183 / 658
5
Thermo Scienti c Poster Note
•
PN63781_E 03/13S
For Research Use Only. Not for use in diagnostic procedures.
All trademarks are the property of Thermo Fisher Scientific and it
This information is not intended to encourage use of these produ
intellectual property rights of others.
5
6
7
8
Time (min)
0
50
100
0
50
100
0
50
100
0
50
100
0
50
100
0
50
100
RT:7.75
RT:7.09
RT:7.39
RT:6.36
RT:6.12
RT:4.74
erone
THG
Oxymesterone
Clostebol
Fluoxymesterone
3-Hydroxystanozolol
**
6
β
-Hydroxyfluoxymesterone
LOQ
LOD
g/mL
<1 ng/mL
g/mL
< 1 ng/mL
g/mL
<1 ng/mL
g/mL
<1 ng/mL
g/mL
<1 ng/mL
g/mL
<1 ng/mL
g/mL
<1 ng/mL
g/mL
<1 ng/mL
g/mL
<1 ng/mL
g/mL
<1 ng/mL
g/mL
<1 ng/mL
g/mL
<1 ng/mL
g/mL
<1 ng/mL
g/mL
<1ng/mL
g/mL
<1 ng/mL
g/mL
<1 ng/mL
g/mL
<1 ng/mL
g/mL
3 ng/mL
Method Precision
QC samples with concentrations across calibration range (2 ng/mL, 15 ng/mL, 90 ng/mL,
450 ng/mL) were prepared in blank oral fluid. QC samples were analyzed in 5 replicates in 3
separate batches to obtain intra- and inter- assay precision (Table 3).
ccuracy of 5 ppm at LOQ
Matrix Effect
Matrix effects (Table 4) were evaluated by spiking blank oral fluid with all analytes at
concentrations of 2 ng/mL, 10 ng/mL, 100 ng/mL and analyzing these samples with
calibration standards prepared in solvent.
Analyte
2 ng/mL
10 ng/mL
100 ng/mL
Clenbuterol
121
131
107
19-Norandrosterone
101
123
101
Nandrolone
ND
97.7
93.5
Methandrosterone
95.0
104
103
6
β
-Hydroxyboldenone
102
92.4
94.3
Boldenone
101
103
99.6
DHEA
100
127
115
Oxandrolone
93.5
124
109
Testosterone
90.5
105
96.8
Epitestosterone
78.5
99.8
102
Formestane
90.5
92.6
95.3
Stanozolol
80.0
81.5
92.8
THG
94.0
100
95.9
Oxymesterone
89.0
109
113
Clostebol
99.7
110
118
Fluoxymesterone
96.5
101
104
3-Hydroxystanozolol
93.5
92.0
105
6
β
-Hydroxufluoxymesterone
80.5*
102
104
ND: not detected; *concentration (1.61 ng/mL) below LOQ
Donor Samples
Testosterone and Epitestosterone in negative teste
Testosterone- 0.206 ng/mL (extrapolated)
Epitestost
Quantifier
Qualifier
Compounds detected in selected positive tested sa
lab with protein precipitation method.
Conclusion
We developed sensitive and robust quantitative s
anabolic steroids in human oral fluid.
• Implementation of the ultra high resolution Q E
collect MS2 spectra and ion ratio confirmation r
compound identification.
• Method was validated using LLE for sample pre
analytes in positive tested samples processed
provided by collaborator laboratory.
Testosterone- 2270 ng/mL (extrapolated)
Epitestoste
Boldenone- 7500 ng/mL (extrapolated)
Methandro
Testosterone 1.6 ng/mL
Epitestoste
Oxandrolo
Clenbuterol 11 ng/mL
Quantifier
Qualifier
Quantifier
Quantifier
Qualifier
Qualifier
Table 3. Intra-assay and inter-assay results
Table 4. Percent recovery in spiked blank oral fluid
Acknowledgement
We would like to thank Erica Guice, Research Dir
Laboratory, for scientific advice and for providing
References
1. Draft SANCO 1805/2000 Rev.1 [Revised Com
laying down performance criteria for analytical me
residues there of in live animals and animals prod
Quantifier
Qualifier
Analyte
Intra assay
Inter assay
2 ng/mL 15 ng/mL 90 ng/mL 450 ng/mL 2 ng/mL 15 ng/mL 90 ng/mL 450 ng/mL
Clenbuterol
<10.5
<3.3
<6.2
<15.1
12.6
8.4
5.8
11.0
19-Norandrosterone
<12.4 <11.6
<12.5
17.9
16.3
9.4
12.0
14.1
Nandrolone
NA <14.2
<12.3
<13.0
NA
12.7
10.0
10.4
Methandrosterone
<13.1 <11.9
<13.9
<18.3
11.5
12.7
13.9
17.3
6
β
-Hydroxyboldenone
<7.9
<13.3
<11.5
<20.0
14.5
10.8
9.5
13.6
Boldenone
<15.1
<9.4
<11.1
<12.9
12.6
11.3
16.1
18.2
DHEA
<16.6 <13.4
<10.5
<9.7
14.2
10.2
10.2
8.9
Oxandrolone
<11.0 <14.4
<12.9
<19.9
10.6
10.4
10.2
13.7
Testosterone
<14.6
<9.0
<11.9
<19.1
11.5
7.6
9.5
16.7
Epitestosterone
<16.4 <14.4
<10.8
<13.2
14.3
9.8
7.7
8.3
Formestane
<10.4 <10.6
<10.0
<18.1
18.7
13.5
14.3
19.9
Stanozolol
<20.9 <10.9
<10.5
<15.2
19.9
10.9
8.2
13.1
THG
<19.5 <10.1
<11.0
<16.9
16.3
11.1
7.5
13.4
Oxymesterone
<25.0
<12.3
<6.0
<15.0
24.5
9.0
4.9
12.6
Clostebol
<14.8 <12.4
<10.3
<12.8
14.1
11.0
6.6
9.7
Fluoxymesterone
<18.0
<9.6
<11.6
<19.2
24.0
9.0
7.5
14.0
3-Hydroxystanozolol
<15.1
<5.0
<5.3
<12.5
24.8
8.0
5.8
11.0
6
β
-Hydroxyfluoxymesterone
NA
<12.8
<6.5
<13.7
NA
9.1
9.4
14.2