International Journal of Mass Spectrometry and Ion Processes, 123 (1993) 125-131 Elsevier Science Publishers B.V., Amsterdam

125

The laser desorption/laser ionization mass spectra of some methylated xanthines and the laser desorption of caffeine and theophylline from thin layer chromatography plates

Kevin Rogers, John Milnes and John Gormally

The Department of Chemistry and Applied Chemistry, The University of Salford, Sarford M5 4WT (UK)

(First received 22 July 1992; in final form 11 September 1992)

Abstract

Laser desorption/laser ionization time-of-flight mass spectra of caffeine, theophylline, theobromine and xanthine are reported. These mass spectra are compared with published spectra obtained using electron impact ionization. Mass spectra of caffeine and theophylline obtained by IR laser desorption from thin layer chromatography plates are also described. The laser desorption of materials from thin layer chromatography plates is discussed.

Keywords: laser desorption; laser ionization; thin layer chromatography; caf&ine; time of flight.

Introduction

Caffeine (1,3,7_trimethylxanthine, (I)) is a trimethylated xanthine derivative that is contained in beverages such as coffee and tea. The metabolism of this compound in humans and in animal species has been the subject of detailed studies [1,2]. The related dimethylated compound, theophylline (1,3- dimethylxanthine (II)), is used as a drug in the treatment of some cardio-pulmonary diseases. Both theophylline and theobromine (3,7-dimethyl- xanthine (III)) have been identified as metabolites of caffeine in humans and animals [l].

Analytical procedures used in the study of samples containing these compounds have fre-

Correspondence to: J. Gormally, The Department of Chemis- try and Applied Chemistry, The University of Salford, Salford MS 4WT, UK.

quently made use of mass spectrometry combined with a pre-separation technique such as liquid

B, B, B,

I CH, CH, CH,

II CH3 CH, H

III H CH, CH,

R2 IV H H H

chromatography [ 11, thin layer chromatography (TLC) [3,4] or gas chromatography [5,6]. The polar nature of underivatized dimethylxanthines renders them unsuitable for direct analysis using gas chro- matography-mass spectrometry (GC-MS) [6] but GC-MS is routinely performed after derivatiz- ation, usually alkylation. Theophylline, theobro- mine and paraxanthine (1,7_dimethylxanthine) have been separated by TLC [5].

In this study, we have recorded the laser desorp- tion/laser ionization mass spectra of caffeine,

‘0168-l 176/93/$06.00 0 1993 Elsevier Science Publishers B.V. All rights reserved

126 K. Rogers et aLlInt. J. Mass Spectrom. Ion Processes 123 (1993) 125-131

theophylline, theobromine and xanthine (IV) using an ionization wavelength of 266nm. The purpose of this was to ensure that these compounds can be ionized easily at this wavelength and to compare the spectra obtained with those resulting from electron impact (EI) ionization. We have also obtained mass spectra of caffeine and theophylline by direct laser desorption from a TLC plate. Laser desorption mass spectrometry from TLC media has been demonstrated previously [7-91 but published reports are few and it is a technique that has yet to be fully explored. TLC is a simple and very useful separation procedure for many compounds that, for reasons of low volatility, are not directly amenable to such techniques as gas chromato- graphy. The combination of TLC with laser de- sorption shows promise of becoming a useful tool in the analysis of compounds in this class.

Experimental

The laser desorption/laser ionization time-of- flight mass spectrometer than was used in this study has been described previously [lo]. Desorbed neutral molecules were entrained in a pulsed (150 ,us) supersonic jet of argon or carbon dioxide and transferred to the ionization region of the mass spectrometer where ionization was effected by the output of a second pulsed laser. Laser desorption was accomplished using a pulsed carbon dioxide laser (Pulse Systems LP-30). Various pulse energies were employed ranging from 20 to 100 mJ and the beam was focussed to a spot of 1.3 mm diameter giving mean energy densities at the sample in the range 1.5-7.5 J crnm2. The positioning of the sample in relation to the supersonic jet is indicated in Fig. 1. Ionization was accomplished using the fourth harmonic (266nm) output of a Nd : YAG laser (Quanta Ray DCR-3). Pulse energies were in the range l-2 mJ in pulses of about 10 ns duration generated at a repetition rate of 5 Hz. The ionizing beam was focussed into the mass spectrometer using a cylindrical lens and power densities in the ionization region were estimated to be in the range 1.5 x lo6 to 3 x lo6 W cmp2. Ions were detected by

a channel plate electron multiplier the output of which was fed directly into a LeCroy 9450 digitiz- ing oscilloscope (2.5 ns between points) without pre-amplification.

