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Anal. Chem. 1992, 6 4 , 238-239
(13) Rawdon, M. G. Anal. Chem. 1984, 56, 831-832. (14) Stiller, S.W.; Johnston. M. V. Anal. Chem. 1987, 59,567-572; Appi. Spectrosc. 1987, 4 1 , 1351-1357. (15) Keller. R. A.; Nogar, N. S. Appl. Opt. 1984, 2 3 , 2146-2151. (16) Anderson, B. D.; Johnston, M. V. Appl. Spectrosc. 1987, 4 1 , 1358-1 361, (17) Rainie, D. E.; Markides, K. E.; Lee, M. L.; Goates, S. R. Anal. Chem. 1989, 61,1178-1181. (18) Guthrie, E. J.; Schwartz, H. E. J . Chromtogr. Sci. 1986, 2 4 , 236-241. (19) Goates, S. R. Ph.D. Thesis, The University of Michigan, 1981. (20) Goates, S. R.; Linford M. R. Unpublished results, Brigham Young University.
(21) Imasaka, T.; Fukuoka, H.; Hayahi, T.; Ishlbashi. N. Anal. Chim. Acta 1984. 156. 111-120. (22) Bouzou, C.;Jouvet, J. B.; Leblond, J. 9.; Millie, Ph.; Tramer, A. Chem. Phys. Lett. 1983, 97, 161-166. (23) Amirav, A.; Even, U.; Jortner, J. Chem. Phys. 1980, 51, 31-42. (24) Leutwyler, S. J. Chem. Phys. 1984, 8 1 , 5480-5493. (25) Dushman. S. Scientific FounoWons of Vacuum Technique, 2nd ed.; Lafferty, J. M.. Ed.; John Wlley and Sons: New York, 1962; pp 82-86. (26) Goates. S. R.; Bartell, L. S. J .Chem. Phys. 1982, 77, 1866-1873.
RECEIVED for review July 29,1991. Accepted October 11,1991.
Derivatization in Trace Organic Analysis: Selection of an Inert Solvent Kariman Allam, Samy Abdel-Baky, and Roger W. Giese* Department of Pharmaceutical Sciences in the College of Pharmacy and Allied Health Professions, and Barnett Institute of Chemical Analysis and Materials Science, Northeastern University, Boston, Massachusetts 02115
INTRODUCTION Generally, a solvent is selected which dissolves the reactants and optimizes the yield of the desired product in a derivatization or chemical reaction in trace organic analysis. If interferences are then encountered in the subsequent detection of the product, additional sample clean-up steps are commonly added to the procedure before or after the reaction step. Here we point out that solvent inertness should be investigated, prior to incorporating extra clean-up steps, when interferences are encountered in trace organic analysis involving derivatization. This is because the solvent itself (including its impurities, which tend to be less in a more inert solvent) may be participating in the reaction, contributing to the interferences that are observed. While a small degree of reaction between the reactants and solvent can go unnoticed under ordinary circumstances, it can build up significant noise relative to the analyte in a trace procedure. Interferences are an important problem in trace analysis. Fortunately, solubility considerations can be less of a concern in trace analysis because of the small amounts of the analyte and reagents. This tends to increase the opportunity to employ a more inert solvent as lower amounts of analyte are determined. Changing a solvent is more attractive than increasing sample clean-up to reduce interferences. It seems that this consideration for derivatization in trace organic analysis should be obvious, but an examination of the relevant literature suggests that it is little appreciated or practiced. This is in spite of many examples in the general literature of solvents participating in chemical reactions, including some analytical reactions (e.g. ref 1). In this note we present the results of an experiment which demonstrate the usefulness of selecting an inert solvent for a derivatization reaction in a method utilizing gas chromatography with detection by electron capture negative-ion mass spectrometry (GC-ECNI-MS). EXPERIMENTAL SECTION Materials. 2,3-Pyrenedicarboxylic acid was prepared as described ( 2 ) . Pentafluorobenzyl bromide was purchased from Aldrich (Milwaukee, WI), and triethylamine was from Pierce (Rockford, E). Hexane, acetonitrile, ethyl acetate, toluene, and tetrahydrofuran (at least 99.9% pure according to the supplier) were purchased from American Burdick and Jackson (American Scientific Products, Boyton, MA). Silica gel (40pm) was purchased from J. T. Baker (Phillipsburg, NJ). All glassware was silanized. *Address reprint requests to this author.
