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detremine testosterone by LC/MS/MS from human body.
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Clinical Research use only, Not for use in Diagnostics Procedure
Kevin He and Marta Kozak
Clinical Research Applications Group
Thermo Fisher Scientific San Jose, CA
Quantitation of Testosterone Using
Q Exactive Mass Spectrometer - Comparison of
APCI, APPI and HESI Sources
2 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
3 Clinical Research use only, Not for use in Diagnostics Procedure
Instrumentation
• LC system: Accela 600 pump
• Autosampler: Thermal Pal
• Q ExactiveTM mass spectrometer equipped with APCI (or HESI, APPI) + Ion Max TM source
4 Clinical Research use only, Not for use in Diagnostics Procedure
Calibration Curve Standards
• Calibration standards were prepared in 50% MeOH/water.
• Injection volume: 50 µL
Sample Testosterone
(pg/mL)
Testosterone-3-C13
(pg/mL)
Blank+IS 0 100
Cal1 5 100
Cal2 10 100
Cal3 20 100
Cal4 50 100
Cal5 100 100
Cal6 250 100
Cal7 500 100
5 Clinical Research use only, Not for use in Diagnostics Procedure
LC Method
• Analytical Column: Hypersil GOLD aQ 100 x 3.0 mm, 5 µm 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 (µµµµL/min)
0 95 5 0 600
0.1 60 40 0 600
0.5 60 40 0 600
3.5 5 95 0 600
4.5 5 95 0 600
4.51 0 0 100 800
5 0 0 100 800
5.1 95 5 0 800
6.5 95 5 0 800
6 Clinical Research use only, Not for use in Diagnostics Procedure
MS Method: APCI source parameters
Q Exactive systemIonization APCI, positive, Ion Max™ source
Vaporizer temperature (ºC) 350
Capillary temperature (ºC) 320
Discharge current (µA) 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
7 Clinical Research use only, Not for use in Diagnostics Procedure
MS Method: APPI source parameters
Q Exactive systemIonization APPI, positive, Ion Max™ source
Vaporizer temperature (ºC) 350
Capillary temperature (ºC) 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: initial position to waste, at 1.5 min position to detector and at 5.5 min position switched back to waste
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.
8 Clinical Research use only, Not for use in Diagnostics Procedure
MS Method: HESI source parameters
Q Exactive systemIonization HESI-II, positive, Ion Max™ source
Vaporizer temperature (ºC) 350
Capillary temperature (ºC) 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: initial position to waste, at 1.5 min position to detector and at 5.5 min position switched back to waste
9 Clinical Research use only, Not for use in Diagnostics Procedure
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:
• Testosterone: 289.2 (fragment ions - 97.0648, 109.0648)
• Testosterone-3-C13: 292.2 (fragment ions -100.0748, 112.0748)
289.2 � 97.0648 289.2 � 109.0648 m/z
10 Clinical Research use only, Not for use in Diagnostics Procedure
Acquisition method parameters: screen capture
11 Clinical Research use only, Not for use in Diagnostics Procedure
LC Gradient: screen capture
12 Clinical Research use only, Not for use in Diagnostics Procedure
IS in 50% MeOH/H2O – 100 pg/mL
2.00 - 6.00
2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5
Time (min)
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100AA: 149119
AA: 320849
AA: 486625
RT: 2.00 - 6.00
2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0
Time (min)
0
10
20
30
40
50
60
70
80
90
100
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
100AA: 180054
AA: 389064
AA: 574722
292.2 � 100.0749 292.2 � 112.0749 m/z
HESI
APPI
APCI
13 Clinical Research use only, Not for use in Diagnostics Procedure
IS signals using different sources (n=6)
0
100000
200000
300000
400000
500000
600000
HESI APPI APCI
100.0748 m/z
112.0748 m/z
14 Clinical Research use only, Not for use in Diagnostics Procedure
Testosterone 5 pg/mL
289.2 � 97.0648
HESI
APPI
