Introduction Conclusions

Methods

Novel Derivatization Strategies for Biomarker Analysis using APLI MS Eduard Deibel1; Ralf Schiewek1; Klaus J. Brockmann2; Thorsten Benter2; Oliver J. Schmitz1

1Analytical Chemistry and 2Physical & Theoretical Chemistry University of Wuppertal, Germany

Institute for Pure and Applied Mass Spectrometry

Derivatization Strategy

Acknowledgement Financial support is gratefully acknowledged:

• German Research Foundation (DFG) within project SCHM 1699/12-1 • Bruker Daltonics

Literature 1) Constapel, M.; Schellenträger, M.; Schmitz,

O. J.; Gäb, S.; Brockmann, K. J.; Giese, R.; Benter, Th. Atmospheric-pressure laser ionization: a novel ionization method for liquid chromatography/mass spectrometry, Rapid Commun. Mass Spectrom. 2005, 19, 326-336.

2) Droste, S.; Schellenträger, M.; Constapel, M.; Gäb, S.; Lorenz, M.; Brockmann, K. J.; Benter, Th.; Lubda, D.; Schmitz, O. J. A silica-based monolithic column in capillary HPLC and CEC coupled with ESI-MS or elctrospray-atmospheric-pressure laser ionization-MS, Electrophoresis 2005, 26, 4098-4103.

3) Schiewek, R.; Schellenträger, M.; Mönnikes, R.; Lorenz, M.; Giese, R.; Brockmann, K. J.; Gäb, S.; Benter, Th.; Schmitz, O. J. Ultrasensitive Determination of Polycyclic Aromatic Compounds with Atmospheric-Pressure Laser Ionization as an Interface for GC/MS, Anal. Chem. 2007, 79, 4135-4140.

4) Schiewek, R.; Mönnikes, R.; Wulf, V.; Gäb, S.; Brockmann, K. J.; Benter, Th.; Schmitz, O. J. A universal ionization label for the APLI-(TOF)MS analysis of small molecules and polymers Angew. Chem. Int. Ed. 2008, 47, 9989-9992.

5) Heppel, C. W.; Heling, A. K.; Richter, E. Ultrasensitive method for the determination of 4-hydroxy-1-(3-pyridyl)-1-butanone-releasing DNA adducts by gas chromatography-high resolution mass spectrometry in mucosal biopsies of the lower esophagus. Anal. Bioanal. Chem. 2009, 393,1525-1530.

6)  Arjunan, P.; Shymasundar, N.; Berlin, K. D.; Najjar, D.; Rockley, M. G. Syntheses of selected .epsilon.-(2- or 9-anthryl)alkanoic acids and certain esters - carbon-13 spin-lattice relaxation time measurements of methyl 5-(2-anthryl)pentanoate and methyl 7-(2-anthryl)heptanoate, J. Org. Chem. 1981, 46 , 626- 629.

For a couple of years we have developed the hyphenation of HPLC, GC, and CEC with APLI and high resolution TOF MS for the analysis of essentially non-polar poly aromatic hydro-carbons1-3. For these compounds APLI is outstandingly sensitive. However, only aromatic compounds are amenable to APLI. This selectivity has both advantages (selectivity in a complex matrix) and disadvantages (severe restriction of the analytical compound range). To overcome these limitations, we have already reported on a derivatization strategy that facilitates ionization of polar non-aromatic compounds in complex matrices without hyphenated techniques or stable-isotope labeled standards4. In this contribution, the derivatization approach is considerably broadened and particularly extended toward various biomarkers.

Experimental Setup MS1: oaTOF MS (micrOTOF, Bruker Daltronics, Bremen, Germany)

Ion Source: home built temperature-controlled multipurpose ion source (TC-MPIS)

Laser System: excimer-laser (KrF, 248 nm) (ATLEX 300 SI, ATL Laser- technik,Wermelskirchen, Germany)

MS2: MALDI-reflectron-TOF mass spectrometer (AXIMA Performance™, Shimadzu Biotech, Manchester, UK)

Separation Technique: HPLC or GC

Synthetic Polymers

AP-REMPI

Free Fatty Acids DNA Adducts

Figure 1) Derivatization Strategy with an ionization marker and APLI.

