Selective detection of alkaloids in MALDI-TOF: the introduction of a novel matrix molecule

ORIGINAL PAPER

Selective detection of alkaloids in MALDI-TOF:

the introduction of a novel matrix molecule

Andreas Schinkovitz&Ghislain Tsague Kenfack&

Denis Seraphin_&Eric Levillain_&Maryléne Dias_&

Pascal Richomme

Received: 24 January 2012 / Revised: 16 March 2012 / Accepted: 19 March 2012 / Published online: 20 April 2012

#Springer-Verlag 2012

Abstract The current manuscript presents 3-[5′-(methyl- thio)-2,2′-bithiophen-5-ylthio]propanenitrile (MT3P), as a novel matrix molecule, which facilitates the selective ioni- zation of alkaloids in matrix-assisted laser desorption/ioni- zation mass spectrometry. Exhibiting strong ionizing properties at low levels of laser energy, MT3P was evaluated on 55 compounds belonging to various chemical families.

The observed molecular ion yields induced by MT3P were compared with those obtained by commercially available matrices such as 1,8-dihydroxy-9,10-dihydroanthracen-9- one, α-cyano-4-hydroxycinnamic acid, 2,2′:5′,2″-terthio- phene and 2,5-dihydroxybenzoic acid. In conclusion, MT3P displayed excellent ionization properties for 23 out of 25 investigated alkaloids, while showing little to no interaction with compounds from different chemical origin.

Further, in comparison to other tested matrices, MT3P

generally facilitated better ionization of alkaloids. Eventual- ly, levels of laser energy were adjusted to obtain spectra with significantly reduced matrix noise.

Keywords Mass spectrometry . MALDI . Aklaloids . Selective detection

Introduction

Matrix-assisted laser desorption ionization (MALDI) repre- sents a powerful technique for the determination of molec- ular weights of organic compounds. The method is broadly applied for the analysis of peptides, proteins, polymers, and other macromolecules. MALDI facilitates a particular soft ionization, minimizing the formation of fragmentation and dimerization products. Furthermore, some matrices show 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 [1–3]. 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 0–600m/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 as important as the sufficient ioniza- tion of analytes by matrix molecules.

In this respect, various strategies have been discussed in order to improve the spectrum quality for low mass mole- cules in MALDI. Some matrices such as 1,8-dihydroxy- Electronic supplementary material The online version of this article

(doi:10.1007/s00216-012-5958-y) contains supplementary material, which is available to authorized users.

A. Schinkovitz (*)

:

:

:

Université d’Angers-Laboratoire SONAS, 16 Boulevard Daviers,

49045 Angers, Cedex 01, France e-mail: pharm@gmx.at

E. Levillain

:

Laboratoire CIMA, Université d’Angers-CNRS, 2 Boulevard Lavoisier,

49045 Angers, Cedex, France A. Schinkovitz

Department of Pharmacognosy, University of Vienna, Althahnstraße 14,

1090 Vienna, Austria

DOI 10.1007/s00216-012-5958-y

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)porphyrine may be utilized, provided they show sufficient ionization of target molecules [4]. Alternatively, so-called “matrix suppression effects”(MSEs) may offer useful strategies for minimizing the formation of matrix noise in MALDI [5, 6]. 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 [7–11]. Furthermore, a simple decrease of applied laser energy may already improve spec- trum qualities but also matrix-to-analyte ratios seem to be of significant relevance [5,6]. In this respect, McCombie and Knochenmuss have introduced the “matrix suppression ef- fect score”(MSE score), a simple but powerful equation for evaluating spectrum qualities [6]. In this equation, the over- all sum of analyte signals is divided by the overall sum of all observed signals (analyte+matrix) yielding a factor between 0 and 1. The MSE score will be further discussed in con- secutive sections of the manuscript.

Studying the chemical properties of bithiophene derivatives, 3-[5′-(methylthio)-2,2′-bithiophen-5-ylthio]propanenitrile (MT3P) was identified as a powerful new matrix mole- cule. Compiling data from extensive analytical studies, the current manuscript presents a compact summary of experiments elucidating its ionization properties and se- lectivity. Overall, MT3P has been tested on 55 com- pounds of various chemical origins. Results were compared with those obtained by the use of commercial matrices such as DIT, CHCA, 2,2′:5′,2″-terthiophene (TER), and 2,5-dihydroxybenzoic acid (DHB). Finally, MSE scores were used to improve spectrum qualities’ by studying the impact of reduced laser energy on analyte and matrix ion formations. Based on these experiments, a general working protocol for MT3P is proposed, providing a good starting point for current and consecutive analytical studies.

