new sensitivity and selectivity gains with the … notes/new_sensitivity...ruo-mkt-11-1287-a new...

RUO-MKT-11-1287-A

New Sensitivity and Selectivity Gains with the QTRAP® 6500 System and SelexION™ Technology for Steroid Analysis For Research Use Only. Not for use in diagnostic procedures.

Michael Jarvis

SCIEX, Canada

2 © 2016 AB Sciex RUO-MKT-11-1287 A

Introduction

• The two major challenges facing steroid researchers (in fact, all

researchers!) using LC/MS/MS are:

‒ Sensitivity

‒ Selectivity


Challenges for LC-MS/MS Analysis of Steroids

Sensitivity:

• Low ionization

efficiencies

Selectivity:

• Common precursor ion

masses

• Similar fragmentation

patterns

• Similar chromatographic

retention properties

• Lots of endogenous

interferences http://en.wikipedia.org/wiki/Steroidogenesis#Steroidogenesis


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 5a-ASD

75% B 80% B

98% B

55% B

(~400pg/mL)

(~500pg/mL)

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

(~4ng/mL)

(~10ng/mL)

Analysis of 6 androgens in 60:40 methanol:water

Similar retention times

Common fragment ions complicate the analysis


Analysis of androstenedione in urine sample

Enzymatic hydrolysis performed, to remove glucuronides

Numerous interferences observed

Androstenedione

For Research Use Only. Not for Use in Diagnostic Procedures.


Agenda

• Pushing the limits of sensitivity with the QTRAP® 6500

LC/MS/MS system

• Enhanced selectivity using SelexION™ ion mobility

technology and LC/MS/MS

SCIEX Triple Quad™ and QTRAP® 6500 LC/MS/MS System


SCIEX Triple Quad™ and QTRAP® 6500 System

• IonDrive™ technology provides a

new level of performance – Up to 10x greater sensitivity

– Up to 6 orders dynamic range

– Improved robustness

• Advanced dual RF electronics

allows 2 modes of operation: – High Sensitivity (5-1250 Da)

– Extended mass range (5-2000 Da)

• Compatible with SelexION™ ion

mobility technology

• 2x faster scanning MRM3

• Same compact footprint as 4500

and 5500 system (32” x 32” x 24”)


IonDrive™ Technology Create more ions. Capture more ions. Detect more ions.

IonDrive™

Turbo V

Source

IonDrive™

High Energy

Detector

IonDrive™

QJet Ion

Guide

10 © 2016 AB Sciex RUO-MKT-11-1287 A

6x signal gain

>4x S/N gain

QTRAP® 5500

system

Actual MRM Sensitivity Gains +ESI mode

QTRAP® 6500

system

Verapamil

m/z 455

Erythromycin

m/z 734

5x signal gain

5x S/N gain

Ginsenoside Rb2

m/z 1101

20x signal gain

>4x S/N gain

Thyroid Research:

Analysis of Free T3 and Free T4

12 © 2016 AB Sciex RUO-MKT-11-1287 A

Thyroid hormones, T3 and T4

• Thyroid hormones circulate in the blood as mostly protein bound

hormone (about 99.9% bound)

• The major binding protein is Thyroxine Binding Globulin (TBG)

and transthyretin. Only the unbound form of the hormones are

biologically active.

• Free T3 (2-4 pg/mL) and free T4 (8-15 pg/mL) are present in

very low concentrations in serum.

O

O

I

I

I

OH

NH2

OH

O

O

I

I

I

I

OH

NH2

OH

3,3,5-triiodothyronine (T3) Thyroxine (T4)

13 © 2016 AB Sciex RUO-MKT-11-1287 A

Sample Preparation

• Free (unbound) fraction of T3 and T4 was isolated from the

protein-bound fraction by ultrafiltration, using a filter having

10kDa molecular-weight cut-off.

• 500uL of serum was subjected to ultrafiltration at 30C, for 60

minutes.