APCI
2.00 - 6.00 SM: 3B
2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0
Time (min)
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
MA: 4785
RT: 2.00 - 6.00 SM: 3B
2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0
Time (min)
0
10
20
30
40
50
60
70
80
90
100
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
MA: 3720
289.2 � 109.0648 m/z
15 Clinical Research use only, Not for use in Diagnostics Procedure
Testosterone 10 pg/mL
289.2 � 97.0648
HESI
APPI
APCI
2.00 - 6.00 SM: 3B
2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0
Time (min)
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
AA: 3024
AA: 7995
RT: 2.00 - 6.00 SM: 3B
2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0
Time (min)
0
10
20
30
40
50
60
70
80
90
100
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
AA: 5515
AA: 13408
289.2 � 109.0648 m/z
16 Clinical Research use only, Not for use in Diagnostics Procedure
Testosterone 20 pg/mL
289.2 � 97.0648
HESI
APPI
APCI
2.00 - 6.00 SM: 3B
2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0
Time (min)
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
MA: 10810
MA: 32233
RT: 2.00 - 6.00 SM: 3B
2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0
Time (min)
0
10
20
30
40
50
60
70
80
90
100
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
AA: 14275
MA: 22946
289.2 � 109.0648 m/z
17 Clinical Research use only, Not for use in Diagnostics Procedure
Testosterone 50 pg/mL
289.2 � 97.0648
HESI
APPI
APCI
2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0
Time (min)
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
MA: 10860
AA: 38892
AA: 97531
2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0
Time (min)
0
10
20
30
40
50
60
70
80
90
100
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
MA: 17425
MA: 45872
MA: 82318
289.2 � 109.0648 m/z
18 Clinical Research use only, Not for use in Diagnostics Procedure
Testosterone 100 pg/mL
289.2 � 97.0648 289.2 � 109.0648 m/z
HESI
APPI
APCI
2.00 - 6.00 SM: 5B
2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0
Time (min)
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100MA: 25313
AA: 73646
AA: 185688
RT: 2.00 - 6.00 SM: 5B
2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0
Time (min)
0
10
20
30
40
50
60
70
80
90
100
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
100MA: 36196
MA: 92064
MA: 204055
19 Clinical Research use only, Not for use in Diagnostics Procedure
Testosterone 250 pg/mL
289.2 � 97.0648
HESI
APPI
APCI
2.00 - 6.00 SM: 5B
2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0
Time (min)
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100AA: 151683
AA: 211707
AA: 490293
RT: 2.00 - 6.00 SM: 5B
2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0
Time (min)
0
10
20
30
40
50
60
70
80
90
100
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
100AA: 173866
AA: 250155
AA: 604848
289.2 � 109.0648 m/z
20 Clinical Research use only, Not for use in Diagnostics Procedure
Testosterone Calibration Curve (APCI, 97.0648 m/z)
Concentration (pg/mL)
testo97Y = -0.00821055+0.00437969*X R^2 = 0.9949 W: 1/X
0 20 40 60 80 100 120 140 160 180 200 220 240 260
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
Are
a R
atio
Specified
(pg/mL)
Diffe.
(%)
5 5.76
10 10.35
20 -7.95
50 -3.07
100 -9.68
250 4.59
21 Clinical Research use only, Not for use in Diagnostics Procedure
Testosterone Calibration Curve (APPI, 97.0648 m/z)
Concentration (pg/mL)
Specified
(pg/mL)
Diffe.
(%)
5 3.69
10 -7.64
20 -2.58
50 4.19
100 4.57
250 -2.23
testo97Y = -0.0141408+0.00371641*X R^2 = 0.9985 W: 1/X
0 20 40 60 80 100 120 140 160 180 200 220 240 260
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
0.80
0.85
0.90
0.95
1.00A
rea R
atio
22 Clinical Research use only, Not for use in Diagnostics Procedure
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.
