POSTER NOTE 64927

A Simple and Fast Solid Phase Extraction Method

for Analysis of Eleven Steroids in Serum Using
A Simple and Fast Solid Phase Extractio
LC-MS/MS Xiaolei Xie, Joe Di Bussolo, Thomas Carrell, Marta Kozak, Thermo Fisher Scientific, San J

Authors ABSTRACT
Xiaolei Xie, Joe Di Bussolo, Purpose: To develop a simple and fast sample preparation method for LC-MS/MS analysis of 11 steroids in serum for
clinical research.
Thomas Carrell, and Marta Kozak,
Methods: A solid phase extraction (SPE) method was developed on a Thermo Scientific™ SOLAµ™ HRP 96-well plate
Thermo Fisher Scientific, to simultaneously extract 11 steroids from serum (11-deoxycortisol, 17-OH progesterone, aldosterone, androstenedione,
corticosterone, cortisol, cortisone, estradiol, estrone, progesterone and testosterone). Extracted compounds were
San Jose, CA, USA separated on a reverse phase column chromatographically followed by analysis on a Thermo Scientific ™ TSQ
Quantiva™ triple quadrupole mass spectrometer.
Results: The entire SPE process takes less than 20 minutes, and no pre-conditioning, evaporation or reconstitution is
required. Comparing to conventional SPE method which involves those steps, our method is simpler and faster. The
recovery rate ranged from 42% (aldosterone) to 95% (testosterone). In neat solution, lower limit of quantitation (LOQ) of
androstenedione is 1 pg/mL. LOQ of testosterone is 2 pg/mL. LOQ of 11-deoxycortisol, 17-OH progesterone, cortisone,
estradiol, estrone, and progesterone is 5 pg/mL. LOQ of aldosterone, corticosterone, and cortisol is 10 pg/mL.

INTRODUCTION
Over the past few years there has been a growing interest to use LC-MS/MS method to measure steroids in serum. LC-
MS/MS offers the potential for more reliable measurement compared to other detection systems, such as
immunoassays. However, the challenges are time-consuming sample pre-treatment, matrix interference and isobars
separation. We developed a simple and fast SPE sample preparation method for an 11-steroid panel and evaluated the
analytical performance on an LC-triple quadrupole MS/MS system.

MATERIALS AND METHODS

Sample Preparation
SPE was performed on a SOLAµ HRP 96-well plate. Calibrators/QCs were spiked into neat solution or charcoal stripped
serum and mixed with water and methanol. The mixture was loaded directly onto the SPE plate; no preconditioning was
required. After washing the plate with 30% methanol, the elution was performed with two volumes of 25 µL of methanol
each. Eluates were diluted with 50 µL water. 50 µL of the diluted eluate was injected for LC-MS/MS analysis.
 serum and mixed with water and methanol. The mixture was loaded directly onto the SPE plate; no preconditioning was
Estradiol-d5 3.3 1 Negative
required. After washing the plate with 30% methanol, the elution was performed with two volumes of 25 µL of methanol
each. Eluates were diluted with 50 µL water. 50 µL of the diluted eluate was injected for LC-MS/MS analysis. Estradiol-d5 3.3 1 Negative
Estrone 3.4 1 Negative
Estrone 3.4 1 Negative
Estrone-13C3 3.3 1 Negative
Estrone-13C3 3.3 1 Negative
Progesterone 3.7 1 Positive
Progesterone 3.7 1 Positive
Progesterone-d9 3.7 1 Positive
Progesterone-d9 3.7 1 Positive
Testosterone 3.4 1 Positive
Testosterone 3.4 1 Positive
Testosterone-13C3 3.4 1 Positive
Testosterone-13C3 3.4 1 Positive

Figure 4. SRM transitions (two for each analyte/IS)

RESULTS
RT: 0.00 - 4.21
RT: 3.27 NL: 1.33E4
100 11-dexoycort
Base Peak m/z= 96.50-97.
c ESI SRM ms2 347.300
5 pg/mL
[96.999-97.001,
3.46 3.65
50
3.16 347.3 -> 97
108.999-109.001] MS
0331_neat_10

Figure 1. SPE process to extract 11 steroids from plasma. 0

RT: 3.80 NL: 1.52E5
100
Base Peak m/z= 108.50-10
17-OH-P
+ c ESI SRM ms2 331.200
[96.999-97.001,
50 5 pg/mL
109.099-109.101] MS ICIS
331.2 -> 109
0331_neat_5
Test Method(s) 3.39

