New Developments of Laser Desorption Ionization Mass

Spectrometry in Plant Analysis

Andreas Schinkovitz, Denis Séraphin and Pascal Richomme

University of Angers, SONAS-IFR QUASAV, UFR des Sciences Pharmaceutiques

16, Bd Daviers 49100 Angers, France

Keywords : laser desorption ionization mass spectrometry secondary metabolites

Abstract

The structural identification of natural products is one of the major focus of analytic chemistry research. Mass Spectrometry (MS) has long been used to obtain molecular weights and further molecular formulas. In the past, former ionization sources such as electronic impact unfortunately limited MS analysis to volatile, polar and thermostable compounds. However, recent developments in soft ionization techniques such as electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI) or laser desorption/ionization (LDI) have gradually extended MS analysis to polar and/or thermolabile compounds. As far as small natural compounds are concerned, LDI sources are still seldom used due to specific technical limitations.

Indeed the photo ionization process of LDI is generally assisted by a matrix, i. e a small and strong UV chromophore, in the so-called matrix assisted laser desorption/ionization (MALDI) process. A MALDI process therefore induces the formation of numerous matrix ions which commonly appear in the range from 0 to

600 Da, and consequently interfere with molecular ions originating from most natural products. For that reason the correct signal assignment is highly impaired in that

2 critical region of interest. Since LDI is not only a soft ionization process but also a quite sensitive technique yielding high resolution spectra when coupled to a time of flight (TOF) analyzer, different attempts have been made to adapt the technique to natural products. Three of them will be more specifically discussed in this book chapter: LDI on neat gold surfaces obtained by physical vapor diffusion (PVD), desorption/ionization on self assembled monolayers surfaces (DIAMS) and use of specific matrixes for selective detection of alkaloids.

1. INTRODUCTION : HISTORICAL BACKGROUND

The development of matrix assisted laser desorption ionization and related methods dates back to the late seventies when it was shown that the irradiation of small organic molecules by a pulsed laser at high intensity leads to the formation of ions that could be analyzed through mass spectrometry (Posthumus et al., 1978). Most generally using an UV laser as the irradiation source, this observation led to the conception of the so-called laser desorption/ionization (LDI) source. Later on, this soft ionization source was coupled with a time of flight detector (TOF) in LDI-TOF mass spectrometers. From that time on it was possible to produce and characterize ions originating from nonvolatile and/or thermolabile compounds. However it also became clear, that not every entity could enter the LDI process thus limiting the technique to very few and particular compounds. An important step forward was taken during the eighties by introducing a so called “matrix”, i. e. a small organic molecule carrying a strong UV chromophore, to generalize the LDI process (Karas et al. 1988; Tanaka et al. 1988). This concept was called matrix assisted laser desorption/ionization (MALDI, Figure 1) and extended the range of mass

3 spectrometry (MS) applications to bioanalytics as well as synthetic polymers. Ever since MALDI MS has gained immense popularity and by today represents one of the most commonly applied analytical techniques for the analysis of large molecules.

2. LASER DESORPTION IONIZATION MASS SPECTROMETRY

2.1 Laser Desorption Ionization (LDI)

Apart from MALDI, further LDI applications were contemporaneously developed over time. With substantial progress made in nanotechnologies, matrix free LDI techniques, such as “surface assisted laser desorption ionization” (SALDI) MS were established. In this method classical matrix molecules are replaced by nanostructured materials such as germanium, carbon and particularly silicon (Chen et al. 2006; Law et al. 2010, 2011). It has been shown that under optimized conditions SALDI may facilitate the detection of selected compounds in the low femtomole range (Chen et al. 2006).[A] Further nanostructured metal or transitional metal derivatives i.e. silver, titanium nitride and gold could be used in a similar way

(McLean et al. 2005; Schuerenberg et al. 1999).

Depending on their specific properties, these materials may be utilized as free nanoparticles, or as surface material attached on a sample carrier (Figure 2). A good

4 example for the practical application of the latter is represented by the spectrum of quinidine displayed in Figure 3, which was obtained by gold film assisted LDI

(GFLDI). In that case a thin gold layer (35nm) was deposited onto the sample carrier by physical vapor deposition (PVD) a method that facilitates a particularly uniform surface formation.

As shown in Figure 3 solely analyte signals were detected representing the ideal case of a LDI spectrum.

Nevertheless like any other technique both LDI and MALDI have their specific benefits and drawback and choosing a suitable system might be difficult even for advanced users. Therefore some of the essential aspects for choosing a working method will be discussed.

2.2 Matrix Assisted LDI (MALDI)

2.2.1 The concept

The concept of matrix assisted laser desorption ionization mass spectrometry

(MALDI MS, Figure 1) was firstly introduced by Hillenkamp and Karas in the mid- eighties. Since then MALDI MS has become a powerful analytical tool in mass spectrometry. Although hyphenations with chromatographic techniques are not available at the moment, MALDI MS represents a very sensitive technique of soft ionization. Further when coupled with a TOF analyzer, the method facilitates accurate mass analysis and consequently access to molecular formulas.

However, while the method has been successfully applied to characterize large organic molecules such as proteins, sugars and polymers its utilization for small molecules (≤ 600 Da) is significantly impaired by the co-formation of matrix ions preventing the correct signal assignment in the area of interest (0 - 600 m/z) in mass spectra (Figure 4).

The matrix — most generally a small UV-absorbing compound — plays a key-role in

MALDI by absorbing laser energy and directing it to the analyte molecules. This leads to ion formation through complex processes, which have been intensively studied and for which various mechanisms have been proposed (Beavis et al. 1989,

1992). In principle the latter can be divided into two groups: Processes which are taking place immediately after laser irradiation (5-20 ns, primary ion formation) and those occurring at a later stage (0.2-5 µsec) in the MALDI plume (secondary ion formation). In any case, ionization is based on the interaction of matrix and analyte molecules after laser irradiation, inducing the formation of their respective ions. Most commonly utilized matrices such as the popular 2,5-dihydroxybenzoic acid (DHB) or

α-cyano-4-hydroxycinnamic acid (CHCA) acid (Figure 5) but also many others, display a molecular mass in the range of 0 to 600. The formation of matrix ions therefore severely impairs the detection of small molecules in that mass region. For that reason MALDI-TOF mass spectrometry is predominately applied to the analysis macromolecules.

