Testosteron LC MS MS APPI

Quantitation of Testosterone Using
Q Exactive Mass Spectrometer - Comparison of

APCI, APPI and HESI Sources
Kevin He and Marta Kozak

Clinical Research Applications Group
Thermo Fisher Scientific San Jose, CA
Clinical Research use only, Not for use in Diagnostics Procedure
Instrumentation
LC system: Accela 600 pump
Q Exactive equipped with APCI + Ion Max TM source
Instrumentation
LC system: Accela 600 pump
Autosampler: Thermal Pal
Q ExactiveTM mass spectrometer equipped with APCI (or HESI, APPI) +
Ion Max TM source
Calibration Curve Standards

Calibration standards were prepared in 50% MeOH/water.
Injection volume: 50 L
Sample
Testosterone
(pg/mL)
Testosterone-3-C13
(pg/mL)
Blank+IS
100
Cal1
100
Cal2
10
100
Cal3
20
100
Cal4
50
100
Cal5
100
100
Cal6
250
100
Cal7
500
100
LC Method
Analytical Column: Hypersil GOLD aQ 100 x 3.0 mm, 5 m particle size
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 (
L/min)
95
600
0.1
60
40
600
0.5
60
40
600
3.5
95
600
4.5
95
600
4.51
100
800
100
800
5.1
95
800
6.5
95
800
MS Method: APCI source parameters

Q Exactive system
Ionization
APCI, positive, Ion Max source
Vaporizer temperature (C)
350
Capillary temperature (C)
320
Discharge current (A)
5.0
Sheath gas (units)
10
Aux gas (units)
20
S-lens Level
60
Probe position
between B and C
Divert valve:
initial position to waste, at 1.5 min position to

detector and at 5.5 min position switched back to waste
MS Method: APPI source parameters

Q Exactive system
Ionization
APPI, positive, Ion Max source
350
320
Spray voltage (V)
4000
Sheath gas (units)
10
Aux gas (units)
20
S-lens Level
60
Probe position
between B and C
Divert valve:

We did not test the APPI method with teed-in dopant infusion, but it seemed that testosterone could
ionize sufficiently well without it under the current conditions.
MS Method: HESI source parameters

Q Exactive system
Ionization
HESI-II, positive, Ion Max source
350
320
Spray voltage (V)
4000
Sheath gas (units)
10
Aux gas (units)
20
S-lens Level
60
Probe position
between B and C
Divert valve:

MS acquisition method
Acquisition mode: HCD fragmentation with collision energy set to 40%
NCE
Precursor isolation width: 2 mu
Resolution: 35K @ 200 m/z
AGC Target: 1e5
Max injection time: 120ms
Inclusion list:
289.2 97.0648
289.2 109.0648
Testosterone: 289.2 (fragment ions - 97.0648, 109.0648)
Testosterone-3-C13: 292.2 (fragment ions -100.0748, 112.0748)
m/z
Acquisition method parameters: screen capture
10
LC Gradient: screen capture
11
IS in 50% MeOH/H2O 100 pg/mL

