testosterone using q exactive - orbitrap • centrifuge at 4200 rpm for 5 min • place tubes in the...
TRANSCRIPT
Clinical Research use only, Not for use Diagnostics Procedure
Quantitative analysis of Testosterone in
plasma using Q Exactive
Kevin He and Marta Kozak
Clinical Research Applications Group
Thermo Fisher Scientific San Jose, CA
2 Clinical Research use only, Not for use Diagnostics Procedure
Instrumentation
• LC system: Accela 600 pump
• Q Exactive equipped with APCI + Ion Max TM source
3 Clinical Research use only, Not for use Diagnostics Procedure
Calibration Curve Standards Preparation
• Calibration standards were prepared in house in charcoal stripped serum (8-14) and in water (1-7).
SampleTestosterone
(pg/mL)
Testosterone-d3
(pg/mL)
Testo On
Column
(pg)
1 0 100 0
2 10 100 1
3 20 100 2
4 50 100 5
5 100 100 10
6 250 100 25
7 500 100 50
8 0 100 0
9 10 100 1
10 20 100 2
11 50 100 5
12 100 100 10
13 250 100 25
14 500 100 50
1-7: water 8-14: CSS
4 Clinical Research use only, Not for use Diagnostics Procedure
Modified Sample Preparation Procedure
• Place 200 uL of plasma sample into 16x100 mm borosilicate glass tubes
• To 200 uL plasma add 10 uL of 2 ng/mL working I.S. in 50:50 MeOH:Di water, and 10 uL testosterone
(various concentrations in MeOH/MeCN)
• Vortex briefly
• Add 1 mL of MTBE
• Cover with aluminum foil
• Vortex-mix for 90 sec
• Let stand for 5 min at room temperature
• Centrifuge at 4200 rpm for 5 min
• Place tubes in the -30 deg C freezer for 30 min
• Pour the upper layer into the 2nd labeled 16x100 mm glass tube – make sure that the maximum volume is
recovered
• Dry down the tubes in the Turbo-vap for 20 min at 37 deg C
• Reconstitute with 100 uL of 50:50 MeOH:DI water
• Vortex for 60 sec
• Injection volume: 50 µL
5 Clinical Research use only, Not for use Diagnostics Procedure
LC Method
• Analytical Column: Hypersil GOLD aQ 100 x 2.1 mm, 5µm
• Mobile Phase
� A: 5% MeOH in DI water
� B: MeOH
� C: ACN/IPA/Acetone: 45/45/10
• Autosampler wash solution #1: Water
• Autosampler wash solution #2: MeOH
• LC Gradient
Time(min) %A %B %C Flow (µµµµL/min)
0 95 5 0 400
0.1 60 40 0 400
0.5 60 40 0 400
3.5 5 95 0 400
4.5 5 95 0 400
4.51 0 0 100 600
5 0 0 100 600
5.1 95 5 0 600
6.5 95 5 0 600
6 Clinical Research use only, Not for use Diagnostics Procedure
Autosampler method
7 Clinical Research use only, Not for use Diagnostics Procedure
MS Method: source parameters
Q Exactive systemIonization APCI, positive, Ion Max™ source
Vaporizer temperature (deg C) 350
Capillary temperature (deg C) 320
Discharge current (µA) 5.0
Sheath gas (units) 10
Aux gas (units) 20
Probe position C
Divert valve: initial position to waste, at 3 min position to detector and at 5 min position switched back to waste
8 Clinical Research use only, Not for use Diagnostics Procedure
MS acquisition method
• Acquisition mode: HCD fragmentation with collision energy set to 40 eV
• Precursor isolation width: 2 mu
• Resolution: 70K @ 200 m/z
• AGC Target: 1e5
• Max injection time: 100ms
9 Clinical Research use only, Not for use Diagnostics Procedure
Acquisition method parameters: screen capture
10 Clinical Research use only, Not for use Diagnostics Procedure
Testosterone processing parameters (97 m/z ion)
97 m/z ion
from 289.2 m/z
parent ion.
97 m/z ion
from 289.2 m/z
parent ion.
Mass window:
3 ppm
Mass window:
3 ppm
11 Clinical Research use only, Not for use Diagnostics Procedure
Testosterone processing parameters (109 m/z ion)
109 m/z ion
from 289.2 m/z
parent ion.
