On the coupling of ion-exchange chromatography to surface-activated chemical ionization in the...

RAPID COMMUNICATIONS IN MASS SPECTROMETRY

Rapid Commun. Mass Spectrom. 2008; 22: 2134–2138

ublished online in Wiley InterScience (www.in
P
RCM

Letter to the Editor

Scheme 1. (a) M3G, (b) M6G, and (c) most abundant fragment ion structures. The

To the Editor-in-Chief

Sir,

On the coupling of ion-exchange

chromatography to surface-activated

chemical ionization in the analysis of

highly polar metabolites in diluted

urine samples

Ion-exchange chromatography (IEC) is

a widely employed technique used in

the analysis of highly polar com-

pounds.1 This technique allows the

analysis of those compounds that

usually are not/poorly retained on

reversed-phase (RP) chromatographic

columns. Basically, the analytes are

retained by means of a binding inter-

action between the analyte and the

stationary phase and are eluted by

using a solution containing ions that

compete with the analyte in the

column binding or by pH gradient.

This chromatographic approach is

rarely coupled to mass spectrometry

atmospheric pressure ionization (API)

sources due to the occurrence of

in-source discharge phenomena typi-

cally observed in the presence of high

concentrations of buffer and salts

needed for the high voltages of API

sources (electrospray ionization (ESI;

4000–6000 V) and atmospheric pres-

sure chemical ionization (APCI;

1000–6000 V)). Moreover, the presence

of salts and buffer can lead to matrix

ionization suppression effects. In other

words the analytes are not ionized.

These in-source discharge effects lead

to analyte degradation with conse-

quent severe loss of sensitivity.2 Thus,

RP chromatography is used to avoid

these problems.3–5 However, con-

ditions required to retain highly

polar/ionic compounds in RP mode

(i.e. high percentage of water in the

mobile phase) account for poor ioniz-

ation efficiency in ESI mode and, even

worse, for relevant matrix effects

(signal suppression/background en-

hancement) due to coeluting matrix

interferents. For these reasons, a pre-

purification step (e.g. solid-phase

extraction (SPE)), is usually necessary

in order to avoid the matrix effect.4,5

In previous investigations, a surface-

activated chemical ionization (SACI)6,7

source was employed to analyze com-

pounds in matrix sample extracts

without the need for pre-purification.8

The ionization mechanism of SACI is

based on the interaction of the eluent

neutral species with a bipolar moment

(e.g. H2O or neutral salts CH3COONa)

with a metallic surface. A low potential

terscience.wiley.com) DOI: 10.1002/rcm.3590

m/z values of the precursor and fragment io

difference between the surface and the

inlet to the mass spectrometer (50–

700 V) is used so as to create a reactive

environment that makes possible

the production of the analyte ions.9

The low potential difference leads to

better focusing of the analyte ions

inside the mass spectrometer and

allows the process to work in the

presence of salts and buffers avoiding

in-source discharge phenomena and

the consequent degradation in spec-

trum quality.6,7,9 Moreover, working at

high flow rates the cloud of neutral

reactive species surrounding the sur-

face strongly increases leading to high

ns are also reported.

Copyright # 2008 John Wiley & Sons, Ltd.

Figure 1. (a) APCI, (b) ESI, and (c) SACI direct infusion full scan spectra of

morphine-3-glucoronide. The solution concentration was 50 ng/mL. The direct infu-

sion flow rate was 10mL/min.

Letter to the Editor 2135

ionization efficiency. This allows the

direct analysis of diluted biological

samples (e.g. urine, hair extracts)

by liquid chromatography/mass spec-

trometry (LC/MS) and tandem

mass spectrometry (LC/MS/MS)

approaches.8,10–13 Looking at the

results, this technology seems to be

in principle highly effective when used

in conjunction with ion-exchange

chromatography (IEC) due to its ability

to work in the presence of salts and

buffers. It must be emphasized that

this chromatographic approach per-

mits the avoidance of the use of organic

solvent (methanol, acetonitrile) for the

analyte elution, with consequent

reductions in costs and lower health

risks for operators.

