Multimatrix Variation Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry as a Tool for Determining the Bonding of Nitrogen Atoms in Alkaloids

The reactivity of alkaloids in dehydrogenation was investigated using multimatrix variation matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) of over 20 different alkaloids with six matrices. The dehydrogenated molecular ions [M – H]+ generated by in-source decay were detected in the MALDI mass spectra of some types of alkaloids such as reserpine. The dehydrogenation proceeded at the cyclic tertiary amine rather than double-bonded nitrogen atoms and indole rings involved in the electron-delocalized systems. The stable protonated primary amines hindered dehydrogenation. The laser-induced dehydrogenation correlated with the chemical properties and structures of alkaloids. Alkaloids were classified into three types by the ratio of dehydrogenation by comparing the relative abundances of [M – H]+, [M]•+, and [M + H]+ ions in α-cyano-4-hydroxycinnamic acid and 5-formylsalicylic acid matrices. Structural isomers were also discriminated by this method of analyzing the three molecular ions’ ratio using multimatrix variation MALDI-MS.


Journal of the American Society for Mass Spectrometry pubs.acs.org/jasms
Research Article mass spectrum. The laser irradiation power was controlled with an attenuation filter of the instrument, and the power values were shown as percentage (%) of the laser output (100%). The experimental laser powers were optimized without signal broadening and overirradiation in each analyte, and each matrix is summarized in Supporting Information Table SI-2.
The optimized laser powers tended to be dependent on the matrices mainly. Sample Preparation. All six matrices (DHB, 18 CHCA, 19 DAN, 20 ASA, 21 FSA, 22 and NSA, 22 their structures are presented in Supporting Information SI-3), each 50 mM, were dissolved in 60% methanol solution by sonication in hot water. All analytes with 5 mM concentrations were dissolved and/or suspended in 60% methanol solution by the same procedure. Five microliter aliquots of the analytes were premixed with 50 μL of the matrix and 45 μL of 60% methanol (total volume of each solution was 100 μL) where the analyte and matrix concentrations were 0.25 mM. One microliter aliquots of the premixed solutions were deposited on the target plate. The analytes of auxin metabolites, indole, indole-3-ethanol, indole-3-acetic acid, indole-3-acetamide, and tryptamine were measured in the higher concentration of the premixed solution because they were small molecules close to the matrix. A total of 20 μL aliquots of each analyte were premixed with the same aliquots of 50 mM CHCA, and 1 μL  Figure 2B). Therefore, the dehydrogenation did not proceed in the galloyl group. On the other hand, the dehydrogenated ion [M − H] + was generated from 3, and its spectrum was similar to those of 1 and 2. Therefore, the indole ring was not involved in the dehydrogenation because 1−5 compounds have the same indole ring (Figure 1). At the same time, the double bond near the nitrogen in 4 and 5 inhibited their dehydrogenation, certifying that the C−N−C single bond in the ring is needed for the alkaloid dehydrogenation in in-source decay MALDI. The single-bonded nitrogen atom was protonated followed by the removal of the hydrogen atoms bonded to the α-carbon and nitrogen atoms.
Therefore, the dehydrogenation of alkaloids was dependent on their structure, namely the type of nitrogen atom bonds (single or double). Therefore, the laser-induced dehydrogenation of alkaloids provided useful information to predict the structure of alkaloids.
Matrix Effects on Dehydrogenation of Alkaloids.  23 The peptides with disulfide bonds were reduced by DAN, and the [M + H + 2H] + ions were detected in the DAN-MALDI mass spectra previously. 23 The dehydrogenation of 1 was mostly suppressed in the DAN-MALDI mass spectrum, and the [M + H] + ion was the most abundant as shown in Figure 3(B) due to the DAN reducing property. 23 The [M − H] + and [M + H] + ions were generated with almost the same abundance in the DHB-MALDI mass spectrum of 1 as shown in Figure 3(C). The [M + H] + ion were slightly stronger with ASA as compared to that without the matrix, which indicated the reduction of 1. The CHCA-and FSA-MALDI mass spectra of 1 were almost the same as that without matrix-MALDI. The NSA matrix accelerated the dehydrogenation of 1 because NSA showed hydrogen-accepting ability. In the oxidation/reduction study of peptides in the MALDI process, DAN, ASA, and DHB matrices possess hydrogen-donating properties, while FSA and NSA are hydrogen acceptors, and CHCA may play the role of both acceptor and donor. 22 The tendency was similar, but the [M − H] + ions of alkaloids were different from the peptides in MALDI-MS. This was because the oxidation/reduction processes in the MALDI were dependent on the analyte's properties.
The tendency to shift from [M + H] + to [M − H] + in 1 was almost the same as in 2 and 3 in the series of MALDI-MS with six matrices (see Supporting Information SI-5 and -6). On the other hand, there were no differences in the series of six-matrix MALDI mass spectra of 4 and 5 (see Supporting Information SI-7 and -8) and the [M + H] + ions were predominantly detected, indicating that the molecular ions of 4 and 5 were no reactive to these matrices. The laser-induced alkaloid shifts indicated their oxidation/reduction reactivity. The mass shifts of the molecular ions were sensitive to the specific structure of alkaloids 1, 2, and 3 and not to 4 and 5. Therefore, the dehydrogenation of alkaloids in MALDI-MS was dependent on their structure.