109 m/z ion
from 289.2 m/z
parent ion.
12 Clinical Research use only, Not for use Diagnostics Procedure
Testosterone processing parameters (both ions)
Both ions from
289.2 m/z
parent ion.
Both ions from
289.2 m/z
parent ion.
Ion ratio applied.Ion ratio applied.
13 Clinical Research use only, Not for use Diagnostics Procedure
Testosterone-d3 processing parameters (both ions)
Both ions from
292.2 m/z
parent ion.
Both ions from
292.2 m/z
parent ion.
Ion ratio applied.Ion ratio applied.
14 Clinical Research use only, Not for use Diagnostics Procedure
Testo-97Y = 0.0404358+0.00884573*X R^2 = 0.9933 W: 1/X
0 50 100 150 200 250 300 350 400 450 500 550
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
Are
a R
atio
Calibration curve in CSS (97 m/z ion)
Standard % Difference
10 pg/mL 8.76
20 pg/mL 2.63
50 pg/mL -8.94
100 pg/mL 2.89
250 pg/mL -10.76
500 pg/mL 5.41
15 Clinical Research use only, Not for use Diagnostics Procedure
Testo-109Y = 0.0970157+0.00839124*X R^2 = 0.9980 W: 1/X
0 50 100 150 200 250 300 350 400 450 500 550
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
3.0
3.2
3.4
3.6
3.8
4.0
4.2
4.4
4.6
4.8
Are
a R
atio
Calibration curve in CSS (109 m/z ion)
Standard % Difference
10 pg/mL 0.97
20 pg/mL 7.45
50 pg/mL -5.78
100 pg/mL -0.29
250 pg/mL -5.35
500 pg/mL 2.99
16 Clinical Research use only, Not for use Diagnostics Procedure
TestoY = 0.0568976+0.0087523*X R^2 = 0.9935 W: 1/X
0 50 100 150 200 250 300 350 400 450 500 550
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
Are
a R
atio
Calibration curve in CSS (both ions)
Standard % Difference
10 pg/mL 11.27
20 pg/mL -5.50
50 pg/mL -3.41
100 pg/mL 3.57
250 pg/mL -11.09
500 pg/mL 5.17
17 Clinical Research use only, Not for use Diagnostics Procedure
97 m/z fragment ion (CSS, left: Testo; right: Testo-d3)
RT: 3.70 - 4.50 SM: 5B
3.8 4.0 4.2 4.4
Time (min)
0
50
100
0
50
100
0
50
100
0
50
100
0
50
100
0
50
100
0
50
100
RT: 4.15AA: 855806SN: 861
RT: 4.12AA: 392960SN: 451
RT: 4.05AA: 156654SN: 281
RT: 4.05AA: 82432SN: 148
RT: 4.06AA: 32309SN: 85
RT: 4.10AA: 21209SN: 74
RT: 4.13AA: 5093SN: 29
NL: 1.25E5
m/z= 97.0649-97.0659 F: FTMS + p APCI corona Full ms2 [email protected] [50.00-315.00] MS ICIS 14
NL: 5.21E4
m/z= 97.0649-97.0659 F: FTMS + p APCI corona Full ms2 [email protected] [50.00-315.00] MS ICIS 13
NL: 2.54E4
m/z= 97.0649-97.0659 F: FTMS + p APCI corona Full ms2 [email protected] [50.00-315.00] MS ICIS 12
NL: 1.26E4
m/z= 97.0649-97.0659 F: FTMS + p APCI corona Full ms2 [email protected] [50.00-315.00] MS ICIS 11
NL: 5.98E3
m/z= 97.0649-97.0659 F: FTMS + p APCI corona Full ms2 [email protected] [50.00-315.00] MS ICIS 10
NL: 4.41E3
m/z= 97.0649-97.0659 F: FTMS + p APCI corona Full ms2 [email protected] [50.00-315.00] MS ICIS 09