2.2.2 Attempts to minimize matrix peaks - Desorption/ionization on self- assembled monolayer surfaces (DIAMS)

Successful attempts to reduce or eliminate matrix noise by lowering applied laser energy or modifying the analyte to matrix mixing ratio have been reported (Beavis et al., 1989; Strupat et al., 1991; Ayorinde et al., 1999). According to these studies, matrix to analyte ratios of ≤100 and low laser energy powers may induce a so called

“matrix suppression effect” (MSE). Unfortunately an efficient MSE is frequently accompanied by an “analyte suppression effect” as both mechanisms share a common underlying principle (Knochenmuss et al., 1996; McGombie et al., 2004).

This is of particular relevance when several analytes are present in the same sample.

Consequently MSE may yield cleaner spectra, but signal intensities of certain analytes might be significantly reduced or even entirely suppressed.

Over the last years desorption/ionization on self-assembled monolayer surfaces

(DIAMS) has been described as an alternative method for eliminating matrix noise from MS spectra (Sanguinet et al., 2006; Bounichou et al., 2008). This method replaces classical matrix molecules by light absorbing self-assembled monolayers

(SAMs), which are covalently bond to a gold surface (Figure 6).

In order to facilitate optimal energy transmission these SAMs are equipped with a strong UV chromophore, i. e. a bithiophene moiety, exhibiting an absorption maximum in the range of the emission wavelength of a standard nitrogen laser

(337 nm). In details, SAMs for DIAMS were elaborated from a 5’-S-substituted 5-S-

7 methyl-2,2’-bithiophene as the redox chromophore, linked to a spacer terminated by a thiol, the anchoring functional group to the gold surface. The spacer must be carefully designed to provide high stability of the SAMs. Moreover, the synthesis of the organic precursors may be easily adapted for studying various SAMs, with modified properties. Following this approach, the concept of click chemistry recently developed by Sharpless (Rostovtsev et al., 2002) and facilitating high yield reactions under soft conditions was applied for the synthesis of the latest generation of SAMs for DIAMS. Indeed, interlocking azidoalkylbithiophenes and alkynthiols of various lengths led, by a convergent click reaction, to triazolobithiophene thiols. With a triazol ring in the linker that might exihibit π-π stacking, the corresponding SAMs showed high surface coverage and stability (Kenfack et al., 2011).

As previously mentioned the bithiophene moiety with its specific absorption properties represents a core feature of DIAMS. In principle the light absorbing monolayer is designed to act like a classical matrix by taking up laser energy and subsequently directing it to the sample but without getting desorbed from the gold surface. Consequently no matrix ions should be present in the plume, yielding spectra solely exhibiting analyte signals.

Hitherto DIAMS MS was successfully applied for the detection of fatty acids, glycerides and salicylic acid (Bounichou et al., 2008). One report directly compared signal intensities of triglycerides obtained by DIAMS and gold film assisted desorption ionization (GFDI). Out of eight analytes five displayed stronger analyte signals in GFDI, while two showed better results in DIAMS. For one sample no difference was detected. From the authors´ experience, the alkaloid totum extracted from Thalictrum flavum L. (Renonculaceae) was successfully detected by DIAMS.

The "common meadow-rue" or "yellow meadow-rue" is a herb of 0.5 to 1.5 m height

8 growing in Europe in tallgrass meadows, ditches, marshes and other habitats close to water bodies (Ropivia et al., 2010). The isoquinolines shown in Figure 7 presents key compounds of the crude extract as well as LC sub-fractions which could be directly indentified by DIAMS-TOF MS without further processing.

However it has also been observed that, contrary to classical MALDI-TOF spectrometry, DIAMS-TOF mass spectra frequently suffer from low signal to noise ratios as shown by the spectrum of the indole alkaloid yohimbine (Figure 8).

Therefore a study on the elucidation of ion formation processes occurring on the surface of light absorbing SAMs was performed. Ion yields obtained by free and immobilized matrix molecules as well as those generated by GFDI (vide supra) were compared (Schinkovitz et al., 2011). In short, this carefully conducted study confirmed that the formation of strong analyte signals is essentially linked to the presence of "free" matrix molecules. Unfortunately immobilizing the latter redirected the absorbed laser energy away from the analyte molecules. This effect was inversely correlated with the surface coverage of SAMs and could be quantified through cyclic voltammetry (Figure 9).

However, seen from another perspective the phenomenon may offer potential applications for analyzing particularly sensitive sample material. The impact of laser light, indeed, may be limited to just the extent necessary for inducing ionization while minimizing dimerization or degradation processes. Further the very organized surface structure of self-assembled monolayers seems to facilitate a most homogeneous distribution of sample material. Consequently, and unlike in classical

MALDI, the observed MS signals acquired from different locations of the sample deposition area tentatively showed little variation in signal intensity This aspect might be of high interest for quantitative applications.

