International Journal of Mass Spectrometry and Ion Processes, 115 (1992) 219-233 Elsevier Science Publishers B.V., Amsterdam

219

The laser desorption/laser ionization mass spectra of some indole derivatives and alkaloids

Kevin Rogers, John Milnes and John Gormally Department of Chemistry and Applied Chemistry, The University of Salford, Salford MS 4 WT (UK)

(First received 16 January 1992; in final form 28 February 1992)

ABSTRACT

The laser desorption and laser ionization mass spectra of some indole derivatives and alkaloids are described with particular reference to their modes of fragmentation. Mass spectra of yohimbine, reserpine, quinine and quinidine are presented. Full experimental details are given.

Keywords: laser desorption; laser ionization; indole derivatives; alkaloids; fragmentation.

INTRODUCTION

The laser desorption of neutral atoms or molecules followed by their multiphoton ionization (MPI) using a second laser has been utilised in several mass spectrometers that have been described in recent years [l-9]. This technique is sensitive, it can be selective for the species that are ionized, and the extent of fragmentation can be varied readily by changing the power density of the ionizing laser beam. Often, it is possible to obtain mass spectra that contain peaks arising from the molecular ion alone or from the molecular ion accompanied by a small number of fragment ions. Time-of-flight mass analysers have been used as they are well suited to pulsed desorption and ionization methods. This type of mass analyser has several desirable features such as its high transmission and very wide mass range. However, it also has limited mass resolution although this can be improved significantly by using ion reflection techniques [5]. Whilst MPI can give high intensity molecular ion peaks, the resolution of the time-of-flight mass analyser is not, in general, adequate to allow the deduction of a molecular formula from this information alone. In principle, knowledge of the ionization wavelength should assist in identifying a sample. However, at present, not enough is known about the high resolution gas phase absorption spectra of large organic molecules to allow this feature of the technique to be used effectively for identification

Correspondence to: J. Gormally, Department of Chemistry and Applied Chemistry, Univer- sity of Salford, Salford MS 4WT, UK.

0168-l 176/92/$05.00 0 1992 Elsevier Science Publishers B.V. All rights reserved.

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DETECTOR

DESORBING

IONIZING

Fig. 1. The apparatus used: the inset shows the positioning of the sample rod in relation to the supersonic beam. The cooling of desorbed molecules could be improved by cementing a narrow plate to the underside of the sample rod as indicated in the inset.

purposes. In this study we have recorded MPI mass spectra at low and high ionization laser intensities with a view to examining their fragmentation features. Much emphasis has been given to the prominence of the molecular ion in such spectra. When the aim is to determine the presence of a compound of known structure, facile detection of the molecular ion is clearly important. However, fragmentation patterns provide additional information that is helpful in determining the composition of unknown samples. The substances that we have looked at are simple indole derivatives and their mass spectra have been compared with those of more complex alkaloids that contain structures related to those found in the indoles.

EXPERIMENTAL

The layout of the apparatus is shown in Fig. 1. Its operation is similar to that of the laser mass spectrometer described by Lubman and Tembreull[4,6]. Neutral molecules were desorbed by a pulsed carbon dioxide laser from the surface of a glass rod coated with sample material. Desorbed molecules were then entrained in a supersonic beam of argon and transferred through a skimmer into the ionization region of a time-of-flight mass spectrometer.

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Resonant two-photon ionization was effected by the absorption of a 5 ns pulse of ultraviolet radiation that had been generated by a Nd-YAG pumped dye laser system with frequency doubled output. Ions were detected by a channel plate electron multiplier, the output of which was fed directly into a digitizing oscilloscope.

A low pressure carbon dioxide laser (Pulse Systems LP-30) was used for desorption. The peak power densities produced by this laser are considerably lower than those available from a TEA laser of similar pulse energy. This laser produces pulses of approximately 25 ,US duration and 100 mJ energy at a wavelength of 10.6 pm and it was triggered at a repetition rate of 5 Hz. In our experiments on the simple indole derivatives, which were easily desorbed and ionized, the laser beam was attenuated to around 40 mJ by passing it through thin sheets of a plastic material such as polyethylene. In the desorption of the larger alkaloid molecules, the desorption pulse energy was about 100 mJ. The beam was focussed onto the sample using ZnSe optics and the irradiated spot had a diameter of about 2mm.