Figure 3) Analysis of free fatty acids in sun flower oil with heptadecanoic acid as an internal standard and derivatization with anthracene methanol. Syringe injection.

2

OH

RO

OH

R

R

OH

NH2

N,N'-dicyclohexylcarbodiimide4-(dimethylamino)pyridine

dichloromethane35°C, 2h

N,N'-dicyclohexylcarbodiimide4-(dimethylamino)pyridine

dichloromethane35°C, 2h

N,N'-dicyclohexylcarbodiimide1-hydroxybenzotriazole

dichloromethane35°C, 2h

OOH

O

1

O

O R

OO

OR

ONH

OR

N

OH

OH

N

OH

O

To reduce the cost of the optimization a substitute (PHP) for the expensive POB and PHB (products of hydrolysis) was used.

N

OH

POB (pyridyl oxobutyl) PHB (pyridyl hydroxybutyl) PHP (pyridyl hydroxypropyl)

10 12 14 160

1x103

2x103

3x103

4x103

Cou

nts

t / min

m/z = 369.5 (S/N = 275)

O O

N

PHP-AMAA M = 369.46 g/mol

Figure 4) Metabolism of nicotine and formation of DNA adducts5 Figure 5) GC-APLI-(TOF)MS analysis of PHP after extraction from hydrolysed and spiked (50 pmol PHP) 500 µg DNA solution and derivatization with AMAA6. 1 µL of the derivatization solution was injected on the coloumn, which results in 50 fmol derivatized PHP on column with a S/N of 275.

230 235 240 245 250 255 260 265 270 275 λ /nm

toluene ethylbenzene propylbenzene butylbenzene hexylbenzene decylbenzene

CH2O

OHn

O

O

OH

+

O

O

OO

CH2

H2C

CH3n

16

H2C

H3C 16

APLI MALDI

Carbodiimide���4-DMAP���35 °C, 1 h

Figure 7) Analysis of Brij 72 with APLI and MALDI with 2,5-dihydroxybenzoic acid as matrix and silver trifluoroacetate as cationizing agent. While the signals in APLI-(TOF)MS arose from the radical cations, in MALDI-(TOF)MS the silver adducts of the analytes were formed.

Figure 8) APLI-(TOF)MS analysis (syringe injection) of PEG 2000 after derivatization with an anthracene label.

Figure 6) Derivatization of Brij 35 with an anthracene label.

The REMPI spectra of the derivatized analytes differ only marginally from the spectrum of the ionization label 1 (Figure 1). The comparison of the spectrum of anthracene with spectra of 1 and labeled analytes suggests that the presence of a C atom directly adjacent to the ring system shifts the spectrum to the red, whereas more distant groups do not significantly affect the absorption features.

It thus follows that the ionization yield for labeled analytes is virtually identical to that of the ionization marker. Naturally, the derivatization yield can be different for different analytes, but instead of expensive stable-isotope labeled standards, one or more homologous compounds can be added to the sample in a known amount, since similar derivatization yields are usually achieved in a homologous series. This was verified by the analysis of three fatty acids, palmitic, heptadecanoic, and stearic acid. Equimolar amounts of each acid were derivatized with 2 (Figure 1) and analyzed with APLI-(TOF)MS. The detected counts of these acids were 155 513, 150 620, and 153 296, respectively, which results in an average of 153 343 ± 2493 (1.6% standard deviation). The derivatization yield for these compounds was 67 %.

Figure 2) Comparison of various AP-REMPI spectra.

In summary, we have demonstrated that derivatization strategies significantly broaden the analytical applicability of APLI-MS. First, the range of ionizable analytes is not restricted by their spectroscopic features (and thus aromatic hydro-carbons) but solely by the presence of reactive anchor groups and the availability of suitable APLI labels. We are currently developing synthesis strategies for various APLI labels that specifically target reactive analyte sites. Secondly, once tagged with a specific APLI active label, the complex exhibits spectroscopic features virtually identical to those of the label itself. This leads to advantages for the quantitative analysis of compounds in complex matrices.