292.2 100.0749
RT: 2.00 - 6.00
90
80
80
70
70
60
60
50
50
40
40
30
30
20
20
10
10
Relative Abundance
0
100
AA: 149119
100
90
HESI
0
100
AA: 389064
90
90
80
80
70
70
60
60
50
50
40
40
30
30
20
20
10
AA: 320849
APPI
10
0
100
0
100
AA: 574722
90
90
80
80
70
70
60
60
50
50
40
40
30
30
20
20
10
10
0
2.0
m/z
2.00 - 6.00
AA: 180054
100
12
292.2 112.0749
2.5
3.0
3.5
4.0
4.5
Time (min)
5.0
5.5
6.0
0
2.0
AA: 486625
APCI
2.5
3.0
3.5
4.0
4.5
Time (min)
5.0
5.5
IS signals using different sources (n=6)
600000
100.0748 m/z
500000
112.0748 m/z
400000
300000
200000
100000
0
HESI
13
APPI
APCI
Testosterone 5 pg/mL
289.2 97.0648
289.2 109.0648
Relative Abundance
RT: 2.00 - 6.00 SM: 3B
2.00 - 6.00 SM: 3B
100
100
90
90
80
80
70
70
60
60
50
50
40
40
30
30
20
20
10
10
0
100
0
100
90
90
80
80
70
70
60
60
50
50
40
40
30
30
20
20
10
14
HESI
APPI
10
0
100
0
100
MA: 3720
90
90
80
80
70
70
60
60
50
50
40
40
30
30
20
20
10
10
0
2.0
m/z
2.5
3.0
3.5
4.0
4.5
Time (min)
5.0
5.5
6.0
0
2.0
MA: 4785
APCI
2.5
3.0
3.5
4.0
4.5
Time (min)
5.0
5.5
6.0
289.2 97.0648
289.2 109.0648
RT: 2.00 - 6.00 SM: 3B
2.00 - 6.00 SM: 3B
100
100
90
90
80
80
70
70
60
60
50
50
40
40
30
30
20
20
10
10
Relative Abundance
0
100
90
80
80
70
70
60
60
50
50
40
40
30
30
20
20
10
AA: 3024
APPI
10
0
100
15
HESI
0
100
AA: 5515
90
0
100
AA: 13408
90
90
80
80
70
70
60
60
50
50
40
40
30
30
20
20
10
10
0
2.0
m/z
2.5
3.0
3.5
4.0
4.5
Time (min)
5.0
5.5
6.0
0
2.0
AA: 7995
APCI
2.5
3.0
3.5
4.0
4.5
Time (min)
5.0
5.5
6.0
289.2 97.0648
289.2 109.0648
RT: 2.00 - 6.00 SM: 3B
2.00 - 6.00 SM: 3B
100
100
90
90
80
80
70
70
60
60
50
50
40
40
30
30
20
20
10
10
Relative Abundance
0
100
90
80
80
70
70
60
60
50
50
40
40
30
30
20
20
10
MA: 10810
APPI
10
0
100
16
HESI
0
100
AA: 14275
90
0
100
MA: 22946
90
90
80
80
70
70
60
60
50
50
40
40
30
30
20
20
10
10
0
2.0
m/z
2.5
3.0
3.5
4.0
4.5
Time (min)
5.0
5.5
6.0
0
2.0
MA: 32233
APCI
2.5
3.0
3.5
4.0
4.5
Time (min)
5.0
5.5
6.0
289.2 97.0648
289.2 109.0648
100
100
90
90
80
80
70
50
40
40
30
30
20
20
10
10
Relative Abundance
0
100
0
100
MA: 45872
90
90
80
80
70
70
60
60
50
50
40
40
30
30
20
20
10
AA: 38892
APPI
10
0
100
17
HESI
60
50
0
100
MA: 82318
90
90
80
80
70
70
60
60
50
50
40
40
30
30
20
20
10
10
0
2.0
MA: 10860
70
MA: 17425
60
m/z
2.5
3.0
3.5
4.0
4.5
Time (min)
5.0
5.5
6.0
0
2.0
AA: 97531
APCI
2.5
3.0
3.5
4.0
4.5
Time (min)
5.0
5.5
6.0

289.2 97.0648
289.2 109.0648
RT: 2.00 - 6.00 SM: 5B
2.00 - 6.00 SM: 5B

MA: 36196
100
90
80
80
70
70
60
60
50
50
40
40
30
30
20
20
10
10
Relative Abundance
0
100
HESI
0
100
MA: 92064
90
90
80
80
70
70
60
60
50
50
40
40
30
30
20
20
10
AA: 73646
APPI
10
0
100
18
MA: 25313
100
90
0
100
MA: 204055
90
90
80
80
70
70
60
60
50
50
40
40
30
30
20
20
10
10
0
2.0
m/z
2.5
3.0
3.5
4.0
4.5
Time (min)
5.0
5.5
6.0
0
2.0
AA: 185688
APCI
2.5
3.0
3.5
4.0
4.5
Time (min)
5.0
5.5
6.0

289.2 97.0648
289.2 109.0648
RT: 2.00 - 6.00 SM: 5B
2.00 - 6.00 SM: 5B

AA: 173866
100
90
80
80
70
70
60
60
50
50
40
40
30
30
20
20
10
10
Relative Abundance
0
100
HESI
0
100
AA: 250155
90
90
80
80
70
70
60
60
50
50
40
40
30
30
20
20
10
AA: 211707
APPI
10
0
100
19
AA: 151683
100
90
0
100
AA: 604848
90
90
80
80
70
70
60
60
50
50
40
40
30
30
20
20
10
10
0
2.0
m/z
2.5
3.0
3.5
4.0
4.5
Time (min)
5.0
5.5
6.0
0
2.0
AA: 490293
APCI
2.5
3.0
3.5
4.0
4.5
Time (min)
5.0
5.5
6.0
Testosterone Calibration Curve (APCI, 97.0648

m/z)
testo97
Y = -0.00821055+0.00437969*X R^2 = 0.9949 W: 1/X
1.2
1.1
1.0
0.9
Area Ratio
0.8
0.7
Specified
(pg/mL)
0.6
0.5
0.4
Diffe.
(%)
5.76
10
10.35
20
-7.95
50
-3.07
100
-9.68
250
4.59
0.3
0.2
0.1
0.0
0
20
40
60
80
100
120
140
160
180
200
220
240
260
Concentration (pg/mL)
20
Testosterone Calibration Curve (APPI, 97.0648

