Anal. Chem. 1988, 60, 720-722
720
CORRESPONDENCE Electrochemistry/Thermospray/Tandem Mass Spectrometry in the Study of Biooxidation of Purines Sir: Redox reactions play an important role in the bioactivation and biodegradation of drugs and other xenobiotics. For example, purine drugs such as the successful antineoplastic agent 6-thiopurine are oxidatively degraded by enzymes of purine metabolism to inactive metabolites ( I , 2 ) . Electrochemical methods in combination with HPLC, molecular spectroscopy, and mass spectrometry are valuable in studies of redox and related chemical reactivity of biologically active compounds such as purine drugs and can provide insight into biodegradation and/or bioactivation reactions (3-10). Time resolution of fast electrochemical measurements allows detection of reaction intermediates as well as products. However, for structural confirmation independent methods are necessary. When mass spectrometry is used off-line, only electrochemically generated compounds of sufficient stability can be analyzed (9, 10). In this study the redox reactivity of two purines, uric acid and 6-thioxanthine, was investigated by on-line electrochemistry/ thermospray/ tandem mass spectrometry (EC/TSP/MS/MS), which allowed direct identification of the intermediates and products formed in the oxidation reactions of these compounds. The enzymatic and electrochemical oxidation pathways of both compounds have been previously characterized by using derivatization and off-line GC/MS analysis (3-5, 9, IO). The results reported here were obtained with an ESA coulometric electrochemical cell which was coupled via a Vestec thermospray interface to a Finnigan Mat TSQ45 tandem quadrupole mass spectrometer equipped with an INCOS data system. The coulometric cell had a cell volume of 5 p L , 12-cm2reticulated vitreous carbon working electrode, and palladium counter and reference electrodes. The large ratio of the working electrode area to cell volume provides for a high electrochemical conversion efficiency (estimated ca. 80%). The thermospray probe tip and source block temperatures were 250 and 290 "C, respectively. The conditions for TSP/MS were scan range mlz 125-300 in 0.3 s, electron multiplier voltage 1000 V, and preamplifier gain lo8 V A-'. This scan range limit was set to avoid background interference from the m / z 119 (CH3COOHCH3C00)-ion. For MS/MS, the scan range was m / z 15-300 in 0.3 s, electron multiplier voltage 1600 V, preamplifier gain los V A-l, collision energy 25 eV, and N2 collision gas pressure 1.8 mTorr. Typically, 20 scans were averaged for both the MS and MS/MS scan modes. The mobile phase was 0.1 M ammonium acetate (pH - 7 ) a t a flow rate of 2.0 mL/min. Twenty-five microliters of a 300 pM solution was injected. The potential of on-line electrochemistry/thermospray/ mass spectrometry (EC/TSP/MS) in the study of electrode processes has recently been demonstrated by Heitbaum et al. (11). In these experiments the electrooxidation of N,N-dimethylaniline at a Pt electrode produced dimers and trimers with subsequent analysis by mass spectrometry. The results reported here demonstrate that on-line electrochemistry/ thermospray/tandem mass spectrometry can provide valuable information about biologically relevant redox reactivity of drugs and other xenobiotics. In our studies the use of TSP/MS/MS provided structurally informative daughter
spectra of standards for comparison to the EC/TSP/MS/MS results. This allowed identification of intermediates and products based on their characteristic daughter spectra, a feature important to the identification of structurally related metabolites (12, 13). Table I summarizes the results that were obtained when the oxidation of uric acid was investigated by EC/TSP/ MS/MS and Figure 1 summarizes the electrochemical oxidation pathway, intermediates, and products identified by EC/TSP/MS/MS in this study. At pH 7.0 uric acid is known (3-5) to be oxidized electrochemically at ca. +0.4 V vs SCE with the formation of the final products allantoin, 5hydroxyhydantoin-5-carboxamide,and, at pH e7.0, alloxan monohydrate; an imine alcohol with a half-life of ca. 3 min is an intermediate in the