RUO-MKT-18-1166-A

LCMSMS Solutions For Steroid Analysis

Dan Blake

Manager, Clinical Applications

For Research Use Only. Not for use in diagnostic procedures.

2 © 2016 SCIEX 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

Special considerations for steroid analysis

4 © 2016 SCIEX RUO-MKT-18-1166 A

• 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 ‒ 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.

5 © 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

6 © 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

Testosterone iMethod™

8 © 2016 SCIEX RUO-MKT-18-1166 A

• 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

9 © 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.

10 © 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.

Online Extraction with 2DLC and simple sample

processing

Testosterone iMethod™

12 © 2016 SCIEX RUO-MKT-18-1166 A

• 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

13 © 2016 SCIEX RUO-MKT-18-1166 A

Switching Valve

TRAPPING COLUMN Analytical Column

Valve position A:

(Analyte Loading onto Trapping Column)

10

6

9

8

7 5

1 2

3

4

GRADIENT PUMPS A/B

AUTOSAMPLER

VALVE ISOCRATIC LOAD PUMP C

WASTE WASTE

6 5

Valve position B:

(Analyte Elution from SPE Column onto

Analytical Column)

MS System

Proposed Flow and Plumbing diagram for

2DLC Steroids method

Column Oven

Switching Valve:

Time(min) Valve

Position

0.0 A

1.0 B

5.5 A

14 © 2016 SCIEX RUO-MKT-18-1166 A

Extracted Steroid sample, approx 0.15nmol/L, Analysed by 2DLC approach

Testosterone

17-OH-P

11-Deoxycortisol

Androstenedione

Low Level Estrogen iMethod™

16 © 2016 SCIEX RUO-MKT-18-1166 A

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.

17 © 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 ‒ 500 µL sample volume required

‒ Liquid-liquid extraction without derivatization

‒ Clinically relevant dynamic range of 5-500 pg/mL

‒ 6 minute total run time

18 © 2016 SCIEX RUO-MKT-18-1166 A

Estrone and Estradiol: Lower Limit of Quantitation

Estrone 5 pg/mL LLOQ, S/N=22 Estradiol 5 pg/mL LLOQ, S/N=19

19 © 2016 SCIEX RUO-MKT-18-1166 A

Estrone and Estradiol: Precision and Accuracy

Sample (pg/mL) %CV % Accuracy

Day 1-3 Day 1-3

LLOQ (5) 9 104

Low QC (15) 3 104

High QC (100) 3 101

ULOQ (500) 3 100

•Estrone (inter-day)

•LLOQ CV’s ≤ 9% ,QC’s CV’s ≤ 5%

•Accuracy deviation ≤ 4%

Sample (pg/mL) %CV % Accuracy

Day 1-3 Day 1-3

LLOQ (5) 10 101

Low QC (15) 4 96

High QC (100) 5 102

ULOQ (500) 4 101

Estrone (E1) Estradiol (E2)

•Estradiol (inter-day)

•LLOQ CV’s ≤ 10% ,QC’s CV’s ≤ 5%

•Accuracy deviation ≤ 4%

20 © 2016 SCIEX RUO-MKT-18-1166 A

Estrone and Estradiol: Linearity

Clinically relevant dynamic range 5-500 pg/ml, R≤ 0.999

Estrone

Estradiol

21 © 2016 SCIEX RUO-MKT-18-1166 A

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

Improving Selectivity with Ion Mobility

23 © 2016 SCIEX RUO-MKT-18-1166 A

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.

24 © 2016 SCIEX RUO-MKT-18-1166 A

SelexION™ Ion Mobility Device Components:

New hardware

Existing ion path

25 © 2016 SCIEX RUO-MKT-18-1166 A

SelexION™ Ion Mobility Device Components:

Robust, easy-to-install, hardware components:

No tools required

No cables

No need to break vacuum

Installation in about 2 minutes

1. Orifice Plate 2. Ion Mobility Cell 3. Curtain Plate

26 © 2016 SCIEX RUO-MKT-18-1166 A

0

0

=0

t1

t2

)0(

)0()()(

K

KEKE

Tuning the SelexION™ Ion Mobility Device

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).

Compensation

Voltage (COV)

Separation

Voltage (SV)Compensation

Voltage (COV)

Separation

Voltage (SV)

ions in

transport gas to mass

spectrometer

27 © 2016 SCIEX RUO-MKT-18-1166 A

Tuning the SelexION™ Ion Mobility Device

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)

-14 -12 -10 -8 -6 -4 -2 0 2 4 6 8 10 12 14 16 180%

50%

100%

Rel. I

nt.

(%

)

Compensation Voltage (CV)

-14 -12 -10 -8 -6 -4 -2 0 2 4 6 8 10 12 14 16 180%

50%

100%

Rel. I

nt.

(%

)

Compensation Voltage (CV)

The ion residence time, and

hence the resolution of the ion

mobility device, can be controlled.

CV peak widths range from

approximately 0.5 – 2.5 V FWHM.

28 © 2016 SCIEX RUO-MKT-18-1166 A

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... Max. 8.7e4 cps.