The ionizing laser system consists of a Q-switched, frequency doubled Nd-YAG laser (Quanta-Ray DCR3) pumping a dye laser (Quanta-Ray PDL2) the output of which can be frequency doubled in a Quanta-Ray WEX wavelength extending unit. The output beam was focussed into the ionization region of the time-of-flight mass spectrometer by a cylindrical lens of 300 mm focal length so that the focal line was perpendicular to the flight tube axis. The approximate dimensions of this line were 2.2mm x 0.6mm. The laser dyes used in the experiments described here allowed the wavelength to be varied from 275 nm to 300nm and pulse energies in the range 0.1 to 2mJ were typical. A pulse energy of 1 mJ corresponded to a power density at the laser focus of approximately 1.5 x 1O’W cme2.

The pulsed valve (General Valve Corporation) was supplied with argon at a backing pressure that could be varied between 1 and 8atm. For most experiments, the backing pressure was 5 atm and the valve was pulsed open for a period of 150~s. The valve was mounted on top of the desorption housing and its position could be adjusted along three axes.

The desorption housing was machined from a solid cylinder of aluminium. It had an internal diameter of 0.29m, a volume of 12dm3 and was pumped by a 6in oil diffusion pump fitted with a liquid nitrogen trap. In practice, it was found that clean, background-free mass spectra could be obtained when the cold traps on the diffusion pumps were not charged with coolant. The relatively large volume of the desorption housing coupled with fast pumping allowed pressures of around 5 x 10-6Torr to be maintained in the flight tube when the pulsed valve was operating, even with a skimmer orifice diameter of up to 8 mm. Various types of skimmer have been tried with orifice diameters down to 1 mm (Beam Dynamics Inc, Minneapolis) but the

222

_//” _ CH$OOH

INDOLE-SACETIC ACID

a) b)

Fig. 2. (a) The molecular ion peak of indole acetic acid of normal isotopic composition. The smaller peak to the right and one mass unit higher arises from molecular ions containing one 13C atom. (b) A mass spectrum of tryptamine obtained from a single laser shot.

one that we have used most frequently was fabricated from two razor blades that were arranged to present a slot-shaped aperture of 2mm width to the supersonic beam. The type of skimmer used did not have a significant effect upon the performance of the apparatus. With the pulsed valve switched off, the pressure in the flight tube was around 5 x 10e7Torr.

The flight tube of the time-of-flight mass spectrometer is 1.35m long and is operated without an inner liner and at ground potential. Pumping of the flight tube is accomplished using two 4in oil diffusion pumps, one of which has no cold trap and is situated directly below the ionization region (Fig. 1). The potentials applied to the three-electrode ion source have been calculated using the Wiley-McLaren [lo] space focussing condition. Mass resolution is dependent upon the position and shape of the ionizing beam and these parameters have changed slightly following the use of the laser for other purposes. At its best, the mass resolution obtained was estimated to be in the region of 600 and the sharper ion peaks had a temporal width of between 8 and 20ns (Fig. 2a).

Ions were detected by an 18 mm diameter channel plate electron multiplier, the output of which was fed into a LeCroy 9450 digitizing oscilloscope (temporal resolution of 2.5 ns between points) that was set in averaging mode.

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Typically, 20 spectra were averaged in order to reduce baseline noise but, often, a good mass spectrum could be obtained from a single shot (Fig. 2b). The oscilloscope was interfaced to a personal computer for the archiving of data.

Various dimensions relating to the equipment as used in these experiments are listed below.

