ionisation in the absence of high voltage using sfc-ms: a...

Ionisation in the absence of

high voltage using SFC-MS:

a route to greater sensitivity

Mohini Thite

Email: mat@

soto

n.ac.uk

BMSS M

eeting -11th Sept 2007

2

Outline

•W

hat is SF

C?

•Couplin

g SF

C-M

S

•Exper

imen

tal Des

ign

•Det

ection

•ESI-A

PCI

•SF

C conditions

•Pre

ssure

•Modifier

•Flow rate

BMSS M


3

What is SF

C?

•Su

per

critical fluid chro

matogr

aphy

•Mobile

phas

e in super

critical state

•Mobile

phas

e: non polar ca

rbon dioxide

•Modifier

: polar org

anic solven

t to

impro

ve

chro

matogr

aphy

•Polar stationar

y phas

es

•e.

g. silica

, am

ine, 2-E

P

•Consider

ed as norm

al phas

e te

chnique

BMSS M


4

SFC-M

S Couplin

g

Ref

: M. G

arzo

tti,

M. H

amda

n, J

. Chr

omat

ogr

B,7

70

(2

00

2)

53

-61

.

Pump port

Knauer

Detector

Fluid control

module

Connec

ted to

hea

t ex

chan

ger

CO

2MeO

H

Chec

k va

lve

Autosampler

10uL loop

10mm cell

Column port

TCM-

Heater

control

module

Peak

detection

module

Mixer

Make-up

from

HPLC

Pump

Mass

Spectrometer

Valco

Tee

Valco

Tee

PEEK tubing

BMSS M


5

MS Det

ection

•Initial studies utilis

ed ESI

•O

ptimisation o

f API fo

r SF

C

•O

bse

rvation o

f sa

mples ionising in the ab

sence

of

high voltag

es

•Sp

ecific te

st compounds an

alys

ed to pro

be th

is

ionisation m

echan

ism

BMSS M


6

What is ca

using ionisation?

•API so

urc

e co

nfig

ura

tion dep

enden

t?

•ESI c

f.APCI

•Chro

matogr

aphic sys

tem spec

ific?

•HPLC c

f.SF

C

BMSS M


7

Is ionisation related

to?

•Char

ge res

idual m

odel?

•Addition o

f ac

id

•Ther

mosp

ray?

•Addition o

f am

monium ace

tate

•So

nic spra

y ionisation?

•Pre

ssure

chan

ge

•% m

odifier

chan

ged

•Neb

ulis

er gas

Ref

: C. Das

s, Principles an

d Pra

ctice of Biologica

l Mas

s Sp

ectromet

ry, Jo

hn W

iley & Sons, Inc., New

York

, 2001.

BMSS M


8

Exper

imen

tal co

nditions

OH

O O

N

STD2. Oxybutynin

O

N

STD1. Diphenhydramine

O

OOO

O

NN H

O

O

OO

STD4. Reserpine

HO

N

HO

STD3. Terfenadine

H

H

H

H

H

H

43

21

HV: 0kV

CV: 0V

HV: 0kV

CV: 20V

HV: 3.5kV

CV: 0V

HV: 3.5kV

CV: 20V

No make-up

HV: 3.5kV

CV: 0V

HV: 3.5kV

CV: 0V

HV: 3.5kV

CV: 0V

HV: 0kV

CV: 20V

HV: 0kV

CV: 20V

HV: 0kV

CV: 20V

HV: 0kV

CV: 0V

HV: 3.5kV

CV: 20V

Methanol + 0.1%

HCOOH

HV: 0kV

CV: 0V

HV: 3.5kV

CV: 20V

Methanol + 0.1%

NH4OAc

HV: 0kV

CV: 0V

HV: 3.5kV

CV: 20V

Pure methanol

BMSS M


9

ESI res

erpine HV o

n/o

ffReserpine (ESI)