2.2.3 Specific matrixes for small compounds : A new concept starting from

MT3P

Following the rapid expansion of proteomic studies, considerable efforts were made for the development and application of MALDI-TOF MS, while fundamental understanding of underlying processes is still limited. This is because MALDI is a quite complex phenomenon, where sample material embedded into a solid mostly crystalline matrix is instantly vaporized and consecutively diluting from a condensed state to a very thin aerosol. MALDI ionization itself predominately takes place in the gaseous phase and is known to be particularly soft, minimizing the formation of fragmentation and dimerization products. Consequently, as far as matrixes are concerned, MALDI-TOF was developed based on empirical approaches. Indeed early efforts were made to identify « good matrices ». During these experiments putative candidates were briefly tested on a few analytes and given qualitative ratings. Nowadays, actually very few of them are of common use. They were

10 identified early on (Knochenmuss, 2006) and are today mainly applied to the analysis of peptides, proteins, polymers and other macromolecules. Further, certain matrixes show some substrate specificity, another favorable feature of MALDI, which may allow the selective detection of target molecules in complex mixtures. Hitherto, the latter has been primarily reported for cinnamic acid based matrices targeting large molecules such as, peptides, proteins or lipids (Beavis et al., 1989, 1992; Strupat et al., 1991). In contrast very little is known about small molecules, as their analysis is generally impaired by the co-formation of matrix ions. Many of most commonly utilized MALDI matrices exhibit their molecular ions in the range of 0-600 m/z, therefore this region is highly critical for correct assignment of analyte and matrix signals. Thus, with respect to small molecules, the reduction of matrix noise is almost equally important as the sufficient ionization of analytes by matrix molecules. Various strategies have been discussed in order to improve spectrum qualities for low mass molecules in MALDI. Some matrices (Figure 5) such as 1,8-dihydroxy-9,10- dihydroanthracen-9-one (DIT) produce fewer signals than others e.g. α-cyano-4- hydroxycinnamic acid (CHCA). Further compounds of higher molecular weights such as meso-tetrakis(pentafluorophenyl)porphyrin may be utilized, provided they show sufficient ionization of target molecules (Ayorinde et al., 1999). Alternatively so-called

“matrix suppression effects” (MSEs, vide supra) may offer useful strategies to minimize the formation of matrix noise in MALDI (Knochenmuss et al., 1996;

McCombie et al., 2004). These effects have been intensively discussed in literature, and approaches to limit matrix noise reach from specifically adapted matrix molecules or mixtures of matrices to matrix noise inhibiting additives (Guo et al.,

2002; Vaidyanathan et al., 2006; Komori et al., 2009; Fujita et al., 2010). Further a simple decrease of applied laser energy may already improve spectrum qualities, but

11 also matrix to analyte ratios seem to be of significant relevance. Following this approach, McCombie and Knochenmuss have introduced the “matrix suppression effect score” (MSE score), a simple but powerful equation for evaluating spectrum qualities which provides a valuable tool for spectrum optimization. In this equation the sum of all analyte related signals are divided by the sum of all detected signals

(analyte + matrix) yielding a factor between 0 and 1. MSE scores close to 1 indicate strong analyte signals with low matrix noise, while factors around 0 represent high matrix noise and low analyte signals.

With these elements in mind we decided to explore a new concept of LDI, i.e. matrixes that would specifically interact with compounds of a selected chemical family. With focus on secondary metabolites from natural products, alkaloids present a most interesting analytical target, as these compounds are associated with a wide range of therapeutic and toxic activities. Alkaloids are well known to show dipolar interactions with nitriles and this particular behavior is successfully exploited for their separation on cyano bonded phases HPLC columns. Thus, coupling a propanenitrile to a bithiophen derivative that strongly absorbs UV laser light at 337 nm (the typical wavelength of a standard nitrogen laser commonly utilized in MALDI-TOF instruments) yielded 3-[5'-(methylthio)-2,2'-bithiophen-5-ylthio]propanenitrile (MT3P,

Figure 5). According to recent experiments (Schinkovitz et al., 2012) the compound represents a powerful new matrix molecule exhibiting an unusual specificity towards alkaloids. So far MT3P was tested on 55 compounds of various chemical origin and results were compared with those obtained by the use of commercial matrices such as dithranol (DIT), CHCA, 2,2’:5’,2’’-terthiophene (TER) and 2,5-dihydroxybenzoic acid (DHB) (Figure 5 and Table 1).

Apart from alkaloids the selection of natural products included coumarins, terpenes, flavonoids, carotenoids, steroids and peptides. Results from this survey are summarized in Table 1, providing a general overview of the ionization properties of

MT3P. Alkaloids almost constantly displayed intense molecular ion signals. Solely nicotine and theobromine were not or weakly detected by MT3P. With regards to ion formation, [M+H]+ was observed for any investigated alkaloid except for claviculine.

The latter displayed a pseudomolecular ion of [M-H]+. On first sight the formation of

[M-H]+ may appear quite unusual, but actually it represents a standard ion originating from photoionization process associated with MALDI (Zenobi et al., 1998).

Beside others, the list of successfully tested alkaloids contained highly biologically active compounds such as codeine, aconitine, L-hyoscyamine, emetin and many more. Figure 10 displays the spectrum of quinidine as single compound (B) as well as the simultaneous detection of colchicine, L- hyoscyamine and senecionine (A) .

As further shown in Figure 10 and similarly to many MALDI matrices, MT3P exhibits its matrix ions within a mass range of 0-600 m/z. Most prominent signals were observed at 297 m/z (M+) and at 243 m/z (fragmentation product resulting from the cleavage of the S-C bond in position 3). Further two minor signals showed up at 312 and 351 m/z respectively. Nevertheless, the intensity of observed alkaloid molecular ions was mostly superior and these features could be used for the accurate calibration of the TOF analyzer, providing high resolution measurements.

Consequently alkaloids were easily and precisely detected even if their molecular ions showed up in the vicinity of associated matrix signals.

Any of the successfully tested alkaloids exhibited strong molecular ions at a relatively low level of applied laser energy (10% =15.6 µJ). Furthermore, with respect to these alkaloids, MT3P usually showed superior ionizing properties in comparison to commonly utilized matrices such as DIT, CHCA, 2,2’:5’,2’’-terthiophene (TER), and

DHB (Figure 5). Solely CHCA was able to ionize as many alkaloids as MT3P, but generally exhibited weaker analyte signals than MT3P. For the majority of these cases (16 out of 20) the observed difference was significant (P values: 0.046 to ≤

0.001). Also berberine, boldine, pilocarpine and sparteine showed stronger signal intensities when analyzed by MT3P but in comparison to CHCA the difference was not statistically significant. Six alkaloids namely codeine, L-hyoscyamin, nicotine, thaligosidine, theobromine and strychnine showed better ionization when CHCA but only for L-hyoscyamine, nicotine and theobromine the observed difference was significant.