Sample probe axis to pulsed valve (variable) 1.5 mm Sample to supersonic beam axis (variable) 4mm Pulsed valve to skimmer orifice (variable) 38 mm Pulsed valve to ionization position (variable) 145mm Backing plate to first grid (fixed) 11 mm First grid to second grid (fixed) 5.5mm Backing plate to ionization position (variable) 5.5 mm Early experiments in which dichlorotoluene vapour was introduced into the

argon supply to the pulsed valve indicated that a considerable degree of cooling of the sample molecules was being achieved. In these experiments, the wavelength of the ionizing laser had to be carefully adjusted within f 0.05 nm of the absorption wavelength at 279.17 nm to obtain a mass spectrum. The degree of cooling obtained with laser desorbed molecules was much less and, in some cases, it was possible to vary the ionizing laser wavelength by several nanometers and still obtain an acceptable mass spectrum. One reason for this is that some desorbed molecules could enter the molecular beam at positions up to 38 mm from the nozzle and, at some of these points, most of the cooling that arises from the expansion has already taken place. By cementing a thin steel plate to the underside of the sample rod, as indicated in Fig. 1, we could restrict this late entry into the molecular beam, leading to an improvement in wavelength selectivity. At present, we do not have facilities that would allow us to perform a wavelength scan conveniently and we have not been able to examine more closely the optical absorption linewidths or the experimental parameters that affect them. However, even in the absence of optimal cooling, the use of a supersonic beam does retain the advantage of presenting to the ionization region molecules that have only a small component of velocity in the direction of the flight tube axis, thus enhancing the mass resolution attainable.

RESULTS

Mass spectra of indole butyric acid obtained at three different ionization pulse energies are shown in Fig. 3. All three spectra show a strong molecular ion peak at m/z 203. This is the dominant peak at low ionization energies. The small peak to the right of the molecular ion peak is 14 mass units higher and is attributed to the methyl ester of indole butyric acid (samples were prepared

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1 - ’ CH2CH2CH2COOH

H

a) 31NDOEBUlYRICAClD ,ru ,_ II II

b)

I

203

27

143

130

115

C) 102,103

Fig. 3. Mass spectra of indole butyric acid obtained at laser pulse energies of (a) 0.08 mJ, (b) 0.6 mJ and (c) 2.0 m.l. The laser wavelength was 280 nm. In this and subsequent spectra, the full width of the trace corresponds to a time of 50~s.

by dissolving indole butyric acid in methanol and coating onto the sample rod). Another strong peak at m/z 130 is found in all spectra. It has been suggested by Beynon [l 11, on the basis of stability considerations, that the species responsible for this peak results from a rearrangement yielding a stable ion with the quinolinium structure:

m ‘I’ ’ N’ J.

H’ QUINOLINIUM ION

We have no evidence that the peak at m/z 130 arises from an ion with this structure but it seems probable that its origin is the same as that of the peak at m/z 130 found in the electron impact mass spectra of indole derivatives. Further fragmentation of this ion gives rise to the phenyl cation at m/z 77 together with smaller fragments down to carbon. A small peak at m/z 143 is seen in all three spectra. The structure of the ion responsible for this peak is uncertain but its mass corresponds to that of the ion at m/z 130, postulated

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1 - ”

CHz=“z’J”z

I v

I 160

130

103

I 17 15

C) I

30

Fig. 4. Mass spectra of tryptamine obtained at laser pulse energies of (a) 0.1 mJ, (b) 0.5 mJ and (c) 2.0mJ. The laser wavelength was 280nm.

by Beynon to be the quinolinium ion, in which a hydrogen atom has been replaced by a CH, group. Mass spectra of indole acetic acid exhibited all of the features described above with the exception that the peak at m/z 143 was absent. The mass spectra of both compounds also contain low intensity peaks at m/z values of 102 and 103. The peak at m/z 103 is very common in the electron impact mass spectra of substituted indoles [11,12]. It has been suggested that it arises from the removal of HCN from the quinolinium ion [12] and that it is a useful indicator of the presence of the indole ring system [l 11. This feature could easily be missed in the spectra generated by laser ionization, especially in the presence of baseline noise, but we have found it to be present in the spectra of all the simple indole compounds examined. At higher ionization energies, an intense peak is observed at m/z 27. The relatively high intensity of peaks due to low mass ions at high pulse energies is one of the features that distinguishes these spectra from those obtained by electron impact ionization.

