endogenous steroids analysis in plasma by lc/ms - … 2... · endogenous steroids analysis in...

Endogenous Steroids Analysis in Plasma by LC/MS

Analysis by LC-MS/MS

for Clinical Research

Andre Szczesniewski

Senior Applications Chemist

Agilent Technologies

May 1, 2014 1

For Research Use Only. Not for

use in diagnostic procedures.

• Introduction

• 13 Steroids Panel Analysis

• Sample Preparation & Methodology

• Results

• Conclusion

• Questions

2

Overview


use in diagnostic procedures. May 1, 2014

3

Why Do Clinical Research Laboratories Adopt LC/MS?

High Selectivity

High Sensitivity

Accurate Quantification

No Sample Derivatization

Measure Analytes and their Metabolites Simultaneously

Easier Research Method Development vs. Antibody-based Techniques



May 1, 2014

4

Cholesterol

Pregnenolone Progesterone Deoxycorticosterone Corticosterone

Aldosterone

17α-OH Pregnenolone 17α-OH Progesterone Cortisol

Testosterone

Dihydrotestosterone

Cortisone

Estrone Estradiol Dehydroepiandrosterone

Androstenediol

11-Deoxycortisol

Androstenedione

Estriol

Cholesterol Steroidogenesis Analysis of 13 Steroids for research

2



May 1, 2014

Structures and Molecular Weights Steroidogenesis

5

Cortisone

C21H28O5

M.W. 360.2

Aldosterone

C21H28O5

M.W. 360.2

11-Deoxycortisol

C21H30O4

M.W. 346.2

Corticosterone

C21H30O4

M.W. 346.2

Cortisol

C21H30O5

M.W. 362.46

Androstenedion

e

C19H26O2

M.W. 286.2

DHEA

C19H28O2

M.W. 288.2

17-Hydroxyprogesterone

C21H30O3

M.W. 330.2

Dihydortestosterone

C19H30O2

M.W. 290.2

Progesterone

C21H30O2

M.W. 314.2

Pregnenolone

C21H32O2

M.W. 316.2

17-OH-Pregnenolone

C21H32O3

M.W. 332.2

Testosterone

C19H28O2

M.W. 288.2

5-delta Steroids

0

2



May 1, 2014

May 1, 2014

http://en.wikipedia.org/wiki/File:Aldosterone-2D-skeletal.svg

//upload.wikimedia.org/wikipedia/commons/4/4d/Cortodoxone.png

//upload.wikimedia.org/wikipedia/commons/a/ac/Corticosterone-2D-skeletal.svg

http://en.wikipedia.org/wiki/File:Androstendion.svg

//upload.wikimedia.org/wikipedia/commons/0/0b/DHEA.png

//upload.wikimedia.org/wikipedia/commons/f/fc/17-Hydroxyprogesterone.svg

//upload.wikimedia.org/wikipedia/commons/c/cd/Androstanolone.svg

//upload.wikimedia.org/wikipedia/commons/6/6d/Progesteron.svg

//upload.wikimedia.org/wikipedia/commons/1/14/Pregnenolone.svg

//upload.wikimedia.org/wikipedia/commons/8/87/17-Hydroxypregnenolone.svg

//upload.wikimedia.org/wikipedia/commons/c/ce/Testosteron.svg

Research Method Development Objectives

6

Comprehensive, quantitative method for the analysis of major cholesterol steroidal metabolites for use in the clinical research laboratories