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5Time, min

0.00

5000.00

1.00e4

1.50e4

2.00e4

2.50e4

3.00e4

3.50e4

4.00e4

4.50e4

5.00e4

5.50e4

6.00e4

6.50e4

7.00e4

7.50e4

8.00e4

8.50e4

9.00e4

9.50e4

1.00e5

1.05e5

1.10e5

1.15e5

1.20e5

1.25e5

1.29e5

Inte

ns

ity

, c

ps

3.17

ASD

T DHT

AND

DHEA 5-ASD

75% B 80% B

98% B

55% B

(~400pg/mL)

(~500pg/mL)

(~30ng/mL) (~10ng/mL)

(~4ng/mL)

(~10ng/mL)

Mixture of 6 androgens in 60:40 Methanol:Water

Sample chromatogram from QTRAP® 5500 LC/MS/MS system

29 © 2016 SCIEX RUO-MKT-18-1166 A

Structures of two androgens, DHEA and T

• Testosterone, C19H28O2

O

CH3

CH3OH

H

HH

O

CH3

CH3

OH

H

HH

Dehydroepiandrosterone (DHEA), C19H28O2

Low ionization efficiencies

Common precursor ion masses (m/z 289)

Similar fragmentation patterns

Similar chromatographic retention properties

30 © 2016 SCIEX RUO-MKT-18-1166 A

MS/MS of DHEA and Testosterone (m/z 289)

(A) MS/MS fragmentation pattern (Collision Energy ramp) for DHEA

MS/MS fragmentation pattern (Collision Energy ramp) for Testosterone (B)

O

CH3

CH3OH

H

HH

O

CH3

CH3

OH

H

HH

Used for MRM

transition

Used for MRM

transition

31 © 2016 SCIEX RUO-MKT-18-1166 A

Resolving DHEA and Testosterone using the SelexION™ Ion Mobility Device

a) There is a significant interference in the MRM channel for DHEA, caused by

the presence of testosterone

DHEA (289.0/271.1)

DHEA (289.0/253)

Interference caused by the

presence of Testosterone (a) No interference caused by the

presence of Testosterone

DHEA (289.0/271.1)

DHEA (289.0/253)

(b)

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.

SelexION™ device “off” SelexION™ device “on”

32 © 2016 SCIEX RUO-MKT-18-1166 A

Liquid-Liquid Extraction (LLE) – LC-MS/MS

289/109 289/97

Testosterone

peak in

female serum

Testosterone

peak in

female serum Interference

Interference

Area ratio ~ 23

Expected ratio

=1.23

x

Liquid-Liquid Extraction (LLE) – LC-MS/MS with SelexION™ Technology

289/109 289/97

Area ratio =

1.28

Expected ratio

=1.23

33 © 2016 SCIEX RUO-MKT-18-1166 A

Protein Precipitation (PPT) – LC-MS/MS

289/109 289/97 Testosterone

peak in

female serum

Testosterone

peak in

female serum

Protein Precipitation (PPT) – LC-MS/MS with SelexION™ Technology

289/109 289/97

34 © 2016 SCIEX RUO-MKT-18-1166 A

STD Curve in Serum Sample using the SelexION™Ion Mobility Device (sample preparation = PPT)

4 female

subjects

Sample Neat (n=28) Patient PPT (n=25) Patient LLE (n=25)

Average Ion Ratio

(97/109) 1.23 1.28 1.24

STDEV 0.04 0.07 0.04

%CV 3.44 5.47 3.12

Accuracy 96- 105%

Sample Neat (N=28) Serum PPT (N=25) Serum LLE (N=25)

4 serum samples

from females

35 © 2016 SCIEX RUO-MKT-18-1166 A

SelexIon performance for Estrogen Analysis Serum (40 uL) by PPT and no SelexIon™

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) Max. 6.8e4 cps.

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5Time, min

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

In

te

ns

it

y,

c

ps

XIC of -MRM (3 pairs): 271.200/145.100 Da ID: E2 from Sa... offset by -2.05 min

Max. 3710.6 cps.

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0

200

400

600

800

1000

In

te

ns

ity

, c

ps

8.72

7.827.98

XIC of -MRM (3 pairs): 269.200/145.100 Da ID: E1 from Sa... offset by -2.05 min

Max. 1.4e4 cps.

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

1000

2000

3000

4000

5000

6000

7000

8000

9000

In

te

ns

ity

, c

ps 8.68

7.76

E2

E1

36 © 2016 SCIEX RUO-MKT-18-1166 A

XIC of -MRM (3 pairs): 269.200/145.100 Da ID: E1 from Sample 6 (plasma 15ppt) of 111222-Plasma15-demifast.wiff (Turbo Spray) Max. 2313.0 cps.

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5Time, min

0

500

1000

1500

2000

2313

In

te

ns

it

y,

c

ps

8.66

XIC of -MRM (3 pairs): 271.200/145.100 Da ID: E2 from Sa... Max. 289.3 cps.

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0

50

100

150

200

250

289

In

te

ns

ity

, c

ps

8.64

XIC of -MRM (3 pairs): 269.200/145.100 Da ID: E1 from Sa... Max. 2313.0 cps.

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

0

500

1000

1500

2000

2313In

te

ns

ity

, c

ps

8.66E2

E1

SelexIon performance for Estrogen Analysis Serum (40 uL) by PPT with SelexIon™

37 © 2016 SCIEX RUO-MKT-18-1166 A

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

Steroid Panel iMethod™

39 © 2016 SCIEX RUO-MKT-18-1166 A

• 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 25-

Hydroxyvitamin 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

40 © 2016 SCIEX RUO-MKT-18-1166 A

Steroid Panel iMethod™, Extracted sample at ULOQ

41 © 2016 SCIEX RUO-MKT-18-1166 A

Performance Data, Steroid Panel iMethod™

42 © 2016 SCIEX RUO-MKT-18-1166 A

• SCIEX offers several solutions for steroid analysis

• Various forces will determine which approach is needed ‒ 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

43 © 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.