The mass spectra for tryptamine are shown in Fig. 4. The MPI mass spectra of this compound and of other indoles have been reported by Tembreull and Lubman [13,14] with findings similar to our own. At low ionization energies

226

b)

Fig. 5. Mass spectra of tetrahydrocarbazole obtained at laser pulse energies of (a) 0.12 mJ, (b) 0.4mJ and (c) l.OmJ. The laser wavelength was 280nm.

the dominant peak is due to the molecular ion at m/z 160. However, in this case, we also find a peak at m/z 30 corresponding to the ion CH,NHl that results from the cleavage of the side-chain in the tryptamine molecule [ 141. No similar small fragment ion resulting from this cleavage was found in the mass spectra of indole acetic acid or indole butyric acid. Peaks due to the ion at m/z 130 and the phenyl cation are also very evident but there is no evidence of the species at m/z 143 that was observed in the spectra of indole butyric acid.

In Fig. 5 the mass spectra obtained with tetrahydrocarbazole are shown. Again, the molecular ion peak is dominant at low ionization energies. Apart from this, the most conspicuous feature of these spectra is the peak at m/z 143 that is much larger than that at m/z 130. It is possible that this peak arises from a rearrangement analogous to that which has been suggested to give rise to the quinolinium ion in other compounds.

The results of these studies of the mass spectra of simple indole derivatives can be summarised as follows. At low laser ionization energies, the molecular ion is the dominant peak in the spectra. Between the molecular ion peak and the peak due to the phenyl cation at m/z 77 are found a number of other peaks that appear to be characteristic of the indole ring system. Two such peaks

227

bl fiEs.3~~~

173

156

Fig. 6. (a) Mass spectra of yohimbine at laser pulse energies of 1 .O mJ and 3.5 mJ. In the higher energy spectrum, the parent ion peak is also shown expanded by a factor of 100 in the horizontal direction. (b) The mass spectra of reset-pine at laser pulse energies of 0.1 mJ and 1.6 mJ. These spectra were obtained at a wavelength of 280 nm but similar traces could be obtained at any wavelength between 280nm and 290nm.

appear at m/z 130 or at m/z 143. In the cases of tetrahydrocarbazole and indole butyric acid, both peaks are found. In addition, smaller peaks are found at m/z values of 102, 103 and 115. Peaks in this region are of relatively low intensity and this is probably a consequence of them arising from species that will themselves absorb at the ionization laser wavelength and thereby undergo further fragmentation. From m/z 77 downwards, live distinct groups of peaks are seen that represent characteristic fragments of the phenyl ring. These findings are qualitatively similar to what has been observed in the electron impact mass spectra of similar compounds. The main differences are the relatively high intensity of the molecular ion peak that is observed at low laser pulse energies and the high intensities of peaks arising from the formation of low mass ions at high pulse energies.

The mass spectra of the indole alkaloids yohimbine and reset-pine are given in Fig. 6. The high energy spectra appear to be very congested but the mass resolution is much better than this appearance suggests. In order to maximise the amount of information that can be displayed on the oscilloscope screen, a data compaction algorithm is employed with the result that peaks appear

228

wider than they actually are. The molecular ion peak of yohimbine is shown expanded horizontally by a factor of 100 in the higher energy spectrum of Fig. 6a. Flight times and mass values were determined from peaks that had been expanded in this way. For these compounds, the wavelength dependence of the ionization cross-section was weak, indicating that effective internal cooling of the molecules had not been attained. It was possible to vary the ionization wavelength over a range of 15 nm and still obtain satisfactory mass spectra. A very similar observation has been made by Tembreull and Lubman [13] in their studies of simpler indole-based compounds. However, more intense and fragmented spectra were obtained at wavelengths around 290 nm than could be obtained at lower wavelengths for a fixed laser pulse energy.