Experimental section

Reagents

Matrix reagents DIT and TER were purchased from Avoca- do Research Chemicals Ltd. (Morecambe, UK); compounds CHCA and DHB were purchased from Sigma-Aldrich (Steinheim, Germany). 3-[5′-(Methylthio)-2,2′-bithiophen- 5-ylthio]propanenitrile was synthesized according to a pre- viously described procedure [12]. Analytes 1–55 were iso- lated within phytochemical research projects or generously provided by collaborating groups.

Sample stock solutions

Stock solutions were prepared at a concentration of 858μM.

Depending on the solubility of the analytes, methanol (MeOH), or mixtures of MeOH and dichloromethane (DCM) were used. For any sample solution, one equivalent of stock solution was diluted with two equivalents of matrix solution. The final concentration of analytes in the analyte–

matrix solution was 286μM. Then, 0.70μL of this solution were spotted onto the sample carrier yielding an amount of 200.2 pmol analyte molecules per sample spot. Samples were air-dried on a standard MALDI steel plate before being analyzed.

Matrix preparation of MT3P, DIT, and TER

Ten milligrams of each compound were dissolved in 1 mL DCM. Two equivalents of each solution were separately mixed with one equivalent of sample stock solution, yield- ing a final matrix concentration of 25.80 mM for MT3P, 29.50 mM for DIT, and 26.84 mM for TER. Eventually, 0.70μL of each sample–matrix mixture was spotted on the MALDI plate.

Matrix preparation of CHCA

α-Cyano-4-hydroxycinnamic acid was dissolved in a mix- ture of 70 % acetonitrile (ACN), 30 % water, and 0.1 % trifluoroacetic acid (TFA) until saturation was reached (so- lution A). The suspension was centrifuged, and the super- natant was diluted by 1:2 with a second solution (30 % ACN, 70 % water, 0.1 % TFA) yielding solution B. After vortex agitation, two equivalents of solution B were added to one equivalent of sample stock solution yielding working solution C. Eventually, 0.70μL of solution C were spotted on the MALDI plate. Verifying the absolute matrix concen- tration, 100μL of solution B were dried, yielding 1.30 mg (SD, 0.02, n03) of CHCA. The final concentration of CHCA in the sample–matrix (solution C) solution was cal- culated to be 45.81 mM.

Matrix preparation of DHB

First, 72.56 mg of DHB were dissolved in 10 mL ACN. One equivalent of this solution was diluted with two equivalents of sample stock solution, yielding a final matrix concentra- tion of 33.33 mM. Eventually, 0.70μL of the sample–matrix mixture were spotted on the MALDI plate.

Mass spectrometry and instrument settings

If not stated differently, all experiments were carried out in the linear positive mode on a Bruker Biflex III time of flight

