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Anal. Chem. 1984, 56, 2138-2141
Confirmation of Ethylene Dibromide in Fruits and Grains by Mass Spectrometry Thomas Cairns,* Emil G. Siegmund, Gregory M. Doose, and Harvey K. Hundley
Department of Health and H u m a n Services, Food and Drug Administration, Office of Regulatory Affairs, 1521 West Pic0 Boulevard, Los Angeles, California 90015 Tom Barry and Glenn Petzinger
Department of Health and H u m a n Services, Food and Drug Administration, Office of Regulatory Affairs, 830 3rd Avenue, Brooklyn, New York 11232
60 mL/min hydrogen; column inlet 200 "C; column temperature 90 "C with additional operation parameters as previously stated (5)) were used. ( b ) Gas ChromatographlMass Spectrometer. A Finnigan
Gas chromatography/mass spectrometry techniques uslng chemlcal lonlratlon and electron Impact have been employed to conflrm the presence of ethylene dlbromlde In frults and gralns determlned by electron capture detection gas chromatrography. Interferences from both the solvent and coextractables have been mlnlmlzed to permlt a determlnatlon uslng only two Ions detected In the correct experlmental lntenslty ratlo belonging to the monobromoethylene carbonlum fragment Ion. A complementary measurement under negative Ion methane chemlcal lonlratlon has determlned the bromide Ion to be present.
Model 3300 or 4021 with INCOS Data System was used. Operating conditions were as follows: 180 cm x 2 mm glass column packed with 3% or 10% OV-17 or OV-225 on Chromosorb W(HP), using methane (CI) or helium (EI); column inlet 220 "C; column temperature 70 "C. Sample Preparation. Twenty five grams of ground pineapple, citrus, or papaya was prepared according to the steam distillation method (4),20 g of flour by the 4-day soak in hexane method (3), and 20 g of oats by the 2-day acetone-water soak method (2).
The need to continually assess the status of existing potential hazardous chemicals has a positive impact on man and the environment. In recent years, there has been a strongly recognized concern for both the environmental and toxicological effects of a wide spectrum of halogenated hydrocarbons, e.g., DDT, dieldrin, Mirex, Kepone, polychlorinated biphenyls (PCBs), dioxins (TCDD), and polybrominated biphenyls (PBBs). The latest candidate to receive this intense scrutiny is ethylene dibromide (EDB) (1). Primarily used as a soil and grain fumigant, its somewhat ubiquitous presence in portions of the food chain has now initiated a widespread analytical investigation into its exact incidences. A comprehensive survey of various products (including but not limited to milk, finished grain products, flour, citrus fruit, pineapples, mangoes, and papayas) has been under way by the U.S. Food and Drug Administration to assist the Environmental Protection Agency (EPA)in determining the recommended levels to be permitted in various foods. More urgently, however, is the demonstration of confirmatory techniques for positive findings from existing extraction and/or partition methods (2-4) in different matrices a t parts-per-billion levels. This paper addresses the analytical background and issues involved with the criteria for routine confirmation by gas chromatography/mass spectrometry (GCIMS). EXPERIMENTAL SECTION Reagents. (a) Ethylene dibromide reference standard from EPA, lot no. AE27, was used. (b) Ethylene dibromide solutions were all prepared in ketone-free ethyl acetate or hexane at concentration levels of 60 pg/fiL or lower. Apparatus. (a) Gas Chromatographs. Gas chromatographs equipped with a 63Nielectron capture detector with a 180 cm X 2 mm glass column packed with 10% OV-17 on 100/120 mesh Gas Chrom Q (operating conditions: column flow 30 mL/min argon/5% methane; column inlet 220 "C; detector temperature 300 "C; column temperature 90 "C) or equipped with a Hall Model 700A electrolytic conductivity detector in the halogen mode with a 120 cm X 2 mm glass column packed with 20% Carbowax 20m on 80/ 100 mesh Supelcoport (operating conditions: column flow