Contrary to its excellent ionizing properties of alkaloids, MT3P did barely interact with any compound of different chemical origin. Seemingly there was strong ionization of pentamethoxyflavone and amentoflavone but these compounds could also be ionized by simple LDI, without any matrix support, since they both include a strong UV chromophore. A contrario, digitoxine which did not show any LDI could be ionized by

MT3P, but this required a higher laser energy (20 % or 23.2 µJ) and led to a crowded spectrum thus exhibiting a low MSE score (≤ 0.05). In this respect the selective setting of applied laser energy provides a useful tool for suppressing the ionization of compounds such as digitoxine that require higher amounts of energy.

The precise mode of action of MT3P still remains unknown but certain structural features may provide useful hints for its elucidation. Considering that solely MT3P and CHCA showed significant interaction with alkaloids, it may be speculated whether the presence of a single free nitrile group enhances the ionization of alkaloids. On the other hand, MT3P unlike CHCA did not show any ionization of peptides, suggesting that the observed selectivity could be due to a more complex mechanism.

Apart from its ionizing properties an “optimized” MALDI matrix should exhibit strong analyte signals together with reduced formation of matrix ions. Therefore MT3P and

CHCA, representing the best working matrixes for alkaloids, were directly compared in terms of their respective MSE scores. These Experiments showed that MT3P exhibited cleaner spectra with reduced matrix noise for 18 out of 25 alkaloid samples

(Table 2).

Subsequent experiments further revealed that MT3P should ideally be utilized at energy levels between 5-15% (11.8-19.4 µJ). Within this power range, alkaloids displayed strong molecular ions while the formation of matrix ions was still limited.

As previously discussed, MSEs are linked to matrix to analyte mixing rations as well as applied laser energy (Knochenmuss et al., 1996; McCombie et al., 2004).

Considering that many alkaloids displayed very strong molecular ions when exposed to a laser energy of 10% (15.6 µJ) a decrease in laser power could result in less matrix noise while still providing sufficient intensities of analyte signals. Therefore 14 alkaloids of various chemical structures were subsequently analyzed at a reduced level of laser energy of 5% (11.8 µJ) (Figure 11, A). As expected, signal intensities

15 decreased with declining laser energy, but any compound was still clearly detectable.

In return MSE scores constantly increased, and for some compounds such as fumarithine (0.3 to 0.5). thalicberine (0.56-0.79) or emetine (0.59-0.85) the raise was particularly remarkable (Figure 11, B).

Exceptionally two compounds showed unchanged (sparteine) or slightly declining

MSE scores (pilocarpine) when analyzed at 5% of laser energy. Obviously these alkaloids responded more sensitively to a reduction of energy than MT3P, and should be analyzed at higher levels of laser energy. Overall, no clear correlation between the extent of signal decay and the raise of MSE scores could be observed. A substantial decrease in signal intensities did not necessarily imply a remarkable increase of MSE scores and vice versa. Eventually it can be concluded that a reduction of laser energy generally improved spectrum qualities, but the extent of the effect was hardly predictable. At that point it shall be mentioned that all spectra were acquired at a certain analyte and matrix concentration. As mentioned earlier, MSE are highly depending on sample to matrix mixing ratios and changing the latter may have significantly altered observed MSE scores. This also applies to Table 2 were

MTP3 and CHCA were used at concentrations of 25.80 Mm and 45.81 mM respectively. In this respect the precise evaluation of the impact of sample to matrix mixing ratios for MT3P represents an interesting subject of research and is currently being studied.

3. CONCLUSION

The selective detection of alkaloids by MALDI-TOF has been continuously as well as intensively discussed in the literature (Sun et al., 1998; Lopez-Legentil et al., 2005;

Cheng et al., 2006; Wu et al., 2007, 2007; Araoz et al., 2008; Feng et al., 2009; Lu et al., 2010), certainly because alkaloids represent highly active natural and/or synthetic substances. Compounds such codeine and its derivatives are commonly used in medicinal applications, while others like strychnine, emetine and aconitine exhibit severe toxic effects. Therefore their precise and accurate identification in crude plant material, medicinal preparations or dietary supplements is highly demanded and subject of contemporary research. While some aromatic alkaloids such as ascididemine can be ionized by simple LDI (Lopez-Legentil et al., 2005) most reports mention CHCA and TER as preferred matrices for MALDI experiments (Wu et al.,

2005; Cheng et al., 2006; Wu et al., 2007, 2007; Lu et al., 2010; Liu et al., 2010) .

Additionally 7-mercapto-4-methylcoumarin has been described for the detection of arecoline and arecaidine but no further alkaloids were tested (Feng et al., 2009).

Considering these findings, MT3P represents a most valuable addition to the pool of available matrix molecules. Its enhanced and selective ionizing properties facilitate high quality MALDI spectra of alkaloids at exceptionally low levels of laser energy.

Consequently it may also be particularly suitable for the analysis of unstable compounds. Future studies and experiments may help to further improve the current working protocol allowing a gain in sensitivity as well as MSE scores. Finally, bithiophene based molecules may represent an entire new group of matrix molecules. Starting from MT3P, chemical modifications may alter their ionizing profile and create specifically adapted molecules for various MALDI applications. This

17 outlook represents a challenging but most promising perspective for future experiments.

Acknowledgements: The authors would like to thank Prof. Rudolf Bauer, head of the Department of Pharmacognosy (Institute of Pharmaceutical Sciences. University of Graz) and Dr. Séverine Derbré, Assistant Processor at SONAS (Laboratoire des

Substances d'Origine Naturelle et Analogues Structuraux, Université d’Angers) for providing sample material of E-notopterol, isoimperatorine and pregnolone as well as thalicberine, thaligosidine and thalfoetidine respectively. Further, we would like to thank Dr. Eric Levillain, director of research at the Centre National de la Recherche

Scientifique (CNRS) in Angers for his most valuable input and practical advice within the DIAMS project. Last but not least we would like thank Dr. Ingrid Freuze

(University of Angers), for her valuable help during the data acquisition process and

Dr. Ghislain Kenfack formerly PhD student at SONAS for conducting the synthesis of

MT3P.