NL: 1.19E3
m/z= 97.0649-97.0659 F: FTMS + p APCI corona Full ms2 [email protected] [50.00-315.00] MS ICIS 08
RT: 3.70 - 4.50 SM: 5B
3.8 4.0 4.2 4.4
Time (min)
0
50
100
0
50
100
0
50
100
0
50
100
0
50
100
0
50
100
0
50
100
RT: 4.14AA: 177542SN: 196
RT: 4.11AA: 189995SN: 166
RT: 4.04AA: 162983SN: 165
RT: 4.04AA: 163899SN: 183
RT: 4.05AA: 150270SN: 176
RT: 4.11AA: 164334SN: 251
RT: 4.11AA: 150535SN: 243
NL: 2.65E4
m/z= 97.0649-97.0659 F: FTMS + p APCI corona Full ms2 [email protected] [50.00-315.00] MS ICIS 14
NL: 2.43E4
m/z= 97.0649-97.0659 F: FTMS + p APCI corona Full ms2 [email protected] [50.00-315.00] MS ICIS 13
NL: 2.61E4
m/z= 97.0649-97.0659 F: FTMS + p APCI corona Full ms2 [email protected] [50.00-315.00] MS ICIS 12
NL: 2.55E4
m/z= 97.0649-97.0659 F: FTMS + p APCI corona Full ms2 [email protected] [50.00-315.00] MS ICIS 11
NL: 2.67E4
m/z= 97.0649-97.0659 F: FTMS + p APCI corona Full ms2 [email protected] [50.00-315.00] MS ICIS 10
NL: 2.66E4
m/z= 97.0649-97.0659 F: FTMS + p APCI corona Full ms2 [email protected] [50.00-315.00] MS ICIS 09
NL: 2.38E4
m/z= 97.0649-97.0659 F: FTMS + p APCI corona Full ms2 [email protected] [50.00-315.00] MS ICIS 08
10 pg/mL
20 pg/mL
50 pg/mL
100 pg/mL
250 pg/mL
500 pg/mL
100 pg/mL100 pg/mL
18 Clinical Research use only, Not for use Diagnostics Procedure
RT: 3.70 - 4.50 SM: 5B
3.8 4.0 4.2 4.4
Time (min)
0
50
100
0
50
100
0
50
100
0
50
100
0
50
100
0
50
100
0
50
100
RT: 4.15AA: 784312SN: 1054
RT: 4.12AA: 381337SN: 352
RT: 4.05AA: 149668SN: 251
RT: 4.05AA: 75236SN: 124
RT: 4.06AA: 38873SN: 50
RT: 4.12AA: 20337SN: 33
RT: 4.13AA: 6971SN: 21
NL: 1.11E5
m/z= 109.0643-109.0653 F: FTMS + p APCI corona Full ms2 [email protected] [50.00-315.00] MS ICIS 14
NL: 4.73E4
m/z= 109.0643-109.0653 F: FTMS + p APCI corona Full ms2 [email protected] [50.00-315.00] MS ICIS 13
NL: 2.34E4
m/z= 109.0643-109.0653 F: FTMS + p APCI corona Full ms2 [email protected] [50.00-315.00] MS ICIS 12
NL: 1.15E4
m/z= 109.0643-109.0653 F: FTMS + p APCI corona Full ms2 [email protected] [50.00-315.00] MS ICIS 11
NL: 5.11E3
m/z= 109.0643-109.0653 F: FTMS + p APCI corona Full ms2 [email protected] [50.00-315.00] MS ICIS 10
NL: 3.35E3
m/z= 109.0643-109.0653 F: FTMS + p APCI corona Full ms2 [email protected] [50.00-315.00] MS ICIS 09
NL: 1.50E3
m/z= 109.0643-109.0653 F: FTMS + p APCI corona Full ms2 [email protected] [50.00-315.00] MS ICIS 08
RT: 3.70 - 4.50 SM: 5B
3.8 4.0 4.2 4.4
Time (min)
0
50
100
0
50
100
0
50
100
0
50
100
0
50
100
0
50
100
0
50
100
RT: 4.14AA: 167514SN: 164
4.353.80 3.95
RT: 4.11AA: 175646SN: 132
4.273.883.78 4.38
RT: 4.04AA: 146377SN: 150
RT: 4.04AA: 163055SN: 195
3.75 4.374.253.88
RT: 4.05AA: 146661SN: 178
RT: 4.11AA: 181988SN: 157
3.98 4.32 4.383.80
RT: 4.11AA: 129563SN: 139
