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
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
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
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
5 Clinical Research use only, Not for use in Diagnostics Procedure
Exactive System Introduction
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)
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
Makarov A. Anal. Chem. 2000, 72, 1156-1162.
� Characteristic frequencies:• Frequency of rotation ωφ• Frequency of radial oscillations ω
r
• Frequency of axial oscillations ωz
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
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
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
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
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)
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
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
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
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
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
45
50
55
60
65
70
75
80
85
90
95
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
90
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
90
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
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lati
ve
In
ten
sit
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
90
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
60
65
70
75
80
85
90
95
100
Re
lati
ve In
ten
sit
y
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%
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
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
20 Clinical Research use only, Not for use in Diagnostics Procedure
Blinded Sample Results Comparison
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
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
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
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
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ve
Ab
un
da
nc
e
0
20
40
60
80
100
Re
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0
20
40
60
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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
25 Clinical Research use only, Not for use in Diagnostics Procedure
Appendix
25 OH Vit D Analysis
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.
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
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
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
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
31 Clinical Research use only, Not for use in Diagnostics Procedure
25-OH-Vit D - Autosampler method
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