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FAB-MS MRM methods (7, 15, 34, 35).
ACKNOWLEDGMENT The authors acknowledge gratefully the support of Drs. Francisco, Haruff, and Berryman (Department of Pathology) for pituitary tissue, Genevieve Fridland for editing and artwork assistance, Deanna Reid and Dianne Cubbins for typing assistance, and Dianne Cubbins for data analysis. Registry No. ME, 58569-55-4.
LITERATURE CITED (1) Krieger, D. T., Brownstein, M. J.. Martin, J. 8. Eds. Brain Peptides; Wiley: New York. 1983; 1032 pp, Faull, K.: Tatemoto, K.; Evans, C. J.; (2) Roth, K. A.; Makk. G.: Beck. 0.; Barchas, J. D. Regul. f e p t . 1985, 72, 185-199. (3) Roth. K. A.: Lorenz, R. G.;McKeel, D. W.; Leykam, J.; Barchas. J. D.; Tyler, A. N. J . Clln. Endocrinol. Metabol. 1988, 6 6 , 804-810. (4) Eipper, B. A.: Mains, R. E. J. Biol. Chem. 1982, 257, 4907-4915. (5) Higa. T.; Wood, G.;Desiderio. D. M. Int. J. f e p t . Protein Res. 1989, 3 3 , 446-451. (6) Orth, D. M.; Gullemin, R.; Ling, N.; Nicholson, W. E. J . Clin. fndocrinol. Metab. 1978. 46, 849-852. (7) Desiderio, D. M. Analysis of neuropeptides by liquid chromatography and mass spectrometry; Elsevier: Amsterdam, 1984; 235 pp. (8) Voyksner, R. D.; Pack, T. W. Biomed. Environ. Mass SDectrom. 1989, 18, 897-903. (9) Bennett, H. P. J. J. Chromatogr. 1986, 359, 383-390. (10) Wiedamann, K.: Teschemacher, H. fharm. Res. 1986, 3 , 142-149. (11) Higa, T.; Desiderio, D. M. Int. J. Pept. Protein Res. 1989, 3 3 , 250-255. (12) Simon. E. J. Future Directions in Opioid PeptMes: Molecubr fharmacology. Biosynthesis, and Analysis; Rapaka, R. S., Hawks, R. L., Eds.; NIDA: Rockville, MD, 1986; pp. 155-174. (13) Desiderio, D. M.: Fridland, G. H.; Francisco, J. T.; Sacks, H.; Robertson, J. T.; Cezayirli, R. C.; Killmar, J.; Lahren, C. Clin. Chem. 1988, 3 4 , 1104-1107. (14) Boarder, M. R.; Weber, E.; Evans, C. J.; Erdelyi, E.; Barchas, J. J. Neurochem 1983, 40. 1517-1522.
(15) Desiderio, D. M. Mass spectrometry of biologically important neurcpeptides I n Mass Spectrometry of PeptMes: DesMerlo, D. M., Ed.; CRC Press: Boca Raton, FL., 1990. (16) Mifune, M.; Krehbiel, D. K.; Stobaugh, J. F.; Riley, C. M. J. Chromat o g . 1989, 496, 55-70, (17) Tanzer, F. S.;Tolun. E.;Fridland, G. H.; Dass, C.; Killmar, J.; Tinsley, P. W.; Desiderio, D. M. Int. J. fept. Protein Res. 1988,32, 117-122. (18) Dass, C.; Fridland, G. H.; Tinsley, P. W.; Killmar, J. T.; Desiderio, D. M. Int. J . Pept. Protein Res. 1989, 34, 81-87. (19) Liu, D.; Desiderio, D. M.; Wood, G.; Dass, C. J. Chromatogr. 1989, 500, 395-412. (20) Fridbnd. G. H.; Desiderio, D. M. J. Chromatogr. 1986, 379, 251-268. (21) Desiderio, D. M.; Cunningham, M. D. J. Liq. Chromatogr. 1981, 4 , 721-733. (22) Desiderio, D. M.; Laughter, J. S.; Katakuse, I.; Kai, M.; Trimble, J. Comput. Enhanced Spectrosc., 1984, 2 , 21. (23) Itoh, M.; Hagiwara, D.; Kamiya, T. Tetrahedron Lett. 1975, 49, 4393. (24) Garner, G. V.; Gordon, D. B.; Tetler. L. W. Org. Mass Spectrom. 1983, 18, 486-488. (25) Dass, C.;Desiderio. D. M. Int. J. Mass SDectrom. Ion Processes 1989, 9 2 , 267-287. Tinsley, P. W.; Fridland, G. H.; Killmar, J. T.; Desiderio, D. M. Peptides 1989 .- - -, 9 - , 1373-1378 .- . - .- . - .
