LCMSMS Solutions For Steroid Analysis
Dan Blake
Manager, Clinical Applications
For Research Use Only. Not for use in diagnostic procedures.
RUO-MKT-18-1166-A
Agenda
• Special considerations for steroid analysis
• Methods of Steroid analysis 1: Simple & Fast
‒ Testosterone iMethod™
‒ Online Extraction with 2DLC and simple sample processing
‒ Low Level Estrogen iMethod™
• Improving Selectivity with Ion Mobility
• Methods of Steroid Analysis 2: Extending the coverage
‒ Steroid Panel iMethod™
‒ Commercial Kits
• Summary of and conclusions from the different approaches
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© 2016 SCIEX RUO-MKT-18-1166 A
Special considerations for steroid
analysis
• Traditional immunoassays, while convenient, are unreliable for
reasons previously discussed
• Varying sample volumes available
‒ Paediatric/neonatal samples
• Pressures of sample number and turnaround time demand a
fast and easy to setup method
• Again, similar pressures require the use of rapid, automatable
extraction procedures
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Ideally some form of protein precipitation
Liquid Liquid extraction is an option but not appropriate for all laboratories
SPE is an option but can be costly
Derivitisation, for example with dansyl chloride, is an option for challenging
steroids but should be considered as a last resort.
© 2016 SCIEX RUO-MKT-18-1166 A
• What Steroids to analyse?
 The LC/MS/MS method should give us as much information as
possible
 However, increasing the number of analytes could potentially
increase the complexity of the assay
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© 2016 SCIEX RUO-MKT-18-1166 A
The “ideal” LC/MS/MS Steroid Application
• The convenience of an immunoassay style kit approach
• Sensitive enough to assay the lowest level steroids
‒ The “which instrument” part of the question
• Able to cope with sample volume and throughput demands
• The highest control
‒ Wide dynamic range standard curve
‒ Deuterated internal standards, for each analyte if possible
‒ Ability to utilise traceable Quality Control material
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© 2016 SCIEX RUO-MKT-18-1166 A
Testosterone iMethod™
• Testosterone is one of the most widely measured steroids
• Measurement of testosterone is used to research a variety of
disorders, such as congenital adrenal hyperplasia and
hypogonadism.
• Measurement of the typical levels in men is straightforward;
however, measurement of the low levels present in women and
children can be especially challenging.
• SCIEX provides solutions for routine analysis for both
circumstances.
‒ Simple male testosterone assay on the API3200™ platform using protein
precipitation
‒ Low level (female) assay on the API5000™ or Triple Quad 5500™ using
more thorough sample preparation
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© 2016 SCIEX RUO-MKT-18-1166 A
Testosterone iMethod™, API3200™
Data from a 3.4 nMol/L testosterone sample analyzed using an SCIEX API
3200™ LC/MS/MS System.
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© 2016 SCIEX RUO-MKT-18-1166 A
Testosterone iMethod™, API5000™
Data from a 0.069 nMol/L testosterone sample analyzed using an SCIEX
API 5000™ LC/MS/MS System.
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© 2016 SCIEX RUO-MKT-18-1166 A
Testosterone iMethod™
Online Extraction with 2DLC and simple sample
processing
• A simple method for analysis of a small number of routinely
measured steroids has been proposed
• The method employs a simple protein precipitation with direct
injection onto a 2DLC system (no need for offline sample
concentration)
• The remainder of the sample cleanup process is performed on a
trapping column, followed by a reversed flow gradient onto a
common C18 HPLC column, with a total runtime of 5 minutes
• As a consequence, sample preparation time is kept to a
minimum and sample throughput is maximised
• The trap column is reusable, with a lifetime in excess of 1000
injections
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© 2016 SCIEX RUO-MKT-18-1166 A
Proposed Flow and Plumbing diagram for
2DLC Steroids method
GRADIENT PUMPS A/B
Column Oven
TRAPPING
COLUMN
MS System
Analytical Column
Switching
Valve
5
AUTOSAMPLER
VALVE
6
ISOCRATIC LOAD PUMP C
1
10
2
9
3
Valve position A:
(Analyte Loading onto Trapping Column)
Switching Valve:
Time(min)
Valve
Position
4
8
7
5
6
WASTE
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© 2016 SCIEX RUO-MKT-18-1166 A
WASTE
Valve position B:
0.0
A
(Analyte Elution from SPE Column onto
Analytical Column)
1.0
B
5.5
A
Extracted Steroid sample, approx 0.15nmol/L, Analysed
by 2DLC approach
Testosterone
17-OH-P
11-Deoxycortisol
Androstenedione
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© 2016 SCIEX RUO-MKT-18-1166 A
Low Level Estrogen iMethod™
The Importance of Estrogens
• Estrogen is an entire class of related female sex hormones: estrone (E1)
estradiol (E2), and estriol (E3)
• Estrone (E1) is widespread throughout the body. It is the only one of
estrogens that’s present in any amount in women after menopause.
