Ion exchange chromatography coupled to SACI for Ion exchange chromatography coupled to SACI for the analysis of high polar low molecular weight drugsthe analysis of high polar low molecular weight drugs

Sara Crotti, ISB srlSara Crotti, ISB srl

XIV Discussion group on Mass SpectrometryXIV Discussion group on Mass Spectrometry

Surface Activated Chemical Ionization

Ion Exchange Chromatography (IEC)Ion Exchange Chromatography (IEC)

TimeTime

Ionic

str

ength

Ionic

str

ength

Elution by means of salt of pH gradient

For high polar compounds

IEC coupled to Mass Spectrometry:IEC coupled to Mass Spectrometry:

ESI&APCI

Discharge phenomena for Discharge phenomena for the high voltages in API the high voltages in API

sourcessources

SACI No discharge phenomena (low No discharge phenomena (low

voltage source)voltage source)

Few application because of Few application because of analyte degradation and ion analyte degradation and ion

suppression phenomenasuppression phenomena

Never applied till nowNever applied till now

Aim: IEC Analysis of M3G and M6GAim: IEC Analysis of M3G and M6G

Optimal source: APCI, ESI or SACI? Optimal source: APCI, ESI or SACI?

Morphine 3-glucoronide Morphine 3-glucoronide (M3G)(M3G)

Morphine 6-glucoronide Morphine 6-glucoronide (M6G)(M6G)

Preliminary infusion of M3G 50ng/ml:Preliminary infusion of M3G 50ng/ml:

In source In source fragmentationfragmentation

MW 461 MW 285

0

2.0

3.0

4.0

5 x105

150 200 250 300 350 400 450 500 550 m/z

286

1.0

Morphine-3- Glucuronide in source fragment

Inte

nsit

y (

cou

nts

/s)

APCIAPCI

Inte

nsit

y (

cou

nts

/s)

462

3.0

4.0

5.0

6 x105

2.0

1.0

0

150 200 250 350 400 450 550 m/z300 500

[M+H]+

Source noise Source noise

Preliminary infusion of M3G 50ng/ml:Preliminary infusion of M3G 50ng/ml:

ESIESI

0.0

2.0

4.0

6.0

x105

150 200 250 300 350 400 450 500 550 m/z

462

286

8

Morphine-3- Glucuronide in source fragment

[M+H]+

Preliminary infusion of M3G 50ng/ml:Preliminary infusion of M3G 50ng/ml:In

ten

sit

y (

cou

nts

/s)

SACISACI

M3G and M6G separation with a linear gradient 5-M3G and M6G separation with a linear gradient 5-100% B100% B

AA: H: H22OO

BB: H: H22O + 0.5% formic ac. + O + 0.5% formic ac. + 10mM ammonium acetate10mM ammonium acetate

SACI

ESI

Cation Exchange Cation Exchange Chromatography Chromatography

(CEC)(CEC)

Injection of Injection of

500pg500pg

Chromatographic analysis:Chromatographic analysis:

2 4 6 8 10 12Time [min]

0

200

400

600

800

Inte

nsit

iy (

cou

nts

/s)

Inte

nsit

iy (

cou

nts

/s)

2 4 6 8 10 12Time [min]

0.0

0.5

1.0

1.5

2.05

x10 10.11

8.42

Morphine-6- Glucuronide

Morphine-3- Glucuronide

Results :Results :

SACI

ESI

Signal suppressionSignal suppression(in source current (in source current

~~40000nA) 40000nA)

Good signalGood signal(in source current (in source current

~~224nA) 4nA)

SACI urine sample analysis:SACI urine sample analysis:

Urine Urine samplesample

Dil. 1:5Dil. 1:5In HIn H2200

1010l injl inj

2 4 6 8 10 12

Time [min]

0.0

0.5

1.0

1.5

2.0x10510.15

8.48

M3G

M6G

Inte

nsit

iy

EIC m/z 462 m/z 286

Matrix effect:Matrix effect:

20 negative 20 negative

urine spiked urine spiked

vs vs

Standard Standard

solutionsolution

% Var=% Var=

(Au-As)/As*100(Au-As)/As*100

sample%Area variation urine vs Water

samples

1 -6

2 -8

3 -6

4 -1

5 -5

6 -7

7 -4

8 -4

9 -5

10 -5

11 -5

12 -5

13 -0,5

14 -2

15 -1

16 -1

17 -4

18 -3

19 -3

20 -2

Conclusions:Conclusions:

SACI is the only source suitable to coupling ion SACI is the only source suitable to coupling ion exchange chromatography with mass spectrometryexchange chromatography with mass spectrometry

Analysis in urine is performed by SACI without any Analysis in urine is performed by SACI without any sample pre-tratment tanks to a low matrix effectsample pre-tratment tanks to a low matrix effect

Good separation of analytes obtained with CECGood separation of analytes obtained with CEC

Future steps:Future steps:

Next experiments with potential and salt Next experiments with potential and salt concentration variation concentration variation

Application of LC-CEC-SACI-MS for screening of Application of LC-CEC-SACI-MS for screening of other addictive drug metabolites in biological other addictive drug metabolites in biological

sample. sample.

Where do you can find us ?Where do you can find us ?

Web site: http://www.isbiotechnology.comWeb site: http://www.isbiotechnology.com