The MS/MS conditions were as follows:
Ion Polarity: Positive Ion Mode
Vaporizer Temperature: 525 °C
Capillary Temperature: 350 °C
Discharge Current: 5.0 uA
Sheath Gas Pressure (N
2
): 35 units
Auxiliary Gas Pressure (N
2
): 10 units
Scan Type: Unit Resolution
Scan Time: 0.050 s
Analyte
Parent Ion (Q1)
Product Ion (Q3)
Collision Tube Lens
Testosterone
289.201
97.118
23
113
Testosterone
289.201
109.114
25
113
Testosterone IS 292.216
97.111
21
92
Testosterone IS 292.216
109.097
26
92
Table 2: List of SRM transitions and their parameters
Results and Discussion
Representative-SRM chromatograms for Testosterone at
5 ng/dL and 200 ng/dL are shown in Figures 1 and 2,
respectively. Clearly identifiable and quantifiable peaks
were observed.
The method precision was evaluated by analyzing
patient sample pools at concentrations of 20 ng/dl, 50 ng/dL,
140 ng/dL and 1000 ng/dL. Intra-assay variability was
determined by processing and analyzing twenty replicates
of the lowest two QC sample pools. Inter-assay variability
was determined by processing and analyzing two replicates
of each QC sample pools in six different batches. Intra-assay
and inter-assay precision results are displayed in Table 3
as % CV.
Precision
Intra-assay
Inter-assay
QC Level 1 (n=20)
8.43%
N/A
QC Level 2 (n=20)
7.33%
N/A
QC Level 1 (n=12)
N/A
15.70%
QC Level 2 (n=12)
N/A
12.29%
QC Level 3 (n=12)
N/A
2.91%
QC Level 4 (n=12)
N/A
5.71%
Table 3: Intra and Inter assay precision
Time (min)
Loading Flow (µL/min)
Loading A% Loading B% Loading C% Eluting Flow (µL/min)
Eluting A% Eluting B%
0.00
1.0
100
0.5
60
40
0.67
1.0
90
10
0.5
60
40
1.67
0.20
90
10
0.3
60
40
1.77
0.15
20
80
0.6
60
40
2.77
1.00
20
80
1.00
100
5.02
2.00
100
1.20
100
6.02
2.00
100
0.80
60
40
Table 1: HPLC Method
Figure 1: 5 ng/dL Testosterone standard with deuterated internal standard
1...,52,53,54,55,56,57,58,59,60,61 63,64,65,66,67,68,69,70,71,72,...374