Determination of Analysis by LC-MS/MS Endogenous … · Endogenous Steroids in Biomatrices Analysis...

Determination of

Endogenous Steroids in

Biomatrices

Analysis by LC-MS/MS

for Clinical Research

Andre Szczesniewski

Senior Applications Chemist

Agilent Technologies

November 29, 2012

November 29, 2012 1

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

• Introduction

• 13 Steroids Panel Analysis

• Sample Preparation & Methodology

• Results

• Questions Break

• Estradiol and Estrone Analysis

• Sample Preparation and Methodology

• Results

• Summary

• Questions

November 29, 2012 2

Overview


November 29, 2012 3

• Sensitivity

• Detection limits down to picomolar levels

• Specificity

• Compound specific response reduces false positives or negatives

• Selectivity

• Differentiate interferences from matrix, background and other noise

• Quantitation

• Wide linear dynamic range and accurate quantitation

• Throughput

• Short analysis time and multiple analytes in a single test

Why Labs Are Interested in LC/MS?


November 29, 2012 4

Cholesterol Metabolism Steroidogenesis


What is Multiple Reaction Monitoring (MRM)?

November 29, 2012 5

170 210 250 290

210

222

268 280 165

Spectrum with

background

ions (from ESI)

Q1 lets only

target ion 210

pass through

190 210

210

Collision cell

breaks ion 210

apart

150 170 190 210

210 158

191

Q3 monitors only

characteristic

fragment 158

from ion 210 for

quantitation

160

158

no chemical background

Ions


• Introduction



• Results

• Questions Break



• Results

• Summary

• Questions

November 29, 2012 6


Method Development Objectives

November 29, 2012 7

• 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 workflow

• Easily modifiable to accommodate and adjust

changing research requirements


November 29, 2012 8

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


Structures and Molecular Weights Steroidogenesis

November 29, 2012 9

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


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

• Introduction



• Results

• Questions Break



• Results

• Summary

• Questions

November 29, 2012 10


Sample Preparation Liquid-Liquid Extraction


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

Method Parameters 1290 UHPLC System


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


6460 QQQ MS conditions

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.

Method Parameters 6460 QQQ System



Method Parameters MRM Transition Parameters for Analytes

Comound 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



Comound 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

Method Parameters MRM Transition Parameters for Internal Standards


• Introduction



• Results

• Questions Break



• Results

• Summary

• Questions



Chromatography 13 Steroid Panel


Co

rtis

ol

Ald

os

tero

ne

Co

rtis

on

e

Co

rtic

oste

ron

e

11

-De

ox

yc

ort

iso

l

17a-

Hyd

rox

yp

rog

es

tero

ne

Pre

gn

en

olo

ne

17

-Hyd

rox

yp

reg

nen

olo

ne

Pro

ge

ste

ron

e

Dih

yd

rote

sto

ste

ron

e

DH

EA

Te

sto

ste

ron

e

An

dro

ste

ne

dio

ne

5-delta Steroids


Chromatography Isobaric Pairs


Ald

os

tero

ne

Co

rtis

on

e

Co

rtic

os

tero

ne

11-D

eo

xyc

ort

iso

l

Te

sto

ste

ron

e

DH

EA


• Poroshell 120 Columns have:

• 80-90% efficiency of sub 2um

• At ~40-50% lower pressure

• 2X efficiency of 3.5um (totally porous)

• A 2um frit to reduce clogging

• A 600 bar pressure limit for HPLC or

UHPLC use

• The superficially porous particle is 2.7um with

a solid core (1.7um) and porous outer layer

with a 0.5um diffusion path

19

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

enhancing productivity in LC and LC/MS

1.7um

0.5um

0.5um

Poroshell 120 Columns For HPLC and UHPLC


Diflusinal in Plasma

0

20

40

60

80

100

120

140

160

180

200

220

240

260

280

300

320

340

360

380

400

1 501 1001 1501 2001 2501

Injections

Pre

ssu

re

0

20000

40000

60000

80000

100000

120000

140000

160000

180000

200000

Eff

icie

ncy (

N)

End Press

Plates

Column: Poroshell 120 EC-C18, 3.0 x 50mm, 2.7um LC: Agilent 1200 RRLC (SL)

Sample: Precipitated Plasma: 2 parts Plasma: 7 Parts 20/80 Water-MeCN w/0.1 % Formic Acid with 1 Part Diflusinal

in 50/50 Water-MeCN 10 ug/ml (Final concentration Diflusinal 1 ug/ml) Shaken and allowed to settle 10 minutes

Not Centrifuged/ Not Filtered Injection Volume: 1ul injections

Solvent A: Water w/0.1 % TFA

Solvent B: MeCN w/0.08 % TFA

Flow Rate 1 ml/min 1 ul injection

Time % B

0 20

0.5 90

0.6 90

1.1 20

2.5 20

Good lifetime with

plasma samples!