Liu, D.; Desiderio, D. M. J. Chromatogr. 1987, 422, 61-71. Roepstorff, P.; Fohlmann, J. Biomed. Mass Spectrom. 1984, 1 7 , 601. Feistner, G. J.; Hojrup, P.; Evans, C. J.; Barofsky, D. F.; Faull, K. F.; Roepstorff, P. Proc. Natl. Acad. Sci. U . S . A . 1989, 8 6 , 6013-6017. Kim, C.; Cheng, R . J. Chromatogr. 1989, 494, 67-76. Tolun, E.; Dass. C.; Desiderio, D. M. Rapid Commun. Mass Spectrom. 1987, 1 , 77-79. Desiderio, D. M.; Kai, M. Biomed. Mass Spectrom. 1983, 10, 471-479. Tomer, K. B. Mass Spectrom. Rev. 1989, 8 , 445-482. Desiderio, D. M.; Fridland, G. H. In Mass Spectrometry in Biomedical Research: Gaskell, S . , Ed.; Wiley: New York, 1986; pp 443-448. Lovelace. J. L.; Kusmierz, J. J.: Desiderio, D. M. J. Chromatogr. Biomed . Appi ., in press.
RECEIVEDfor review July 12,1990. Accepted August 2, 1990. This research was supported by NIH (GM 26666).
Laser Ionization Gas ChromatographylMass Spectrometry of TetraethyItin Steven M. Colby, Michael Stewart, and James P. Reilly* Department of Chemistry, Indiana University, Bloomington, Indiana 47405
A new method for detecting tetraethyltin that combines the high efflclency of laser Ionization, the selectlvlty of capillary column gas chromatography, and the large throughput of a tlme-of-flight mass spectrometer Is demonstrated. Detection limits of 2.5 pg of tetraethyHln/mL or 1.5 fg of tetraethyltin absolute have been obtalned. These values correspond to 1.3 pg of lln/mL or 750 ag of tin absolute. This method should be generalizable to other elements.
INTRODUCTION The use of organotin compounds has increased dramatically in the last 30 years (1-3). They find applications in catalysts, biocides, antifouling paints, and as stabilizers for poly(viny1 chloride). Their roles in biocides and paints have had significant environmental impact, particularly in estuaries near harbors ( I , 2, 4-10). Approximately 10% of the organotin compounds used as biocides and in antifouling paints enter the aquatic environment (11). Recent legislation, limiting the use of organotins in paint, is not expected to reduce their environmental impact in the near term because of the large number of vessels to which they have already been applied.
Paint chips scraped from these vessels are expected to be a continuing source of contamination (2). Once organotins have entered the environment they can have long term chronic effects on marine life (2, 10). The toxicity of organotin compounds is roughly dependent on the number of alkyl groups, with the greatest number, 3 and 4, being the most toxic (2, 9,11). The magnitude of the toxicity with regard to individual species and the probability that a compound will be incorporated in the food chain are determined by the identity of the alkyl groups (9,12). Among the most visible effects is shell deformation of the oyster Crassostrea virginica and development of male sexual organs in the female dogwhelk Nucella lapillus (1, 2). Growth retardation of some bivalves occurs with organotin concentrations as low as 100 pg/mL (5). In the environment, organotins are partitioned into several different media (sediment, biota, and water layers) (8, 13). Each of these must be analyzed to properly model the bioaccumulation processes and fully understand the lifetime of the environmental impact (9). The analysis of organotin compounds at the concentrations found within these media requires techniques that can measure concentrations as low as 1 pg/mL in water and less than 1 ng/g in sediments (9,14). New, very sensitive methods that can accomplish this need to be developed.