In the mass spectra of yohimbine and reserpine, the molecular ion gives rise to the dominant peak at low ionization laser energies. At higher energies, the mass spectrum of yohimbine has many peaks in common with the mass spectrum of tetrahydrocarbazole. In particular, we find peaks at m/z values of 102, 115, 130 and 143, along with an intense peak at m/z 27. There is a notable absence of intense peaks in the m/z range from 223 up to the molecular ion peak, and the MPI mass spectrum shows only a small M - 1 peak at m/z 353 in contrast to the electron impact spectrum of this compound in which the base peak is at M - 1 [15,16] (M represents the molecular ion). The origin of this species in the electron impact spectrum has been attributed to the loss of hydrogen from the 3 carbon atom (Fig. 6) in the yohimbine ring system [15]. The peak at M + 1 has an intensity that is 24.6% of the molecular ion peak. From the known isotopic abundance of 13C we would expect this figure to be 23.3 1%. The difference between these two values can be accounted for by the limitations upon accuracy resulting from the vertical and horizontal resolutions of the oscilloscope (eight bits and 2.5 ns respectively). Other characteristic peaks that are found in both the electron impact and MPI spectra of yohimbine are at m/z values of 156, 169, 170 and 184. Structures for the ions responsible for these peaks have been suggested [ 151. However, in the MPI spectrum, these peaks are of lower intensity relative to the molecular ion peak.

The mass spectrum of reserpine is given in Fig. 6b. The molecular ion peak is seen at m/z 608. Other peaks that have been observed in the electron impact mass spectrum [ 171 of this compound were also found. This spectrum contains many of the peaks found in the spectrum of yohimbine with additional peaks that are 30 mass units higher. These peaks are listed in Table 1, they appear at m/z 172 and above in the spectrum of reserpine and probably arise from the presence of the 1 1-methoxy group on the indole ring system in this molecule.

The mass spectra of quinine and quinidine are given in Fig. 7. These two compounds have the same structural formulae but differ in their stereochemis- try and have different physical properties. Both compounds can be ionized

229

TABLE 1

Masses in the spectrum of reserpine that are 30 mass units higher than corresponding peaks found in the spectrum of yohimbine

Yohimbine Reset-pine

223 253 211 241 184 214 170 200 169 199 156 186 143 173 142 172

efficiently at 290nm and have very similar mass spectra with the exception that the ion at m/z 136 gives rise to a much more intense peak in the spectrum of quinine than in that of quinidine. This difference is particularly noticeable in the low energy spectra and could be used to distinguish between these two compounds. The electron impact mass spectra of these two compounds show

Fig. 7. (a) Mass spectra of quinine at laser pulse energies of 0.2 mJ and 1.1 mJ. (b) Mass spectra of quinidine at laser pulse energies of 0.1 mJ and 1.1 mJ. These spectra were obtained at a laser wavelength of 290 nm.

230

similar differences [16] but to a lesser degree. MPI mass spectra can often be used to distinguish between isomers [9] and the differences between quinine and quinidine are very clear. At a laser pulse energy of 1.1 mJ, the peak at m/z

136 is 7.5 times more intense in quinine than in quinidine. However, the relative intensities of peaks can vary widely with the power density of the ionizing laser beam. As laser beams do not have a uniform power density across their cross-section and, at any position within the cross-section, the power changes with time during the pulse, it is difficult to specify what the effective power density is. As a result, it is not possible to quote peak inten- sities as a percentage of a base peak height in a useful way. However, gross differences in peak intensities, such as those illustrated in Fig. 7, can readily be seen from an inspection of the spectra although a satisfactory way of quantifying these differences has yet to be devised.