0
RT: 3.03 NL: 6.54E2
100

Relative Abundance
3.33 Aldosteron
Base Peak m/z= 188.60-18
- c ESI SRM ms2 359.300

50
3.36
10 pg/mL
[189.099-189.101,
331.099-331.101] MS ICIS
2.94
3.48 359.3 -> 189
0331_neat_10

0
RT: 3.36 NL: 1.04E4
100
Base Peak m/z= 96.50-97.
Androstenedi
c ESI SRM ms2 287.200

50
3.07
3.11
3.67
1 pg/mL
[96.999-97.001,
109.099-109.101] MS ICIS
2.89 287.2 -> 97
neat_1

0
RT: 3.24 NL: 6.81E3
100 3.47
Corticostero
Base Peak m/z= 120.60-12
+ c ESI SRM ms2 347.300

50
3.08 3.61 10 pg/mL
[121.099-121.101,
147.099-147.101] MS nea
347.3 -> 121
0
RT: 3.12 NL: 5.86E3
100
Cortisol
Base Peak m/z= 120.60-12
+ c ESI SRM ms2 363.200

50
2.90
3.43
10 pg/mL
[121.099-121.101,
309.199-309.201] MS nea
363.2 -> 309
0
0 1 2 3 4
Time (min)

Figure 5. Chromatograms on the lower limit of quantita

Figure 2. Thermo Scientific™ UltiMate™ 3000 HPLC and TSQ Quantiva MS with heated ESI source
11-dexoycortisol

5 – 10,000 pg/mL

Androstenedione

1 – 10,000 pg/mL

Figure 3. LC gradient (Column: Thermo Scientific™ Accucore™ RP-MS, 2.6 µm, 100 X 2.1 mm)
2
 RT RT Window Precursor Collision
Compound (min) (min) Polarity (m/z) Product (m/z) Energy (V) RF Lens (V)
for 11-deoxycortisol 3.2 1 Positive 347.3 97 25 72 Cortisol
11-deoxycortisol 3.2 1 Positive 347.3 109 28 72
plate 11-Deoxycortisol-d5 3.2 1 Positive 352.3 100 25 71
one,
10 – 10,000 pg/mL
11-Deoxycortisol-d5 3.2 1 Positive 352.3 113 30 71
17-OH-P 3.4 1 Positive 331.2 97 24 69
17-OH-P 3.4 1 Positive 331.2 109.1 27 69
17OH-P-d8 3.4 1 Positive 339.3 100 26 70
is
e 17OH-P-d8 3.4 1 Positive 339.3 113 29 70
Q) of Aldosterone 3 1 Negative 359.3 189.1 17 69
one, Aldosterone 3 1 Negative 359.3 331.1 15 69
Androstenedione 3.3 1 Positive 287.2 97 23 64
Androstenedione 3.3 1 Positive 287.2 109.1 25 64
Androstenedione-
13C3 3.3 1 Positive 290.2 100.1 22 65
Androstenedione-
LC- 13C3 3.3 1 Positive 290.2 112 25 65
Estradiol
s Corticosterone 3.2 1 Positive 347.3 121.1 25 68
the Corticosterone 3.2 1 Positive 347.3 147.1 26 68
5 – 10,000 pg/mL
Cortisol 3 1 Positive 363.2 121.1 26 71
Cortisol 3 1 Positive 363.2 309.2 20 71
Cortisol-d4 3 1 Positive 367.3 121.1 26 72
Cortisol-d4 3 1 Positive 367.3 175.1 27 72
Cortisone 3 1 Positive 361.2 121.1 31 78
Cortisone 3 1 Positive 361.2 163 24 78
Estradiol 3.3 1 Negative 271.2 145 41 108
pped Estradiol 3.3 1 Negative 271.2 183 40 108
was
Estradiol-d5 3.3 1 Negative 276.1 147 40 115
nol
Estradiol-d5 3.3 1 Negative 276.1 187 41 115
Estrone 3.4 1 Negative 269.2 145.1 40 90
Estrone 3.4 1 Negative 269.2 183.1 40 90 Progesterone
Estrone-13C3 3.3 1 Negative 272.2 146 54 106
Estrone-13C3 3.3 1 Negative 272.2 148.1 39 106 5 – 10,000 pg/mL
Progesterone 3.7 1 Positive 315.3 97.1 22 68
Progesterone 3.7 1 Positive 315.3 109.1 25 68
Progesterone-d9 3.7 1 Positive 324.5 100.1 22 67
Progesterone-d9 3.7 1 Positive 324.5 113 27 67
Testosterone 3.4 1 Positive 289.3 97 22 64
Testosterone 3.4 1 Positive 289.3 109.1 25 64
Testosterone-13C3 3.4 1 Positive 292.3 100 23 66
Testosterone-13C3 3.4 1 Positive 292.3 112 25 66
Figure 6. Linearity range
Figure 4. SRM transitions (two for each analyte/IS)