Compound MT3P STD MT3P D T STD DIT CHCA STD CHCA TER STD TER DHB STD DHB MF MT3P vs. CHCA

Acetylsalicylic acid nd nd nd nd nd C21H26N2O3

Aconitine* 14.00 4.86 0.22 0.09 3.90 1.17 0.48 0.21 nd C35H68O5 Yes P = 0.002

Amentoflavone 17.68 3.76 0.25 0.10 14.24 7.44 0.18 0.03 nd C22H25NO6 No P = 0.589

Angiotensin II nd nd 10.85 4.49 nd nd C9H6O2 Yes P = 0.002

Atropine* 18.61 5.24 0.04 0.03 9.67 2.72 0.02 0.01 nd C20H20O7 Yes P = 0.009

Aucuparin nd nd nd nd nd C20H24N2O2

β−Caroten 0.24 0.16 0.01 0.00 0.15 0.05 0.69 0.27 nd C27H30O16 No P = 0.198

Benzocaine nd nd 1.51 0.46 nd nd C28H37N5O7 Yes P = 0.002

Berberine* 23.56 5.67 0.34 0.16 18.75 4.48 6.16 5.49 0.01 0.01 C12H8O4 No P = 0.134

Bergaptene 0. 7 0.02 nd 8.72 3.88 nd nd C28H34O15 Yes P = 0.002

Boldine* 11.22 4.32 0.20 0.24 7.94 5.25 0.36 0.30 nd C15H24 No P = 0.265

Caffeic acid 2.48 2.30 nd nd 1.30 0.79 nd C21H20O6 Yes P = 0.002

Caryophyllen nd nd nd nd nd C9H11NO2

Chlorogenic acid nd nd 0.02 0.02 nd nd C30H18O10 Yes P = 0.015

Cholesterol nd nd nd nd nd C14H12O5

Claviculine* 20.43 4.75 0.19 0.11 6.51 1.85 1.74 2.74 nd C9H8O4 Yes P = 0.002

Codeine* 12.07 3.59 0.12 0.08 16.58 3.62 0.65 0.40 nd C4H4O4 No P = 0.056

Compound MT3P STD MT3P DIT STD DIT CHCA STD CHCA TER STD TER DHB STD DHB MF MT3P vs CHCA

Colchicine* 12.11 3.77 0.45 0.21 2.98 1.39 0.12 0.07 nd C16H18O9 Yes P < 0.001

Coumarin nd nd 1.12 0.36 nd nd C9H6O2 Yes P = 0.002

Curcumin 5.41 0.43 0.29 0.15 4.77 2.65 0.05 0.06 nd C9H8O4 No P = 0.573

Digitoxin 8.27 4.07 1.22 0.92 3.34 1.83 4.42 1.67 nd C16H14O4 Yes P = 0.022

1.3-Dipalmitoyl-glycerol nd nd nd nd nd C29H50O

Emetine* 28.02 1.37 0.32 0.45 5.34 2.42 0.18 0.15 nd C21H22O5 Yes P < 0.001

E-Notopterol nd nd nd nd nd C15H10O7

Fumaric acid 0.31 0.00 nd nd nd nd C41H64O13

Fumaritine* 25.72 2.90 0.23 0.10 2.14 2.15 1.29 1.08 nd C18H19NO4 Yes P < 0.001

Geraniol nd nd nd nd nd C29H40N2O4

Glyceryl 1,3-distearate nd nd nd nd nd C27H46O

Harmine* 24.68 3.42 2.39 2.43 9.34 2.83 2.57 1.16 0.01 0.00 C11H16N2O2 Yes P < 0.001

Hesperidin 0.24 0.30 0.08 0.06 0.82 0.67 0.10 0.10 nd Yes P < 0.041

L-hyoscyamine* 11.26 3.02 0.32 0.27 19.38 3.38 0.61 0.30 nd C34H47NO11 Yes P < 0.001

Isoimperatorine nd nd 5.47 13.41 nd nd C21H22N2O2 Yes P = 0.002

Khellin 2.32 1.46 nd 12.95 6.56 nd nd C17H23NO3 Yes P = 0.003

Leucine Enkelphalin nd nd 9.10 2.82 nd nd C10H14N2 Yes P = 0.002

Limogine* 26.03 2.46 0.52 0.19 7.92 1.69 0.09 0.07 nd C20H18NO4 Yes P < 0.001

Compound MT3P STD MT3P DIT STD DIT CHCA STD CHCA TER STD TER DHB STD DHB MF MT3P vs CHCA

Nicotine* 0.43 0.20 0.01 0.01 1.29 0.73 0.01 0.00 nd C21H32O2 Yes P = 0.018

Pentamethoxyflavone 26.00 4.27 2.06 0.36 13.19 5.24 1.70 1.77 0.00 0.00 C13H12N2O Yes P < 0.001

Pilocarpine* 11.03 4.07 0.86 0.80 7.56 2.81 0.03 0.01 nd C19H17NO4 No P = 0.117

Pregnolon nd nd nd nd nd C15H26N2

Quercetine 0.15 0.08 0.05 0.03 14.47 3.41 0.00 0.01 nd C15H10O7 Yes P = 0.002

Quinidine* 20.96 6.16 2.05 1.24 3.08 1.99 0.15 0.11 nd C20H17NO5 Yes P < 0.001

Rutin 3.63 2.24 0.65 0.20 3.92 2.44 0.23 0.19 nd C37H40N2O6 No P = 0.837

Scopolamine* 13.56 2.59 0.10 0.08 3.03 1.92 0.05 0.02 nd C21H19NO5 Yes P < 0.001

Senecionine* 12.05 3.73 0.14 0.08 3.64 2.55 0.21 0.24 nd C37H40N2O7 Yes P < 0.001