In the absence of a suitable method for standardizing intensities, a simple aid to comparing the mass spectra of different molecules is to calculate a cross correlation function defined by

C,,(x) = (l/N) $ R(m) - S(m + x) m=l

where R(m) is assigned a value of one if there is a peak in the reference molecule spectrum at mass number m and a value of zero otherwise and S(m) is similarly defined for the sample molecule spectrum. The number of peaks in the reference spectrum is denoted by N and M, is the highest mass number in this spectrum. A consequence of this definition is that C,,(O) has a value of unity if every peak in the reference spectrum is also present in the sample spectrum and a smaller value otherwise. The function will also have a high value if a large proportion of peaks in the reference are found in the sample shifted by some integral value of x . As defined here, the correlation function takes no account of peak intensities. The reference that we have chosen consists of 15 peaks taken from the spectrum of tetrahydrocarbazole, ranging from m/z 27 to m/z 143. The sample spectrum for yohimbine consisted of 60 peaks ranging from m/z 15 to m/z 368, that of reserpine contained 112 peaks from m/z 12 to m/z 608, and for quinidine 52 peaks ranging in mass from m/z

27 to m/z 326 were used. The only criterion used in selecting sample peaks was that they should be detectable reliably above baseline noise. Peaks in the sample spectra that corresponded to m/z values in excess of 242 made no contribution to calculations. The cross correlation functions for these spectra taken against tetrahydrocarbazole as a reference are shown in Fig. 8. It can be seen that C,,(O) is equal to unity for the yohimbine and reset-pine spectra indicating that all 15 peaks in the reference are found in these samples. For quinidine, the correlation is poor and only six of the reference peaks are found in the sample spectrum. This observation does not add support to the

231

cRS(x)

cRS(x)

I 0

Y

b)

5b

X4

Fig. 8. Correlation functions taken with tetrahydrocarbazole as reference for (a) yohimbine, (b) reserpine and (c) quinidine.

232

suggestion [l l] that the quinolinium structure is formed in the fragmentation of indoles. It is also seen that all reference peaks appear in the spectrum of yohimbine shifted 13 mass units higher and similar shifts of 13,27 and 41 mass units are found in the spectrum of reserpine. To some extent, this latter correspondence is a consequence of the larger number of peaks in the reserpine spectrum. However, these observations do indicate that the spectra of yohimbine and reserpine are more similar to the spectrum of tetrahydrocar- bazole than is the spectrum of quinidine and this reflects similarities in the structures of the former molecules.

CONCLUSIONS

In spite of the apparent complexity of the equipment, obtaining a mass spectrum with this technique can be a very quick and simple task provided that the ionizing laser is tuned to emit in the appropriate wavelength range. Typically, mass spectra were generated by averaging 20 laser shots which could be done in a few seconds. The preparation and loading of the sample into the equipment could be accomplished in about 1Omin. The selective nature of the ionization process effectively eliminates interference from background contamination. Only when the equipment had been used for several weeks with closely related compounds did signs of background spectra appear. In some experiments, we deliberately contaminated samples of indole acetic acid with an equal mass of nicotinic acid and also with an equal mass of a long alkyl chain surfactant (hexadecyltrimethylammonium bromide). For both samples, we observed only the spectrum of indole acetic acid. Peaks arising from ionization of the contaminant or from ion attachment to neutral contaminant molecules were not found. This is an important feature of the technique as many of the fragment ions that might have a diagnostic value are of low intensity and it is necessary to be able to distinguish them from background.

The relatively high intensity of molecular ion peaks is a characteristic of the method that has been noted previously [6,14] and it was evident in our work. However, some useful fragment peaks were often of quite low intensity and a degree of signal averaging was required to detect them reliably. The probable reason for this is that these peaks (e.g. m/z 130 and 143) arise from ions that are themselves absorbing and efficiently fragmented at the wavelength used.

Varying the energy of the ionizing laser pulse had the predictable effect of changing the extent of fragmentation. At high pulse energies, the relative height of a molecular ion peak declined and fragment peaks became more intense. However, even at energies that gave rise to extensive fragmentation, molecular ion peaks remained conspicuous features of the spectra. Low

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energy spectra were generally better resolved, probably because the effects of ion-ion repulsion were less significant under these conditions. Whilst being able to vary the degree of fragmentation easily does have advantages, at present the technique suffers from the absence of a satisfactory way of standardizing the ionization conditions which would allow relative peak intensities to be specified quantitatively.

ACKNOWLEDGEMENTS

We thank the Science and Engineering Research Council and the University of Salford Research Committee for the provision of facilities used in this work.

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