• 25uL of internal standard was added to 100uL of the ultrafiltrate

• 50uL was directly injected onto LC/MS/MS system

14 © 2016 AB Sciex RUO-MKT-11-1287 A

MS Method

• Triple Quad™ 6500 system

• Positive ESI mode

Name Q1 Q3

T3 (1) 651.7 605.8

T3 (2) 651.7 479.0

T4 (1) 777.7 731.6

T4 (2) 777.7 604.8

LC Method

Phenomenex Kinetex C18, 50x4.6mm, 2.6um @ 0.6 mL/min

A = Water + 0.1% acetic acid

B = Methanol + 0.1% acetic acid

5.0 8.0

8.1 10.0 0

Time %B

0 20

5 95

8 95

8.1 20

10 20

15 © 2016 AB Sciex RUO-MKT-11-1287 A

LOQ < 0.5 pg/mL

T3

Blank injection

XIC of +MRM (6 pairs): 651.700/605.800 Da ID: T3 1 from Sample 3 (blank) of Feb22-2014_final.wiff (Turbo Spray IonDrive), S... Max. 2921.9 cps.

5.00 5.05 5.10 5.15 5.20 5.25 5.30 5.35 5.40 5.45 5.50 5.55 5.60 5.65 5.70 5.75 5.80 5.851112 1123 1134 1145 1156 1167 1178 1189 1201 1212 1223 1234 1245 1256 1267 1278 1289 1301

Time, min

0

50

100

150

200

250

300

350

400

450

500

550

600

619

In

te

ns

ity

, c

ps

5.61 5.83

5.885.47

5.79

5.65

5.715.76

5.22

4.975.53

5.38

5.55

XIC of +MRM (6 pairs): 651.700/605.800 Da ID: T3 1 from Sample 6 (0.5pg) of Feb22-2014_final.wiff (Turbo Spray IonDrive), S... Max. 2781.3 cps.

5.05 5.10 5.15 5.20 5.25 5.30 5.35 5.40 5.45 5.50 5.55 5.60 5.65 5.701123 1134 1145 1156 1167 1178 1189 1201 1212 1223 1234 1245 1256 1267

Time, min

0

200

400

600

800

1000

1200

1400

1600

1800

2000

2200

2400

2600

2781

In

te

ns

ity

, c

ps

5.37

5.06 5.68

5.16 5.61 5.635.565.53 5.665.22

XIC of +MRM (6 pairs): 777.700/731.600 Da ID: T4 1 from Sample 6 (0.5pg) of Feb22-2014_final.wiff (Turbo Spray IonDrive), S... Max. 2784.4 cps.

5.40 5.45 5.50 5.55 5.60 5.65 5.70 5.75 5.80 5.85 5.90 5.95 6.00 6.05 6.10 6.15 6.20 6.251201 1212 1223 1234 1245 1256 1267 1278 1289 1301 1312 1323 1334 1345 1356 1367 1378 1389

Time, min

0

100

200

300

400

500

600

700

800

900

1000

1100

1200

1300

1400

1500

1600

1700

1800

In

te

ns

ity

, c

ps

5.74

5.93

6.25

5.97 6.216.02 6.11 6.155.69 6.065.45 6.095.61 5.64 5.855.83

5.38 5.515.43

XIC of +MRM (6 pairs): 777.700/731.600 Da ID: T4 1 from Sample 3 (blank) of Feb22-2014_final.wiff (Turbo Spray IonDrive) Max. 7076.0 cps.

5.40 5.45 5.50 5.55 5.60 5.65 5.70 5.75 5.80 5.85 5.90 5.95 6.00 6.05 6.10 6.15 6.20 6.251201 1212 1223 1234 1245 1256 1267 1278 1289 1301 1312 1323 1334 1345 1356 1367 1378 1389

Time, min

0

50

100

150

200

250

300

350

400

450

500

550

600

650

700

750

800

850

875

In

te

ns

ity

, c

ps

5.72

6.23

5.845.47 6.21

5.77

5.68

6.125.87 6.10

6.155.63

5.66 6.19

5.91

5.805.59

5.99 6.06

T4

Blank injection

T3

0.5 pg/mL (S/N = 48)

T4

0.5 pg/mL (S/N = 60)

16 © 2016 AB Sciex RUO-MKT-11-1287 A

Measurements in serum

T3, sample 2

1.4 pg/mL

T4, sample 5

7.8 pg/mL

T3, sample 4

3.4 pg/mL

T4, sample 4

17.3 pg/mL


Steroid Research:

Analysis of Aldosterone

18 © 2016 AB Sciex RUO-MKT-11-1287 A

5.0

7.5

7.6 10.0 0 1.0

6.0

6.5

MS Method

• Triple Quad™ 6500 system

• Negative ESI mode

Name Q1 Q3

Aldosterone (1) 359.2 189.0

Aldosterone (2) 359.2 331.1

Aldosterone-d7 366.2 338.2

LC Method

Phenomenex Gemini-NX C18, 150x3.0mm, 5um @ 0.5 mL/min

A = Water + 2mM ammonium acetate

B = Methanol + 2mM ammonium acetate Time %B

0 20

1 20

5 70

6 70

6.5 90

7.5 90

7.6 20

10 20

19 © 2016 AB Sciex RUO-MKT-11-1287 A

LOQ = 1 pg/mL

14 pg/mL (39.6 pmol/L) aldosterone in human

serum sample

LOQ = 1 pg/mL (2.8 pmol/L) aldosterone in human serum

14 pg/mL Aldosterone

(unknown sample) 1 pg/mL Aldosterone

(serum calibrator)


20 © 2016 AB Sciex RUO-MKT-11-1287 A

Linear Dynamic Range: 1pg/mL – 1mg/mL Aldosterone

1 mg/mL

6 orders LDR!

1 pg/mL 1 pg/mL

5 pg/mL

10 pg/mL

100 pg/mL

SelexION™ Ion Mobility Technology

22 © 2016 AB Sciex RUO-MKT-11-1287 A

Revolutionizing the way we do LC-MS/MS

• Benefits of ion mobility separations ‒ Separation of isobaric interferences

‒ Removal of chemical background noise

‒ Enables simplified sample preparation

‒ Enables faster LC run-times

‒ Improves LLOQ

‒ Improves data quality

• Features of the SelexION™ device ‒ Installation without breaking vacuum

‒ Ability to operate in “transparent” mode

‒ User-defined resolution setting

‒ Rapid MRM scanning

‒ Enhanced separations using chemical

modifiers

SCIEX Triple Quad™ 6500 system,

equipped with SelexION™ technology

23 © 2016 AB Sciex RUO-MKT-11-1287 A

SelexION™ Ion Mobility Device Components:

New hardware for

SelexION™ device

Existing ion path

24 © 2016 AB Sciex RUO-MKT-11-1287 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

25 © 2016 AB Sciex RUO-MKT-11-1287 A

a0

a0

a=0

t1

t2

)0(

)0()()(

K

KEKE

a

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), a>0

– If an ion’s low-field mobility K(0) is larger than it high-field mobility K(E), a<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

26 © 2016 AB Sciex RUO-MKT-11-1287 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.

31 © 2016 AB Sciex RUO-MKT-11-1287 A

Resolving isobaric prostaglandins with SelexION™ technology

• Optimization of the SelexION™ device for isomers is accomplished by ramping the COV parameter.

• At COV=10, only PGF2a is transmitted. At COV=7, only 8-iso-PGF2a is transmitted.

COV=6

COV=7

COV=8

COV=9

COV=10

COV=11

COV=9

PGF2a

8-iso

COV=6

COV=7

COV=8

COV=9

COV=10

COV=11

COV=9

PGF2a

8-iso

COV=6

COV=7

COV=8

COV=9

COV=10

COV=11

COV=9

PGF2a

8-iso

COV=6

COV=7

COV=8

COV=9

COV=10

COV=11

COV=9

PGF2a

8-iso

O

OH

OH

OH

OH

CH3

PGF2a 8-iso PGF2a

O

OH

OH

OH

OH

CH3

Chromatographic run-time (minutes)

32 © 2016 AB Sciex RUO-MKT-11-1287 A

Resolving isobaric prostaglandins with SelexION™ technology

SelexION™ device

“off”

SelexION™ device “on”

Tuned for PGF2a

SelexION™ device “on”

Tuned for 8-iso-PGF2a

(a)

(b)

(c)