β-Sitosterol nd nd nd nd nd C38H42N2O7

Sparteine* 20.34 5.34 0.92 0.41 15.32 3.63 2.43 2.11 0.00 0.00 C18H25NO5 No P = 0.086

Strychnine* 13.16 5.72 0.55 0.23 13.07 4.29 0.29 0.08 nd C20H21NO5 Yes P < 0.001

Stylopine* 24.84 2.19 0.43 0.39 7.65 1.0 1.22 0.61 nd C19H21NO4 Yes P < 0.001

Thalfoetidine* 5.67 1.82 0.07 0.06 3.51 1.46 1.09 0.96 nd C40H56 Yes P = 0.046

Thalicberine* 20.54 5.22 0.07 0.05 3.90 2.59 0.87 0.78 nd C18H21NO3 Yes P < 0.001

Thaligosidine* 4.40 0.79 0.06 0.02 4.75 1.62 0.12 0.08 nd C17H23NO3

Thebaine* 17.58 3.84 0.37 0.17 10.92 4.81 0.81 0.57 0.00 0.01 Yes P = 0.024

Theobromine* 0.08 0.11 nd 8.58 4.16 0.01 0.01 nd C14H14O3 Yes P = 0.002

Compound MT3P STD MT3P DIT STD DIT CHCA STD CHCA TER STD TER DHB STD DHB MF MT3P vs CHCA

Umbelliferone nd nd nd nd nd C39H76O5

Yohimbine* 16.31 4.32 0.21 0.16 6.35 3.14 1.14 1.01 nd C7H8N4O2 Yes P = 0.001

Table1: Fifty five tested compounds in MALDI-TOF MS with MT3P as the matrix; *Alcaloids; Originally acquired signal intensities for molecular ions were divided by 1250 for illustration purposes. Zero numbers indicate that molecular ions were detected but at low intensities. STD: standard deviation n=6 MF: molecular formula; LP10: linear positive mode, laser energy 10% (15.6 µJ);

LN10: linear negative mode, laser energy 10% (15.6 µJ); nd: Signal not detected. MT3P vs. CHCA: Significance (Yes/No) of observed differences between MT3P and CHCA (t-test).

MSE score

Compound MT3P STDMT3P CHCA STDCHCA

Aconitine 0.83 0.10 0.76 0.23

Atropine 0.30 0.10 0.39 0.13

Berberine 0.64 0.14 0.97 1.86

Boldine 0.46 0.06 0.22 0.19

Claviculine 0.26 0.09 0.18 0.07

Codeine 0.22 0.01 0.14 0.04

Colchicine 0.40 0.05 0.08 0.06

Emetine 0.59 0.08 0.15 0.08

Fumaritine 0.30 0.05 0.22 0.06

Harmine 0.27 0.07 0.35 0.15

L-hyoscyamine 0.58 0.10 0.23 0.06

Limogine 0.27 0.05 0.71 0.10

Nicotine 0.01 0.00 0.06 0.04

Pilocarpine 0.17 0.07 0.12 0.02

Quinidine 0.30 0.04 0.17 0.02

Scopolamine 0.14 0.02 0.12 0.04

Senecionine 0.17 0.01 0.10 0.06

Sparteine 0.32 0.02 0.79 0.52

Strychnine 0.43 0.08 0.44 0.08

Stylopine 0.79 0.12 0.37 0.25

Thalfoetidine 0.56 0.31 0.16 0.10

Thalicberine 0.56 0.16 0.16 0.06

Thaligosidine 0.85 0.13 0.24 0.28

Theobromine 0.00 0.00 0.19 0.06

Yohimbine 0.57 0.28 0.15 0.01

Table 2 : MSE scores of alkaloids utilizing matrices MT3P and

CHCA. All data were acquired in the linear positive mode at a

laser energy of 10% (15.6 µJ). STD: Standard deviation.

References

Beavis RC, Chait BT, Fales HM. 1989. Cinnamic acid derivatives as matrices for ultraviolet laser desorption mass spectrometry of proteins. Rapid Commun Mass Spectrom

3: 432–435.

Araoz R, Guerineau V, Rippka R, Palibroda N, Herdman M, Laprevote O, von Doehren

H, Tandeau de Marsac N, Erhard M. 2008. MALDI-TOF-MS detection of the low molecular weight neurotoxins anatoxin-a and homoanatoxin-a on lyophilized and fresh filaments of axenic Oscillatoria strains. Toxicon 51: 1308-1315.

Ayorinde FO, Hambright P, Porter TN, Keith QL. 1999. Use of meso- tetrakis(pentafluorophenyl)porphyrin as a matrix for low molecular weight alkylphenol ethoxylates in laser desorption/ionization time-of-flight mass spectrometry. Rapid

Commun Mass Spectrom 13: 2474-2479.

Beavis RC, Chaudhary T, Chait BT. 1992. α-Cyano-4-hydroxycinnamic acid as a matrix for matrixassisted laser desorption mass spectromtry. Org Mass Spectrom 27: 156-158.

Bounichou M, Aleveque O, Dias M., Sanguinet L, Levillian E, Rondeau D. 2009. Self- assembled monolayer-assisted mass spectrometry. J Mater Chem 19: 8032-8039.

Bounichou M, Sanguinet L, Elouarzaki K, Aleveque O, Dias M, Levillian E, Rondeau D.

2008. Evaluation of new matrix-free laser/desorption method through statistic studies: comparison of the DIAMS (desorption/ionization on self assembled monolayer surfact) methos with MALDI and TGFA-LDI techniques. J Mass Spectrom 43: 1618-1626.

Chen Y, Vertes A. 2006 Adjustable Fragmentation in Laser Desorption/Ionization from

Laser-Induced Silicon Microcolumn Arrays. Anal Chem 78: 5835-5844.

