JOURNAL OF MASS SPECTROMETRY, VOL. 31, 735-740 (1996)

Mass Spectrometric Investigation of 5,6- and 5,7=Dihydroxytryptamines and their Biological Precursors

Antonella Bertazzo Department of Pharmaceutical Sciences, University of Padova, Via Marzolo 5,I-35131 Padova, Italy

Silvia Catinella and Pietro Traldif CNR, Area della Ricerca, Corso Stati Uniti 4,1-35100 Padova Italy

A mass spectrometric investigation of tryptamine, 5-hydroxytryptamine and 56- and 5,7-dihydroxytryptamine was carried out using two different ionization methods, metastable ion studies and accurate mass measurements. In electron impact (EI) conditions, a series of decomposition processes, clearly related to the structure of neutral molecules, was evidenced, although 5,7dihydroxytryptamine showed severe problems related to its vaporization. The characterization of the two isomeric compounds was possible only by ion trap mass spectrometry. In fast atom bombardment conditions, decomposition pathways analogous to those observed in EI conditions were detected, and new decomposition channels due to NH, and NH, loss indicated that protonation takes place on the primary amine nitrogen atom.

KEYWORDS : dihydroxytryptamines; ion trap mass spectrometry; electron impact ionization; fast atom bombardment

INTRODUCTION

It has been proposed that a defect in the metabolism of 5-hydroxytryptamine (5-HT) (serotonin) leads to the formation of more highly hydroxylated ind~leaminesl-~ such as 5,6- and 5,7-dihydroxytryptamine (5,6- and 5,7- DHT), powerful serotonergic neurotoxins. The dis- covery of this oxidative pathway from 5-HT may provide a chemical basis for understanding certain neurological and psychotic disorders.

In particular, these dihydroxytryptamines, when cen- trally administered, give rise to selective destruction of serotonergic neurons. Pharmacological studies demon- strated that the selectivity of 5,6- and 5,7-DHT is derived from their high-affinity uptake by the serotoner- gic membrane pump. The molecular mechanism by which these dhydroxytryptamines can express their neurodegenerative effect remains u n ~ l e a r . ~ - ' ~

For these reasons, the analytical and spectroscopic characterization of these compounds is of considerable interest. In this context this paper reports the mass spectrometric behaviour of tryptamine (l), 5-HT (2), 5,6- DHT (3) and 5,7-DHT (4), as obtained by two different ionization methods, electron impact (EI) and fast atom bombardment (FAB),ll and mass-analysed ion kinetic

t Author to whom correspondence should be addressed.

energy (MIKE) spectrometry.' Furthermore, in order to characterize better the 5,6- and 5,7-dihydroxyindole isomers, ion trap mass spectrometry (ITMS) was used in energy-resolved mass spectrometric (ERMS) experi- m e n t ~ . ~ ~

EXPERIMENTAL

Compounds 1 (tryptamine ; 3-(aminoethyl)indole), 2 (5- hydroxytryptamine; 3-(2-aminoethyl)-5-hydroxyindole), 3 (5,6-dihydroxytryptamine; 3-(2-aminoethyl)-5,6-dihy- droxyindole) and 4 (5,7-dihydroxytryptamine; 342- aminoethyl)-5,7-dihydroxyindole) were purchased from Sigma-Aldrich (Milan, Italy).

CCC 1076-5174/96/070735-06 0 1996 by John Wiley & Sons, Ltd.

Received 18 January 1996 Accepted 15 March 1996

736 A BERTAZZO, S. CATINELLA AND P. TRALDI

EI and FAB measurements were performed on a VG ZAB 2F instrument.14 EI mass spectra were obtained with an electron energy of 70 eV (208 PA) at a source temperature of 180 "C. Samples 1-3 were introduced directly into the source by means of a direct insertion probe and heated in the range 100-180°C; sample 4 was heated to 450°C. FAB" mass spectra were obtained by bombarding glycerol solutions of 1-4 with 8 keV xenon atoms. Metastable ion studies were per- formed by MIKE spectrometry. l 2 Precursor ion spectra were obtained by B2/E = constant linked scans.15

Collisional spectroscopy13 was performed by 8 keV ions colliding with air in the second free-field region. The gas pressure in the collision cell was such as to reduce the main beam intensity to 40% of its usual value.

Accurate mass measurements were performed by the peak matching technique at 10 OOO resolving power (10%0 valley definition).