m/z)
testo97
Y = -0.0141408+0.00371641*X R^2 = 0.9985 W: 1/X
1.00
0.95
0.90
0.85
0.80
0.75
0.70
Area Ratio
0.65
0.60
0.55
Specified
(pg/mL)
0.50
0.45
Diffe.
(%)
0.40
3.69
0.35
10
-7.64
20
-2.58
50
4.19
0.15
100
4.57
0.10
250
-2.23
0.30
0.25
0.20
0.05
0.00
0
20
40
60
80
100
120
140
160
180
200
220
240
260
Concentration (pg/mL)
21
Conclusion
We have tested the detection of testosterone with LC-MS/MS using three ionization
techniques: HESI, APPI and APCI.
With most of the source parameters consistent, the order of signal intensity of ionized
testosterone is (from highest to lowest): APCI, APPI and HESI.
We can detect 5 pg/mL testosterone in neat solutions with both APPI and APCI, but not
HESI (>50 pg/mL), under the current conditions.
While APPI ranked in the middle in terms of signal intensity, the background signals were
lowest. And we observed excellent calibration curve.
Regardless of the ionization technique, we observe the consistent ion ratio between the
two major fragment ions.
Due to multiple reasons, we did not add dopant for APPI, which might further improve the
performance.
Concentrations of testosterone in this presentation were the straight-up values in the
glass inserts. For real samples, we will have 2 concentration factor and it will improve
LLOQ by ~ 2 fold.
22

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Testosteron LC MS MS APPI

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Quantitation of Testosterone Using

Q Exactive Mass Spectrometer - Comparison of

Kevin He and Marta Kozak

Clinical Research use only, Not for use in Diagnostics Procedure

Clinical Research use only, Not for use in Diagnostics Procedure

Clinical Research use only, Not for use in Diagnostics Procedure

Calibration Curve Standards

Clinical Research use only, Not for use in Diagnostics Procedure

Clinical Research use only, Not for use in Diagnostics Procedure

MS Method: APCI source parameters

APCI, positive, Ion Max source

Vaporizer temperature (C)

Capillary temperature (C)

Discharge current (A)

Sheath gas (units)

Aux gas (units)

initial position to waste, at 1.5 min position to

Clinical Research use only, Not for use in Diagnostics Procedure

MS Method: APPI source parameters

APPI, positive, Ion Max source

Vaporizer temperature (C)

Capillary temperature (C)

Spray voltage (V)

Sheath gas (units)

Aux gas (units)

initial position to waste, at 1.5 min position to

Clinical Research use only, Not for use in Diagnostics Procedure

MS Method: HESI source parameters

HESI-II, positive, Ion Max source

Vaporizer temperature (C)

Capillary temperature (C)

Spray voltage (V)

Sheath gas (units)

Aux gas (units)

initial position to waste, at 1.5 min position to

Clinical Research use only, Not for use in Diagnostics Procedure

Testosterone: 289.2 (fragment ions - 97.0648, 109.0648)

Testosterone-3-C13: 292.2 (fragment ions -100.0748, 112.0748)

Clinical Research use only, Not for use in Diagnostics Procedure

Acquisition method parameters: screen capture

Clinical Research use only, Not for use in Diagnostics Procedure

LC Gradient: screen capture

Clinical Research use only, Not for use in Diagnostics Procedure

IS in 50% MeOH/H2O 100 pg/mL

Clinical Research use only, Not for use in Diagnostics Procedure

IS signals using different sources (n=6)

Clinical Research use only, Not for use in Diagnostics Procedure

RT: 2.00 - 6.00 SM: 3B

2.00 - 6.00 SM: 3B

Clinical Research use only, Not for use in Diagnostics Procedure

RT: 2.00 - 6.00 SM: 3B

2.00 - 6.00 SM: 3B

Clinical Research use only, Not for use in Diagnostics Procedure

RT: 2.00 - 6.00 SM: 3B

2.00 - 6.00 SM: 3B

Clinical Research use only, Not for use in Diagnostics Procedure

Clinical Research use only, Not for use in Diagnostics Procedure

Testosterone 100 pg/mL

RT: 2.00 - 6.00 SM: 5B

2.00 - 6.00 SM: 5B

Clinical Research use only, Not for use in Diagnostics Procedure

Testosterone 250 pg/mL

RT: 2.00 - 6.00 SM: 5B

2.00 - 6.00 SM: 5B