Table1Fifty-fivecompoundsevaluatedbyMALDI-TOFusingfivedifferentmatrixmolecules CompoundMT3PSDMT3PDITSDDITCHCASDCHCATERSDTERDHBSDDHBMFMT3Pvs.CHCAMode AcetylsalicylicacidndndndndndC21H26N2O3LN10 Aconitinea 14.004.860.220.093.901.170.480.21ndC35H68O5YesP00.002LP10 Amentoflavone17.683.760.250.1014.247.440.180.03ndC22H25NO6NoP00.589LP10 AngiotensineIIndnd10.854.49ndndC9H6O2YesP00.002LP10 Atropinea 18.615.240.040.039.672.720.020.01ndC20H20O7YesP00.009LP10 AucuparinndndndndndC20H24N2O2LP10 B-Carotene0.240.160.010.000.150.050.690.27ndC27H30O16NoP00.198LP10 Benzocainendnd1.510.46ndndC28H37N5O7YesP00.002LP10 Berberinea 23.565.670.340.1618.754.486.165.490.010.01C12H8O4NoP00.134LP10 Bergapten0.070.02nd8.723.88ndndC28H34O15YesP00.002LP10 Boldinea11.224.320.200.247.945.250.360.30ndC15H24NoP00.265LP10 Caffeicacid2.482.30ndnd1.300.79ndC21H20O6YesP00.002LN10 CaryophyllenendndndndndC9H11NO2LP15 Chlorogenicacidndnd0.020.02ndndC30H18O10YesP00.015LN10 CholesterolndndndndndC14H12O5LP10 Claviculinea20.434.750.190.116.511.851.742.74ndC9H8O4YesP00.002LP10 Codeinea 12.073.590.120.0816.583.620.650.40ndC4H4O4NoP00.056LP10 Colchicinea 12.113.770.450.212.981.390.120.07ndC16H18O9YesP<0.001LP10 Coumarinndnd1.120.36ndndC9H6O2YesP00.002LP10 Curcumin5.410.430.290.154.772.650.050.06ndC9H8O4NoP00.573LP10 Digitoxin8.274.071.220.923.341.834.421.67ndC16H14O4YesP00.022LP20 1.3-Dipalmitoyl-glycerolndndndndndC29H50OLP10 Emetinea 28.021.370.320.455.342.420.180.15ndC21H22O5YesP<0.001LP10 E-NotopterolndndndndndC15H10O7LP10 Fumaricacid0.310.00ndndndndC41H64O13LN10 Fumaritinea 25.722.900.230.102.142.151.291.08ndC18H19NO4YesP<0.001LP10 GeraniolndndndndndC29H40N2O4LP15 Glyceryl1.3-distearatendndndndndC27H46OLP10 Harminea24.683.422.392.439.342.832.571.160.010.00C11H16N2O2YesP<0.001LP10 Hesperidin0.240.300.080.060.820.670.100.10ndYesP00.041LP15 L-hyoscyaminea11.263.020.320.2719.383.380.610.30ndC34H47NO11YesP<0.001LP10 Isoimperatorinndnd5.4713.41ndndC21H22N2O2YesP00.002LP10 Khellin2.321.46nd12.956.56ndndC17H23NO3YesP00.003LP10 LeucineEnkelphalinendnd9.102.82ndndC10H14N2YesP00.002LP10 Limoginea26.032.460.520.197.921.690.090.07ndC20H18NO4YesP<0.001LP10

Table1(continued) CompoundMT3PSDMT3PDITSDDITCHCASDCHCATERSDTERDHBSDDHBMFMT3Pvs.CHCAMode Nicotinea 0.430.200.010.011.290.730.010.00ndC21H32O2YesP00.018LP10 Pentamethoxyflavone26.004.272.060.3613.195.241.701.770.000.00C13H12N2OYesP<0.001LP10 Pilocarpinea 11.034.070.860.807.562.810.030.01ndC19H17NO4NoP00.117LP10 PregnolonendndndndndC15H26N2LP10 Quercetine0.150.080.050.0314.473.410.000.01ndC15H10O7YesP00.002LP10 Quinidinea 20.966.162.051.243.081.990.150.11ndC20H17NO5YesP<0.001LP10 Rutin3.632.240.650.203.922.440.230.19ndC37H40N2O6NoP00.837LP20 Scopolaminea13.562.590.100.083.031.920.050.02ndC21H19NO5YesP<0.001LP10 Senecioninea12.053.730.140.083.642.550.210.24ndC37H40N2O7YesP<0.001LP10 SitosterolndndndndndC38H42N2O7LP10 Sparteinea20.345.340.920.4115.323.632.432.110.000.00C18H25NO5NoP00.086LP10 Strychninea13.165.720.550.2313.074.290.290.08ndC20H21NO5YesP<0.001LP10 Stylopinea 24.842.190.430.397.651.091.220.61ndC19H21NO4YesP<0.001LP10 Thalfoetidinea 5.671.820.070.063.511.461.090.96ndC40H56YesP00.046LP10 Thalicberina 20.545.220.070.053.902.590.870.78ndC18H21NO3YesP<0.001LP10 Thaligosidinea 4.400.790.060.024.751.620.120.08ndC17H23NO3LP10 Thebainea 17.583.840.370.1710.924.810.810.570.000.01YesP00.024LP10 Theobrominea 0.080.11nd8.584.160.010.01ndC14H14O3YesP00.002LP10 UmbelliferonendndndndndC39H76O5LP10 Yohimbina 16.314.320.210.166.353.141.141.01ndC7H8N4O2YesP00.001LP10 Originallyacquiredsignalintensitiesformolecularionsweredividedby1,250forillustrationpurposes.Zeronumbersindicatethatmolecularionsweredetectedbutatlowintensities MT3Pvs.CHCA:significance(Yes/No)ofobserveddifferencesbetweenMT3PandCHCA(ttest) SDstandarddeviationn06,MFmolecularformula,LP10linearpositivemode,laserenergy10%(15.6μJ),LN10linearnegativemode,laserenergy10%(15.6μJ),ndsignalnotdetected a Alkaloids