4.27 4.453.983.77
NL: 2.37E4
m/z= 109.0643-109.0653 F: FTMS + p APCI corona Full ms2 [email protected] [50.00-315.00] MS ICIS 14
NL: 2.03E4
m/z= 109.0643-109.0653 F: FTMS + p APCI corona Full ms2 [email protected] [50.00-315.00] MS ICIS 13
NL: 2.27E4
m/z= 109.0643-109.0653 F: FTMS + p APCI corona Full ms2 [email protected] [50.00-315.00] MS ICIS 12
NL: 2.44E4
m/z= 109.0643-109.0653 F: FTMS + p APCI corona Full ms2 [email protected] [50.00-315.00] MS ICIS 11
NL: 2.52E4
m/z= 109.0643-109.0653 F: FTMS + p APCI corona Full ms2 [email protected] [50.00-315.00] MS ICIS 10
NL: 2.39E4
m/z= 109.0643-109.0653 F: FTMS + p APCI corona Full ms2 [email protected] [50.00-315.00] MS ICIS 09
NL: 2.16E4
m/z= 109.0643-109.0653 F: FTMS + p APCI corona Full ms2 [email protected] [50.00-315.00] MS ICIS 08
109 m/z fragment ion (CSS, left: Testo; right: Testo-d3)
10 pg/mL
20 pg/mL
50 pg/mL
100 pg/mL
250 pg/mL
500 pg/mL
100 pg/mL100 pg/mL
19 Clinical Research use only, Not for use Diagnostics Procedure
Testo-97Y = 0.0268377+0.00888146*X R^2 = 0.9991 W: 1/X
0 50 100 150 200 250 300 350 400 450 500 550
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
3.0
3.2
3.4
3.6
3.8
4.0
4.2
4.4
4.6
4.8
Are
a R
atio
Calibration curve in H2O (97 m/z ion)
Standard % Difference
10 pg/mL -0.98
20 pg/mL 3.29
50 pg/mL -6.12
100 pg/mL 2.05
250 pg/mL 3.36
500 pg/mL -1.59
20 Clinical Research use only, Not for use Diagnostics Procedure
Testo-109Y = 0.036356+0.00864486*X R^2 = 0.9982 W: 1/X
0 50 100 150 200 250 300 350 400 450 500 550
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
3.0
3.2
3.4
3.6
3.8
4.0
4.2
4.4
4.6
Are
a R
atio
Standard % Difference
10 pg/mL 6.72
20 pg/mL -8.61
50 pg/mL -6.72
100 pg/mL 9.02
250 pg/mL 1.02
500 pg/mL -1.43
Calibration curve in H2O (109 m/z ion)
21 Clinical Research use only, Not for use Diagnostics Procedure
TestoY = 0.0300348+0.00876047*X R^2 = 0.9995 W: 1/X
0 50 100 150 200 250 300 350 400 450 500 550
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
3.0
3.2
3.4
3.6
3.8
4.0
4.2
4.4
4.6
4.8
Are
a R
atio
Calibration curve in H2O (both ions)
Standard % Difference
10 pg/mL NA
20 pg/mL -2.27
50 pg/mL -2.30
100 pg/mL 5.04
250 pg/mL 0.43
500 pg/mL -0.90
22 Clinical Research use only, Not for use Diagnostics Procedure
97 m/z fragment ion (H2O, left: Testo; right: Testo-d3)
10 pg/mL
20 pg/mL
50 pg/mL
100 pg/mL
250 pg/mL
500 pg/mL
100 pg/mL100 pg/mL
RT: 3.70 - 4.50 SM: 5B
3.8 4.0 4.2 4.4
Time (min)
0
50
100
0
50
100
0
50
100
0
50
100
0
50
100
0
50
100
0
50
100
RT: 4.15AA: 731519SN: 1262
RT: 4.23AA: 338779SN: 419
RT: 4.13AA: 127859SN: 218
RT: 4.20AA: 68755SN: 114
RT: 4.12AA: 31493SN: 41
RT: 4.13AA: 18119SN: 173
RT: 4.16AA: 4012SN: 60
NL: 1.17E5
m/z= 97.0649-97.0659 F: FTMS + p APCI corona Full ms2 [email protected] [50.00-315.00] MS ICIS 07
