [350.00-420.00] MS sample_a4
NL: 1.74E4
m/z= 395.3285-395.3325 F: FTMS {0,0} + p APCI Full ms [350.00-420.00] MS sample_a5
C:\Xcalibur\...\Rawfiles\Sample_A_001 7/14/2009 3:25:28 PM
RT: 0.00 - 2.00 SM: 5B
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0
Time (min)
0
20
40
60
80
100
Re
lati
ve
Ab
un
da
nc
e
0
20
40
60
80
100
Re
lati
ve
Ab
un
da
nc
e
0
20
40
60
80
100
Re
lati
ve
Ab
un
da
nc
e
0.79
0.84
0.951.00
1.18
1.451.24 1.620.03 1.67 1.84 1.940.460.25 0.620.30
0.81
0.76
0.85
0.94
1.011.06
1.15
1.461.26 1.650.20 1.800.570.02 0.39 1.96
0.79
0.84
0.93
1.011.03
1.15
1.25 1.46 1.59 1.66 1.890.07 0.660.560.26 0.420.15
NL: 8.68E4
TIC F: + c APCI SRM ms2 395.290 [269.093-269.143, 377.175-377.225] MS Sample_A_001
NL: 8.68E4
TIC F: + c APCI SRM ms2 395.290 [269.093-269.143, 377.175-377.225] MS sample_a_002
NL: 7.09E4
TIC F: + c APCI SRM ms2 395.290 [269.093-269.143, 377.175-377.225] MS sample_a_003
Exactive<LOQ
Access<LOQ
Note: 5 injections on Exactive and 3 injections on Access
See appendix for other 6 samples
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25 Clinical Research use only, Not for use in Diagnostics Procedure
Appendix
25 OH Vit D Analysis
![Page 26: Quantitative Analysis of Vit D Metabolites in Human Plasma ... quantitation of 25-OH-Vit D2 and 25-OH-Vit D3 in human plasma • Exactive@Thermo - Off line sample prep and 2 min gradient](https://reader034.fdocuments.in/reader034/viewer/2022050718/5e16fd3a28cbb144061fb54d/html5/thumbnails/26.jpg)
26 Clinical Research use only, Not for use in Diagnostics Procedure
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.
![Page 27: Quantitative Analysis of Vit D Metabolites in Human Plasma ... quantitation of 25-OH-Vit D2 and 25-OH-Vit D3 in human plasma • Exactive@Thermo - Off line sample prep and 2 min gradient](https://reader034.fdocuments.in/reader034/viewer/2022050718/5e16fd3a28cbb144061fb54d/html5/thumbnails/27.jpg)
27 Clinical Research use only, Not for use in Diagnostics Procedure
Method validation
Endogenous quantity in
blank plasma measured using IS alone
25-OH-D3 = 16.5 ng/mL25-OH-D2 = 0.8 ng/mL
Total quantity in
QC sample minus =
Intra assay variability - 5 replicates of each QC sampleInter assay variability - 5 replicates of each QC sample in 3
different batches
Confirm method accuracy
Actual spiked
amount in QC sample
![Page 28: Quantitative Analysis of Vit D Metabolites in Human Plasma ... quantitation of 25-OH-Vit D2 and 25-OH-Vit D3 in human plasma • Exactive@Thermo - Off line sample prep and 2 min gradient](https://reader034.fdocuments.in/reader034/viewer/2022050718/5e16fd3a28cbb144061fb54d/html5/thumbnails/28.jpg)
28 Clinical Research use only, Not for use in Diagnostics Procedure
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
![Page 29: Quantitative Analysis of Vit D Metabolites in Human Plasma ... quantitation of 25-OH-Vit D2 and 25-OH-Vit D3 in human plasma • Exactive@Thermo - Off line sample prep and 2 min gradient](https://reader034.fdocuments.in/reader034/viewer/2022050718/5e16fd3a28cbb144061fb54d/html5/thumbnails/29.jpg)
29 Clinical Research use only, Not for use in Diagnostics Procedure
25-OH-Vit D - Exactive Method
Polarity: Positive
Microscans: 1Resolution: Ultra High (100 000)Target: BalancedMax Inject time: 250 Scan range: 350-420
Divert valve: switch events 4
1-0.95 min – to waste0.95-1.4 – to detector1.4-1.9 – to waste1.9-2.0 – to detector
![Page 30: Quantitative Analysis of Vit D Metabolites in Human Plasma ... quantitation of 25-OH-Vit D2 and 25-OH-Vit D3 in human plasma • Exactive@Thermo - Off line sample prep and 2 min gradient](https://reader034.fdocuments.in/reader034/viewer/2022050718/5e16fd3a28cbb144061fb54d/html5/thumbnails/30.jpg)
30 Clinical Research use only, Not for use in Diagnostics Procedure
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
• GradientTime (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
![Page 31: Quantitative Analysis of Vit D Metabolites in Human Plasma ... quantitation of 25-OH-Vit D2 and 25-OH-Vit D3 in human plasma • Exactive@Thermo - Off line sample prep and 2 min gradient](https://reader034.fdocuments.in/reader034/viewer/2022050718/5e16fd3a28cbb144061fb54d/html5/thumbnails/31.jpg)
31 Clinical Research use only, Not for use in Diagnostics Procedure
25-OH-Vit D - Autosampler method