Simple and economical sample preparation

Robust and reliable research workflow

Easily modifiable to accommodate and adjust changing research requirements

May 1, 2014



Sample Preparation Liquid-Liquid Extraction

7

500 μL of plasma or serum + 100 μL ISTD at 5ng/ml

Vortex 30 seconds

Add 5 mL MTBE*

Vortex/mix 5 minutes

Transfer MTBE layer (top) to a test tube

then evaporate under N2 at 35 °C

Reconstitute in 100 μL in 30% Methanol

Analyze by LC-MS/MS

* Methyl tert-butyl ether

May 1, 2014



LC Research Method Parameters 1290 UHPLC System

8

Injection volume: 10 mL

Autosampler temp: 5 ºC

Column Temp: 50 ºC

Needle Wash: 50%IPA : 25% MeOH : 25% H2O

Column: Poroshell 120 EC-C18, 2.1x50 mm, 2.7 mm

Pump Flow: 0.4 mL/min

Mobile Phase: A = Water + 5mM NH4FA

B = Methanol + 5mM NH4FA

Gradient: Time %B

0.00 10

0.50 10

8.00 40

9.00 60

9.50 60

9.60 95

10.4 95

10.5 10

Stop time – 10.5 min

Post time – 1.00 min

May 1, 2014



9

Ion Mode: Jetstream, Positive

ESI conditions

Drying Gas Temperature: 250 ºC

Drying Gas Flow: 11 L/min

Nebulizer Pressure: 35 psi

Sheath Gas Temperature: 350 ºC

Sheath Gas Flow: 11 L/min

Capillary: 3000 V

Nozzle Voltage: 0 V

EMV: 300 V

Resolution: MS1 - Unit, MS2 – Unit

Dwell Time 20 ms

Cell Acceleration 3 V

• The selection of the spray chamber conditions for a multi-component method always involves

a trade-off between the optimum settings of the individual compounds.

MS Research Method Parameters 6460 QQQ System

May 1, 2014



10

MS/MS Research Method Parameters MRM Transition Parameters for Analytes

Compound Name Precursor Ion Product Ion Quant-Qual

Frag (V) CE (V) Quant-Qual

11-Doxycortisol 347.2 109.1 - 97.1 95 28 - 28

17a-Hydroxypregnenolone 315.2 297.1 - 255.1 93 2 - 7

17a-Hydroxyprogesterone 331.2 109.1 - 97.1 100 27 - 27

Aldosterone 361.2 343.2 - 315.2 100 15 - 16

Androstenedione 287.2 109.1 - 97.1 100 25 - 17

Corticosterone 347.2 329.2 - 121.1 110 12 - 24

Cortisol 363.2 121.1 - 91.1 105 24 - 60

Cortisone 361.2 163.1 - 121.1 120 20 - 32

DHEA 289.2 271.2 - 231.2 95 2 - 20

Dihydrotestosterone 291.2 255.2 - 91.1 120 12 - 52

Pregnenolone 317.2 299.2 - 159.1 102 4 - 16

Progesterone 315.2 109.1 - 97.1 100 25 - 20

Testosterone 289.2 109.1 - 97.1 100 25 - 25

May 1, 2014



11

Compound Name Precursor Ion Product Ion Frag (V) CE (V) 11-Doxycortisol-D5 352.2 100.1 95 28

17a-Hydroxypregnenolone-13C2-D2 319.2 301.1 93 2

17a-Hydroxyprogesterone-D8 339.2 100.1 100 27

Aldosterone-D8 369.2 351.2 100 15

Androstenedione-3C13 290.2 100.1 100 17

Corticosterone-D8 355.2 337.2 110 12

Cortisol-D4 367.2 121.1 105 24

Cortisone-D7 369.2 169.1 120 20

DHEA-D5 294.2 258.2 95 2

Dihydrotestosterone-D3 294.2 258.2 120 12

Pregnenolone-13C2-D2 321.2 303.2 102 4

Progesterone-D9 324.2 100.1 100 20

Testosterone-D3 292.2 97.1 100 25

Research Method Parameters MRM Transition Parameters for Internal Standards

May 1, 2014



Chromatography 13 Steroid Panel for Research

12

Co

rtis

ol

Ald

oste

ron

e

Co

rtis

on

e

Co

rtic

oste

ron

e

11-D

eo

xyco

rtis

ol

17a-

Hyd

roxyp

rog

este

ron

e

Pre

gn

en

olo

ne

17-H

yd

roxyp

reg

nen

olo

ne

Pro

geste

ron

e

Dih

yd

rote

sto

ste

ron

e

DH

EA

Testo

ste

ron

e

An

dro

ste

ned

ion

e

5-delta Steroids

May 1, 2014



Chromatography Isobaric Pairs Need for Baseline Separation

13

Ald

oste

ron

e

Co

rtis

on

e

Co

rtic

oste

ron

e

11

-Deo

xyco

rtis

ol

Testo

ste

ron

e

DH

EA



Limit Of Detection Example: Testosterone at 5 pg/mL 50 femtograms injected on Column

14

Blank Injection

LOD at 5 pg/mL

or

50 fg on column

S/N=5.6



Androstenedione Calibration: 10pg/mL-100 ng/mL Regular Scale vs. Log Scale

1 2 3 4 5

Linearity of 5 Orders of Magnitude

15

For Research Use Only. Not for use

in diagnostic procedures.