Energy-resolved mass spectra were obtained using ITMS', (Finnigan MAT, San Jose, CA, USA) working in the presence of helium as buffer gas to reach a final pressure of 1.33 x lop2 Pa at a trap temperature of 120°C. The selected ions were isolated by a two-step procedure17 by applying consecutive positive and nega- tive d.c. voltages. Collisional experiments were per- formed by applying a supplementary as. voltage ('tickle voltage') to the two electrode end-caps, ranging from 500 to 750 mV.

RESULTS AND DISCUSSION

The EI mass spectra of compounds 1-4 are reported in Table 1 and the MIKE spectra of the related M+' species (if present) are shown in Fig. 1.

On the basis of these data, if follows that tryptamine (1) undergoes the fragmentation pattern shown in Scheme 1. The most abundant peaks are at m/z 131 and 130 and originate from CH,-CH, bond cleavage with and without H rearrangement, respectively. The col- lisionally activated decomposition (CAD) MIKE spec- trum of the species at m/z 131 is identical with that obtained for M" of 3-methylindole, proving that the ion at m/z 131 has the same structure as the 3- methylindole molecular ion. The same ion species are present in the CAD MIKE spectrum of M", and under such conditions the formation of ions at m/z 131 is favoured (see Fig. 1). This was expected, owing to the nature of the fragmentation pathway occurring as a result of H rearrangement.

Further primary decomposition is detected in the CAD MIKE spectrum only, and leads to the ions at m/z 133. It probably originates from cleavage of the indole ring with loss of HCN, as already described for other indole derivatives.'s Further less abundant ions at m/z 116 and 117 are due to the loss of the whole side-chain. While both ions are present in the mass spectrum (see Table l), in the CAD MIKE spectrum of M" of 1 only [M - C,H,N]+ ions (m/z 116) are detectable. Finally, two ions at m/z 103 and 102 are present in the EI spec- trum. Linked scan experiments at B2/E = constant per- formed on these ions showed that they originate from

Table 1. EI mass spectra of compounds 1-4

Compound mp (relative intensity (%))

1 161 (3), 160 (20), 132 (7), 131 (80), 130 (loo), 129 (6), 128 (4), 117 (2), 116 (3), 103 (13), 102 (9). 101 (2), 89 (2). 77 (3). 76 (22), 75 (4), 63 (4). 52 (2). 51 (6)

177 (3), 176 (28). 174 (3), 160 (2), 158 (3). 148 (lo), 147 (78). 146 (100). 145 (7), 133 (2), 131 (12). 129 (3), 119 (5). 117 (9), 116 (3). 105 (5), 91 (1 1)- 90 (4), 89 (7). 81 (61, 80 (6). 77 (81, 65 (9), 63 (7),52 (5), 51 (7). 50 (4)

192 (25), 191 (3). 186 (7), 178 (6), 177 (5). 176 (5). 171 (4), 170 (4), 164 (5), 163 (66), 162 (100). 161 (7), 150 (9). 149 (70). 148 (11). 146 (7). 133 (7). 132 (8), 131 (5). 130 (26), 129 (4). 120 (9), 119 (4), 118 (7), 116 (12). 114 (90). 113 (60). 11 1 (95). 105 (17), 103 (34), 99 (7), 97 (32). 96 (7). 93 (16). 92 (6). 91 (9). 89 (12), 86 (16). 84 (66). 83 (92). 81 (10). 78 (15), 76 (87). 75 (31)

131 (2), 130 (7), 1 1 4 (loo), 1 1 2 (56), 105 (6). 104 (5). 98 (6). 94 (13), 91 (5), 85 (12), 83 (54), 81 (6), 78 (6), 77 (20). 76 (loo), 73 (7 ) , 71 (lo), 70 (17). 69 (35), 68 (1 l), 67 (9), 66 (96), 64 (33), 63 (98), 62 (50), 60 (91), 59 (24), 57 (16), 56 (30), 55 (20). 54 (38), 53 (15), 52 (26), 51 (1 1)

2

3

4

the ion at m/z 133 (see Scheme 1). Accurate mass mea- surements gave values of 103.0543 (+0.002) and 102.0459 ( f 0.002), corresponding to elemental formulae CsH7 (calc. 103.0546) and C,H, (calc. 102.0468), respec- tively. For the ions at m/z 133, we therefore propose structure a (Scheme l), which accounts for the above experimental data.

In the case of serotonin (5-HT, 2), analogous EI- induced decomposition occurs. The ions due to CH3N and CH4N' losses, leading to ions at m/z 147 and 146, are still present and give rise to the most abundant

160

f02 116 158,

192

569 EW) 893

Figure 1. MIKE spectra of M+' of compounds 1 (tryptamine), 2 (5-hydroxytryptamine) and 3 (5.6-dihydroxytryptamine).