In this application, two highly

hydrophilic morphine metabolites

(morphine-3-glucoronide and morphine-

6-glucoronide), weakly retained using

the classical RP columns, have been

analyzed in urine samples by

using cation-exchange chromatog-

raphy (CEC) coupled to ESI, APCI

and SACI sources to verify and com-

pare the compatibility of these

ionization approaches with the IEC

techniques. Moreover, different chro-

matographic conditions were investi-

gated to reduce the matrix effect and

consequently to analyze directly urine

samples, avoiding pre-purification

steps.

Standard morphine-3-glucuronide

(M3G; Scheme 1(a)), morphine-6-

glucuronide (M6G; Scheme 1(b)), for-

mic acid and ammonium formate were

purchased from Sigma Aldrich (Milan,

Italy).

The direct infusion MS and MS/MS

spectra were achieved by analyzing a

50 ng/mL aqueous analyte solution.

The direct infusion sample flow was

10mL/min

Negative urine samples were spiked

with a known amount of standard

compounds (50 ng/mL for each ana-

lyte) and used to test the stability of the

developed LC-CEC/MS/MS method.

Positive urine samples were provided

from drug-addicted subjects. These

solutions were diluted (1:5) with dis-

tilled water and directly analyzed by

means of the LC-CEC/ESI- and

SACI-MS/MS approaches. A volume

of 10mL of the diluted analyte solution

was injected for each analysis.


An Ultimate 3000 apparatus (Dio-

nex, Sunnyvale, CA, USA) was used

for liquid chromatography (LC).

The chromatographic column was a

cation-exchange BioBasic SCX

(50� 2.1 mm, 5mm, 300 A). The HPLC

gradient was performed using two

eluents at a flow rate of 250mL/min.

The eluents were: (A) H2O and

(B) H2Oþ 0.5% formic acid þ100 mmol/L ammonium acetate.

Solution B (5%) was maintained for

5 min, then a linear gradient was used

passing from 5 to 100% of B in 3 min.

Solution B was maintained for 10 min

at 100%, and then re- equilibrated back

to the starting conditions after 2 min.

Thus, chromatographic analysis was

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performed in 20 min, but the mass

chromatogram acquisition time was

set to 27 min to allow the chromato-

graphic column to re-equilibrate. A

divert valve was used to direct the

eluent flow to waste for the first 7 min

of the analysis; then it was switched to

direct the eluent flow to the mass

spectrometer for another 10 min and

after which the valve was switched

again to the waste position.

The ESI, APCI and SACI sources

were employed to obtain direct infu-

sion spectra and LC/MS/MS chroma-

tograms.

MS inlet capillary voltage was 4000 V.

The entrance capillary temperature was

2508C. The flow of nebulizing gas

d Commun. Mass Spectrom. 2008; 22: 2134–2138

DOI: 10.1002/rcm

Figure 2. (a) APCI, (b) ESI, and (c) SACI direct infusion full scan spectra of

morphine-3-glucoronide obtained after background noise subtraction. The solution

concentration was 50 ng/mL. The direct infusion flow rate was 10mL/min.

2136 Letter to the Editor

(nitrogen) was 9.0 L/min. The drying

gas flow rate was 6.0 L/min.

APCI vaporizer temperature was

4008C and the entrance capillary

temperature was 1508C. The APCI

needle current was 4000 nA. The flow

rate of nebulizing gas (nitrogen) was

9.0 L/min. The drying gas flow rate

was 5.0 L/min. The MS inlet capillary

voltage was set to 4000 V.

SACI vaporizer temperature was

4008C and the MS capillary tempera-

ture was 2508C. The surface voltage

was 50 V. The surface temperature, as

measured by an optical pyrometer,

was 1108C. The flow rate of nebulizing

gas (nitrogen) was 9.0 L/min. The

drying gas flow rate was 2.0 L/min.

The MS inlet capillary was set to 650 V.

Themassspectrawereobtainedusing

an HCTultra ion trap mass analyzer

(Bruker Daltonics, Bremen, Germany).

The maximum number of ions per scan

was 100 000, with an average of three

microscans per spectrum. The ion

charge control (ICC) was on.

LC/ESI and SACI mass chromato-

grams were obtained using the

tandem mass spectrometry (MS/MS)

approach. The isolation width of the

ions was three mass units. The collision

energy was 100% of its maximum

value (2 V peak-to-peak). The mass

spectra were acquired in positive ion

mode.

