Quantitative Determination of Testosterone in
Plasma Using Unique Automated Online Sample
Preparation and LC-MS/MS
Marta Kozak, Thermo Fisher Scientific, San Jose, CA, USA
For Research Use Only. Not for use in diagnostic procedures.
Introduction
Testosterone quantitative methods require a limit of
quantitation of 10 pg/mL in plasma. The commonly-used
liquid-liquid extraction (LLE) technique for sample
preparation consists of multiple steps (including
evaporation). It is neither time- nor cost-efficient. We have
developed a fast and cost-efficient online sample
preparation method implementing Thermo Scientific
TurboFlow technology for clinical research purposes.
Goal
The goal is to develop an automated, interference-free LC-
MS/MS method to quantitate testosterone with a low limit
of quantitation (LOQ) in plasma. The method utilizes the
analytical speed of the Thermo Scientific Transcend
system, powered by TurboFlow
™
automated, online
sample preparation technology, coupled with a triple stage
quadrupole mass spectrometer.
Experimental
Sample Preparation
A 100 µL aliquot of plasma was mixed with 100 µL of
methanol containing testosterone-d3 (internal standard)
and precipitated in ice for 10-15 minutes. The resulting
plasma was centrifuged at 12,000 rpm for 10 minutes at
4 °C. Calibrators were prepared in double charcoal-
stripped plasma at six concentration levels from 10 pg/mL
to 500 pg/mL.
HPLC
HPLC analysis was performed using the Transcend
™
TLX-1 system. Plasma samples were extracted using a
TurboFlow
™
Cyclone P column (1 x 50 mm).
Chromatographic separation was performed using a
Thermo Scientific Hypersil GOLD aQ column
(100 x 2.1 mm, 5µm). A gradient liquid chromatography
method was used.
Mass Spectrometry
MS analysis was carried out on a Thermo Scientific TSQ
Vantage triple stage quadrupole mass spectrometer with a
Thermo Scientific Ion MAX source and atmospheric
pressure chemical ionization (APCI) probe in the positive
ionization mode. The selective reaction monitoring (SRM)
mode was used for mass spectrometry detection.
Results and Discussion
Figure 1 shows the linear calibration curve for
testosterone. The R
2
value is 0.9999, which indicates an
excellent linear fit over the dynamic range of 10 – 500
pg/mL. Figure 2 shows the results for four unknown
samples. Table 1 compares these results to the averaged
results from three other laboratories. The outside labs
used either SPE or LLE sample preparation. Ion ratios
were used for confirmation.
Conclusion
An automated method utilizing online sample preparation
coupled with a triple stage quadrupole mass spectrometer
met analytical requirements. The results correlated well
with conventional (LLE) sample preparation methods. The
method is interference-free and robust. The entire analysis
in plasma samples can be done in 10 minutes, with a
quantitation limit of 10 pg/mL and linearity range from
10 to 500 pg/mL. Analytical throughput can be increased
by implementing a 2-channel (TLX-2) or 4-channel (TLX-
4) column multiplexing system in clinical research.
Method performance summary
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Key Words
• Transcend
System
• TSQ Vantage
• Endocrinology
• Clinical Research
• TurboFlow
Technology
Application
Note: 490b
Y=0.0187183+0.00269886*X R^2 = 0.9999 W: 1/X
0
50
100
150
200
250
300
350
400
450
500
550
pg/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
0.75
0.80
0.85
0.90
0.95
1.00
1.05
1.10
1.15
1.20
1.25
1.30
1.35
1.40
1.45
1.50
AreaRatio
-0.45
500pg/mL
0.83
250pg/mL
1.35
100pg/mL
-3.03
50pg/mL
1.89
20pg/mL
-0.80
10pg/mL
%Difference
Standard
Figure 1: Testosterone
calibration curve
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Relative Abundance
RT:4.26
AA:15393
SN:1004
3.38
4.05
4.44
3.55
4.52
3.80
4.694.81
6.69
4.88
2.95
6.43
2.75
2.11
5.06
5.82
6.34
2.352.52
5.63
5.47
3.22
6.05
6.82
M – 364 pg/mL
0
1
2
3
4
5
6
7
8
9
10
Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Relative Abundance
RT:4.21
AA:15145
SN:900
3.98
3.62
3.34
4.38
4.50
4.83
5.40
4.90
8.609.07
9.61
3.23
45.2
76.0
1.59
0.48
5.98
99.9
22.8
0.92
6.10
1.81
7.61
7.05
6.58
N – 384 pg/mL
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Relative Abundance
RT:4.23
AA:28142
SN:1479
3.34
4.03
4.47
3.473.543.79
4.534.734.81
5.56
4.96
2.63
5.32
2.31
6.39
3.23
6.72
49.2
01.2
5.745.83
6.98
6.15
O – 546 pg/mL
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Relative Abundance
RT:4.24
AA:3692
SN:292
3.44
3.54
4.08
4.05
3.39
3.59
3.33
4.39
3.85
4.534.77
4.84
2.82
2.46
4.94
5.82
3.15
5.12
6.77
5.67
2.012.16
5.46
6.14
5.43
2.90
2.68
6.25
6.74
5.98
R* – 184 pg/mL
Figure 2: SRM
chromatograms for
four unknown samples
Table 1: Comparison of results for four unknown samples
Sample
Current Method (pg/mL) Other Laboratories (pg/mL)
M
36
32
N
38
36
O
55
61
R*
18
13
R* is a gel-tube sample.
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