• Estradiol (E2) is the primary sex hormone of childbearing women. It is
formed from developing ovarian follicles. Estradiol is responsible for
female characteristics and sexual functioning. Also, estradiol is
important to women’s bone health. Estradiol contributes to most
gynecologic problems and even female cancers.
• Estriol (E3) is made from the placenta. It’s important only during
pregnancy.
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© 2016 SCIEX RUO-MKT-18-1166 A
Estrone and Estradiol:
Conditions:
• Instrumentation
‒ API 5000™ and SCIEX Triple Quad™ 5500 Mass Spectrometers
‒ Shimadzu Prominence 20A and Agilent 1200 integrated systems
• Method Information
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500 µL sample volume required
Liquid-liquid extraction without derivatization
Clinically relevant dynamic range of 5-500 pg/mL
6 minute total run time
© 2016 SCIEX RUO-MKT-18-1166 A
Estrone and Estradiol: Lower Limit of Quantitation
Estrone 5 pg/mL LLOQ, S/N=22
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Estradiol 5 pg/mL LLOQ, S/N=19
Estrone and Estradiol: Precision and Accuracy
Estrone (E1)
Sample
(pg/mL)
Estradiol (E2)
%CV
% Accuracy
Day 1-3
Day 1-3
LLOQ (5)
9
104
Low QC (15)
3
High QC (100)
ULOQ (500)
Sample
(pg/mL)
%CV
% Accuracy
Day 1-3
Day 1-3
LLOQ (5)
10
101
104
Low QC (15)
4
96
3
101
High QC (100)
5
102
3
100
ULOQ (500)
4
101
•Estrone (inter-day)
•Estradiol (inter-day)
•LLOQ CV’s ≤ 9% ,QC’s CV’s ≤ 5%
•LLOQ CV’s ≤ 10% ,QC’s CV’s ≤ 5%
•Accuracy deviation ≤ 4%
•Accuracy deviation ≤ 4%
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© 2016 SCIEX RUO-MKT-18-1166 A
Estrone and Estradiol: Linearity
Estrone
Estradiol
Clinically relevant dynamic range 5-500 pg/ml, R≤ 0.999
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Estrone and Estradiol: Summary
• The method was verified internally across laboratories for
analytical analysis
• LLOQ with suitable signal to noise (20)
• Inter-day Precision and Accuracy
‒ LLOQ CVs ≤ 10% ,QCs CV’s ≤ 5%
‒ Accuracy Deviation ±4%
• Control samples at clinically relevant low and high
concentrations (5-500 pg/mL) with linear response r ≤ 0.999
• Liquid-Liquid extraction without derivatization
• 6 minutes run time
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© 2016 SCIEX RUO-MKT-18-1166 A
Improving Selectivity with Ion
Mobility
Ion Mobility to improve selectivity
• There are many different ways to achieve improved selectivity in applications of
LC-MS/MS, including:
‒ Improved chromatography
‒ Tandem mass spectrometry (MS/MS, or even higher MS3)
‒ High-resolution mass spectrometry
‒ More up-front sample cleanup
• In many applications of LC-MS/MS, mass measurements alone (even high
resolution) cannot provide sufficient selectivity
‒ Isobaric interferences may have the same “exact mass” as analyte ions
‒ Interferences may have common fragment ions
‒ Chemical background may pose a challenge
• The SelexION™ ion mobility technology provides an orthogonal means of
separating / filtering ions prior to, and independent of, MS/MS analysis.
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SelexION™ Ion Mobility Device Components:
Existing ion path
New hardware
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SelexION™ Ion Mobility Device Components:
1. Orifice Plate
2. Ion Mobility Cell
Robust, easy-to-install, hardware components:




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No tools required
No cables
No need to break vacuum
Installation in about 2 minutes
© 2016 SCIEX RUO-MKT-18-1166 A
3. Curtain Plate
Tuning the SelexION™ Ion Mobility Device
t2
ions in
transport gas

=0 0
to mass
spectrometer
0
t1
Separation
Voltage (SV)
Compensation
Voltage (COV)
 (E) 
K ( E )  K (0)
K (0)
 Ions migrate towards one of the planar electrodes
– If an ion’s high-field mobility K(E) is larger than its low-field mobility K(0), >0
– If an ion’s low-field mobility K(0) is larger than it high-field mobility K(E), <0
 Any ion can be steered back onto the center-line, by application of
a compound-specific DC compensation voltage (CV).