The signal/noise (S/N) ratio was

calculated using the root mean square

(RMS) algorithm. An extensive descrip-

tion of this S/N calculation approach

has been reported elsewhere.14 Hystar

software (Bruker Daltonics) wasused to

acquire the data and DataAnalysis

software (Bruker Daltonics) was used

to elaborate them.

Preliminary results were performed by

direct infusion of two 50 ng/mL of M3G

(Scheme 1(a)) and M6G (Scheme 1(b))

aqueous solutions. APCI, ESI and SACI

direct infusion spectra of M3G are

shown in Figs. 1(a), 1(b) and 1(c). It

must be emphasized that the direct

infusion full scan and MS/MS spectra

of M6G exhibit the same behaviour

observed for M3G but the ionization

efficiency is about 10 times higher. In

the case of APCI (Fig. 1(a)) only an

abundant ion at m/z 286 was detected

at this concentration level due to in-

source compound fragmentation

(Scheme 1(c)). Thus, the compound is


fully fragmented in the ionization

source, probably due to the highly

destructive effect of the corona dis-

charge, with consequent loss of speci-

ficity. In the case of ESI (Fig. 1(b)) an

abundant ion ([MþH]þ) at m/z 462

(counts/s 6.0� 105; S/N ratio of the

[MþH]Rion: 18) was detected. In the

case of SACI (Fig. 1(c)), a high ioniz-

ation efficiency was achieved (counts/

s 8� 105; S/N ratio of the [MþH]Rion:

33) even if the fragment ion at m/z 286,

probably due to in-source thermal

decomposition or to the well-known

surface-induced dissociation (SID)

effect,16 was observed. To better com-

pare the delta in ionization efficiency of

the different sources noise subtraction

Rapi

was applied to all samples. Figures 2

(a)– 2(c) show the APCI, ESI and SACI

spectra, respectively, obtained after

noise subtraction. As can be seen, even

in this case the higher signal intensity

was achieved using SACI (Fig. 2(c);

7.5� 104) with respect to that achieved

using ESI (Fig. 2(b); 5.5� 104). How-

ever, even without background noise

subtraction, as usual a lower chemical

noise was observed in the mass spec-

trum obtained under SACI conditions.

Thus, ESI and SACI were chosen,

due to their best performance, in

order to develop a LC-CEC/MS/MS

approach to analyze these polar com-

pounds. The ESI and SACI cation

exchange – ion extraction – tandem


DOI: 10.1002/rcm

Figure 3. Cation exchange – ion extraction – tandem mass chromatograms

obtained of morphine-3-glucoronide and morphine-6-glucoronide by using (a) ESI

(water standard solution), (b) SACI (water standard solution), and (c) SACI (addicted

subject urine sample). A volume of 10mL was injected for each analysis.

Letter to the Editor 2137

mass chromatograms of M3G and

M6G obtained by injecting 10mL of a

50 ng/mL standard solution, corre-

sponding to 500 pg injected, in water

are shown in Figs. 3(a) and 3(b). The

collisionally generated MS/MS frag-

ment ion at m/z 286 was monitored

(Scheme 1(c)). As can be seen, the two

compounds are detected only under

SACI conditions (retention times

8.42 min for M3G and 10.11 min for

M6G). This phenomenon is probably

due to the ion suppression effect

obtained under cation-exchange

elution conditions. In fact, the two

compounds elute with 100% of mobile

phase B (H2Oþ 0.5% formic acidþ100 mmol/L ammonium formate). It

must be emphasized that, by using ESI

and working with 100% B buffer

conditions, a high in-source discharge


current is achieved (40000 nA; instru-

ment current limit) while in the case of

SACI a definitely lower value (24 nA) is

achieved. This behaviour suggests that

the degradation of analytes is achieved

under high discharge conditions, as

observed in the APCI case. On the

other hand it is possible that a higher

matrix ionization suppression effect

takes place under ESI conditions.

Moreover, the chromatographic peaks

of the two isomers are well resolved

under CEC conditions. Thus, they can

be separately quantified even if they

exhibit the same fragmentation beha-

viour.