NL: 5.56E4
m/z= 97.0649-97.0659 F: FTMS + p APCI corona Full ms2 [email protected] [50.00-315.00] MS ICIS 06
NL: 1.96E4
m/z= 97.0649-97.0659 F: FTMS + p APCI corona Full ms2 [email protected] [50.00-315.00] MS ICIS 05
NL: 1.06E4
m/z= 97.0649-97.0659 F: FTMS + p APCI corona Full ms2 [email protected] [50.00-315.00] MS ICIS 04
NL: 5.37E3
m/z= 97.0649-97.0659 F: FTMS + p APCI corona Full ms2 [email protected] [50.00-315.00] MS ICIS 03
NL: 3.29E3
m/z= 97.0649-97.0659 F: FTMS + p APCI corona Full ms2 [email protected] [50.00-315.00] MS ICIS 02
NL: 9.40E2
m/z= 97.0649-97.0659 F: FTMS + p APCI corona Full ms2 [email protected] [50.00-315.00] MS ICIS 01
RT: 3.70 - 4.50 SM: 5B
3.8 4.0 4.2 4.4
Time (min)
0
50
100
0
50
100
0
50
100
0
50
100
0
50
100
0
50
100
0
50
100
RT: 4.14AA: 174109SN: 580
RT: 4.22AA: 154715SN: 390
RT: 4.12AA: 151383SN: 298
RT: 4.20AA: 153346SN: 317
RT: 4.11AA: 167461SN: 387
RT: 4.12AA: 152585SN: 407
RT: 4.15AA: 143067SN: 234
NL: 2.87E4
m/z= 97.0649-97.0659 F: FTMS + p APCI corona Full ms2 [email protected] [50.00-315.00] MS ICIS 07
NL: 2.44E4
m/z= 97.0649-97.0659 F: FTMS + p APCI corona Full ms2 [email protected] [50.00-315.00] MS ICIS 06
NL: 2.29E4
m/z= 97.0649-97.0659 F: FTMS + p APCI corona Full ms2 [email protected] [50.00-315.00] MS ICIS 05
NL: 2.52E4
m/z= 97.0649-97.0659 F: FTMS + p APCI corona Full ms2 [email protected] [50.00-315.00] MS ICIS 04
NL: 2.61E4
m/z= 97.0649-97.0659 F: FTMS + p APCI corona Full ms2 [email protected] [50.00-315.00] MS ICIS 03
NL: 2.65E4
m/z= 97.0649-97.0659 F: FTMS + p APCI corona Full ms2 [email protected] [50.00-315.00] MS ICIS 02
NL: 2.24E4
m/z= 97.0649-97.0659 F: FTMS + p APCI corona Full ms2 [email protected] [50.00-315.00] MS ICIS 01
23 Clinical Research use only, Not for use Diagnostics Procedure
109 m/z fragment ion (H2O, left: Testo; right: Testo-d3)
10 pg/mL
20 pg/mL
50 pg/mL
100 pg/mL
250 pg/mL
500 pg/mL
100 pg/mL100 pg/mL
RT: 3.70 - 4.50 SM: 5B
3.8 4.0 4.2 4.4
Time (min)
0
50
100
0
50
100
0
50
100
0
50
100
0
50
100
0
50
100
0
50
100
RT: 4.15AA: 668828SN: 1146
RT: 4.22AA: 309183SN: 851
RT: 4.13AA: 121461SN: 229
RT: 4.20AA: 61020SN: 192
RT: 4.10AA: 26842SN: 166
4.29 4.44
RT: 4.13AA: 18598SN: 222
4.243.96 4.44
RT: 4.16AA: 5012SN: 31
NL: 1.06E5
m/z= 109.0643-109.0653 F: FTMS + p APCI corona Full ms2 [email protected] [50.00-315.00] MS ICIS 07
NL: 4.81E4
m/z= 109.0643-109.0653 F: FTMS + p APCI corona Full ms2 [email protected] [50.00-315.00] MS ICIS 06
NL: 1.77E4
m/z= 109.0643-109.0653 F: FTMS + p APCI corona Full ms2 [email protected] [50.00-315.00] MS ICIS 05
NL: 1.04E4
m/z= 109.0643-109.0653 F: FTMS + p APCI corona Full ms2 [email protected] [50.00-315.00] MS ICIS 04
NL: 4.57E3