Make-up solvent

MeOH

+NH4OAc

+HCOOH

None

Peak area

01e+4

2e+4

3e+4

4e+4

5e+4

6e+4

HV = 3.5kV

HV = 0kV

4No m

ake-up

solvent(M

+H)+

HV =

3.5kV

(M+H)+

HV =

0kV

BMSS M


10

ESI and A

PCI

Reserpine (ESI)

Make-up solvent

MeOH

+NH4OAc

+HCOOH

None

Peak area 01e+4

2e+4

3e+4

4e+4

5e+4

6e+4

HV = 3.5kV

HV = 0kV

Resepine (APCI)

Make-up solvent

MeOH

+NH4OAc

+HCOOH

None

Peak area

02e+2

4e+2

6e+2

8e+2

1e+3

1e+3

HV= 3.5kV

HV= 0kV

NH4OAc

NH4OAc

No m

ake-up

solvent

No m

ake-up

solvent

BMSS M


11

Summar

y of ESI and A

PCI

•Compar

able/Impro

ved ionisation in abse

nce

of high

voltag

e

•Io

nisation suppre

ssed

with addition o

f am

monium

acet

ate

•Fo

rmic acid does

not en

han

ce ionisation

•Data fo

r APCI so

urc

e co

mpar

able w

ith ESI sourc

e

•Io

nisation indep

enden

t of ca

pillar

y type

BMSS M


12

Pre

ssure

and m

odifier

(b) Pressure : 100 Bar

% M

odifier

10% M

eOH20% M

eOH50% M

eOH

Peak Area 0.0

2.0e+3

4.0e+3

6.0e+3

8.0e+3

1.0e+4

1.2e+4

1.4e+4

HV : On

HV : Off

(a) Pressure : 75 Bar

% Modifier

10% M

eOH20% M

eOH50% M

eOH

Peak Area 0.0

2.0e+3

4.0e+3

6.0e+3

8.0e+3

1.0e+4

1.2e+4

1.4e+4

(c) Pressure: 150 Bar

% Modifier

10% MeOH20% M

eOH50% M

eOH

Peak Area 0.0

2.0e+3

4.0e+3

6.0e+3

8.0e+3

1.0e+4

1.2e+4

1.4e+4

HV : On

HV : Off

HV : On

HV : Off

•Mixtu

re o

f te

rfen

adine, o

xyb

utynin and ery

thro

myc

in

analys

ed at va

rying pre

ssure

and m

odifier

per

centage

•O

ptimum conditions fo

r max

imum sen

sitivity in

pre

sence

or ab

sence

of high voltag

e:Pre

ssure

100bar

and m

odifier

30% M

eOH

BMSS M


13

Oxybutynin

Flow rate in m

L/m

in

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

Peak Area

02e+5

4e+5

6e+5

8e+5

HV = 3.5kV

HV = 0kV

Nicotinanilide

Flow rate in m

L/m

in

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

Peak Area

01e+5

2e+5

3e+5

4e+5

HV = 3.5kV

HV = 0kV

Mebendazole

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

01e+5

2e+5

3e+5

4e+5

5e+5

HV = 3.5kV

HV = 0kV

Peak Area

Flow rate in m

L/m

in

Reserpine

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

02e+5

4e+5

6e+5

8e+5

HV = 3.5kV

HV = 0kV

Peak Area

Flow rate in m

L/m

in

Oxybutynin

Flow rate in m

L/m

in

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

Peak Area

02e+5

4e+5

6e+5

8e+5

HV = 3.5kV

HV = 0kV

Flow rate

BMSS M


14

High flow rate (5

mL/m

in)

•Im

pro

vemen

t in sen

sitivity upon incr

easing th

e distance

bet

wee

n the ESI nee

dle tip and the sa

mple cone

Mebendazole

Distance of needle and sampling cone

default

plus 1 mm

plus 2 mm

Peak Area

02e+4

4e+4

6e+4

8e+4

1e+5

1e+5

1e+5

HV = 3.5kV

HV = 0kV

BMSS M


15

Modifier

effec

tComparison of acetonitrile and m

ethanol modifiers

Oxybutynin

Nicotinanilide

Mebendazole

Reserpine

Peak Area 01e+5

2e+5

3e+5

4e+5

HV: On (MeCN)