reaction (9, 10). As can be seen from Table I and Figure 1,all three products and the imine alcohol the intermediate previously identified (9,10) are observed after uric acid oxidation at potentials >+0.2 V. Both positive and negative thermospray mass spectra of uric acid did not contain any ions which were not present in the background thermospray spectra (12). However, a t pH -4 uric acid did yield a weak [M - HI- ion, whose daughter spectrum included m/z 167 (100%) and m / z 124 (60%). Similar purine compounds (e.g., 6-thioxanthine, also reported here) typically provide intense negative ion thermospray mass spectra (mainly [M - HI-) as well as ca. 10 times less intense positive ion spectra (mainly [M + HI+). Alloxan monohydrate was the only compound that did not yield an [M H]+ or an [M - HI- ion; rather, it produces a [M - 44 + CH,COO]- ion, an acetate adduct of a decarboxylation product. As can be seen from Figure 1 and Table I, the ions 139- and 141' indicate the formation of the decarboxylation product (MW 140) of the imine alcohol, which hydrolyzes to produce allantoin. The ion a t m / z 134 presumably corresponds to the [M NH,]' adduct of 5-hydroxyhydantoin, the decomposition product of 5-hydroxyhydantoin-5-carboxamide; the corresponding [M H]+ and [M - HI- ions were not detected since they would be present below the m / z 125, the lower limit of the scan range. The 142- ion corresponds to a compound whose structure has not yet been determined. The assignments in Table I were established based on the MS/MS daughter spectra of available standards. The agreement between daughter ion abundances of standards and electrochemically generated products was generally f20% relative abundance. Table I also summarizes results that were obtained in the EC/TSP/MS/MS of 6-thioxanthine. This thiopurine is known to be oxidized only a t the thio group at potentials >+0.2 and +0.7 V is accompanied by the disappearance of the ions characteristic of 6-thioxanthine. The structure of the compound responsible for the negative ion at mlz 142 is a t present uncertain. As demonstrated here, the on-line combination of electrochemistry with thermospray/tandem mass spectrometry shows
Department of Chemistry University of Florida Gainesville, Florida 32611
RECEIVED for review July 17,1987. Accepted November 24, 1987. We acknowledge ESA, Inc., for the loan of the electrochemical cell. This work was supported, in part, by grants from the U.S. Army Chemical Research, Development, and Engineering Center (R.A.Y., No. DAAA15-85-C-0034),Research Corporation (A.B.T.), NIH (A.B.T., through Grant GM 35341-01A2),and the Division of Sponsored Research at the University of Florida (A.B.T.).
Indirect Fluorometric Detection of Anions in Thin-Layer Chromatography Sir: Thin-layer chromatography (TLC) is a broadly applicable separation technique. With the recent growth of interest and products for high-performance liquid chromatography (HPLC), TLC has not received the attention it deserves. A major factor is that the approach is still not highly “instrumental” compared to HPLC, both in the separation step and the detection step, even though much progress has been made ( 1 ) . TLC provides many advantages as an analytical technique. It is easily adapted for two-dimensional separation (Z),for “whole-column” detection ( 3 ) , and for handling multiple samples. As a complementary technique to HPLC, one can use TLC as an initial screening step to
optimize conditions. This is because of the ease of equilibration with a new mobile phase, the speed of development, the freedom from contamination and carry over, the applicability to highly retained solutes, and the low cost of stationary and mobile phases used. Detection in TLC lags behind the corresponding technology in HPLC. The two-dimensional geometry of the spots limits the path length available for absorption photometry, although nanogram levels are readily detectable. The sensitivity problem can be partially solved by using photothermal spectroscopy ( 4 ) or photoacoustic spectroscopy ( 5 ) . Nonlinearity still exists because of the scattering nature of the
0003-2700/88/0360-0722$01.50/00 1988 American Chemical Society
