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7/28/2019 SSI-008
1/4
Summary
Sensitivity was tested for four steroids on the LCMS-
8030 triple quadrupole mass spectrometer coupled
with a Nexera UHPLC.
Background
Steroids are powerful hormones that are needed fornormal biological activity but may also be present in
the environment. Water contaminated with steroids
poses a serious environmental and public health
threat. Sensitive and rapid methods are needed for
accurate quantitative analysis. UHPLC-MS-MS meth-
ods for analysis of these compounds were developed
and the sensitivity for each analyte was tested.
Method
Standards for four steroids were obtained at a concen
tration of 1 mg/mL in methanol. The
standards were: Testosterone (T4), Progesterone (P4
Estradiol (E2), and Ethinylestradiol (EE2). Standards
were diluted in 50/50 water/acetonitrile for optimizatio
of mass spectrometry parameters.
Multiple Reaction Monitoring (MRM) was used for
analysis. Collision energy, quadrupole prerod bias
voltage, and other MS parameters were optimized by
flow injection of the individual standards. T4 and P4
ionized most efficiently using a 0.1% formic
acid/acetonitrile mobile phase using the DUIS dual io
Figure 1: Structures of tested steroidsTable 1: Dilution levels (ng/mL)
Table 2: MRM parameters
Testosterone Progesterone
Estradiol Ethinylestradiol
O
OH
H
H
H
O
O
H
H
H
HO
OH
H
H
H
HO
OH
H
H
H
Type Event# +/- Compound Name (m/z) Dwell Time (msec) Q1 Pre Bias(V) CE Q3 Pre Bias(V)
MRM 1 + Testosterone 288.9>97 80 -14 -25 -10
MRM 2 + Progesterone 315.4>109.1 80 -12 -30 -20
Type Event# +/- Compound Name (m/z) Dwell Time (msec) Q1 Pre Bias(V) CE Q3 Pre Bias(V)
MRM 1 - Estradiol 271.4>183.1 50 13 41 30
MRM 2 - Ethinylestradiol 295.4>145 50 14 47 28
Measurement Time (min)
0-2
0-2
Measurement Time (min)
0-2
0-2
Level T4 P4 E2 EE2
L1 370.3 370.3 10000 10000
L2 123.5 123.5 3333 3333
L3 41.17 41.17 1111 1111
L4 13.72 13.72 370.3 370.3
L5 4.57 4.57 123.5 123.5
L6 1.52 1.52 41.17 41.17
L7 0.582 0.582 13.72 13.72
L8 4.57 4.57
Steroids: Testosterone, Progesterone, Estradiol,
Ethinylestradiol
ApplicationNews
No. SSI-008
Liquid Chromatography Mass Spectrometry
7/28/2019 SSI-008
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spray (ESI with corona discharge) source in positive
mode. The MRM transition ofm/z288.997 was
monitored for T4 and the transition ofm/z315.4109.1 was used for P4.
E2 and EE2 ionized most efficiently using a
water/acetonitrile mobile phase with atmospheric
pressure chemical ionization (APCI) in negative mode.
Therefore these compounds were analyzed in a
separate method. The MRM transitions for E2 and EE2
were 271.4183.1 and 295.4145.0 respectively.
A Shimadzu Shimpack-XR ODS III column (1.6 m, 2
50 mm) was used for analysis. A binary gradient of
0.1% formic acid and acetonitrile was used for T4 an
P4 while a binary gradient of water and acetonitrilewas used for E2 and EE2. The gradient began at 55
acetonitrile and increased to 95% over 1 minute. Aft
a 6 second isocratic hold, the column was
re-equilibrated for approximately 1 minute. The colu
temperature was 40 C and the injection volume wa
10 L.
Calibration curves were prepared by serial dilution o
mixed standard as shown in Table 1.
110504.SetEPAx01_
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P4 r2 = 0.999 T4 r 2 = 0.991
Figure 2: Calibration curves and selected mass chromatograms for P4 and T4
P4 Level 7
P4 Level 2
T4 0.065 ng/mL
T4 Level 2
0 100 200 300 Conc.0.0
1.0
2.0
3.0
Area(x100,000)
0 100 200 300 Conc0.00
0.25
0.50
0.75
1.00
Area(x1,000,000)
0.0 0.5 1.0 1.5
0.0
1.0
2.0
3.0
4.0
5.0(x100)
1:315.40>109.10(+)
0.
992
0.5 1.0 1.5
0.0
0.5
1.0
1.5
(x100)2:288.90>97.00(+)
0.
616
110504.SetEPA
x01_
8_
EPAMIX2L12_
008.lcd
0.0 0.5 1.0 1.5
0.00
0.25
0.50
0.75
1.00
(x100,000)1:315.40>109.10(+)
0.
998
110504.SetEPAx01_
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029.lcd
110504.SetE
PAx01_
29_
EPAMIX2L05_
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0.5 1.0 1.5
0.0
0.5
1.0
1.5
2.0
2.5
3.0(x100,000)
2:288.90>97.00(+)
0.
620
110504.SetEPAx01.
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Results and Discussion
Representative chromatograms of the T4/P4 andE2/EE2 methods are shown in Figures 2 and 3. The
limit of detection (LOD, S/N > 3) for P4 was between
0.194 and 0.582 ng/mL using a 10 L injection, and the
limit of quantitation (LOQ, S/N >10) was approximately
0.5 ng/mL. For T4, the LOD was approximately
0.065 ng/mL and the LOQ was approximately
0.2 ng/mL. For T4 and P4, the calibration curve was
constructed between 0.194 and 370 ng/mL. The curves
were linear in the tested range (r2 > 0.99).
For E2, the LOD was approximately 1.5 ng/mL and th
LOQ was approximately 4.5 ng/mL. The LOD and LOfor EE2 was similar. The calibration curves were
constructed from 4.57 ng/mL up to 10 g/mL, and the
curves were both linear (r2 > 0.99)
Conclusion
Rapid and sensitive methods for
determination of four steroids was developed using
UHPLC-MS-MS.
E2 r2 > 0.999 EE2 r2 > 0.999
Figure 3: Calibration curves and selected mass chromatograms for E2 and EE2
E2 1.52 ng/mL
E2 Level 3
EE2 1.52 ng/mL
EE2 Level 3
0 2500 5000 7500 Conc.0.0
1.0
2.0
Area(x100,000)
0 2500 5000 7500 Conc.0.0
0.5
1.0
1.5
Area(x100,000)
110505.SetEP
Ax02.lcb
110505.SetEPA
x02.lcb
0.0 0.5 1.0 1.5
0.0
1.0
2.0
3.0
4.0
(x10)1:271.40>183.10(-)
0.
209
0.0 0.5 1.0 1.5
0.0
0.5
1.0
1.5
2.0
2.5
(x10,000)1:271.40>183.10(-)
0.
214
110
505.SetEPAx02_
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etEPAx02_
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011.lcd
0.5 1.0 1.5
0.00
0.25
0.50
0.75
1.00
1.25
1.50(x10,000)
2:295.40>145.00(-)
0.
234
0.5 1.0 1.5
0.0
0.5
1.0
1.5
2.0
2.5
(x10)2:295.40>145.00(-)
0.
234
110505.SetE
PAx02_
10_
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First Edition: November 20
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pplicationews
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