MS OF DIHYDROXYTRYPTAMINES AND THEIR PRECURSORS 737

a+ H

d z 116

l+*

-CH N ' 1, M+'

+ 2 mlz160

QCS H

d z 130 J l+- 0-JCH3 '

H

mlz 131

[M-2H] + *

dz 1SB

~ C H - C H ~ - C H ~ - N H ~ +

f--)

idz 133

Fr-Hl+ dz 159

o$ H

1

H a

m/z 133

ac-cH' d z 103 mlz 102

Scheme 1

peaks in the mass spectrum (see Table 1, Fig. 1 and Scheme 2).

We undertook a CAD MIKE experiment in order to investigate the possible structure of the ionic species at m/z 146. The related spectrum, reported in Fig. 2, shows only two main losses, the most favoured of which is due to CO elimination, typical behaviour of phenol- containing species, leading to ions at m/z 118. Instead, the other decomposition pathway, consisting of an H,O loss, cannot be explained by a species maintaining the 5-hydroxyindole structure. We therefore propose alter- native structures, such as those reported in Scheme 2, for these ions. In particular, structure b may account for the detected H,O loss.

The primary CHN loss, observed for 1, is in this case accompanied by loss of CN' radical, leading to the ion at m/z 150. Both fragment ions can only be detected under CAD MIKE conditions.

When subjected to EI analysis, 3 and 4 behave in a very different manner from each other. Compound 3, heated to 180 "C, easily vaporizes, revealing an EI mass spectrum clearly related to its structure. The molecular ion at m/z 192 is easily detectable (see Table 1) and the primary fragments due to CH,N and CH,N' losses at m/z 163 and 162 still represent the most abundant ions present in the spectrum. In contrast, 4 does not show any simple vaporization process; only by heating the sample to 320-330 "C could some ions be detected, but neither molecular ions nor typical fragment ions were observed. Hence the results from 4 (Table 1) have very little analytical significance, because it must be considered that the mass spectrum is not of 4 but of its pyrolysis products.

Compound 3, for which both EI and CAD MIKE

spectra were obtained (see Table 1 and Fig. l), behaves differently from 1 and 2. The CAD MIKE spectrum of the M+ ' shows only one major decomposition product at m/z 163. For 1 and 2 a family of peaks due to CH,N', CH,N and CHN losses were detectable, and in the case of 3 only CH,N loss was observed and no competitive decomposition pathways were present (see Fig. 1).

In order to overcome the problem of the vaporization of 4, we thought it would be interesting to perform a series of experiments on 3 and 4 using the same ioniza- tion method but a different mass spectrometric approach, i.e. ITMS. Once introduced close to the ion trap, the sample undergoes fast heating owing to infra- red irradiation from the heating system of the instru- ment housing. This kind of sample vaporization has proved to be highly effective." In fact, both 3 and 4 easily vaporized under these conditions, leading to the spectra shown in Fig. 3. In this case, the molecular ion became detectable, even if of low abundance, also for 4. Using this approach, the severe problems encountered in measurements performed on the ZAB instrument were overcome; Fig. 3 shows that the two isomeric compounds lead to very similar spectra, with only a few differences in the relative abundances of product ions (m/z 91,92,134 and 135).

Product ion mass spectra, obtained by resonance excitation of the molecular species, did not lead to any significantly different results: in both cases, at a tickling voltage of 750 mV, only minor differences in the relative abundances of the ions at m/z 149, 132, 122, 120 and 108 were detected. For more significant results, it was thought of interest to perform energy-resolved mass spectrometric experiments. This method has been suc- cessfully applied in the characterization of isomeric

738 A BERTAZZO. S. CATINELLA A N D P. TRALDI

[M-H]+

m/z 175

1 +'

H

d z 147

HowcH2 - - O m - / 4' c-- - H20y-JJ H H

b dz 146

0 4 2 a H H

Scheme 2

compounds by using an ITMS instrument.20 By such an instrumental approach, CAD can be performed applying a supplementary ax. voltage (usually called a tickle voltage) between the two end-cap electrodes. When the tickle frequency is equal to the secular fre- quency of the axial component of motion of the selected ions, resonant excitation takes place. Ions peak up kinetic energy and, following multiple collisions with

118

the buffer gas, dissociate and give rise to fragment ions. Various methods can be employed to deposit a range of energies upon activation in order to achieve ERMS

but the most effective approach is that based on the variation of 'tickle' voltage, adopted in the present case. The plots of the abundances of the above discussed product ions us. 'tickle' voltage are shown in Fig. 4 for 3 and 4. The plots show that, in the case of 4,

146

Figure 2. MIKE spectrum of ionic species at m/z 146 El-generated from compound 2 (5-hydroxytryptamine).