Compound SPE
RESULTS 11-deoxycortisol
11-Deoxycortisol-d5
RT: 0.00 - 4.21
RT: 3.27 NL: 1.33E4
RT: 2.30 - 4.21
RT: 3.08 NL: 2.62E3
17-OH-P
100 11-dexoycortisol
Base Peak m/z= 96.50-97.50 F: +
100
Base Peak m/z= 162.50-163.5
17OH-P-d8
c ESI SRM ms2 347.300
5 pg/mL 80 Cortisone
c ESI SRM ms2 361.200
[121.099-121.101,
[96.999-97.001, 3.35
50 3.46 3.65
347.3 -> 97.0
108.999-109.001] MS 60
2.95 3.26 3.42 5 pg/mL
162.999-163.001] MS neat_5 Aldosterone
3.16 2.84 2.87
0331_neat_10 3.48
40
2.72
361.2 -> 163.0 Androstenedione
0 20
RT: 3.80 NL: 1.52E5
100
Base Peak m/z= 108.50-109.50 F:
17-OH-P
+ c ESI SRM ms2 331.200
0
RT: 3.34 NL: 4.81E2 Androstenedione-13C3
100
Base Peak m/z= 182.50-183.5
[96.999-97.001,
50 5 pg/mL
109.099-109.101] MS ICIS 80 Estradiol
c ESI SRM ms2 271.200 Corticosterone
[144.999-145.001,
3.39 331.2 -> 109.1
0331_neat_5
60 5 pg/mL
182.999-183.001] MS ICIS ne
Cortisol
0
RT: 3.03 NL: 6.54E2
40 271.2 -> 183.0 Cortisol-d4
100
Relative Abundance

3.33 Aldosterone
Base Peak m/z= 188.60-189.60 F:
- c ESI SRM ms2 359.300
20
Cortisone
0
3.36
10 pg/mL
[189.099-189.101, RT: 3.37 NL: 6.17E2
50 331.099-331.101] MS ICIS 100
Base Peak m/z= 144.50-145.5 Estradiol
2.94
359.3 -> 189.1
Relative Abundance

3.48 0331_neat_10
80 Estrone
c ESI SRM ms2 269.200
[145.099-145.101, Estradiol-d5
0 60 5 pg/mL
183.099-183.101] MS ICIS

100
RT: 3.36 NL: 1.04E4
Base Peak m/z= 96.50-97.50 F: + 40
0331_neat_5
269.2 -> 183.1 Estrone
Androstenedione
c ESI SRM ms2 287.200
20 Estrone-13C3
50
3.07
3.11
3.67
1 pg/mL
[96.999-97.001,
109.099-109.101] MS ICIS
2.89 287.2 -> 97.0
neat_1
0
100
RT: 3.97 NL: 3.98E4 Progesterone
Base Peak m/z= 108.60-109.6

0 80 c ESI SRM ms2 315.300

Progesterone
[97.099-97.101, 109.099-109.
Progesterone-d9
RT: 3.24 NL: 6.81E3
100 3.47
Corticosterone
Base Peak m/z= 120.60-121.60 F: 60
5 pg/mL
MS neat_5
Testosterone
+ c ESI SRM ms2 347.300
3.08 3.61 10 pg/mL
[121.099-121.101,
40
3.73 315.3 -> 109.1 Testosterone-13C3
50 147.099-147.101] MS neat_10 20 3.24 3.33 4.09
347.3 -> 121.1 3.48
0
RT: 3.48 NL: 8.88E4
0
RT: 3.12 NL: 5.86E3
100
3.10 TIC F: + c ESI SRM ms2 289.3 Figure 7. Recovery rate and lower limit of quantitat
100 80 Testosterone
[96.999-97.001, 109.099-109.
Cortisol
Base Peak m/z= 120.60-121.60 F: MS 0331_neat_10
+ c ESI SRM ms2 363.200 60 2 pg/mL
2.90
3.43
10 pg/mL
[121.099-121.101,
50 309.199-309.201] MS neat_10 40 3.31 3.68 3.88
289.3 -> 97.0
363.2 -> 309.2 20