(TOF) mass spectrometer (Bruker Daltonik, Bremen, Ger- many) equipped with a 337 nm pulsed nitrogen laser (model VSL-337i, Laser Sciences Inc., Boston, MA) at a laser energy of 10 % (15.6μJ). A stainless steel plate was used as sample carrier. Spectra were acquired within a mass range of 20–2,000m/z. Acceleration voltage was set to 19 kV, pulse ion extraction was 200 ns, and laser frequency was 5 ns.

Data collection

For each sample, data from 30 separate acquisition points evenly scattered on the sample deposition area were acquired. Each acquisition point was irradiated by 30 laser shots, and data from five acquisition points (150 laser irradiations) were summed and stored as individual spectra. The process was repeated six times, yielding an overall amount of 900 single laser acquisitions. Finally, the signal intensities of six spectra were averaged, and statistical calculations (standard deviations, t tests) were performed. The robustness of the acquisition method was validated by comparing the averaged signal intensities of five randomly chosen alkaloid samples analyzed by MT3P on different days. Any sample showed compara- ble signal intensities on different days and observed differences were never statically significant (t test

comparison, P00.426–0.692). These data are presented in Fig. 4A-E in the Electronic supplementary material of the manuscript.

Data analysis and MSE score calculations

All data were processed using Bruker Daltonics Flex Anal- ysis 2.0 software. Statistical calculations were performed by SigmaPlot version 11 (Systat Software Inc.). MSE score calculations were performed for selected alkaloid spectra originating from matrix MT3P and CHCA. MSE score calculations were done according the equation of McCombie et al.: MSE score¼ P

Analyte Signals

PAnalyte SignalsþP

Matrix Signals

[6] Signals associated to a signal-to-noise ratio of 100 or below were neglected.

The equation yields a factor between 0 and 1, providing a good indication for the quality of the spectrum. A score close to 1 indicates strong analyte signals and low matrix noise, while numbers close to 0 indicate intense matrix noise and comparatively weak analyte signals. Spectra exhibiting low MSE scores could still be analyzed provided there was no direct overlap of matrix and analyte signals. This was verified by the acquisition of a blank spectrum for each matrix molecule. Compounds exhibiting MSE scores of less than 0.05 were considered not detectable.

Fig. 1 MALDI-TOF spectra of selected alkaloids analyzed by using MT3P as matrix.A Simultaneous detection of toxic alkaloidsL-hyoscyamine, senecionine, and colchicine.B Spectrum of quinidine as single compound

Results and discussion

The MALDI properties of MT3P were discovered while studying the energy transfer of self-assembling monolayers and their possible application in laser desorption-mass spec- trometry (LD-MS) [13, 14]. Early experiments showed strong ionization of alkaloids when MT3P was used as matrix molecule. Based on these findings, the compound was further tested on 55 different compounds of diverse chemical origin. Apart from alkaloids, the selection included coumarins, terpenes, flavonoids, carotenoids, steroids, and peptides. Results from this survey are summarized in Ta- ble 1, providing an overview about the general ionization properties of MT3P. Because of space limitations, Table1 solely indicates trivial names of the analyzed compounds.

Systematic CAS names can be found in Table S1presented in the supplemental section of the manuscript. Except for two samples, alkaloids constantly displayed intense

molecular ion signals. Solely nicotine and theobromine were not or weakly detected by MT3P.