20

Poroshell 120 Resists Plugging with 2µm Frit Use with Plasma or Similar Matrices



Aldosterone

Cortisol Testosterone

Calibration Curves Representative Steroids

17-Hydroxypregnenolone


Results Summary of Analytical Performance

22

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

• Introduction



• Results

• Questions Break



• Results

• Summary

• Questions



Questions

24


• Introduction



• Results

• Questions Break



• Results

• Summary

• Questions



Method Development Objectives


• Simple, quantitative method for the analysis of 17ß-

Estradiol (E2) and Estrone (E1) for use in clinical

research laboratories

• Reliable and solid workflow

• Utilize state of the art LC/MS instrumentation with

superior sensitivity to meet challenging limits of

detection


Cholesterol Steroidogenesis Analysis of Estrogens


Cholesterol

Pregnenolone Progesterone Deoxycorticosterone Corticosterone

Aldosterone

17α-OH Pregnenolone 17α-OH Progesterone Cortisol

Testosterone

Dihydrotestosterone

Cortisone

Estrone Estradiol Dehydroepiandrosterone

Androstenediol

11-Deoxycortisol

Androstenedione

Estriol


Estrogens Estradiol and Estrone


17ß-Estradiol

C18H24O2

M.W. 272.2

Estrone

C18H22O2

M.W. 270.2


17ß-Estradiol Dealing with a Difficult Analysis

Analytical Challenge

• Low level detection of a compound lacking of highly ionizable

functional groups

Current Approaches

• Immunoaffinity Extraction

• Solid-Phase Extraction

Drawbacks

• Costly

• Time-Consuming

• Introduce Variability


OHCH3

H

H

H

OH


Increased Ion Generation Agilent JetStream Technology


•Thermal confinement of

nebulizer spray

•Efficient desolvation to

create gas phase ions

•Creates an ion rich zone


Increased Sensitivity Using Ion Funnel Technology Agilent 6490 QQQ


Hexabore for

greater

sampling

Removal of

carrier gas

and neutrals

Higher ion

yield


• Introduction



• Results

• Questions Break



• Results

• Summary

• Questions



Sample Preparation Liquid-Liquid Extraction (LLE) and Derivatization


500 µL of serum + ISTD

Vortex 20 sec, let it stand 5 minutes in room temp.

Add 50 µL of 100 mM Sodium Biocarbonate at pH 10.5

and 50 µL of 1mg/mL Dansyl Chloride

Incubate at 60ºC for 5 minutes

Add 6 mL of 3:2 of Hexane:Dichloromethane, vortex 60

sec then centriufuge at 3000 rpm for 10 min

Collect organic layer (top), dry down with N2 at 40ºC

Add 100 µL of water and analyze by LC-MS/MS


Estradiol Derivatization Dansyl Chloride


SO O

NCH3 CH3

CH3OH

H

H H

OOHCH3

H

H

H

OH

Estradiol Dansyl Chloride

+

Dansyl-Estradiol


On-Sample Cleanup System Lab Bench Layout


1: Loading pump

2: Analytical pump

3: Autosampler

4: Column compartment

1

3

4

2


Online Sample Cleanup System Diagram Forward Flush


Trapping Column

Waste

Loading

Pump

(1260)

+ALS

QQQ MS

Analytical Column

Analytical

Pump

(1290)


Online Sample Cleanup Operation Loading, Cleaning, Transferring and Analyzing


Trapping

Column

Waste

Loading

Pump

(1260)

+ALS

QQQ MS

Transfer Position

Analytical

Column

Analytical

Pump

(1290)

Trapping

Column

Waste

Loading

Pump

(1260)

+ALS

QQQ MS

Loading/Analysis Position

Analytical

Pump

(1290)

Analytical

Column


Injection volume 40 µL

Needle Wash flushport (methanol:water 75:25), 5 seconds

Injector Temp 4 °C

Columns Loading: Zorbax XDB-C18, 2.1 x 30mm, 3.5 µm

Analysis: Poroshell EC-18, 2.1 x 50 mm, 2.7 µm

Column Temp 50 °C

Switching Valve 0.0 min Pos1

1.9 min Pos 2

2.4 min Pos 1

Stop Time 7.7 minutes

Post Time 0.3 minute

Mobile Phase Chanel A = H2O + 5mM Ammonium Formate

Chanel B = Methanol + 5mM Ammonium Formate

Pump Gradients

Pump 1 Pump 2

LC Parameters 1290 HPLC With Online Sample Cleanup


Time Flow % Solvent B

0.0 0.3 50

2.0 0.3 70

4.0 0.3 75

6.2 0.3 78

6.3 0.6 98

7.5 0.6 98

7.6 0.6 50

7.7 0.3 50

Time Flow % Solvent B

0.0 0.5 50

2.0 0.5 70

5.0 0.5 75

5.1 0.5 98

7.5 0.5 98

7.6 0.5 50


Polarity Positive

Drying Gas Temp 150⁰C

Drying Gas Flow 17 L/min

Nebulizer Pressure 45 psi

Sheath Gas Temp 400 °C

Sheath Gas Flow 12 L/min

Capillary Voltage 4500 V

Nozzle Voltage 500V

MS Method Parameters 6490 QQQ System


Compound Precursor Product Frag (V) Dwell (msec) CE (V) Cell Acc Estradiol 506.3 171.1 380 100 41 3 Estradiol-d5 511.3 171.1 380 100 41 3 Estrone 504.3 171.1 380 100 35 3 Estrone-d4 508.3 171.1 380 100 35 3