16

Aldosterone

Cortisol Testosterone

Calibration Curves Representative Steroids

17-Hydroxypregnenolone



Results Summary of Analytical Performance

17

Compound R2 Level Conc.

Compound R2 Level Conc.

(pg/mL) (pg/mL)

Aldosterone 0.998

LOD 5

DHEA 0.995

LOD 250

LLOQ 10 LLOQ 500

ULOQ 500000 ULOQ 100000

Cortisone 0.998

LOD 5

17α-Hydroxyprogesterone 0.996

LOD 10

LLOQ 10 LLOQ 25

ULOQ 1000000 ULOQ 100000

Cortisol 0.999

LOD 1

17-Hydroxypregnenolone 0.995

LOD 250

LLOQ 5 LLOQ 500

ULOQ 1000000 ULOQ 250000

Corticosterone 0.996 LOD 5

Dihydrotestosterone 0.997 LOD 5

LLOQ 10 LLOQ 10

ULOQ 100000 ULOQ 500000

11-Deoxycortisol 0.995

LOD 2.5

Progesterone 0.995

LOD 25

LLOQ 5 LLOQ 50

ULOQ 500000 ULOQ 500000

Androstenedione 0.999

LOD 2.5

Pregnenolone 0.999

LOD 1000

LLOQ 5 LLOQ 2500

ULOQ 1000000 ULOQ 500000

Testosterone 0.996

LOD 5

LLOQ 10

ULOQ 500000



Conclusions

18

• Comprehensive analytical research method for steroids profile and a

highly sensitive and robust research method for low-level quantitation

of 13 steroids panel has been developed

• Sample preparation consisted of a straight forward LLE

• Steroid Panel LLOQs were in the low pg/ml range while 5-Δ steroids

were in high pg/mL. Dynamic range of up to 5 orders of magnitude

with R2 ≥ 0.995

• Number of steroids and analysis time can be reduced according to

research needs.



Questions

19

Thank You!

Acknowledgements

Kevin McCann Agilent Technologies

Applications Specialist



Vitamin D Metabolite Analysis in Serum by LC/MS

Rory M Doyle

Manager-Clinical/Forensic AE AFO

Agilent Technologies, Inc

Wilmington, DE



Agenda

• Researching Vitamin D metabolites

• LCMSMS reserach methods for the accurate analysis of

Vitamin D metabolites and isoforms

• How automated, online sample clean-up can be used to reduce offline sample preparation

• Technological advances enabling the analysis of low-level Vitamin D metabolites

• Quantitative data analysis



Rapid Analysis of Vitamin D Metabolites for Research

• 25-OH, epimers, 1,25-(OH)2, and more

• Chromatic separation of isoforms for each metabolite

• Internal standard corrected quantification

• Multi-point calibration curve covering a wide dynamic range

• Secondary qualifier ion for each analyte

Dietary Intake

7-Dehydrocholesterol

Skin Liver

Kidney

Vitamin D

25-OH Vitamin D

Gut

1,25-(OH)2 Vitamin D



C H 2

H

C H 3

CH 3

CH 3

C H 3

O H O H

A Few Metabolites of Vitamin D

O H

O H

Vitamin D325-OH Vitamin D3

3-epi-25-OH Vitamin D3

1,25-(OH)2 Vitamin D3

25-OH Vitamin D2

3-epi-25-OH Vitamin D2

1,25-(OH)2 Vitamin D2



Protein Precipitation for 25-OH Metabolite Research Analysis

Sample

Methanol

precipitate centrifuge analyze



Cleaner Samples with Online Sample Cleanup

Column A

Column B

Mass Spec

Pump 1

ALS

Pump 2

Waste

1

6

5 4

3

2

Column A

Column B

Mass Spec

Pump 1

ALS

Pump 2

Waste

1

6

5 4

3

2

Position 1 (Port 1 > 2) Position 2 (Port 1 > 6)



Poroshell 120 Columns for HPLC and UHPLC

Poroshell 120 Columns have:

• 80-90% efficiency of sub-2µm columns

• ~40-50% lower pressure

• 2x efficiency of 3.5µm (totally porous)