m/z= 109.0643-109.0653 F: FTMS + p APCI corona Full ms2 [email protected] [50.00-315.00] MS ICIS 03
NL: 3.79E3
m/z= 109.0643-109.0653 F: FTMS + p APCI corona Full ms2 [email protected] [50.00-315.00] MS ICIS 02
NL: 9.85E2
m/z= 109.0643-109.0653 F: FTMS + p APCI corona Full ms2 [email protected] [50.00-315.00] MS ICIS 01
RT: 3.70 - 4.50 SM: 5B
3.8 4.0 4.2 4.4
Time (min)
0
50
100
0
50
100
0
50
100
0
50
100
0
50
100
0
50
100
0
50
100
RT: 4.14AA: 157317SN: 316
RT: 4.22AA: 136573SN: 265
4.393.983.77 3.92
RT: 4.12AA: 141279SN: 294
RT: 4.20AA: 149539SN: 286
RT: 4.11AA: 149154SN: 489
4.34 4.473.78 3.83
RT: 4.12AA: 130006SN: 366
4.34 4.473.78 3.87
RT: 4.15AA: 138849SN: 208
NL: 2.49E4
m/z= 109.0643-109.0653 F: FTMS + p APCI corona Full ms2 [email protected] [50.00-315.00] MS ICIS 07
NL: 2.21E4
m/z= 109.0643-109.0653 F: FTMS + p APCI corona Full ms2 [email protected] [50.00-315.00] MS ICIS 06
NL: 2.08E4
m/z= 109.0643-109.0653 F: FTMS + p APCI corona Full ms2 [email protected] [50.00-315.00] MS ICIS 05
NL: 2.31E4
m/z= 109.0643-109.0653 F: FTMS + p APCI corona Full ms2 [email protected] [50.00-315.00] MS ICIS 04
NL: 2.38E4
m/z= 109.0643-109.0653 F: FTMS + p APCI corona Full ms2 [email protected] [50.00-315.00] MS ICIS 03
NL: 2.23E4
m/z= 109.0643-109.0653 F: FTMS + p APCI corona Full ms2 [email protected] [50.00-315.00] MS ICIS 02
NL: 2.15E4
m/z= 109.0643-109.0653 F: FTMS + p APCI corona Full ms2 [email protected] [50.00-315.00] MS ICIS 01
24 Clinical Research use only, Not for use Diagnostics Procedure
20 pg/mL Testosterone (and I.S. 100 pg/mL) in CSS
RT: 3.93 - 4.25
3.95 4.00 4.05 4.10 4.15 4.20 4.25
Time (min)
0
20
40
60
80
100
Re
lative
Ab
un
da
nce
0
20
40
60
80
100
Re
lative
Ab
un
da
nce
0
20
40
60
80
100
Re
lative
Ab
un
da
nce
0
20
40
60
80
100
Re
lative
Ab
un
da
nce
RT: 4.07AA: 32298SN: 33
RT: 4.05AA: 155314SN: 169
RT: 4.07AA: 40108SN: 40
RT: 4.05AA: 147559SN: 165
NL: 7.43E3
m/z= 97.0649-97.0659 F: FTMS + p APCI corona Full ms2 [email protected] [50.00-315.00] MS ICIS 10
NL: 3.08E4
m/z= 97.0649-97.0659 F: FTMS + p APCI corona Full ms2 [email protected] [50.00-315.00] MS ICIS 10
NL: 6.38E3
m/z= 109.0647-109.0657 F: FTMS + p APCI corona Full ms2 [email protected] [50.00-315.00] MS ICIS 10
NL: 3.21E4
m/z= 109.0647-109.0657 F: FTMS + p APCI corona Full ms2 [email protected] [50.00-315.00] MS ICIS 10
10 #572 RT: 4.07 AV: 1 NL: 2.98E4F: FTMS + p APCI corona Full ms2 [email protected] [50.00-315.00]
95 100 105 110
m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lative
Ab
un
da
nce
97.0654R=108800
109.0652R=103900
107.0859R=9030095.0861
R=99500 105.0703R=7860098.1048
R=64000100.2727R=62200
Testo
Testo
Testo-d3
Testo-d3
97 m/z ion97 m/z ion
109 m/z ion109 m/z ion
15 scans in 6 sec, at 100K resolution for fragments
