development of a sensitive method of analysis for the ... · pdf filequantitation of...

Jens-Jakob Karlsson/Discovery DMPK 22-Nov-11 2nd Nordic MS Symposium

Development of a sensitive method of analysis for the

quantitation of neurotransmitters i microdialysis samples using

the Xevo TQ-S mass spectrometer


Why measure neurotransmitters?

• Neurotransmitters are important biomarkers

associated with various neurological

disorders.

• Neurotransmitters are involved in a variety

of regulating systems such as stress and

learning1.

• Enzymatic hydrolysis of acetylcholine in the

synaptic cleft is fast and quickly metabolises

acetylcholine to choline and acetate by

acetylcholinesterase1.

• Acetylcholine concentration in extracellular

fluid as low as 100 pM

1 Bergquist J, Sciubisz A, Kaczor A, Silberring J. J. Neurosci. Methods 2002; 113: 1.


Why use mass spectrometry?

• Two years ago HPLC-ECD was used at H. Lundbeck for quantitation of acetylcholine.

• The switch to LC-MS had an immediate positive impact on cost and assay quality (precision).

Basal Acetylcholine levels in rat mPFC

0

1

2

3

4

5

6

Young Old

Age

ng

/ml

*

Statistical difference on basal extracellular

acetylcholine levels in young and aged animals,

p = 0.024.


A fast and sensitive method of

analysis for acetylcholine

Objectives

• Lower limit of quantitation 100 pM

– LLOQ of old method: 700 pM

• Short runtime

– Old method had a runtime of 12 min.

Means

• A more sensitive MS (Xevo TQ-S)

• HPLC Waters Acquity UPLC

– Need to find a suitable column

• Reduction of background noise

– Salts in Ringer solution

– Mobile phase

• Minimise ion-suppresion

– Check elution of salts and other

interfering compounds with Waters

Radar® Technology.


Decision tree for HILIC method

development

Acetylcholine chloride


Column selection: Waters UPLC BEH HILIC

• Waters UPLC BEH HILIC First choice, works well in our

generic HILIC method (protein precipitated samples, low water content).

Unbonded 1.7 µm Bridged Ethylene Hybrid [BEH] particle, giving good pH stability in the range 1-12.

Peak shape very sensitive to water content in sample.

10 ng/mL acetylcholine in A: ACN:MeOH (75:25),

B: Ringer:ACN (1:9), C: Ringer:ACN (1:4), D:

Ringer:ACN (1:3), E: Ringer:ACN (1:2), F:

Ringer:ACN (1:1).


Column selection: Waters UPLC BEH Amide

• Waters UPLC BEH Amide

Also a HILIC column

Utilize a trifunctionally-bonded

amide phase.

Good sensitivity despite dilution

Small sample volume needed

Time resolvation

Re-analysis possible

Sample for analysis on other

system/other

neurotransmitters

10 ng/mL acetylcholine in A: ACN:MeOH (75:25), B:

Ringer:ACN (1:9), C: Ringer:ACN (1:4), D: Ringer:ACN (1:3),

E: Ringer:ACN (1:2), F: Ringer:ACN (1:1).

O

O

O

Si

NH2

OBEH

LINKER


Column selection: Merck Sequant ZIC-HILIC

• Merck/Sequant ZIC-HILIC

Zwitter-ionic bonded silica.

Almost insensitive to water content in sample.

No sample preparation.

5 µL of a 0.5 ng/mL acetylcholine on a ZIC-HILIC

column.


Column selection: ability to separate

acetylcholine from endogenous (3-carboxy-

propyl)trimethylammonium

Acetylcholine chloride

1 Zhu, Y et al. Rapid Commun. Mass Spectrom 14, 1695-1700 (2000).

A: 2.5 pg/mL acetylcholine eluting at 0.61 min. and 0.5 ng/mL (3-

carboxypropyl)-trimethylammonium eluting at 1.26 min. Column:

50 mm UPLC BEH Amide column.


Separation from eluting salts and

other interfering compounds

ZIC HILIC column. Upper trace (A): scanning function.

Lower trace (B): MRM function (acetylcholine).