• A 2µm frit to reduce clogging

• A 600 bar pressure limit for HPLC or UHPLC

• The superficially porous particle is 2.7um with a

solid core (1.7um) and porous outer layer with a

0.5um diffusion path

Poroshell 120 is a high efficiency, high resolution column choice for

enhancing productivity in LC and LC/MS

1.7µm

0.5µm

0.5µm



Chromatographic 2D Separation of Metabolites

x104

0

1

2

3

4

5

6

Acquisition Time (min)

0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 3 3.2 3.4 3.6

Co

un

ts 1 25-OH Vitamin D2

2 25-OH Vitamin D3 1

2



Accurate 1D Quantification of 25-OH Vitamin D for Research

25-OH Vitamin D2

Concentration (ng/ml)

0 10 20 30 40 50 60 70 80 90 100

X10-1

0

1

2

3

4

y = 0.0049 * x + 0.0025

R^2 = 0.999Type:Linear, Origin:Ignore, Weight:1/x

Re

lative

Re

sp

on

se

25-OH Vitamin D3


0 10 20 30 40 50 60 70 80 90 100

0.0

0.2

0.4

0.6

0.8

1.0

y = 0.010 * x + 0.0038

R^2 = 0.999Type:Linear, Origin:Ignore, Weight:1/x

Re

lative

Re

sp

on

se


2 2.5 3

x102

0.6

0.8

1.0

Co

un

ts

D2 LOQ

1.25 ng/ml


2 2.5 3

x102

0.5

1.0

1.5

2.0

Co

un

ts

D3 LOQ

1.25 ng/ml

For Research Use Only. Not for use

in diagnostic procedures.

Chromatographic 1D Separation of 3-Epi-25-Hydroxy-Vitamin D using a PFP column



Analytical Range for Vitamin D Metabolites in Research

y = 0.0024x + 0.0006 R² = 0.9993

0

0.1

0.2

0.3

0 20 40 60 80 100 120

Rela

tive

Re

sp

on

se


Analytical Range for

1,25-(OH)2 Vitamin D

(low pg/ml)



Quantification of 1,25-(OH)2 Vitamin D for Research Dealing with a Difficult Analysis

Accurate and reliable quantification for research of low level analytes

such as 1,25-(OH)2 Vitamin D can be challenging, costly and time-

consuming.

Through the implementation of novel and cutting-edge technologies,

Agilent makes these research analyses a reality.



Agilent Jet Stream

• Thermal confinement of

ESI plume

• Efficient desolvation to

create gas phase ions

• Creates an ion rich zone

Hexabore Capillary

• 6 capillary inlets

• Samples ~10 times

more ion rich gas from

the source

• Captures the majority of

the gas from the source

region

Dual Ion Funnel

• Removes the gas but

captures the ions

• Removes neutral noise

• Extends turbo pump life

Capture up to 10 x more ions

MS Inlet

Microflow LC/MS Nebulizer

Heated Sheath Gas

Thermal Gradient Focusing Region

Heat Sink with Active Cooling

iFunnel Technology Revolutionizes Ion Sampling



Liquid-Liquid Extraction for 1,25-(OH)2 Metabolite Analysis

Sample

Hexane

extract transfer

organic analyze evaporate reconstitute



Analysis of 1,25-(OH)2 Vitamin D

x103

0.2

0.6

1.0

1.4


7 8 9 10 11 12

Co

un

ts

1,25-OH Vitamin D3

Other Metabolites

• LOQ of 5 pg/mL

• Chromatographic separation from isobaric metabolites

• Possible to quantitate other metabolites for research



Immuno-cleanup of 1,25-Dihydroxy-Vitamin D3/D2

1-25DiOH Vit D3 1-25DiOH Vit D2



Accurate Quantitation of 1,25-(OH)2 Vitamin D for Research

Concentration (pg/ml)

0 50 100 150 200 250 300 350 400 450 500

0

0.2

0.4

0.6

0.8

1.0

1.2

1.4 y = 0.0025 * x + 0.053

R^2 = 0.998

Re

lative

Re

sp

on

se


8.6 8.8 9.0 9.2 9.4 9.6 9.8 10 10.2 10.4

x102

0.5

1.0

1.5

2.0

2.5

3.0

3.5

Counts

1,25-OH Vitamin D3

10 pg/mL



MassHunter Quantitative Analysis for Research Batch at a Glance and Reporting



Conclusions

• Agilent offers robust solutions to enable the reliable quantification of abundant and low-level Vitamin D metabolites for research

• Proven technology reduces your workload by minimizing hands-on sample preparation

• Agilent’s MassHunter software is optimized for

researchers' data review and reporting workflow



Multiply LC/MS Productivity

The New Agilent StreamSelect

LC/MS System

Maria VanDamme

StreamSelect Product Manager

Agilent Technologies, Inc.