Caffeine was obtained from Hopkin and Williams, theophylline and xanthine were obtained from Aldrich Chemical Co and theobromine was obtained from BDH. The four compounds were used as supplied. Samples were prepared for laser desorption by making a slurry of each compound in methanol and coating onto 1 mm diameter glass rods. Sample was desorbed from the curved surface of the rod.

The TLC plates used were supplied by Camlab. These plates had a plastic backing and a 0.25mm thick coating of silica gel containing a fluorescent indicator. Plastic-backed plates were chosen instead of the glass-backed variety because they are more easily cut up into narrow strips which fa- cilitated their mounting in the desorption housing of the mass spectrometer.

Samples of caffeine and theophylline to be used for development on TLC plates were dissolved in a 50 : 50 chloroform/methanol solvent mixture at a concentration of 10mgmll’. One ~1 of each solution was deposited on the TLC plate using a microlitre syringe and developed with a mobile phase made up by mixing methanol and ammonia solution in proportions of 99 : 1. The sample spot was allowed to run 5-6cm up the plate before removal from the development vessel. After drying, a strip of dimensions 50 mm x 3 mm containing the sample spot was cut from the plate perpendicular to the direction of development and cemented to a glass rod as shown in Fig. 1. The amount of sample in the area of the TLC plate irradiated during desorption was estimated to be about lpg.

Results

Laser desorption/laser ionization mass spectra of caffeine, theophylline, theobromine and xanthine are shown in Fig. 2. These mass spectra are averages of five shots for caffeine and theophyl- line and 20 shots for theobromine and xanthine.

K. Rogers et al./Int. J. Mass Spectrom. Ion Processes 123 (1993) 125-131 127

Deaorption Beam f------- pulsed Valve

TLC Plate

I I I I I \

l-5 Skimmer

$ -

I--

Region

Fig. 1. The positioning of the sample in relation to the pulsed valve, skimmer and ionization region of the mass spectrometer. To obtain mass spectra from compounds on TLC plates, strips of the developed plate 3 mm in width were cemented to the glass sample rod and

material was desorbed into the supersonic jet issuing from the pulsed valve.

The peak heights obtained for the latter two compounds were somewhat lower than those obtained for caffeine and theophylline and the greater number of shots was required to give mass spectra showing similar signal-to-noise ratios for the four compounds. Desorption and ionization pulse energies were the same for the four compounds (100 and 2 mJ respectively). Peak heights for the molecular ion were 378mV for caffeine, 400 mV for theophylline, 116 mV for theo- bromine and 72mV for xanthine. The spectra of theobromine and xanthine show evidence of cluster formation, a feature that is common to all four of

these compounds. The mass spectrum of theobro- mine was obtained using carbon dioxide at a backing pressure of 3.5 atmos. as the carrier gas in the pulsed valve. Similar mass spectra were obtained with argon as the carrier gas at high backing pressures. The molecular ion of theobro- mine is seen at m/z 180 but there are also peaks at m/z values of 360,540 and 720 due to the formation of dimers, trimers and quadramers. In addition, there are smaller peaks at m/z values greater than that of the molecular ion arising from clusters formed with carrier gas molecules and with water. The formation of clusters within supersonic jets is

128 K. Rogers et al./Inr. J. Mass Spectrom. Ion Processes I23 (1993) 125-131

I CAFFEINE II THEOPHYLLINE

67 15

III THEOBROMINE I’.’ XANTHINE

P-v . V 540 720

360

67

Ii0

10 Time (pee)

20 30 40

180

109

54 I 152

Time (psec) 10 20 30 40 I I

Fig. 2. The laser desorption/laser ionization mass spectra of caffeine, theophylline, theobromine and xanthine. For each spectrum the ionizing pulse energy was 2 mJ at a wavelength of 266 nm and the energy of the desorption pulse was 100 ~LT. Theobromine and xanthine spectra were recorded using higher vertical sensitivity than was used for caffeine and theophylline. Heights of the molecular ion peaks

are given in the text.

not uncommon but this particular class of compounds appears to form clusters very easily. These effects can be removed completely by using argon as the carrier gas at a backing pressure not exceeding 2atm as was done to obtain the mass spectra of caffeine and theophylline that are shown in Fig. 2.