Indole and Auxin Metabolites. Indole 6 has a nitrogen atom, which is not linked by a double bond but by C−N−C single bonds in the structural formula. However, dehydrogenation did not proceed in the indole ring. Therefore, we studied the properties of indole compounds 6−10 ( Figure 1 Table 1 and SI 9−13. A molecular ion is generated in the high aromaticity structure part. 8 The chemical properties of indole are determined by the amine group not possessing Lewis basicity because the lone-pair electron on the nitrogen atom is involved in electron delocalization in the aromatic ring. It was reasonable to assume that dehydrogenation does not proceed in the indole rings. The lone pair electron of nitrogen had to be free and not involved in aromaticity or a delocalized electron system for the laser-induced dehydrogenation of alkaloids. Indole alkaloids such as auxin or indole-3-acetic acid 7, indole-3-ethanol 8, and indole-3-acetamide 9 can be ionized due to their aromaticity as both the molecular ion [M] •+ and the protonated molecule [M + H] + are observed in the spectra with the CHCA matrix (Supporting Information SI 9−13).  Table 1 (Supporting Information SI-15−27). From the abundance of the dehydrogenated ion [M − H] + , the reactivity of alkaloids was categorized into three categories roughly�the highly reactive ones such as 1−3 and 11−16 (Table 1), the middle reactive ones such as 21 and 22, and the low reactive ones such as 4, 5, and 17−20 and 6−10 ( Table 1). The dehydrogenated ions [M − H] + were generated by MALDI from the cyclic and single bonded amines of alkaloids such as emetine 11, ajmalicine 12, indapamide 13, brucine 14, vincamine 15, and antazoline 16. None of them contains the primary amine as the stable protonated site. The sulfonamide group in 13 does not possess the basicity due to the sulfo groups and it was not the stable protonated site. The proton dehydrogenation process is linked to the reactive nitrogen atoms.
On the other hand, epinastine 17 and lisinopril 18 contain the stable protonated site in their structure such as the basic primary amine. They were not dehydrogenated even though they contain the other reactive nitrogen atoms. Camptothecin 19 and 7-ethyl-10-hydroxycamptothecin 20 have a quinoline possessing the same basicity as the protonated site, suppressing the dehydrogenation. Additionally, the electron-withdrawing groups neighboring the nitrogen hindered the dehydrogenation because the lone pair of the nitrogen atom did not induce the reaction directly. Cinchonidine 21 also involves a quinoline, but the tertiary amine is without the neighboring electron-withdrawing groups. Diltiazem 22 contains the noncyclic or cyclic tertiary amines, and the cyclic one neighbors the aromatic ring and ketone group. Their dehydrogenation proceeded weakly as shown in Table 1. The partial dehydrogenation was observed with 21 and 22. We compared the alkaloid mass shifts in the MALDI-MS spectra with CHCA and FSA matrices ( Table 1). The dehydrogenation with FSA proceeded better than that with CHCA in MALDI of 1 (Figure 3). Compounds 21 and 22 were clearly classified as middle reactive alkaloids following the alkaloid mass shift tables with the CHCA and FSA matrices. The dehydrogenation was the reaction of the alkaloid analytes and matrices. The matrices controlled the reactivity of alkaloids under laser irradiation, which was a new function of matrix in  The ion intensities were normalized to the highest ion as 100% between the three ions. The data are the averages of the relative ion intensities of the five times measurements. The standard deviations are summarized in SI-27. The asterisks mean that the signals of the matrix and alkaloids were overlapped as shown in SI-10 (7) and SI-12 (9). MALDI-MS. Here, it is important for us to investigate new functional matrices such as a reaction inducer to develop more fine structure analysis.
Discrimination of Structural Isomers of Alkaloids. Alkaloids 2 and 15 had the same molecular formula (C 21 H 26 N 2 O 3 ) (Supporting Information SI-2) and were structural isomers. We tried to discriminate between 2 and 15 using the alkaloid reactivity of dehydrogenation in various matrix MALDI-MS (see Supporting Information SI-5 and -19). The reactivity of 2 was 44% of the [M − H] + intensity, and it was higher than that of 15 (24%) in CHCA-MALDI mass spectra (Table 1). In addition, the isomeric alkaloids 2 and 15 were clearly discriminated in the FSA-MALDI mass spectra; the [M] •+ ion of 15 was predominant and the [M − H] + ion of 2 was predominant ( Table 1 and Supporting Information SI-5 and -19). The nitrogen atom of the indole ring in 2 was a secondary amine and that of 15 was a tertiary amine. Compound 15 had higher aromaticity at the indole ring than 2. Thus, the molecular ion [M] •+ of 15 was generated predominantly. The alkaloid reactivity analysis using matrixvariation MALDI-MS was useful for the discrimination of the structural isomers of alkaloids.

■ CONCLUSION
The dehydrogenation of alkaloids proceeded by MALDI as their in-source decay reaction. The alkaloids were classified into those with high, middle, and low reactivity according to their molecular related ions. The ion abundance ratio of [M − H] + , [M] •+ , and [M + H] + was taken as the indicator of alkaloid reactivity with the multimatrices variation MALDI-MS. The dehydrogenation of alkaloids was used for the prediction of their structure around the nitrogen bondings. The alkaloid isomers were also discriminated based on the reactivity difference between alkaloid and matrices. ■ ASSOCIATED CONTENT