Multiply LC/MS Productivity Agilent StreamSelect LC/MS System

• Dual-stream capacity increases throughput up to two-fold

• More samples on fewer instruments through efficient MS utilization

• Excellent analytical performance for accurate & reproducible quantitation for research

• Simple implementation with intuitive automation software

• Fully integrated solution allows intelligent error handling

41



Traditional LC/MS Analyses in Series for Researchers

0.0 2.5 25.0 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5

injection 1 injection 2 injection 3 injection 4 injection 5

A significant portion of the chromatographic run contains no peaks of interest

5 analyses in 25 minutes

42



Offset

Agilent StreamSelect LC/MS Solution Analyses in Parallel for Researchers

44

Str

eam

1

Str

eam

2

Mass S

pectr

om

ete

r V

iew

Idle

0.0 2.5 25.0 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5

Nearly 10 analyses in the same 25 minutes!

No idle time, MS is used efficiently



QQQ

StreamSelect Research Workflow

1

1 2

LC 1

2

1 2

LC 2 SSV

Calibrators and QC samples

Run on both streams

Unknowns

Run on either LC1 or LC2

Autosampler

46



S1 S2

Dramatically Increase Throughput for Researchers

Standard LC/MS StreamSelect LC/MS

12 1

2

3 9

8

7 6

4

10

5

11

8 hours to complete the batch 4.5 hours to complete the batch

48



Quantitative and Retention Time Reproducibility

50

R2 >0.999

Results Experimental

Combined calibration curves for

25-OH Vitamin D3 across both

LC systems

Retention time reproducibility of

25-OH Vitamin D3 for research over 500 injections across both LCs

Application Note 5991-2900EN

RSD <10%



Batch Submission Simple 3-Step Process

The simulation shows what a researcher would do to submit a batch

51



StreamSelect Profile Development

Choose Example Data File

Define Peak Elution Time

Create Sample Plate Templates

53



Intelligent Operation Constant Communication

Cals and QC

Unknowns

QQQ

1

1 2

LC 1

2

1 2

LC 2 SSV

• Direct, bi-directional

communication between the

LCs and the MS eliminates

sample loss.

54




Cals and QC

Unknowns

?

QQQ

1

1 2

LC 1

2

1 2

LC 2 SSV




sample loss.

• In this example, the LC running

Stream 1 fails during the

analysis of Sample 29. What

happens to Sample 29?

55




Cals and QC

Unknowns

QQQ

1

1 2

LC 1

2

1 2

LC 2 SSV




sample loss.

• In this example, the LC running

Stream 1 fails during the

analysis of Sample 29. What

happens to Sample 29?

• Sample 29 and all further

samples are seamlessly re-

assigned for analysis on

Stream 2, assuring continued

productivity without any loss of

data.

56



Intelligent Operation Stream Equivalency

0 1000 2000 30000

1000

2000

3000

Stream crossoverFailure on stream 1

Stream 1

Str

ea

m 2

Slope=1.001Intercept=0.008

r2=0.9998

0 1000 2000 30000

1000

2000

3000

Stream crossoverFailure on stream 2

Stream 1S

trea

m 2

Slope=0.989Intercept=0.02

r2=0.99981

Simulated Leak on Stream 1,

samples diverted to Stream 2,

-> equivalent results

Simulated Overpressure on Stream 2,

samples diverted to Stream 1

-> equivalent results

58



Conclusions

• Agilent’s StreamSelect LC/MS Solution is an efficient way

for researchers to increase productivity without sacrificing

quantitative performance

• Intuitive StreamSelect Software is part of a fully integrated

system that provides a simple and robust solution to your

research laboratory’s needs

• Straightforward implementation based on existing methods

and efficient system utilization lower overall costs

60



Questions?

Maria VanDamme

Product Manager StreamSelect LC/MS System

[email protected]

Kevin McCann

Application Specialist StreamSelect LC/MS System

61



mailto:[email protected]

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