Beside others, the list of successfully tested alkaloids contained highly biologically active compounds such as codeine, aconitine, ^L-hyoscyamine, emetine, and many more. Figure1displays the spectrum of quinidine as single compound (B) together with the simultaneous detection of cholchicine, ^L-hyoscyamine, and senecionine (A). Any of these substances showed strong molecular ions at a relative- ly low level of applied laser energy (10 %015.6μJ). Fur- thermore, with respect to these alkaloids, MT3P generally showed equal or superior ionizing properties compared to commonly utilized matrices such as DIT, CHCA, TER, and DHB. Solely CHCA was able to ionize as many alkaloids as MT3P, but, in general, the latter showed better ionizing properties. In the majority of these cases (16 out of 20), the observed difference was significant (Pvalues, 0.046 to

≤0.001). Furthermore, in comparison to CHCA, MTP3 in- duced stronger molecular ion formation for berberine, bol- dine, pilocarpine, and sparteine, but the observed difference was not significant. Four alkaloids namely codeine,^L-hyo- scyamine, thaligosidine, and strychnine showed better ion- ization with CHCA, but only for L-hyoscyamine the observed difference was significant (P00.001).

With respect to matrix noise formation, MT3P yielded better MSE scores than CHCA for these compounds (Table2).

Table 2 MSE score of alkaloids utilizing matrices MT3P and CHCA MSE score

Compound MT3P SD_MT3P CHCA SD_CHCA

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 0.00

Boldine 0.46 0.06 0.22 0.19

Cholchicine 0.40 0.05 0.08 0.06

Claviculine 0.26 0.09 0.18 0.07

Codeine 0.22 0.01 0.14 0.04

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

Limogine 0.27 0.05 0.71 0.10

L-hyoscyamine 0.58 0.10 0.23 0.06

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

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

Thalicberin 0.56 0.16 0.16 0.06

Thaligosidine 0.85 0.13 0.24 0.28

Senecionine 0.17 0.01 0.10 0.06

Theobromine 0.00 0.00 0.19 0.06

Yohimbine 0.57 0.28 0.15 0.01

All data were acquired in the linear positive mode at a laser energy of 10 % (15.6μJ)

SDstandard deviation

Fig. 2 3-[5′-(Methylthio)-2,2′-bithiophen-5-ylthio]propanenitrile (MT3P) and related bithiophene derivatives, which were analyzed for their ionizing properties. Names of compound: 2,2′:5′,2″-terthiophene (TER), 2,2′-bithiophene (BT), 5,5′-bis(methylthio)-2,2′-bithiophene (MetS2BT), and 3,3′-([2,2′-bithiophene]-5,5′-diylbis(sulfanediyl)) dipropanenitrile (NCEtS2BT)

A similar observation was made for the collective sample pool of alkaloids where MT3P generally showed better MSE scores than CHCA (18 out of 25 samples). However, it shall be noted that matrix solutions of MT3P and CHCA were prepared at different concentrations and that an equivalent matrix- to-sample ratio for both matrices might have yielded a different result.

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]⁺appears unusual, but it represents a standard ion originating from the process of photoionization in MALDI [15].

Contrary to its excellent ionizing properties of alkaloids, MT3P did barely interact with any compound of different chemical origin. Seemingly, there was strong ionization with some flavonoids such as pentamethoxyflavone or amento- flavone, but these compounds could also be ionized by simple laser desorption (LD) without any matrix support.

Digitoxin which did not show LD could be ionized by MT3P but required laser energy of at least 20 % (23.2μJ).

Consequently, the observed spectrum was very crowded (MSE score≤0.05) and therefore rejected from further considerations.

Subsequent experiments revealed that MT3P is ideally utilized at energy levels between 5 % and 15 % (11.8–

19.4μJ). Within this range, alkaloids displayed strong mo- lecular ions while the formation of matrix ions was still

limited. Consequently, the selective setting of applied laser energy provides a useful tool for suppressing the ionization of compounds like digitoxin that require higher amounts of energy.

With respect to the structure of MT3P, it is difficult to speculate about key features facilitating its selective ioniz- ing properties. Undoubtedly, the aromatic bithiophene (BT) core (Fig. 2) represents a key element, allowing light ab- sorption at a desired wavelength of 337 nm. However, BT itself did not facilitate any energy transfer to alkaloids or other compounds and therefore could not be used as a MALDI matrix. Similar observations were made for TER which has been reported as working matrix for chlorophyll derivatives [16]. The introduction of two thiomethyl moie- ties at the ortho positions of BT yielded 5,5′-bis(methyl- thio)-2,2′-bithiophene (MetS2BT), which showed significantly enhanced ionizing capabilities. Most substan- tial improvement was gained by the substitution of one methyl group of MetS2BT by a 2-cyanoethyl group, yield- ing the final structure of MT3P.