The laser ionization mass spectrum of caffeine exhibits many of the features found in the EI mass spectrum of this compound. The molecular ion at

differ appreciably and are dependent on the laser pulse power density [lo]. Proposed structures of the

spectra, the relative abundances of these ions do

fragment ions responsible for these peaks have been reported by Houghton [6]. The laser ioniza- tion mass spectrum of theophylline also was found to be similar to that obtained using EI ionization [l 11. The molecular ion peak at m/z 180 and fragment ion peaks at m/z values of 123,95,68 and 53 are found in both mass spectra. These peaks

m/z 194 together with ion peaks at m/z values of appear at m/z values that are 14 u lower than peaks 137, 109, 82, 67 and 55 are common to both types found in the mass spectrum of caffeine. As caffeine of ionization. However, as is usually found in the has a methyl group on the 7-nitrogen and in comparison of laser ionization and EI mass theophylline this position is occupied by hydrogen,

K. Rogers et aLlInt. J. Mass Spectrom. Ion Processes 123 (1993) 125-131 129

this implies that the fragment ions observed derive from the five-membered ring in the xanthine struc- ture in agreement with the scheme proposed by Houghton [6], the first step of which is shown below:

The same scheme can be used to account for the fragment peaks that we observe at m/z values of 137, 109, 82, 67 and 55 in our mass spectrum of theobromine. The fragmentation pattern for xanthine does not fit this scheme as well as the other compounds studied and xanthine was not discussed in ref. 6. The peaks at m/z 109 and 54 can be accounted for but the peak at m/z 136 is 16 u lower than the molecular ion (m/z 152) and suggests loss of oxygen from the molecule. Also, we observe peaks at m/z values of 82 and 40 whereas these would be predicted to occur at m/z 81 and 41 respectively. However, the correspondence between the laser ionization fragmentation behavior and that observed with EI is generally very close and the mass spectra obtained can be rationalized in terms of a fragmentation scheme proposed for the latter form of ionization.

Laser desorption/laser ionization mass spectra of caffeine and theophylline desorbed directly from TLC plates are shown in Fig. 3. We are not aware of a suitable solvent system for TLC of xanthine, which is insoluble in all solvents that we have tried, and theobromine requires a more complex solvent system [5] than that which we used. In the mass spectra of caffeine and theophylline, the molecular ion peak is clearly seen. These spectra were obtained using an ionizing laser pulse energy of 2 mJ for caffeine, i.e. the same as was used to record the mass spectra in Fig. 2, and 1 mJ in the case of theophylline. The molecular ion peak of caffeine desorbed from the TLC plate has an amplitude of

I CAFFEINE

194

II THEOPHYLLINE

Is0

Time (pa) 10 20 30 40

Fig. 3. Mass spectra of caffeine and theophylline obtained by IR laser desorption from a TLC plate followed by multiphoton ionization at 266~1. The desorption pulse energy was 3OmJ (2.3 Jcm-‘) in both cases. Ionization pulse energies were 2mJ for caffeine and 1 mJ for theophylline.

75 mV and is five times smaller than the molecular ion peak in the mass spectrum of caffeine shown in Fig. 2. In addition, there is much less evidence of fragmentation in the spectra obtained from TLC plates. These results indicate that the degree of fragmentation depends not only upon the power density of the ionizing pulse but also upon the conditions of desorption. The spectra shown in Fig. 3 were obtained using a single laser shot. The back- ground that can be seen arises from ionization and fragmentation of contaminants in the ion source rather than electronic noise. This background could still be seen when the desorbing beam was blocked showing that it did not arise from the presence of the fluorescent indicator on the TLC plate or from contamination of the plate. Single

130 K. Rogers et al./Int. J. ikfass Spectrom. Ion Processes I23 (1993) 125-131

shot spectra were taken because it was found that most of the material that is desorbed comes off the TLC plate in the first shot, indicating that desorp- tion is mainly from the surface of the plate. It is likely that the use of plates with a thinner silica layer would increase the surface concentration of analyte and thereby enhance the sensitivity of the technique. Desorption pulse energies of less than 20 mJ (energy density at the sample of 1.5 J crn2) were found to be insufficient to give an observable signal. On raising the desorption pulse energy to 3OmJ, a good signal was obtained with no detect- able damage to the TLC plate. Desorption pulse energies of between 80 and 100mJ did not lead to significantly larger signals but they did produce visible damage to the surface of the plate.