Thus, SACI was chosen, due to its

sensitivity and its ability to operate in

the presence of high amounts of salts

and buffers,6,7,9 to analyze the selected

morphine metabolites, in diluted urine

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samples, without purifying the sample

by SPE, usually employed when work-

ing under RP conditions.

Figure 3(c) shows the LC-CEC/

SACI-MS/MS ion extraction mass

chromatogram obtained by analyzing

the real positive urine sample. The

urine was diluted 1:5 using doubly

distilled water before the analysis. A

volume of 10mL of the diluted mixture

was injected. As can be seen under

SACI conditions, the two compounds

were clearly detected.

Finally, in order to evaluate matrix

effects,8,15 20 different negative urine

solutions were then spiked with each

analyte at 50 ng/mL, and analyzed as

previously described. The % chroma-

tographic peak area variation obtained

by comparing urine and water sample

analyte areas was calculated using

Eqn. (1):

AV ¼ ½ðAu � AwÞ=Aw� � 100 (1)

where AV is the % area variation of the

compounds (M3G or M6G), Au and Aw

are the compound chromatographic

peak areas obtained analyzing urine

and water samples, respectively. The

results clearly show that the chromato-

graphic peaks % area variation among

the selected 20 samples in urine with

respect to that achieved by analyzing

water samples was between �0.5 and

�8%. Thus, a low ion suppression effect

seems to be present. However, in order

to further investigate this phenomenon

a post-column analyte (M3G and M6G)

infusion method was employed.17 Even

in this case the matrix effect observed

was negligible.

In conclusion, SACI technology was

shown to be fully compatible with

the cation-exchange chromatographic

approach in the analysis of M3G and

M6G while APCI and ESI leads to

higher in-source sample fragmentation

due to higher in-source discharge or to

an ionization matrix suppression

effect. These results have been

achieved mainly thanks to the low

potential (50 V) at which SACI oper-

ates, strongly reducing the in-source

discharge phenomenon (ion current

25 nA) that is present when working

under APCI and ESI conditions (about

40000 nA) in the presence of cation

exchange elution phase buffers. On the

other hand, ESI does not lead to

in-source fragmentation effects under


DOI: 10.1002/rcm

2138 Letter to the Editor

pure water solvent conditions but

exhibits a lower ionization efficiency

with respect to SACI. In fact, it

provides a lower signal intensity and

higher chemical noise, reflected in a

lower S/N ratio. Moreover, the SACI

approach allows the use of inexpensive

and non-toxic organic solvents. There-

fore, this technique allows a new way

to obtain the separation and analysis of

highly polar metabolite in urine

samples.

Future developments will be

focused on the application of the

LC-CEC/SACI-MS/MS technique in

the screening of other addictive drug

metabolites in urine samples. In fact

this technique could be used to

advantage for other highly polar

metabolites because of the high poten-

tial of the LC ion exchange technique

coupled to SACI mainly in terms of

chromatographic resolution, matrix

effect reduction and sensitivity.

AcknowledgementsThe authors thank Bruker Daltonics for sup-port. This work was supported by theINGENIO project (Finlombarda spa). Theauthors thank Ms. Karim Jacqueline AmayaMendoza for her essential support.


Simone Cristoni1*, Sara Crotti1, LorenzoZingaro1, Luigi Rossi Bernardi2, RossellaGottardo3, Lucia Politi4, Aldo Polettini3,

and Franco Tagliaro31

ISB – Ion Source & Biotechnologies,Milan, Italy

2Multimedica Laboratories, Milan,Italy

3Universita degli Studi di Verona,Facolta di Medicina e Chirurgia, Dip.

to di Medicina e Sanita Pubblica–Sez.di Medicina Legale, Verona, Italy

4Universita degli Studi di Pavia, Dip.todi Medicina e Sanita Pubblica, Pavia,

Italy

*Correspondence to: S. Cristoni, ISB – IonSource & Biotechnologies, Via Fantoli 16/15, 20138 Milan, Italy.E-mail: [email protected]/grant sponsor: INGENIO project(Finlombarda spa).

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Received 24 December 2007Revised 24 April 2008

Accepted 25 April 2008


DOI: 10.1002/rcm

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