• Introduction



• Results

• Questions Break



• Results

• Summary

• Questions



Derivatized Estradiol Extracted From Serum LLOQ 0.25pg/mL


Injection Area

1 5323

2 4568

3 4574

4 4277

5 5522

6 5237

7 5444

8 5483

9 4615

Average 5004

SD 487

CV (%) 9.7

LOQ: 0.25 pg/mL

10fg on column

Blank


Estradiol Calibration Curve 0.25-100 pg/mL


Concentration (pg/ml)

0 10 20 30 40 50 60 70 80 90 100

x10-1

0

1

2

3

4 y = 0.004 * x + 3.83E-004

R^2 = 0.999

Re

lative

Re

sp

on

se

0 0.2 0.4 0.6 0.8 1.0

x10-3

0

1

2

3

4

Level Expected Average Accuracy (%) SD CV (%)

1 0.25 0.25 82 - 115 0.02 8.45

2 0.50 0.49 81 - 114 0.04 9.01

3 1.00 0.97 88 - 112 0.07 6.81 n = 15; all measurements in pg/ml


Derivatized Estrone Extracted From Serum LLOQ 0.25pg/mL


Injection Area

1 4207

2 4272

3 3703

4 3822

5 3724

6 3842

7 4184

8 4256

9 3879

Average 3988

SD 237

CV (%) 6.0

LOQ: 0.25 pg/mL

10fg on column

Blank


Estrone Calibration Curve 0.25-100 pg/mL


Level Expected Average Accuracy (%) SD CV (%)

1 0.25 0.27 99 - 121 0.02 6.11

2 0.50 0.50 91 - 110 0.03 5.00

3 1.00 0.92 86 - 101 0.04 4.50 n = 15; all measurements in pg/ml

0.0 0.2 0.4 0.6 0.8 1.0

x10-3

0

1

2

3

4

5

Concentration (ng/ml)

0 10 20 30 40 50 60 70 80 90 100

x10-1

0

1

2

3

4

y = 0.004 * x - 1.146E-004

R^2 = 0.997

Re

lative

Re

sp

on

se



• Introduction



• Results

• Questions Break



• Results

• Summary

• Questions


Conclusions


• Comprehensive method for steroids profile and a highly sensitive

and robust method for low-level quantitation of Estradiol and Estrone

has been developed

• Sample preparation consists of a straightforward LLE and addition of

derivatization for estrogens

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

were in high pg/mL. Dynamic range of 4 orders of magnitude with

R2 ≥ 0.995

• LLOQ of 0.25 pg/ml has been achieved for derivatized Estradiol and

Estrone with an R2 ≥ 0.995

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

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

Tips and Tricks


• Mobile phase quality; grade and freshness

• Inline filter between needle seat and injection valve

• Make internal standards in deuterated methanol

• If sensitivity decreases, check cleanness of a spray chamber

and run MS2 scan method to monitor background ions levels

• Divert the flow to waste for the first minute to remove potential

residual salts or unreacted derivatization reagent

• Use needle wash to reduce carryover

• Keep the column compartment closed (cover on) to avoid

variations due to external temperature fluctuations


List of Columns and Supplies


Supplier Part No. Description

Agilent 695775-902 Analytical column: Poroshell 120 EC-C18,

2.1x100 mm, 2.7 µM (Estrogens)

Agilent 820555-901 Trapping column hardware kit (Estrogens)

Agilent 973700-932 Trapping Column: Zorbax Eclipse XDB-C18,

2.1x 30 mm, 3.5 µM (Estrogens)

Agilent 699775-902 Analytical column: Poroshell 120 EC-C18,

2.1x50 mm, 2.7 µM (Panel)

Agilent 5067-4638 1290 Infinity Inline filter, 0.3 µM

Agilent 5182-0556 2.0 mL Amber vials and caps


List of Reagents


Biological Solutions

Human Serum Double Charcoal Stripped, Delipidized Golden West Biologicals

Bovine Serum Albumin Golden West Biologicals

Solvents

Methanol Honeywell

Water Millipore

MTBE Honeywell

Hexane Honnewell

Dichloromethane Honeywell

Other Chemicals

Ammonium Formate Fisher

Dansyl Chloride Sigma-Aldrich

Sodium Bicarbonate Sigma-Aldrich

Standards and Internal Standards Cerillinat, IsoSciences


Questions


Thank You!

Acknowledgements

Kevin McCann Agilent Technologies

Applications Specialist

Anabel Fandino Agilent Technologies

R&D Applications Scientist


Determination of Analysis by LC-MS/MS Endogenous … · Endogenous Steroids in Biomatrices Analysis...

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Transcript of Determination of Analysis by LC-MS/MS Endogenous … · Endogenous Steroids in Biomatrices Analysis...