MS OF DIHYDROXYTRYPTAMINES A N D THEIR PRECURSORS 739

3

INT

40 80 120 160 200 mlz 100% 91

4

INT

. . ' 4b 80 I20 160 200 mlz

Figure 3. lTMS/El spectra of compounds 3 (5.6-dihydroxytryptamine) and 4 (5,7-dihydroxytryptamine).

the formation of ions at m/z 122 is energetically more favoured than for 3: when a tickle voltage of 600 mV is employed, the species at m/z 122 has a relative abun- dance of 25% for 4 but only 8% for 3. In other words, whereas for 3 the abundance of the ions at m/z 122 shows a very small increase with increase in tickle voltage, in the case of 4 its abundance shows a higher slope up to 600 mV, after which it slowly decreases.

Sample 3

Such a behaviour is a good indication that two pro- cesses, different from the energetic point of view, occur for 3 and 4.

In order to investigate the behaviour of the proton- ated molecular species of 1-4, a series of FAB experi- ments were undertaken. The FAB spectra are reported in Table 2 and the CAD MIKE spectra of the related [M + H]+ species are shown in Fig. 5. Under FAB

500 550 600 650 700 750 mV

Sample 4

70 eo n

- d z 192

-x- m h 149

-mh132 - mlz 122 - mlz 120

-*- mh 108

-f- mh192

-x- mlz 149

& I 3 2

-lIlk122 - mh 120

-x- m/z 108

500 550 800 850 700 750 mV

Figure 4. Energy-resolved mass spectra obtained on molecular cation (M+') for dihydroxyindole isomers 3 (5.6-dihydroxyytryptamine) and 4 (5,7-dihydroxytryptamine) at various collision energy amplitudes.

740 A BERTAZZO, S. CATINELLA AND P. TRALDI

~~

Table 2. FAB mass spectra of compounds 1-4

Compound m/z (relative intensity (%))'

1 162 (20), 161 (100). 160 (15), 145 (18). 144 (98), 143 (20). 132 (18), 131 (30), 130 (33), 118 (7), 117 (15),116(5),115(10),77(8),76(4),75(12)

179 (5). 178 (30), 177 (100). 176 (25). 175 (lo), 161 (17). 160 (75). 159 (14). 148 (11). 147 (10). 146 (25). 145 (17), 143 (6). 133 (7). 130 (5)

194 (4). 193 (16). 192 (5), 177 (15), 163 (5). 162 (5),149 (5),115 (12),114(100),113(8)

194 (24). 193 (80), 192 (28), 191 (12), 177 (15), 176 (60), 175 (20), 150 (15). 149 (100). 147 (14), 132 (20). 131 (30). 130 (12). 129 (21)

2

3

4

a Peaks due to matrix ions have been omitted.

conditions, the behaviour of 1-4 was very different from that observed in EI conditions. Surprisingly (considering the lower internal energy deposition present under FAB conditions), the same fragmentation processes related to the side-chain were still seen but new decomposition pathways were activated, consisting of NH,' and NH, losses.

In particular, the latter process was responsible for the most intense peak present in the CAD MIKE spectra of [M + HIf for all compounds. The CH,N loss, highly favoured in EI conditions, in this case leads to a peak with an intensity similar to those due to CH,N' loss and, for 3 and 4, gives rise to signals of negligible intensity. The above results indicate that the protonation site for ail compounds is on the amine nitrogen atom. This hypothesis is in agreement with the proton affinities, which are 8.98 x lo5 J mol-' for eth~larnine?~ 8.63 x lo5 J mol-' for pyrroleZ4 and 8.15 x lo5 J mol-' for phenol.25

It is worth noting that in FAB conditions 3 and 4

E(v) 760 893

Figure 5. MIKE spectra of [M + H]+ of compounds 1 (tryptamine), 2 (5-hydroxytryptamine), 3 (5.6-dihydroxytrypta- mine) and 4 (5,7-dihydroxytryptamine).

lead to clearly different spectra, allowing the character- ization of the two isomers.

In conclusion, the mass spectrometric investigations performed on 1 4 revealed a series of decomposition processes clearly related to the structure of neutral mol- ecules. In particular, isomeric compounds 3 and 4 lead to almost identical EI spectra; even the metastable ion data related to M+' and [M + HI+ do not allow the structural characterization of 3 and 4. ERMS, which is considered to be one of the most powerful methods in structural investigations, allowed the two isomers to be differentiated. In FAB conditions, such a differentiation is achieved with the single spectra.

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