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© 2016 SCIEX RUO-MKT-18-1166 A
Tuning the SelexION™ Ion Mobility Device
Rel. Int. (%)
100%
The ion residence time, and
hence the resolution of the ion
mobility device, can be controlled.
50%
CV peak widths range from
approximately 0.5 – 2.5 V FWHM.
0%
-14
-12
-10
-8
-6
-4
-2
0
Compensation Voltage (CV)
2
4
6
8
10
12
 In infusion mode, the compensation voltage (CV) parameter is
ramped in order to determine the optimized value.
 Note that this is analogous to tuning any other compound-specific
parameter, e.g. the collision energy (CE)
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© 2016 SCIEX RUO-MKT-18-1166 A
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Mixture of 6 androgens in 60:40 Methanol:Water
 Sample chromatogram from QTRAP® 5500 LC/MS/MS system
XIC of +MRM (6 pairs): Exp 1, 287.2/97.0 Da ID: ASD from Sample 15 (Androgens mix 5) of Quant-RestekC18-150msec.wiff (Turbo Spr...
5-ASD
DHEA
1.29e5
1.25e5
(~10ng/mL)
(~30ng/mL)
1.20e5
1.15e5
DHT
T
1.10e5
1.05e5
(~4ng/mL)
(~400pg/mL)
1.00e5
9.50e4
9.00e4
AND
3.17
In te n s ity , c p s
98% B
(~10ng/mL)
8.50e4
8.00e4
7.50e4
ASD
7.00e4
(~500pg/mL)
Max. 8.7e4 cps.
6.50e4
6.00e4
80% B
5.50e4
75% B
5.00e4
4.50e4
4.00e4
3.50e4
3.00e4
2.50e4
55% B
2.00e4
1.50e4
1.00e4
5000.00
0.00
0.0
28
0.5
1.0
1.5
2.0
2.5
© 2016 SCIEX RUO-MKT-18-1166 A
3.0
3.5
4.0
4.5
5.0
Time, min
5.5
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
Structures of two androgens, DHEA and T
• Testosterone, C19H28O2
CH3
O
 Low ionization efficiencies
 Common precursor ion
masses (m/z 289)
CH3
H
 Similar fragmentation
patterns
H
 Similar chromatographic
H
retention properties
HO
 Dehydroepiandrosterone (DHEA), C19H28O2
OH
CH3
CH3
H
O
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© 2016 SCIEX RUO-MKT-18-1166 A
H
H
MS/MS of DHEA and Testosterone (m/z 289)
(A) MS/MS fragmentation pattern (Collision Energy ramp) for DHEA
CH3
CH3
O
H
H
Used for MRM
transition
H
HO
(B) MS/MS fragmentation pattern (Collision Energy ramp) for Testosterone
Used for MRM
transition
OH
CH3
CH3
H
O
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© 2016 SCIEX RUO-MKT-18-1166 A
H
H
Resolving DHEA and Testosterone using the SelexION™
Ion Mobility Device
SelexION™ device “off”
Interference caused by the
presence of Testosterone
SelexION™ device “on”
(a)
(b)
No interference caused by the
presence of Testosterone
DHEA (289.0/271.1)
DHEA (289.0/253)
DHEA (289.0/271.1)
DHEA (289.0/253)
a) There is a significant interference in the MRM channel for DHEA, caused by
the presence of testosterone
b) When the SelexION™ ion mobility device is employed the interference is
completely removed. Thus there is no longer a need for chromatographic
separation, and run-times can be reduced dramatically.
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© 2016 SCIEX RUO-MKT-18-1166 A
Liquid-Liquid Extraction (LLE) – LC-MS/MS
289/97
289/109
Testosterone
peak in
female serum
Interference
Testosterone
peak in
female serum
Interference
Area ratio ~ 23
Expected ratio
=1.23
Liquid-Liquid Extraction (LLE) – LC-MS/MS with SelexION™ Technology
289/109
289/97
Area ratio =
1.28
Expected ratio
=1.23
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© 2016 SCIEX RUO-MKT-18-1166 A
x
Protein Precipitation (PPT) – LC-MS/MS
289/109
Testosterone
peak in
female serum
289/97
Testosterone
peak in
female serum
Protein Precipitation (PPT) – LC-MS/MS with SelexION™ Technology
289/109
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© 2016 SCIEX RUO-MKT-18-1166 A
289/97
STD Curve in Serum Sample using the SelexION™Ion Mobility
Device (sample preparation = PPT)
4 female
4 serum
samples
from females
subjects
Accuracy 96- 105%
Sample
Sample
Average Ion Ratio
(97/109)
STDEV
%CV
34
Neat (N=28)
Serum PPT (N=25)
Serum LLE (N=25)
Neat
(n=28) Patient
PPT (n=25) Patient
LLE (n=25)
© 2016 SCIEX RUO-MKT-18-1166 A
1.23
0.04
3.44
1.28
0.07
5.47
1.24
0.04
3.12
SelexIon performance for Estrogen Analysis
p
s
Serum (40 uL) by PPT and no SelexIon™
Max. 6.8e4 cps.