25 Clinical Research use only, Not for use Diagnostics Procedure
RT: 3.00 - 5.00 SM: 5B
3.0 3.5 4.0 4.5 5.0
Time (min)
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
RT: 4.17AA: 347921SN: 310
3.96
4.73 4.944.403.703.31 3.36
RT: 4.18AA: 216369SN: 209
4.984.694.39
3.30 3.53 3.67 3.943.06
NL: 6.32E4
m/z= 97.0649-97.0659 F: FTMS + p APCI corona Full ms2 [email protected] [50.00-315.00] MS ICIS T2
NL: 3.72E4
m/z= 97.0649-97.0659 F: FTMS + p APCI corona Full ms2 [email protected] [50.00-315.00] MS ICIS T2
RT: 3.00 - 5.00 SM: 5B
3.0 3.5 4.0 4.5 5.0
Time (min)
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
RT: 4.17AA: 311283SN: 380RMS
3.96
4.934.633.723.363.08
RT: 4.17AA: 196492SN: 275RMS
4.76
4.68
4.51
3.07 3.983.823.13 3.73
NL: 5.45E4
m/z= 109.0647-109.0657 F: FTMS + p APCI corona Full ms2 [email protected] [50.00-315.00] MS ICIS T2
NL: 3.58E4
m/z= 109.0647-109.0657 F: FTMS + p APCI corona Full ms2 [email protected] [50.00-315.00] MS ICIS T2
Clinical Plasma sample T2 (left: 97 m/z; right: 109 m/z)
Testo-d3 Testo-d3
Testo Testo
26 Clinical Research use only, Not for use Diagnostics Procedure
In CSS
97.0654 m/z R20.9933 Slope 8.85E-03
in CSS Conc. (pg/mL) % Dif Patient Conc. (ng/dL)
Cal1 10 8.76 T1 15
Cal2 20 2.63 T2 17
Cal3 50 -8.94 T3 156
Cal4 100 2.89 T4 34
Cal5 250 -10.76 T5 8
Cal6 500 5.41
109.0652 m/z R20.998 Slope 8.39E-03
Cal1 10 0.97 T1 13
Cal2 20 7.45 T2 19
Cal3 50 -5.78 T3 156
Cal4 100 -0.29 T4 37
Cal5 250 -5.35 T5 8
Cal6 500 2.99
Both ions R20.9935 Slope 8.75E-03
Cal1 10 11.27 T1 15
Cal2 20 -5.5 T2 17
Cal3 50 -3.41 T3 164
Cal4 100 3.57 T4 36
Cal5 250 -11.09 T5 9
Cal6 500 5.17
27 Clinical Research use only, Not for use Diagnostics Procedure
97.0654 m/z R20.9991 Slope 8.88E-03
in H2O Conc. (pg/mL) % Dif Patient Conc. (ng/dL)
Cal1 10 -0.98 T1 15
Cal2 20 3.29 T2 17
Cal3 50 -6.12 T3 156
Cal4 100 2.05 T4 34
Cal5 250 3.36 T5 8
Cal6 500 -1.59
109.0652 m/z R20.9982 Slope 8.64E-03
Cal1 10 6.72 T1 14
Cal2 20 -8.61 T2 20
Cal3 50 -6.72 T3 152
Cal4 100 9.02 T4 36
Cal5 250 1.02 T5 7
Cal6 500 -1.43
Both ions R20.9995 Slope 8.76E-03
Cal1 10 NA T1 15
Cal2 20 -2.27 T2 18
Cal3 50 -2.3 T3 164
Cal4 100 5.04 T4 36
Cal5 250 0.43 T5 9
Cal6 500 -0.9
In water
28 Clinical Research use only, Not for use Diagnostics Procedure
Donor samples data vs immunoassay results
Sample Fragment 97 Fragment 109 Immunoassay results
ng/dL Calibrators in
water
Calibrators in plasma Calibrators in
water
Calibrators in
plasma
T1 15 15 13 14 21 ng/dl
T2 17 17 19 20 38 ng/dL
T3 156 156 156 152 178 ng/dL
T3 34 34 37 36 62 ng/dL
T5 8 8 8 7 <20 ng/dL
29 Clinical Research use only, Not for use Diagnostics Procedure
Conclusion
• Q Exactive LC-MS/MS system has high sensitivity and specificity needed for
Testosterone analysis in plasma matrix