0003-2700/90/0362-2400$02.50/00 1990 American Chemical Society
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A number of techniques have been applied for detecting tin in sediment and solution (3,12,14-26). All involve a series of extraction and/or derivatization steps before the tin compounds are analyzed. In general, derivatizations involve alkylation or hydride generation and extraction is performed by using nonpolar solvents. The requirements of the extraction and detection methods determine the choice of final organotin derivative. In these experiments we have chosen tetraethyltin as our analyte because it is compatible with gas chromatography. Extraction techniques can be over 90% efficient (8,14,23, 25). Regardless of the preparation steps the sensitivity of the sample analysis depends critically on the final detection scheme. Ion spray MS/MS (3,19), gas chromatography with flame photometric detection (14,16,17,21, 23,26,27), and liquid chromatography with either inductively coupled plasma mass spectrometry (ZO),or laser enhanced ionization (15),have all been employed. Laser ionization gas chromatography/time-of-flight mass spectrometry is an analytical method well suited to the detection of organotin compounds. It has previously been shown that resonant multiphoton ionization can be a highly efficient and selective process. Atoms (28,29)can be ionized somewhat more efficiently than molecules (30) because atomic cross sections are comparatively large and atoms cannot undergo rapid nonradiative excited-state relaxation (31). With sufficient intensity the laser light can function as both the atomizer and ionization source as has been demonstrated a number of times. For example, ArF laser light yields copious quantities of C+ ions when irradiating benzene (32,33). This is possible because the high-energy photons efficiently fragment the molecule into single atoms and the (3s 'P 2p2 'D) transition of the carbon atom is in resonance with the 193-nm light. Similar results have been obtained for organotin compounds by using laser-enhanced ionization (15,29,34). In this case, however, a flame is used as the atomizer and the laser light is only used for exciting the atoms. I t is well-known that organometallic compounds irradiated by high intensity laser pulses fragment to form metal atoms and free radicals (35-39). Therefore any analytical method for detecting these species by laser ionization should be based on observing atomic ions. Detecting atomic rather than molecular ions has two distinct advantages. First, organometallic compounds containing different metallic elements are readily distinguished. Second, sensitivity is improved because the atomic signal is fixed within a limited mass range. In contrast, molecular ions can fragment leading to the distribution of signal over a number of masses. This increases the possibility of detecting background ions and therefore decreases sensitivity. The combination of these factors, along with the high throughput of TOFMS, results in a detection method for organotins that is more sensitive than those previously reported.
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EXPERIMENTAL SECTION Reagents. Tetraethyltin was purchased from Aldrich Chemical Co. Samples were prepared by dilution in Spectranalyzed methylene chloride from Fisher Scientific. Final concentrations were 10 ng/mL and 100 pg/mL of tetraethyltin. Gas chromatography/flame ionization delection (GC/FID) analysis of the sample determined the chromatographic retention time of tetraethyltin and established that the sample contained only one solute detectable with the FID. Blanks using the same solvent, glassware, and syringe were tested to verify that there was no contamination. No attempts were made to account for loss of tetraethyltin due to irreversible adsorption onto glassware or the syringe. All sample injections contained 0.6 pL of the above solutions. Instrumentation. Chromatography was performed on a Varian 3700 gas chromatograph with on-column injection and a 30 m long, 250 pm i.d. fused silica capillary column (Supelco SPB-1). Nitrogen carrier gas was used at a flow rate of =0.6 mL/min. The chromatographic temperature program was started
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Table I. Relevant Tin Atom Transitions transition
-
(8d 3D20 5pz 3Pz) (8d 3F30 5p2 'D2) 6
(8d 'Pio
6
5p2 'Dz)
wavelength, nm 193.412 192.912 193.110