PGF2a

8-iso-PGF2a

PGF2a

8-iso-PGF2a

O

OH

OH

OH

OH

CH3

PGF2a

O

OH

OH

OH

OH

CH3

PGF2a

8-iso PGF2a

O

OH

OH

OH

OH

CH3

8-iso PGF2a

O

OH

OH

OH

OH

CH3

Q1 Q3 CE

353.2 309.2 -28

Q1 Q3 CE CV

353.2 309.2 -28 10.0

Q1 Q3 CE CV

353.2 309.2 -28 7.0

33 © 2016 AB Sciex RUO-MKT-11-1287 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


Analysis of the Neuro-active Steroid Allopregnanolone

35 © 2016 AB Sciex RUO-MKT-11-1287 A

Metabolism of Neurosteroids

Progesterone 5a-Dihydroprogesterone Allopregnanolone

(3a,5a-Tetrahydroprogesterone)

Pregnanolone

(3a,5b-Tetrahydroprogesterone)

Pregnenolone

3b-HSD

5a-reductase

3a-HSD

3a-HSD

5b-Dihydroprogesterone

• The most familiar steroid hormones, commonly measured in blood for research

purposes, are the mineralocorticoids, glucocorticoids and the androgens and

estrogens.

• One alternative pathway that has been of great interest to researchers is the

production of compounds by 5α-reductase , followed by 3α-hydroxysteroid

oxido reductase.

36 © 2016 AB Sciex RUO-MKT-11-1287 A

Currently, the assays in use are an RIA (but supplies of the

antibody are very low) and GC/MS (which is only performed by a

few academic labs).

Availability of the assay is a real backlog in trying to perform

research studies.

• Sensitivity ‒ Potential for increased sensitivity by LC/MS/MS

• Sample prep ‒ 5-day process with GC/MS

‒ Very labor intensive for GC/MS

‒ Many opportunities for errors

‒ Limited number of samples/run

‒ Long sample run time for GC/MS

Allopregnanolone: Why LC-MS/MS?

37 © 2016 AB Sciex RUO-MKT-11-1287 A

Current Method Early days for LC-MS/MS…

10 pg/mL

(31.5 pM)

Sample Name: "THP, 0.5 ng/mL" Sample ID: "" File: "THP LLE calibration curve with IS 2012-10-09.wiff"Peak Name: "THP 1" Mass(es): "301.200/135.100 Da"Comment: "" Annotation: ""

Sample Index: 12

Sample Type: Standard

Concentration: 0.500 ng/mL

Calculated Conc: 0.457 ng/mL

Acq. Date: 2012/10/09

Acq. Time: 5:44:30 PM

Modified: Yes

Proc. Algorithm: Specify Parameters - MQ III

Noise Percentage: 100

Base. Sub. Window: 1.00 min

Peak-Split. Factor: 2

Report Largest Peak: Yes

Min. Peak Height: 0.00 cps

Min. Peak Width: 0.00 sec

Smoothing Width: 3 points

RT Window: 10.0 sec

Expected RT: 7.46 min

Use Relative RT: No

Int. Type: Manual

Retention Time: 7.46 min

Area: 5.73e+005 counts

Height: 2.00e+005 cps

Start Time: 7.43 min

End Time: 7.52 min

7.00 7.05 7.10 7.15 7.20 7.25 7.30 7.35 7.40 7.45 7.50 7.55 7.60 7.65 7.70 7.75 7.80 7.85 7.90 7.951910 1923 1937 1950 1964 1978 1991 2005 2019 2032 2046 2059 2073 2087 2100 2114 2128 2141 2155 2169

Time, min

0.0

2.0e4

4.0e4

6.0e4

8.0e4

1.0e5

1.2e5

1.4e5

1.6e5

1.8e5

2.0e5

2.2e5

2.4e5

2.6e5

2.8e5

3.0e5

3.2e5

3.4e5

3.6e5

3.8e5

4.0e5

4.2e5

Inte

ns

ity

, c

ps

7.56

7.12 7.46

7.35

7.32

7.25

7.41

7.05

7.79

7.67

7.957.89

500 pg/mL

(157.5 pM)

Keys to success

1. Derivatization with the SCIEX Amplifex™ Keto reagent

– Add reagent at room temp; 30 minutes; no need for fume hood

2. Removal of interferences with SelexION™ ion mobility technology

38 © 2016 AB Sciex RUO-MKT-11-1287 A

Plasma samples Collected from pregnant, postpartum and normal females

Liquid-liquid extraction Extract 100uL of plasma, using 1:1 (v:v) ethyl acetate:hexane mixture

Derivatization Add 50uL of SCIEX Amplifex™ Keto reagent to the dried, extracted sample

Allow to react for 1 hour.