c
XIC of -MRM (3 pairs): 275.200/147.100 Da ID: d4-E2 from Sample 1 (PlasmaNew ppt 15) of 111220-NoDms-PlasNew-15.wiff (Turbo Spray)
i t y
,
9000
8000
s
7000
t e
n
6000
5000
I n
4000
3000
2000
1000
0
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
XIC of -MRM (3 pairs): 271.200/145.100 Da ID: E2 from Sa...
offset by -2.05 min
4.0
E2
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
Time, min
Max. 3710.6 cps.
XIC of -MRM (3 pairs): 269.200/145.100 Da ID: E1 from Sa...
offset by -2.05 min
In te n s ity , c p s
In te n s ity , c p s
7.82 7.98
800
600
400
11.0
11.5
Max. 1.4e4 cps.
8000
8.68
E1
7000
6000
5000
4000
3000
2000
200
7.76
1000
0
0.0
35
10.5
9000
8.72
1000
10.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
Time, min
© 2016 SCIEX RUO-MKT-18-1166 A
8.0
9.0
10.0
11.0
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
Time, min
8.0
9.0
10.0
11.0
SelexIon performance for Estrogen Analysis
p
s
Serum (40 uL) by PPT with SelexIon™
Max. 2313.0 cps.
c
XIC of -MRM (3 pairs): 269.200/145.100 Da ID: E1 from Sample 6 (plasma 15ppt) of 111222-Plasma15-demifast.wiff (Turbo Spray)
8.66
i t y
,
2313
n
s
2000
t e
1500
I n
1000
500
0
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
XIC of -MRM (3 pairs): 271.200/145.100 Da ID: E2 from Sa...
8.64
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
Time, min
Max. 289.3 cps.
XIC of -MRM (3 pairs): 269.200/145.100 Da ID: E1 from Sa...
E2
200
150
100
10.5
11.0
11.5
Max. 2313.0 cps.
8.66
E1
2000
1500
1000
500
50
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10.0
2313
250
0
0.0
5.5
In te n s ity , c p s
In te n s ity , c p s
289
5.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
Time, min
© 2016 SCIEX RUO-MKT-18-1166 A
8.0
9.0
10.0
11.0
0
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
Time, min
8.0
9.0
10.0
11.0
Spiked plasma sample analyzed without SelexION™ (A) and with
SelexION™ Technology (B). The light blue marked regions defined
where signal and noise were collected.
E1: S/N (1 sigma): 77
E2: S/N (1 sigma): 27
E1: S/N (1 sigma): 462
E2: S/N (1 sigma): 162
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© 2016 SCIEX RUO-MKT-18-1166 A
Steroid Panel iMethod™
• Increasing the number of compounds analysed in an assay is a
perfect way of maximising potential return on investment
• LC-MS/MS is ideally suited to this approach
• The SCIEX Steroid Panel iMethod™ allows simultaneous
analysis of 10 commonly measured steroids and also 25Hydroxyvitamin D3
• Sample preparation is simple protein precipitation with dilution
• LC separation is required in order to keep the sample
preparation to a minimum, run time is 12.5 minutes
• In order to achieve the required sensitivity for all compounds
assayed, this method is designed for use on the API5000™and
Triple Quad 5500™instruments
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© 2016 SCIEX RUO-MKT-18-1166 A
Steroid Panel iMethod™, Extracted sample at ULOQ
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© 2016 SCIEX RUO-MKT-18-1166 A
Performance Data, Steroid Panel iMethod™
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© 2016 SCIEX RUO-MKT-18-1166 A
• SCIEX offers several solutions for steroid analysis
• Various forces will determine which approach is needed
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Analysis of less challenging steroids
Requirements for analysis of multiple compounds
Sensitivity requirements on challenging steroids
Desire for the convenience of a kit-based assay
Multiplexing with additional applications
• From this, the appropriate method and instrument can be
matched to the analytical requirements
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© 2016 SCIEX RUO-MKT-18-1166 A
Trademarks/Licensing
SCIEX is doing business as SCIEX.
For Research Use Only. Not for use in diagnostic procedures.
© 2016 SCIEX. The trademarks mentioned herein are
the property of SCIEX Pte. Ltd. or their respective owners.
SCIEX™ is being used under license.
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© 2016 SCIEX RUO-MKT-18-1166 A