APLI

Electrospray-Atmospheric Pressure Laser Ionization (ES-APLI): A new interface for LC-MS coupling
Oliver J. Schmitz; Stefan Droste; Marc Schellentrger; Marc Constapel; Ralf Schiewek; Siegmar Gb; Matthias Lorenz; Ana Mangas Suarez; Klaus J. Brockmann; Thorsten Benter

University of Wuppertal
Department of Chemistry
Gauss Str. 20, 42119 Wuppertal, Germany

Introduction
The APLI interface described earlier was based on the design of the original MicroMass Z-Spray ion source, the heated AP probe being used without modification as the inlet stage. This set-up allows coupling with HPLC, but because of bubble formation not with CEC. In addition, pneumatically assisted vaporization by heat renders the analysis of thermally labile compounds difficult. We demonstrate here that silica-based monolithic columns can be used with either capillary HPLC or CEC for coupling with an API mass spectrometer to analyze non-polar compounds such as PAHs. The PAHs were volatilized by a commercial ES interface (nanoflow-ESI from Micromass, Chip-ESI from Advion or a homemade ESI for the micrOTOF), and APLI was employed. With these interface combinations it should in general be possible to analyze thermally labile non-polar aromatic compounds or analytes with a high boiling point without generating thermal decomposition products.

Results
Laser off Laser on
O O R

b
Counts

1.2e+4 8.0e+3 4.0e+3

OH O

R-OH + R-NH 2

N C N O

b
4000

Ethanol

M+

600

Cholesterol
H3C CH 3 H

M+
H

m/z 294
CH3

m/z 649

400
O O O

O O O

counts

2000
200

1200

0.0

d
Counts
800

3.5

4.0

4.5

5.0

5.5

6.0

N O H

counts
0 100 200 300 400 500 600 700
0 100 200 300 400 500 600 700

1
1.2e+5
[M+H]+

Time [min]
1.2e+5
M+

m/z
400

m/z

.
291
counts
O

Dodecyl amine
NH

M+

400

m/z 448
300

Oleic acid
O CH2O

M+

m/z 473

291

166

300

O O

Counts

400

4.0e+4 0.0 100 200 300

4.0e+4 0.0 100 200 300

N C N OH

200

Counts

3 5

Counts

8.0e+4

8.0e+4

200

0 0 20

100

40

100

Time [min]

m/z

m/z

R-COOH

0 100 200 300 400 500 600 700

0 100 200 300 400 500 600 700

m/z

m/z

Methods
HPLC-System: Column: Split column: Injection: CEC-System: Column: Electrolyte: Separation voltage: Injection: MS-System: Microspray-ESI: Capillary voltage: Nebulizing gas N2: Cone gas N2: Sheath flow: MS-System: ESI: Capillary: Nebulizing gas N2: Dry gas N2: Injection: Chip-ESI: Voltage: Injection: L7110 HPLC (Merck) 51.2 cm, 100 m I.D. CapROD RP 18 35 cm, 50 m I.D. fs-capillary 5 L injection loop HP3dCE system (Agilent) 45 cm, 100 m I.D. CapROD RP 18 80 % ACN and 20 % 5 mM ammonium acetate, pH 8.0 30 kV 10 kV, 5 s QTOF-MS Ultima (MicroMass) home-made APLI-source 3 kV 18 psi (CEC) and 22 psi (HPLC) none 1 L/min (99 % MeOH/H2O = 1/1 and 1 % acetic acid) micrOTOF (Bruker Daltonics) home-made APLI-source 6 kV 8.7 psi 2 L/min Syringe injection (flow: 5 L/min) Nanomate 100 (Advion BioSciences Ltd.) 1.5 kV flow: 200 nL/min

Figure 1 Electrospray/Atmospheric-Pressure Laser Ionization-Interface based on the microspray-ESI-source from MicroMass. a) and b) Pictures of the source. The blue arrow indicates the laser beam position (from top). c) Upper panel: The mass trace at 166 is shown with laser turned off and on. Without the laser beam, only the polar catechin is ionized by electrospray, whereas the combination with APLI ionizes the non-polar fluorene as well. The lower panel shows mass spectra for both cases. d) Analysis of fluorene (1), anthracene (2), fluoranthene (3), pyrene (4) and benzo[a]pyrene (5) with CEC-ES/APLI-MS.

Figure 4 Derivatization of several analytes with an aromatic ionization label. a) Derivatives of anthracene are used to label several analytes (amines, alcohols or carboxylic acids) in the presence of a carbodiimide under soft conditions. b) The analyses with APLI-QTOF-MS (heated probe) of derivatized ethanol, cholesterol, dodecyl amine and oleic acid are shown.

Inte ns. x10 5

b
2.0

[M+H]+

M+.
202.1

+MS, 0.1-1.9 min #(7-227)

Conclusions

N
1 63.1

1.5

1.0

0.5

211 .2 149.0 161.1 0.0 150 160 170 180 1 90 200 210 169.2

221 .2

220

m /z

Figure 2 Electrospray/Atmospheric-Pressure Laser Ionization-Interface coupled to the micrOTOF from Bruker. a and b) Pictures of the source. The blue arrow indicates the entrance of the laser beam. c) Simultaneous analysis of nicotine and pyrene with the ES/APLI-TOF-MS. Nicotine is ionized by ESI [M+H]+ and the non-polar pyrene is sprayed by ES but ionized by APLI [M+.].

4.00E+03

c
M+.

chip lens
2.00E+03

The parallel application of the two ionization methods (ESI and APLI) to a mixture containing a PAH (fluorene, pyrene or benzo[a]pyrene) and a polar compound (catechin or nicotine) in acetonitrile was demonstrated on two different MS systems. The PAH is efficiently ionized by APLI, but not by ESI. For catechin or nicotine the exact opposite is true. With the laser turned off, only the quasi-molecular ion of catechin ([M+H]+, m/z = 291) or nicotine ([M+H]+, m/z = 163) is detected; when the laser is switched on, the PAH also appears in the spectra, as the radical cation (M+, m/z = 166, 202 or 252). In addition, a mixture of PAHs consisting of fluorene, anthracene, fluoranthene, pyrene and benzo[a]pyrene was separated by CEC and HPLC on a monolithic column and detected by ES-APLI-MS. We have also shown the use of two ionization labels, both based on anthracene for the derivatization of amines, alcohols or carboxylic acids. Only a few fragments are observed in the spectra. In the future the use of the soft ionization process ES/APLI will test to avoid these fragmentations, and further ionization labels will be synthesized for several APLI combinations (GC, HPLC, CEC).

Laser system:

KrF*, 100 Hz, 18 mJ pulse energy (QTOF Ultima) KrF*, 200 Hz, 12 mJ pulse energy (micrOTOF)

[M+H]+

Acknowledgement
200 250 300 350 400

0.00E+00

100

150

Financial support is gratefully acknowledged: Merck KaG, Ford Motor Company, State of NRW, BUW, MicroMass/Waters, Bruker Daltonics, Studienstiftung des deutschen Volkes.

Figure 3 Chip-ES/APLI coupled to the QTOF-MS. a) Picture of the Chip-ESI system from Advion coupled to the QTOF-MS. The yellow arrows show the laser beam, which is slightly focused in front of the cone by a lens. B) Commercially available chip with 400 jets (for each analysis one jet is needed). c) Simultaneous analysis of nicotine and benzo[a]pyrene with Chip-ES/APLI-QTOF-MS. Nicotine is ionized by Chip-ESI [M+H]+ and the non-polar benzo[a]pyrene is sprayed by Chip-ES, but ionized by APLI [M+.].

For further information: www.apli-ms.com