Cheng Z, Guo Y, Wang H, Chen G. 2006. Qualitative and quantitative analysis of quaternary ammonium alkaloids from Rhizoma Corydalis by matrix-assisted laser desorption/ionization Fourier transform mass spectrometry coupled with a selective precipitation reaction using Reinecke salt. Anal Chim Acta 555: 269-277.

Feng C, Lu C. 2009. A new matrix for analyzing low molecular mass compounds and its application for determination of carcinogenic areca alkaloids by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Anal Chim Acta 649: 230-235.

Fujita T, Fujino T, Hirabayashi K, Korenaga T. 2010. MALDI mass spectrometry using

2.4.6-trihydroxyacetophenone and 2.4-dihydroxyacetophenone with cyclodextrins: suppression of matrix-related ions in low-molecular-weight region. Anal Sci 26: 743-748.

Guo Z, He L. 2007. A binary matrix for background suppression in MALDI-MS of small molecules. Analytical and Bioanalytical Chemistry 387: 1939-1944.

Guo Z, Zhang Q, Zou H, Guo B, Ni J. 2002. A method for the analysis of low-mass

26 molecules by MALDI-TOF mass spectrometry. Anal Chem 74: 1637-1641.

Karas M. and Hillenkamp F. 1988. Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons. Anal. Chem 60: 2299-2301.

Kenfack GT, Schinkovitz A, Babu S, Elouarzaki K, Dias M, Derbre S, Helesbeux J-J,

Levillain E, Richomme P, Seraphin D. 2011. Triazolobithiophene light absorbing self- assembled monolayers: synthesis and mass spectrometry applications. Molecules 16:

8758-8774.

Knochenmuss R, Dubois F, Dale MJ, Zenobi R. 1996. The matrix suppression effect and ionization mechanisms in matrix-assisted laser desorption ionization. Rapid Commun

Mass Spectrom 10: 871-877.

Knochenmuss R. 2006. Ion formation mechanisms in UV-MALDI. Analyst 131: 966–986.

Komori Y, Shima H, Fujino T, Kondo J, Hashimoto K, Korenaga T. 2009. Pronounced selectivity in matrix-assisted laser desorption-ionization mass spectrometry with 2.4.6- trihydroxyacetophenone on a zeolite surface: intensity enhancement of protonated peptides and suppression of matrix-related ions. J Phys Chem C 114: 1593-1600.

Law KP. 2010. Laser desorption/ionization mass spectrometry on nanostructured semiconductor substrates: DIOSTM and QuickMassTM. Int J Mass Spectrom 290: 72-84.

Law KP, Larkin JR. 2011. Recent advances in SALDI-MS techniques and their chemical and bioanalytical applications. Anal Bioanal Chem 399: 2597-2622.

Liu Z, Lu L, Song F, Liu S. 2010. Direct detection method for alkaloid in traditional chinese medicine by adopting matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. China Patent 2009-10067479101644694.

Lopez-Legentil S, Dieckmann R, Bontemps-Subielos N, Turon X, Banaigs B. 2005

Qualitative variation of alkaloids in color morphs of Cystodytes (Ascidiacea). Biochem

Syst Ecol 33: 1107-1119.

Lu L, Yue H, Song F, Tsao R, Liu Z, Liu S. 2010. Rapid profiling of alkaloids in several medicinal herbs by matrix-assisted laser desorption/ionization mass spectrometry. Chem

Res Chin Univ 26: 11-16.

McCombie G, Knochenmuss R. 2004. Small-molecule MALDI using the matrix suppression effect to reduce or eliminate matrix background interferences. Anal Chem 76:

4990-4997.

McLean JA, Stumpo KA, Russell DH. 2005. Size-Selected (2-10 nm) Gold Nanoparticles for Matrix Assisted Laser Desorption Ionization of Peptides. J Am Chem Soc 127: 5304-

5305.

Posthumus MA, Kistemaker PG, Meuzelaar HLC, Ten Noever de Brauw MC. 1978.

Analytical Chemistry 50: 985-991.

Ropivia J, Derbré S, Rouger C, Pagniez P, Le Pape P, Richomme P. 2010. Isoquinolines from the roots of Thalictrum flavum L. and their evaluation as antiparasitic compounds.

Molecules 15: 6476-6484.

Rostovtsev V, Green, L, Fokin, V, Sharpless, K. 2002. A stepwise Huisgen cycloaddition process: copper(I)-catalyzed regioselective ligation of azides and terminal alkynes. Angew

Chem Int Ed 41: 2596-2599.

Sanguinet L, Aleveque O, Blanchard P, Dias M, Levillian E, Rondeau D. 2006.

Desorption/ionization on self-assembled monolayer surfaces (DIAMS). J Mass Spectrom

41: 830-833.

Schinkovitz A, Kenfack GT, Levillain E, Dias M, Helesbeux J-J, Derbre S, Seraphin D,

Richomme P. 2011. Free and immobilized matrix molecules: impairing ionization by quenching secondary ion formation in laser desorption MS. J Mass Spectrom 46: 884-890.

Schinkovitz A, Kenfack GT, Seraphin D, Levillain E, Dias M, Richomme P. 2012.

Selective detection of alkaloids in MALDI-TOF: the introduction of a novel matrix molecule. Anal Bioanal Chem In press. DOI: 10.1007/s00216-012-5958-y

Schuerenberg M, Dreisewerd K, Hillenkamp F. 1999. Laser Desorption/Ionization Mass

Spectrometry of Peptides and Proteins with Particle Suspension Matrixes.

Anal Chem 71: 221-229.

Strupat K, Karas M, Hillenkamp F. 1991. 2.5-Dihydroxybenzoic acid: a new matrix for laser desorption—ionization mass spectrometry. International Journal of Mass

Spectrometry and Ion Processes 111: 89-102.

Sun W, Liu S, Liu Z, Song F, Fang S. 1998. A study of Aconitum alkaloids from aconite roots in Aconitum carmichaeli Debx using matrix-assisted laser desorption/ionization mass spectrometry. Rapid Commun Mass Spectrom 12: 821-824.