Instrumentation. A Model 5988A mass spectrometer from Hewlett Packard (Palo Alto, CA) was connected to a gas chromatograph (Model 5890 Series TI from Hewlett Packard) with a capillary interface kept at 290 "C. The column was a 7- or 12-m fused-silica capillary, Ultra-l,O.2 mm i.d., 0.11-pmfilm thickness (Hewlett Packard). The carrier gas (helium, 99.999%) for gas chromatography and the reagent gas (methane 99.998%) for ionization by electron capture were from Med-Tech (Medford, MA) and filtered through an Oxisorb cartridge (MG Scientific Gases, Branchburg, NJ). Samples were on-column injected with a 10-pL syringe fitted with a fused-silica capillary needle. The instrument was manually tuned, and the conditions for GC-MS were column head pressure 20 psi (linear velocity = 35 cm s-l), ion source pressure 2 Torr, ion source temperature 250 "C, electron energy 240 eV, dwell time 1s, and the cycle time 0.9 s. Detection was by selected ion monitoring at m / z 469. For the GC column, the starting temperature of 140 "C was programmed to 290 "C at 70 "C/min immediately after injection, and the hold time was 8 min. Continuous vortexing at 50 "C was achieved with a Model 4600 Multi-Mixer (placed in an oven) obtained from Lab-Line Instruments, Melrose Park, IL. Method. 2,3-Pyrenedicarboxylicacid (10 pg, 34.5 fmol) in 10 pL of methanol was dried at 60 OC under N2 in a 2-mL Micro-V vial (Supelco). Derivatizing agent (50 pL) was added, consisting of 2 pL of pentafluorobenzyl bromide and 2.5 WLof triethylamine in the reaction solvent (toluene, tetrahydrofuran, or toluene/ acetonitrile, l/l). The vial was capped and kept at 50 "C for 5 h with continuous vortexing, followed by evaporation at 60 "C under NZ. A silica solid-phase extraction column was prepared from a silanized Pasteur pipet (5.5 in.), silanized glass wool, and 100 mg of silica gel. The column was conditioned with 4 X 0.5 mL each of ethyl acetate and hexane. The reaction mixture (containing some solid which was identified as pentafluorobenzyl-triethylammonium bromide by 'H and 13C NMR spectrometry) was transferred to the column with a 9-in. Pasteur pipet using 2 X 0.1 mL of hexane. After the column was washed with 4 X 0.5 mL of hexane, the diester was eluted with 4 X 0.5 mL of ethyl acetate into a 3-mL Reacti-Vial (Pierce). After evaporation at 60 OC under N2, the residue was focused by adding and evaporating 0.1 mL of acetonitrile. Acetonitrile (10 pL) wm added, vortexing was done for 30 s, and 1 pL was injected into the gas chromatograph. RESULTS AND DISCUSSION We are developing an analytical procedure for the determination of diol epoxide polyaromatic hydrocarbon (DE-PAH) DNA adducts by GC-ECNI-MS. Basically, the intended method is to consist of four steps: (1)with a mild acid, hydrolyze the DNA to release a tetrahydrotetrol PAH from the IP-guanine site that has reacted with a corresponding DEPAH (3);(2) oxidize the released tetrahydrotetrol PAH to a
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differences in the reaction of the reagents with the solvent, with the contaminants in the solvent, or (unlikely,we believe) among the reagents in the different solvents. We ruled out a contribution from nonvolatile contaminants in the acetonitrile by evaporating 2 X l mL of this solvent in a vial (which would tend to amplify such contaminants 80-fold) prior to conducting the reaction in 50 p L of toluene/acetonitrile, 1/1, as usual. The broad level of the interfering background signal was essentially unchanged. Visually, the two samples, prior to injection into the GC system, were indistinguishable (pale yellow) whereas the corresponding sample from a reaction in toluene is colorless. Apparently, then, the acetonitrile is reacting more than the toluene with the reagents, giving reaction side products that form the broad background signal in the GC-ECNI-MS. Another general approach for minimizing the level of contamination introduced by a chemical reaction step in a trace procedure is to conduct the reaction with gas-phase reagents in the absence of solvent, e.g. gas-phase reactions for amino acid sequencing (9). However, when a solvent is necessary for an analytical reaction, then an inert one should be considered.