Discussion

Our results show that mass spectra of caffeine, theophylline, theobromine and xanthine can be obtained readily by IR laser desorption followed by multiphoton ionization at a wavelength of 266 nm. The fragmentation behavior observed is very similar to that which has been reported for the EI mass spectra of these compounds. The major features of this behavior can be rationalized in terms of a fragmentation scheme [6] that has been proposed in the literature.

The mass spectra obtained by IR laser desorp- tion from silica coated TLC plates indicate that there is potential for further development of this method. It is of interest to contrast this technique with that reported by Kubis et al. [9] in which a frequency quadrupled Nd:YAG laser was used both to desorb and to ionize material from polya- mide TLC plates in a LAMMA- laser micro- probe mass spectrometer. In this method, laser pulse energies in the range 15-l 8 fl focussed to a spot size of a few micrometers were employed. This arrangement allows probing of the plate surface with high spatial resolution but it also has the result that the amount of analyte desorbed is only a very small fraction of what is contained in the spot on the TLC plate. As TLC spots have dimensions in

the region of a few millimetres, this technique utilizes the amount of sample available very ineffi- ciently. The laser pulse energies quoted correspond to estimated energy densities at the plate of about 150JcmP2. With our arrangement, the larger diameter of the desorption laser focus allows us to desorb a much higher proportion of what is in the spot on the TLC plate and the mean energy densities required are lower by a factor of about 60. In the study by Kubis et al. [9], both positive and negative ion mass spectra were reported. These generally showed the presence of protonated and de- protonated molecular ions respectively. Spectra showing prominent molecular ion peaks were unusual. In contrast, desorption using a carbon dioxide laser and postionization in the UV gives mass spectra containing very prominent molecular ion peaks. The LAMMA approach could have an advantage in that the range of compounds that can be ionized is probably greater than can be achieved in practice using laser postionization but this carries with it the disadvantage of generating background peaks [9] arising from the TLC plate itself. Another feature of the LAMMA method is that combined desorption and ionization of material within the ion source region of the mass spectrometer results in high overall sensitivity compensating to some extent for the small proportion of sample used.

We have chosen to use an ionizing wavelength of

266nm because the frequency quadrupled output of the Nd : YAG laser can be generated at suffi- ciently high power to allow non-resonant, and therefore less selective, ionization. In practice, the ionization pulse power densities that were found to be necessary were not very much higher than those used for resonant two photon ionization. As in- dicated above, there is some advantage in employ- ing a mode of ionization that is less selective when laser ionization is used in combination with a sepa- ration technique such as TLC. This would clearly be advantageous when the absorption spectra of the substances that are separated on the TLC plate are unknown. However, as demonstrated in the work of Li and Lubman [8], selective ionization can be useful when different compounds are incom-

K. Rogers et aLlInt. J. Mass Spectrom. Ion Processes 123 (1993) 125-131 131

pletely separated by TLC. We have estimated that the amount of analyte in the area that was subject- ed to laser desorption was about 1 pg (approxim- ately 5 nmol) but the amount actually desorbed was probably much less than this and the spectra obtained are clearly above the detection threshold. However, the sensitivity of the technique that we have used is far from optimal and could be improved substantially. Our equipment, like that used by others [8,9], has not been designed specific- ally for laser desorption from TLC plates. In par- ticular, we expect significant losses in the transfer of desorbed molecules from the desorption housing to the ion source by the supersonic jet. A reported study on the performance of a laser desorption/ laser ionization mass spectrometer [12] similar to that which we have used indicated that approxim- ately 1% of material entrained in the supersonic jet was transported into the ionization region. We do not have a comparable figure for our equipment but the losses involved are expected to be substan- tial. The transfer of desorbed molecules into the ionization region could be improved significantly be reducing the distance between the sites of de- sorption and ionization. In our equipment, this distance is 143 mm. A second limitation on achiev- able sensitivity is chemical noise. This problem was not evident in the work reported by Li and Lubman [8]. However, the mass spectra used to illustrate their results were of indole derivatives and these compounds can be ionized very efficiently by multi- photon ionization giving a signal that is large relative to the background. As an example, we have found that the mass spectrum of indole acetic acid, obtained under the same conditions as those that we have used to record the mass spectrum of

caffeine, contains a molecular ion peak that is at least five times as large as that found with caffeine. The sensitivity of the technique is very dependent upon the sample used. However, there is consider- able scope for increasing the signal relative to back- ground by an appropriate modification to in- strumental parameters as discussed above with the prospect of significantly enhancing the sensitivity attainable.

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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