1. Clinical Research Use Only Not For Diagnostic Purposes
Validation of PerkinElmer Steroids Kit using Exactive Ultrahigh Resolution LC/MS System
Clinical Research Applications Group Thermo Fisher Scientific San Jose, CA
Clinical Research Use Only Not For Diagnostic Purposes

2. Clinical Research Use Only Not For Diagnostic Purposes
Overview
Results Summary for 10 Steroid Panel in Perkin Elmer Kit
Calibrators
Controls
Internal Standards
Sample Preparation Protocol
Method Details
Conclusion
Appendix
Note: Perkin Elmer Kit is available for sale in Europe only
Due limited reagent availability the evaluation could be
done by running samples in duplicate only.
Clinical Research Use Only Not For Diagnostic Purposes

3. Clinical Research Use Only Not For Diagnostic Purposes
Accuracy and Precision % (2 replicates)
ALDO
ADIONE
CCONE
CORT
11-DELCO
Accuracy
Precision
Cal 1 NA
6.7
21.8
-1.2
7.6
1.5
5.3
-2.8
17.1
Cal 2
0.8
14.8
-3.8
3.1
2.0
5.0
-3.2
3.4
8.0
11.1
Cal 3
-1.4
-0.7
9.3
2.6
2.9
-2.1
1.4
-0.6
7.7
Cal 5
-4.2
4.9
1.8
-3.0
9.6
-3.9
9.0
-8.2
3.0
Cal 6
1.3
9.2
1.7
2.4
5.5
2.1
3.3
4.6
1.2
Cal 7
3.6
24.1
-0.1
7.5
-5.8
10.4
4.4
-1.0
9.7
Control 1
23.3
19.5
-7.3
2.5
-2.4
9.9
-4.6
12.7
-20.5
5.6
Control 2
-6.7
0.1
-12.9
3.2
-1.8
6.9
-4.8
-10.0
8.1
Control 3
0.6
4.8
3.8
0.4
3.7
0.2
7.4
DHEA
DHEAS
PROG
17-OHP
TESTO
-0.9
0.7
10.8
-5.2
9.4
-6.1
43.8
5.7
5.8
-3.3
4.0
11.6
0.9
-8.6
4.5
-1.7
-7.4
13.3
7.9
-4.7
1.1
6.4
6.8
0.0
17.8
13.6
-0.5
12.2
15.2
20.6
10.0
-11.9
16.9
5.4
-13.3
2.3
-0.3
10.9
6.3
-0.8
15.0
4.3
2.7
7.0
2.8
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4. Clinical Research Use Only Not For Diagnostic Purposes
Calibrators
Analytes (ng/mL)
Calibrators L1 L2 L3 L4 L5 L6 L7
Aldosterone
0.03
0.06
0.16
0.39
1.00
2.59
6.23
4-Androstene-3,17-dione
0.08
0.19
0.47
1.16
2.91
7.49
18.20
Corticosterone
0.28
0.70
1.71
4.19
10.80
27.20
65.80
Cortisol
1.14
3.11
7.88
19.10
47.60
122.00
276.00
11-Deoxycortisol
0.07
0.18
1.18
3.03
7.64
18.60
Dehydroepiandrosterone
0.25
0.79
1.88
4.49
11.30
28.70
71.10
Dehydroepiandrosterone sulfate
9.32
24.00
60.40
149.00
386.00
983.00
Progesterone
0.13
0.29
0.68
1.63
4.09
10.50
25.00
17a-Hydroxyprogesterone
0.12
0.30
1.72
4.35
10.90
25.70
Testosterone
0.46
1.17
2.98
7.21
Note: 6 vials lyophilized serum. Concentrations after reconstitution in 2ml DI water. L4 not available
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5. Clinical Research Use Only Not For Diagnostic Purposes
Controls
Controls (ng/mL)
Note: 3 vials lyophilized serum. Concentrations after reconstitution in 2mL DI water.
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6. Clinical Research Use Only Not For Diagnostic Purposes
Internal Standards
Stable Isotope Internal Standards in Lyophilized Form
Note: Int. standards in 1 vial, dry
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7. Clinical Research Use Only Not For Diagnostic Purposes
Sample Preparation
200 uL of DPS uL of serum, calibrator or control
Shake for 15 min
Centrifuge 5 min 13,000 rpm
Dry 175 uL of the supernatant, 15 min at 36C, N2 level 15
Reconstitute with 125 mL of sample reconstitution solution
Shake for 2 min.
Samples stored at -30C before they are ready for injection
Inject 100 mL
DPS - Daily Precipitation Solution
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8. Clinical Research Use Only Not For Diagnostic Purposes
M/Z used for XIC (5 ppm)
Compound
m/z
Internal Standard
ALDO (-)
D7 ALDO
ADIONE
D5 ADIONE
CCONE
D8 CCONE
CORT
D3 CORT
11-DELCO
D5 11-DELCO
DHEA
D5 TESTO
DHEAS
D6 DHEAS
PROG
D9 PROG
17-OHP
D8 17-OHP
TESTO
XIC – Extracted Ion Chromatogram
Clinical Research Use Only Not For Diagnostic Purposes