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Flguro 1. Selectedjon ( m / z 469) GC-ECNI-MS chromatograms of 2,3-bis(pentaflwnobenryl)pyrenedica~~late (peak indicated by the arrow) derived from 10 pg of the corresponding diacid. Derhratlzatbn in ( A ) toluene/acetonib#e, 1/1, and ( B )toluene. After postderhrethatbn cleatwp on a short column packed with silica, the evaporated sample was dissolved in 10 pL of acetonitrile and 1.0 pL was Injected. The capillary GC column was 7 m in length. This column was 3 months OM (400 prior injections at the time of this experlment). Inset: use of a new, 12-m GC column to repeat determlnatlon B.
corresponding dicarboxy-PAH with potassium superoxide (4); (3) convert the dicarboxy-PAH to a corresponding diesterPAH with pentafluorobenzyl bromide (2);and (4)determine the latter by GC-ECNI-MS. 2,3-Pyrenedicarboxylicacid is the model analyte being studied currently to develop steps 3 and 4. As part of our effort to optimize the yield of step 3, the pentafluorobenzylation reaction, we investigated three solvents for this step. While all three solvents turned out to give a high yield of the desired diester-PAH product, there was a significant variation in the GC-ECNI-MS background signal in step 4. Representative selected-ion chromatograms are shown in Figure 1A (toluene/acetonitrile reaction solvent) (5) and 1B (toluene) from reactions applied to 10 pg of 2,3-pyrenedicarboxylic acid. As seen, the signal/noise ratio is much improved when toluene is used rather than a 1/1mixture of toluene and acetonitrile. In toluene, the absolute yield of product obtained from 10 pg of 2,3-pyrenedicarboxylicacid is 87 f 7% (z f SD;n = 5). A new GC column was used to obtain the latter data;a repreeentative chromatogram is shown as an inset in Figure 1. Tetrahydrofuran as a reaction solvent gave an intermediate background signal (data not shown). With this observation, it is interesting that trace esterifications with pentafluorobenzyl bromide are commonly done in acetonitrile (e.g. refs 7 and 8) but apparently never in toluene. The latter solvent obviously deserves more attention for this reaction on the basis of its ability to significantly reduce the level of the interferences when GC-ECNI-MS is used for detection. The variation in the background signal that we have observed by using different solvents in step 3 can be due to
REFERENCES (1) Chou. T.; David, M.; %ha, M.; Giese, R. W.; Vouros, P. Biomed. Mesa Spectrom. 1986, 14, 23-27. (2)' U, W.; Sotirlolcleventls,C.; AWelBaky. S.;Glese. R. W. J . Chrometm.,1991. 588. 273-280. (3) Vahakangas, K.; Haugen, A.; Harris, C. C. Carcinogenesis (London) 1986, 6 , 1109. (4) Sotiriou, C.; Li, W.; Glese, R. W. J . Org. Chem. 1990. 55, 2 159-21 64. ( 5 ) The broad background disappears when an ion of significantly higher mass, e.g. m l z 600, is monkored, but not when ions of comparable or bwer mass are detected. Thus, the broad signal, apparently, is due mostly to injectbnderlved contaminants that elute slowly trom the QC column. Ail bwer ion channels get filled, and the signal is relatively smooth and broad, because of the richness of the low-intenslty "grass" of mass spectral peaks from these Contaminants. Thus the event is analogous to the "peakat-every-mass" phenomenon of standard FABMS ( 6 ) . The background signal was not reduced by changing the QC column. (6) Mcckskey. J. A. Mefhods E r u y m d . 1990, 193, 138 and 219. (7) Roberts, L. J. J . chromat~gr.1984, 287, 155-160. (8) Strife, R. J.; Murphy, R. C. J . Chromatogr. 1984, 305, 3-12. (9) Hewick, R. M.; Hunkapiiler, M. W.; Hood, L. E.; Dreyer, W. J. J . Bld. Chem. 1981, 256, 7990-7997.
RECEIVEDfor review July 18,1991. Accepted October 25,1991. The research described in this note was conducted in part under a contract to the Health Effects Institute (HEI), an organization jointly funded by the United States Environmental Protection Agency (EPA) (Assistance Agreement X-812059) and automotive manufacturers. The specific grant was HE1 Research Agreement 86-82. The contents of this article do not necessarily reflect the views of the HE1 nor do they necessarily reflect the policies of the EPA or automotive manufacturers. Contribution No. 497 from the Barnett Institute.