Interestingly, the presence of a second 2-cyanoethyl group like in the symmetrical compound NCEtS2BT severe- ly impaired the ionizing properties.

As noted earlier, solely MT3P and CHCA showed most significant interaction with alkaloids. Consequently, 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 Fig. 3 Signal intensity vs.

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

Filled triangles: applied laser energy was 10 % (15.6μJ).

Blank circles: applied laser energy was 5 % (11.8μJ)

peptides, suggesting that the observed selectivity is due to a more complex mechanism.

Apart from desirable strong analyte ions formation, ma- trix noise represents a crucial parameter for the quality of a spectrum. Similar to many MALDI matrices, MT3P exhibits its matrix ions within a mass range of 0–600m/z. Most prominent signals were observed at 297m/z([M]⁺) and at 243m/z(fragmentation product resulting from the cleavage of the S–C bond in position 3). Furthermore, two minor signals showed up at 312 and 351m/z, respectively. Despite exhibiting four signals in the low mass region, MT3P facil- itated a straightforward analysis of alkaloids. At the given concentration, the intensity of observed alkaloid molecular ions was mostly superior, but at least in the range of the largest observed matrix signal. Therefore, alkaloids were easily detected even if their molecular ions showed up in the vicinity of matrix signals.

In order to further improve spectrum qualities, potential MSEs were investigated. As previously discussed, MSEs are linked to matrix-to-analyte mixing rations as well as applied laser energy [5,6]. Many alkaloids displayed very strong molecular ions when exposed to a laser energy of 10 % (15.6μJ). Consequently, a decrease of laser energy may result in less matrix noise while still providing suffi- cient intensities of analyte signals. Therefore, 14 alkaloids were subsequently analyzed at a reduced energy level of 5 % (11.8μJ) (Fig.3A). As expected signal intensities decreased with declining laser energy, but any compound could still be clearly detected. In return, MSE scores constantly increased, and for some compounds such as fumaritine (0.3 to 0.5), thalicberin (0.56–0.79), or emetine (0.59–0.85), the raise was particularly remarkable (Fig.3B). The most prominent increase was observed for berberine (0.64 to 0.97). Consider- ing its special structure, this finding is not really surprising.

Unlike the other alkaloids, berberine already carries a charge and consists of a conjugated ring system which promotes the absorption of laser energy. Thus, berberine already shows a strong signal in LD-MS and can be detected without any matrix support.

Exceptionally, two compounds showed unchanged (spar- teine) or slightly declining MSE scores (pilocarpine) when analyzed at 5 % of laser energy. Obviously, these alkaloids respond more sensitive 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 not predictable.

As mentioned earlier, MSE scores are also dependent on sample-to-matrix mixing ratios. As this impact is currently

being addressed in a separate study, acquired results may complement the described working protocol.

The selective detection of alkaloids by MALDI-TOF has been intensively discussed in the literature [17–24], certain- ly 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 prepara- tions, or dietary supplements is highly demanded and a subject of contemporary research. While some aromatic alkaloids such as ascididemin can be ionized by simple LD [23], most reports mention CHCA and TER as preferred matrices for MALDI experiments [17,20–22,24,25]. Ad- ditionally, 7-mercapto-4-methylcoumarin has been de- scribed for the detection of arecoline and arecaidine, but no further alkaloids were tested [18]. In this respect, MT3P represents a most valuable addition to the pool of available matrix molecules. Its enhanced and selective ionizing prop- erties facilitate high-quality MALDI spectra of alkaloids at low levels of laser energy. Consequently, it may also be suitable for the analysis of unstable compounds.

Overall, the current manuscript represents a good start- ing point for working with MT3P, but future studies and experiments may help to further improve the current work- ing protocol. Further, bithiophen-based molecules may represent an entire new group of matrix molecules. Start- ing from MT3P, chemical modifications may alter their ionizing profile and create specifically adapted molecules for various MALDI applications. This outlook represents a challenging but most promising perspective for future experiments.

Acknowledgments Prof. Rudolf Bauer, head of the Department of Pharmacognosy (Institute of Pharmaceutical Sciences. University of Graz), is gratefully acknowledged for providing samples ofE-notopterol, isoimperatorin and pregnolon. Dr. Séverine Derbré (Laboratoire: Sub- stances d’Origine Naturelle et Analogues, Université d’Angers) is grate- fully acknowledged for providing samples of thalicberin and thalfoetidine.

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