HV: Off (MeCN)

HV: On (MeOH)

HV: Off (MeOH)

BMSS M


16

Modifier

effec

t

•Incr

ease

d ionisation w

ith M

eOH m

odifier

compar

ed

to M

eCN

•Is the ionisation efficiency

related

to pre

form

ed ions

in solution?

Comparison of acetonitrile and m

ethanol modifiers

Oxybutynin

Nicotinanilide

Mebendazole

Reserpine

Peak Area 01e+5

2e+5

3e+5

4e+5

HV: On (MeCN)

HV: Off (MeCN)

HV: On (MeOH)

HV: Off (MeOH)

BMSS M


17

High voltag

e pro

file

•Not elec

trosp

ray

•CRM -

Ions fo

rmed

in solution o

r during sp

ray pro

cess

•CRM plus oth

er pro

cesses

, so

nic spra

y

oxybutynin

HV (kV)

-0.5

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

Peak area

02e+5

4e+5

6e+5

8e+5

1e+6

BMSS M


18

Conclusions

•Compounds ionise in the ab

sence

of high voltag

e∴

not elec

trosp

ray ionisation

•Addition o

f am

monium ace

tate

does

not aid

ionisation ∴

not th

ermosp

ray ionisation

•Su

gges

ts ions ar

e fo

rmed

in solution o

r during th

e su

per

sonic jet

expan

sion

BMSS M


19

Conclusions

•MeC

Nas

mak

e-up solven

t or as

modifier

does

not

assist in ionisation, unlik

e MeO

H ∴

supporting CRM?

•Io

nisation o

n FIA

-ESI-M

S with H

V turn

ed o

ff but not as

go

od as SF

C-M

S

•API-MS re

sponse

dep

enden

t on m

odifier

conce

ntration;

this should be take

n into

acc

ount fo

r gr

adient elution

studies

BMSS M


20

HV: Off

HV: On

Conclusions

For sm

all molecu

le analys

isif se

nsitivity is an

issue :

Turn

ing off the elec

trosp

ray high voltag

e co

uld

impro

ve LO

D-

e.g.

oxyb

utynin, re

serp

ine data

21

Ack

now

ledg

emen

ts

Gla

xoSm

ithK

line,

CA

SS, N

ew

Fron

tiers

Nor

th, H

arlo

w,

CM

19

5A

W,

UK

Pfiz

er G

loba

l Res

earc

h an

d D

evel

opm

ent,

Sand

wic

h,

CT

13

9N

J, U

K

Laure Hitzel

Frank Pullen

Met

tler-

Tol

edo

Aut

oche

m, 1

30

E

xecu

tive

Drive

, Sui

te 2

A, N

ewar

k,

DE

19

70

2, U

SA

Clare Paterson

Bob Boughtflower

Paul Oakley

Prin

ceto

n C

hrom

atog

raphy

Inc

., C

ranb

ury,

NJ

08

51

2, U

SA

Jeff Caldwell

G. John Langley

Julie Herniman

Amaury Cazenave Gassiot

Scho

ol o

f C

hem

istr

y,U

nive

rsity

of

Sout

ham

pto

n,SO

17

1B

J, U

K

Gla

xoSm

ithK

line,

Gun

nels

Woo

d R

oad,

Ste

vena

ge, S

G1

2N

Y, U

K

Steve Lane

Evo

tec,

15

1 M

ilton

Pa

rk, A

bing

don,

O

X1

4 4

SD, U

K

Stefan Richardson

22

Tha

nk Y

ou

ionisation in the absence of high voltage using sfc-ms: a...

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