at 40 "C. This was followed by an increase, at a rate of 10 "C/min, to 200 "C. For gas chromatography/mass spectrometry (GC/MS) experiments, a 250 "C interface connected the gas chromatograph and time of flight mass spectrometer. The end of the capillary column was positioned between the first two accelerating grids of a linear time-of-flight mass spectrometer that has been described earlier (30). The mass spectrometer was evacuated through a liquid nitrogen trap by a Varian VHS-6 diffusion pump. The Torr. base pressure, with the carrier gas flowing, was 5.0 X In the mass Spectrometer, ions were accelerated to an energy of 2000 eV. They passed through four 90% transmitting grids (Buckbee Meers) before being detected by a pair of tandem microchannel plates (Galileo). The detector signal was amplified (LeCroy WlOlATB) and then digitized by a high-speed waveform recorder (Biomation 6500). The data were transferred to a microcomputer where software was used to record mass spectra and integrate the ion signal over the isotopic mass envelope of tin. The chromatogram was generated by recording the integrated signal once per second. The light source used for these experiments was a Lumonics TE-861 excimer laser operating at the ArF wavelength of 193 nm with a pulse duration of about 8 ns. The spectral range of this laser is approximately 193.1 to 193.6 nm (41). The profile of the laser light overlaps a number of tin atom transitions (42,43). The closest one to this wavelength range, originating from the ground state, is (6d 3D,0 5p2 3P0) at 194.269 nm. Three others that originate from low lying metastable states are in resonance with the ArF laser. They are listed in Table I. Note that transitions from the 5p2 lDZstate reach autoionizing levels. Atoms in this state can actually be one-photon ionized. Because of the large amount of energy involved in the atomization process, it is likely the 5p2 3P2and 5p2 'D2excited states will be highly populated. The laser beam was apertured and then focused into the source of the mass spectrometer using either a 1000 or 500 mm focal length lens. The rectangular focal spots produced by these lenses were 4.5 X 0.6 mm and 2.5 X 0.4 mm (full width at half maximum), respectively. They were measured by passing a sharp edged blade through the focal plane and monitoring the transmitted light intensity. Their size affects both the mass resolution and the number of sample molecules ionized. The lens was positioned so that the narrow edge of the focal spot intercepted the chromatographic effluent approximately 1.5 mm from the tip of the chromatographic column. The laser energy, as measured with a Gentec ED-500 joulemeter at the exit of the mass spectrometer, was between 1.0 and 6.2 mJ. The pulse repetition rate was 20 Hz.
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RESULTS Figure 1 shows a chromatogram resulting from the injection of 6 pg of tetraethyltin in a 0.6 FL sample. The integrated area of the peak is 26985 relative units; the mean and standard deviation of the background integrated over the same amount of time are 6.2 and 3.8 in the same units, respectively. These data were recorded with a 500-mm lens and an average laser pulse energy of 1.0 mJ. From these results we project a detection limit a t 30 of 3.9 fg of tetraethyltin. Because this was a long and potentially unreliably extrapolation, additional data were recorded for much lower sample concentrations. Chromatograms obtained by injecting 60 fg of tetraethyltin in 0.6 MLof solvent are shown in Figure 2. Figure 2A displays the data obtained by using a 500-mm lens and an average pulse energy of 4.1 mJ. Figure 2B displays that obtained by using a 1000-mm lens and an average pulse energy of 6.2 mJ. A comparison of the results is shown in Table 11. Detection limits of 6.8 and 1.5 fg of tetraethyltin can be projected from parts A and B of Figure 2. This is in reasonable agreement
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ANALYTICAL CHEMISTRY, VOL. 62, NO. 21, NOVEMBER 1, 1990
51 3 C
& I
.-
wi 'Y' I
I ^
c I/)
t
I'
I
1 , 436
I
,
,
'
"
1
476 516 556 596 Retention t i m e ( s e c o n d s )
93
636
Figure 1. Chromatogram resulting from the injection of 6 pg of tetraethyltin.
99 105 1 1 1 "17 " 2 3 129 '35 Mass (CWJ)
Figure 3. ArF laser ionization mass spectrum obtained during the elution of a tetraethyltin peak.
Table 111. Detection Limit Comparison
method LC/laser enhanced ionization (15) GC/flame photometric detection (16) GC/flame photometric detection (17) GC/flame photometric detection (14) GC/flame photometric detection (27) GC/flame photometric detection (26) LC/graphite furnace AAS (12) direct current plasma (18) ionspray tandem mass spectrometry (3, 19) GC/flame ionization-quenching (40) ion pair chromatography/ICP-MS (20) ICP-AAS (18) quartz furnace AAS (22)
-P
Q
x 4 6 0
460
490
490
520
520
550
550
580
580
610
610
current work: GC/laser ionization/time-of-flight MS
Retention t i m e (seconds)
detection sensitivitp concn, absolute, ng/mL Pg 3b 16c
60 8OC
30d 3e 0.04r