Sample preparation

Allopregnanolone Derivatized Allopregnanolone

Steroids are non-polar, and are difficult to ionize

Derivatization enhanced the sensitivity, by improving the ionization efficiency


39 © 2016 AB Sciex RUO-MKT-11-1287 A

MS/MS and Ion Mobility

Ion

mobility MS/MS LC

a0

a0

a=0

t1

t2

Compensation

Voltage (COV)

Separation

Voltage (SV)Compensation

Voltage (COV)

Separation

Voltage (SV)

ions in

transport gasto mass

spectrometer

Q1 Q2 Q3

Mass selection

of parent ion

Fragmentation Mass selection

of fragment ion

To

Detector

• MS/MS separates compounds

based on differences in the

masses of the ions (both

precursor ions and fragment

ions).

Ion mobility separates compounds based on differences in the size/shape of the ionized compounds.

40 © 2016 AB Sciex RUO-MKT-11-1287 A

Assessing the effect of the SelexION™ ion mobility device

Postpartum sample #P5

(without ion mobility)

Postpartum sample #P5

(with ion mobility)

LC MS/MS

Ion

mobility MS/MS LC

allopregnanolone

allopregnanolone


41 © 2016 AB Sciex RUO-MKT-11-1287 A

10 pg/mL

allopregnanolone

(31.5 pM)

50 pg/mL

allopregnanolone

(157.2 pM)

200 pg/mL

allopregnanolone

(628.9 pM)

Sensitivity of the LC-ion mobility-MS/MS method

ALLO derivatized LC MRM calibration curve 2012-10-24 reinjection with ACN MP.rdb (THP 1): "Linear" Regression ("1 / (x * x)" weighting): y = 0.37 x + 0.00146 (r = 0.9994)

0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55Analyte Conc. / IS Conc.

0.00

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

0.10

0.11

0.12

0.13

0.14

0.15

0.16

0.17

0.18

0.19

An

aly

te A

rea

/ I

S A

rea

50 pg/mL 10 pg/mL

500 pg/mL

200 pg/mL

Using this very sensitive method, it was possible to measure <10pg/mL

Only 10uL were injected

The instrument response was linear over the calibration range covering from 10 - 25,000 pg/mL


42 © 2016 AB Sciex RUO-MKT-11-1287 A

Pregnanolone

(3a,5b-THP)

Allopregnanolone

(3a,5a-THP)

Epipregnanolone

(3b,5b-THP)

Epiallopregnanolone

(3b,5a-THP)

Allopregnanolone

Pregnanolone

Epi-allopregnanolone

+ Epi-pregnanolone

Representative chromatogram: “Pregnant” sample


44 © 2016 AB Sciex RUO-MKT-11-1287 A

• Keys to success, for LC-MS/MS method: ‒ Ion mobility separation using the SelexION™ device

‒ Derivatization using the SCIEX Amplifex™ Keto reagent

‒ Highly sensitive SCIEX QTRAP® 6500 LC/MS/MS system

• Great potential for research into: ‒ Detecting individual differences in metabolism of reproductive and

neurosteroid hormones

‒ Predicting individual differences in responses to SSRIs and other

medications.

• The QTRAP® 6500 system, combined with SelexION™ ion

mobility technology, provides researchers with the sensitivity

and selectivity required to measure low-level endogenous

steroids in complex biological matrices.

Conclusions

46 © 2016 AB Sciex RUO-MKT-11-1287 A

Trademarks/Licensing

AB Sciex is doing business as SCIEX.

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

© 2016 AB Sciex. The trademarks mentioned herein are

the property of AB Sciex Pte. Ltd. or their respective owners.

AB SCIEX™ is being used under license.

new sensitivity and selectivity gains with the … notes/new_sensitivity...ruo-mkt-11-1287-a new...

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