Suzuki T, Midonoya H, Shioi Y. 2009. Analysis of chlorophylls and their derivatives by matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry. Anal

Biochem 390: 57-62.

Tanaka K, Waki H, Ido Y, Akita S, Yoshida Y, Yoshida T. 1988. Protein and polymer analyses up to m/z 100 000 by laser ionization time-of-flight mass spectrometry. Rapid

Commun Mass Spectrom 20: 151–153.

Vaidyanathan S, Gaskell S, Goodacre R. 2006. Matrix-suppressed laser desorption/ionisation mass spectrometry and its suitability for metabolome analyses.

Rapid Commun Mass Spectrom 20: 1192-1198.

Wu W, Liang Z, Zhao Z, Cai Z. 2007. Direct analysis of alkaloid profiling in plant tissue by using matrix-assisted laser desorption/ionization mass spectrometry. J Mass Spectrom

42: 58-69.

Wu W, Qiao C, Liang Z, Xu H, Zhao Z, Cai Z. 2007. Alkaloid profiling in crude and processed Strychnos nux-vomica seeds by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. J Pharm Biomed Anal 45: 430-436.

Zenobi R, Knochenmuss R. 1998. Ion formation in mass spectrometry. Mass Spectrom

Rev 17: 337-336.

Figure 1 : Basic principle of MALDI (TOF) MS.

(1) A sample (analyte) is co-crystallized with an excess of matrix on a stainless steel sample plate (2). A laser pulse (a few ns) ionizes matrix molecules. Sample molecules are ionized by charge transfer ([M+H]+, [M+Na]+, [M+K]+…) (3) ions are accelerated by an electric field (4) small ions reach the detector earlier than large ones. A time of flight (TOF) analyzer measures the time required by the ions to reach the detector: the mass to charge ratio of an ion is proportional to the square of its drift time.

Figure 2 : Laser ionization/desorption on a gold surface.

The sample (analyte) is directly crystallized on the sample plate covered with Au.

Figure 3 : GFLDI mass spectrum (positive mode) of Pentamethoxyflavone => manque "O" !!.

Pentamethoxyflavone [C20H20O7 monoisotopic mass: 372.1209] is ionized by GFLDI using a laser power of 40% (38.8 µJ). Soleyly sample related signals are detected.

Figure 4: MALDI-TOF mass spectrum (positive mode) of the alkaloid Harmin => sans "e" (corriger dans figure).

The MALDI-TOF MS of Harmin (C13H12N2O, monosiotopic mass : 212.0950) was recorded using α-cyano-4-hydroxycinnamic acid CHCA (C14H10O3, monosiotopic mass: 179.04259) as the matrix. The low mass region is very crowded and the molecular ion of harmine (M+) => a corriger dans figure (= pas de "[ ]") is buried below a matrix associated signal also showing up at 212 m/z. It is impossible to distinguish between matrix and analyte signals.

Figure 5 : Some matrixes for MALDI-TOF mass spectrometry

DHB : 2,5-dihydroxy benzoic acid, CHCA : α-cyano-4-hydroxycinnamic acid, DIT :

Dithranol (Anthralin), TER : Tertiophene, MT3P : 3-[5'-(methylthio)-2,2'-bithiophen-5- ylthio]propanenitrile.

Figure 6 : The principle of DIAMS MS

Desorption/ionization on self-assembled monolayer surfaces : A self-assembled monolayer (SAM) incorporating the matrix is covalently bound to a gold surface.

Figure 7 : DIAMS mass spectrum (positive mode) of an alkaloid totum extracted from

Thalictrum flavum L. (Renonculaceae).

Key compounds of the crude extract as well as LC sub-fractions : Berberin (1),

Thalicberin (2), Thaligoside (3), Thalfoetidin (4).

354.17 [M]+ 250

200 .]

a.u 150 . [ .

Intens 100

0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 x 103 m/z

Figure 8 : DIAMS-TOF mass spectrum (positive mode) of Yohimbin. => corriger [M]+ en M+

+ Yohimbin : C21H26N2O3 ; calculated monoisotopic mass: 354.1943, observed: M :

354.17. Laser energy 30% (46.8 µJ)

A B

SAM´s Coverage of Gold Surface SAM´s Coverage of Gold Surface

Figure 9 : SAM "quality" (surface coverage) vs MS signal intensity observed for the alkaloid Claviculin.

Claviculin : C18H19NO4, monoisotopic mass : 313.1214. A : As the bithiophene moiety exhibits a reversible redox system, SAM degradation may be monitored by cyclic voltammetry (CV, A). Charge transfers and consequently ion intensities clearly increase with the decay of the SAM surface. On the other, small standard deviation of the signal intensity correlate with high surface coverage and indicates that SAM´s surfaces facilitate an excellent sample distribution, limiting sweet spot phenomena known from classical MALDI (B). This effect might be beneficial for quantitative applications.

=> a corriger L-hyoscyamin (sans "e")... etc + indiquer les numéros de formules + reprendre formule "Figure 12" (et non Wikipedia comme ici !)

Figure 10 : MALDI-TOF spectra (positive mode) of selected alkaloids 1-4 analyzed by using MT3P as matrix.

A: Simultaneous detection of toxic alkaloids L-Hyoscyamin (1), Senecionin (2) and

Colchicine (3).

B: Spectrum of Quinidin (4) as single compound.

(* denotes matrix ions) Unknown Mis en forme: Police :(Par défaut) Times New Roman

Figure 11 : Signal Intensity vs. MSE scores using MT3P as the matrix in MALDI-TOF

Comparison of signal intensities and MSE scores for 14 alkaloids utilizing MT3P at different levels of applied laser energy.

A ▲Signal Intensity at 10% of laser energy (15.6 µJ) ○Signal Intensity at 5% of laser energy (11.8 µJ)

B ▲MSE score at 10% of laser energy (15.6 µJ) ○ MSE score at 5% of laser energy (11.8 µJ)