• Waters Radar® technology was used to ensure separation of acetylcholine from

eluting salts and other interfering compounds.

UPLC BEH Amide column. Upper trace (A): scanning

function. Lower trace (B): MRM function (acetylcholine).


Method robustness

Trend plot of calibration standards covering

5 runs over 2 days and a total of 385

injections. Column: ZIC HILIC.


Method robustness

• 50 injections of the same sample on the UPLC BEH Amide column

Without internal standard: With internal standard (acetyl-ß-methylcholine):


Impact of different brands of salts on

background noise

• Chromatograms of purified water and solutions of individual salts in concentrations

corresponding to the given salt in the microdialysis solution (Ringer solution). Column: ZIC

HILIC.


Impact of different brands of

acetonitrile on background noise

Fluka LC-MS Chromasolv Acetonitrile: Fisher Scientific Optima LC/MS Acetonitrile:

• Using Fisher Scientific Optima LC/MS acetonitrile reduces background noise with around

30 %. Upper trace: 5 µL of 1 pg/mL acetylcholine, Lower trace: Blank sample. Column:

UPLC BEH Amide.

Acetonitrile, LC-MS CHROMASOLV, Fluka, No. 34967

Acetonitrile, Optima LC/MS, Fisher Scientific, No. A955-1


Isocratic vs. gradient elution

Isocratic elution

• No time used for re-equilibrations between

injections.

• Risk of buildup of sample impurities giving

higher background and interferences.

• Using isocratic elution on the ZIC-HILIC

column we have been able to reduce the

run-time from 12 min to 6 min (low flow

HPLC column).

Gradient elution

• Higher resolving Power.

• No risk of buildup of sample impurities giving

higher background and interferences.

• Short run-times can be maintained with high

UPLC flow.

• Run-time reduced from 12 min to 5 min on the

UPLC BEH Amide column.


Lower limit of quantitation for

acetylcholine: UPLC BEH Amide

• Upper trace: 5 µL of 1 pg/mL (before

dilution) acetylcholine, Lower trace: Blank

sample.

• LLOQ: 2 pg/mL (14 pM)


Lower limit of quantitation for

acetylcholine: Sequant ZIC HILIC

• Chromatogram of an injection of 5 µL of

blank sample (Ringer) and 5 pg/mL (34 pM)

acetylcholine.

• Signal-to-noise ratio calculated as the ratio

of the peaks.


Measurement of acetylcholine in

microdialysis samples

Subcutaneous administration of Lu Compound X (Figure A), but not WAY-181187 (Figure B),

showed a significant increase in the extracellular acetylcholine levels.

Ventral hippocampus of freely moving rats

min

-80 -60 -40 -20 0 20 40 60 80 100 120 140 160 180

AC

h in

dia

lysa

te (

% o

f b

ase

line

)

0

20

40

60

80

100

120

140

160

180

200

220

240

260

Vehiclen = 7

Cmpd X, 2.5 mg/kg scn = 8

Cmpd X, 5.0 mg/kg scn = 8

Injection

** ** *

*

*P < 0.05 vs vehicle

Injection

Prefrontal cortex of freely moving ratsWAY-181187 30 mg/kg sc

min

-80 -60 -40 -20 0 20 40 60 80 100 120 140 160 180

AC

h in d

ialy

sate

(%

of base

line)

0

20

40

60

80

100

120

140

160

180

200

220

240

260

Vehiclen = 7

WAY-181187n = 8

Figure A Figure B


Perspectives

Perspectives

• Develop methods for some of the other very

interesting neurotransmitters.

• A more robust set of data for decision

making in the drug development process.

• Quantitation of all these neurotransmitters

using as few different chromatographic

systems as possible.

• Waters Trizaic UPLC system for smaller

samples and the possibility to study fast

signaling events in the brain.

Neurotransmitter Mw (amu) Molecular structure LLOQ needed pM (pg/mL)

Acetylcholine 146.21

100 (15)

GABA 103.12

1000 (100)

Serotonin 176.22

100 (18)

Dopamine 153.18

100 (15)

Glutamate 147.13

50000 (7400)

Aspartate 133.10

50000 (6700)

Norepinephrine 169.18

200 (34)

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