9. Aldosterone (Negative mode)
Cal 2 (64 pg/mL)
6.4 pg on column.
ng/mL
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10. Clinical Research Use Only Not For Diagnostic Purposes
4-Androstene-3,17-dione
Cal 1 (76 pg/mL)
7.6 pg on column.
ng/mL
Clinical Research Use Only Not For Diagnostic Purposes

11. Clinical Research Use Only Not For Diagnostic Purposes
Corticosterone
Cal 1 (283 pg/mL)
28.3 pg on column.
ng/mL
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12. Clinical Research Use Only Not For Diagnostic Purposes
Cortisol
Cal 1 (1.14 ng/mL)
114 pg on column.
ng/mL
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13. Clinical Research Use Only Not For Diagnostic Purposes
11-Deoxycortisol
Cal 1 (70 pg/mL)
7 pg on column
ng/mL
Clinical Research Use Only Not For Diagnostic Purposes

14. Dehydroepiandrosterone
Cal 2 (248 pg/mL)
24.8 pg on column.
ng/mL
Clinical Research Use Only Not For Diagnostic Purposes

15. Dehydroepiandrosterone sulfate
Cal 1 (9.32 ng/mL)
932 pg on column.
ng/mL
Clinical Research Use Only Not For Diagnostic Purposes

16. Clinical Research Use Only Not For Diagnostic Purposes
Progesterone
Cal 1 (128 pg/mL)
12.8 pg on column
ng/mL
Clinical Research Use Only Not For Diagnostic Purposes

17. 17a-Hydroxyprogesterone
Cal 1 (123 pg/mL)
12.3 pg on column
ng/mL
Clinical Research Use Only Not For Diagnostic Purposes

18. Clinical Research Use Only Not For Diagnostic Purposes
Testosterone
Cal 1 (33 pg/mL)
3.3 pg on column.
ng/mL
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19. Clinical Research Use Only Not For Diagnostic Purposes
Conclusions
Exactive LC-MS system is capable of quantitatively analyzing 10 steroids in plasma samples using Perkin Elmer reagents kit
The LOQ and precision determined using 2 replicates indicates a very feasible method for routine analysis
More inter assay performance data is needed to confirm the above findings
Note: Perkin Elmer Kit is available for sale in Europe Only
Clinical Research Use Only Not For Diagnostic Purposes

20. Clinical Research Use Only Not For Diagnostic Purposes
Appendix
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21. LC-MS System Configuration
Exactive with APCI probe
Accela pump
CTC autosampler
Column - Phenomenex Luna (C8), 50 x 3.0 mm, 3 mm
Phenomenex SecuriGuard analytical guard holder kit
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22. Clinical Research Use Only Not For Diagnostic Purposes
LC Method
Column - Phenomenex Luna (C8), 50 x 3.0 mm, 3 mm
Phenomenex SecuriGuard analytical guard holder kit
Column Temperature - Ambient
Mobile Phase and gradient
A – 2% Methanol in water (0.025% PE solvent additive)
B – Methanol (0.025% PE solvent additive)
Time (min) %A %B
Flow (mL/min) 65 35
600 3 4 55 45 12
12.1
100 15
15.1 18
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23. Clinical Research Use Only Not For Diagnostic Purposes
MS Method
APCI probe
Spray voltage: 5000 V (positive or negative polarity)
Vaporizer temp: 350C
Sheath Gas: 21
Aux Gas: 5
Ion Sweep Gas: 0
Capillary temp: 270C
Probe position: Between B and C
Divert valve: first 3 min of analysis time to waste, then to Mass Spectrometer for the rest of acquisition time
2-Segment data acquisition method at 100,000 resolution with max injection time of 250 ms
1st segment (0-6 min): Negative polarity, scanning from 358 to 370 m/z
2nd segment (7-18 min): Positive polarity, scanning from 270 to 370 m/z
Clinical Research Use Only Not For Diagnostic Purposes