0 0
0 1 2 3 4 2.5 3.0 3.5 4.0
Time (min) Time (min)
3
CONCLUSIONS
Figure 5. Chromatograms on the lower limit of quantitation level in neat solution
 A simple and fast SPE method (~20 min) for clinica
serum with recovery rate ranged from 42% to 95%
 0 1 2 3 4 2.5 3.0 3.5 4.0
Time (min) Time (min)

CONCLUSIONS
Figure 5. Chromatograms on the lower limit of quantitation level in neat solution
 A simple and fast SPE method (~20 min) for clini
serum with recovery rate ranged from 42% to 95%
 A sensitive LC-MS/MS analytical method was dem
ranged from 1 pg/mL to 10 pg/mL.

11-dexoycortisol 17-OH-P

5 – 10,000 pg/mL 5 – 10,000 pg/mL TRADEMARKS/LICENSING

© 2016 Thermo Fisher Scientific Inc. All rights reser
its subsidiaries. This information is not intended to e
intellectual property rights of others.

FOR RESEARCH USE ONLY. NOT FOR USE

PO64927-EN

Androstenedione Corticosterone

1 – 10,000 pg/mL 10 – 10,000 pg/mL

roids in Serum Using LC-MS/MS

ens (V)
72 Cortisol Cortisone
72
71
10 – 10,000 pg/mL 5 – 10,000 pg/mL
71
69
69
70
70
69
69
64
64

65

65
Estradiol Estrone
68
68
5 – 10,000 pg/mL 5 – 10,000 pg/mL
71
71
72
72
78
78
108
108
115
115
90
90 Progesterone Testosterone
106
106 5 – 10,000 pg/mL 2 – 10,000 pg/mL
68
68
67
67
64
64
66
66
Figure 6. Linearity range
4

Compound SPE Recovery Rate (%) Lower Limit of Quantitation (pg/mL)

 Figure 6. Linearity range

Compound SPE Recovery Rate (%) Lower Limit of Quantitation (pg/mL)

11-deoxycortisol 63 5
11-Deoxycortisol-d5 64
17-OH-P 84 5
m/z= 162.50-163.5
one
ms2 361.200 17OH-P-d8 83
1.101,
mL
3.001] MS neat_5 Aldosterone 42 10
163.0 Androstenedione 93 1

Androstenedione-13C3 95
m/z= 182.50-183.5

diol
ms2 271.200 Corticosterone 67 10
5.001,

mL
3.001] MS ICIS ne
Cortisol 68 10
183.0 Cortisol-d4 70
Cortisone 71 5
m/z= 144.50-145.5 Estradiol 87 5
ne
ms2 269.200
5.101, Estradiol-d5 88
mL
3.101] MS ICIS
5
183.1 Estrone 89 5
Estrone-13C3 89
Progesterone 84 5
m/z= 108.60-109.6
ms2 315.300
erone
101, 109.099-109.
Progesterone-d9 86
mL Testosterone 95 2
109.1 Testosterone-13C3 97

SI SRM ms2 289.3 Figure 7. Recovery rate and lower limit of quantitation
erone
001, 109.099-109.
eat_10
mL
> 97.0

CONCLUSIONS
 A simple and fast SPE method (~20 min) for clinical research was develop to simultaneously extract 11 steroids from
serum with recovery rate ranged from 42% to 95%.
 A sensitive LC-MS/MS analytical method was demonstrated to analyze 11 steroids with lower limit of quantitation
ranged from 1 pg/mL to 10 pg/mL.

TRADEMARKS/LICENSING
© 2016 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and
its subsidiaries. This information is not intended to encourage use of these products in any manner that might infringe the
intellectual property rights of others.

FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.

PO64927-EN

For research use only. Not for use in diagnostic procedures.

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© 2017 Thermo Fisher Scientifi c Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientifi c and its
subsidiaries unless otherwise specifi ed. PN64927 EN 0217S