DISCUSSION AND RESULTS Because of the widespread use of EDB as a fumigant, a number of analytical procedures have emerged to deal with the various foodstuffs involved (Figure 1). For the determination of EDB in citrus fruit, Clower (4) developed a steam distillation method using ethyl acetate as the carrier solvent. The determinative step was via 63Nielectron capture (EC) detection gas chromatography. While the lower level of detection was l ppb, the purity of the ethyl acetate employed was found to be extremely critical in that some grades exhibited impurity peaks close to the expected relative retention time (RRT) for EDB. While such impurities could be avoided by using a high grade solvent, element-sensitive detectors such as EC and the Hall electrolytic conductivity detector (HECD) are normally blind to other impurities and coextractables that might otherwise interfere with confirmation by mass spectrometry. To illustrate this impurity issue, the chromatograms exhibited in Figure 2 clearly demonstrate the fundamental analytical problem. The reagent blank (Figure 2A) does show several impurity peaks which do not interfere with an EDB reference standard injection (Figure 2B). However, the extract of a pineapple sample (Figure 2C) does demonstrate the increased coextractable profile, probably due to various flavor and essential oils from the fruit itself. Therefore, the sample background can be considered a two-component system, solvent plus coextractables. Admittedly this sample extract was not found to contain any EDB, but the potential for severe interference on confirmation by mass spectrometry (MS) does exist. The dramatic change in elution profile from using a specific element detector to MS detection can cause problems (6). In the case of a positive determination of EDB in papayas (Figure 2D,E) at the 200 ppb level, the resultant elution profile is sufficiently complex to challenge confirmation by MS. AS an alternate to proceeding with MS confirmation, the HECD does offer additional proof of the presence of a halogenated compound a t the correct RRT (Figure 3). The use of two different stationary phases on two different element-sensitive detectors may be sufficient to provide confirmation of the presence of EDB. All samples, however, cannot be successfully analyzed by HECD and an alternate method is required. In
This article not subject to US. Copyright. Published 1984 by the American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 56, NO. 12, OCTOBER 1984
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Scheme I. Proposed Fragmentation Pathway for EDB under Ammonia Chemical Ionization Conditions
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Figure 1. Various methods of analysis of ethylene dibromide (EDB)
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these instances, the confirmation by MS was demanded. In the case of analysis of flour and biscuits Rains and Holder (3)developed a 4-day hexane soak and triple steam distillation with hexane, respectively (Figure 1). Use of pesticide grade hexane still introduced interferences from the solvent itself and coextractables from the sample matrix. A modification of this method was suggested (7)whereby a soak in acetone-water was employed. This particular solvent system is much more polar than hexane and increased the incidences of coextractables into the final organic phase for analysis. The acetone extract, however, has a distinct advantage in that it contains less fat and therefore provides for better chromatography. With this background to the various methods used currently to detect EDB in foodstuffs, the confirmation of positive results via MS is now explained in detail. In the process of characterizing EDB for such confirmation analysis, various ionization techniques were employed. The base peak was observed at mlz 1071109 corresponding to the loss of bromine from the molecular ion. This monobromoethylene cation is probably resonance stabilized by the positive charge distribution shared by the two carbons bridged by the electronegative bromine (Scheme I). These ions were the only abundant ions present in the electron impact (EI) spectrum and the positive ion methane chemical ionization spectrum.
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In the case of ammonia as reagent gas, two additional ions a t mlz 1241126 were evident and have been attributed to an SN2 reaction by neutral ammonia on the fragment ion at mlz 1071109 (8). It therefore seemed analytically sound to devise a strategy for confirmation based on the presence of mlz 1071109 at the correct RRT in the intensity ratio expected for one bromine atom in the carbonium ion, Le., 0.98 within experimental error. With these criteria in hand, examinations
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Flgure 2. 63Nielectron capture chromatograms: (A) solvent blank (ethyl acetate), (B) 90 pg injection (equivalent to 10 ppb), (C) pineapple extract (equivalent to 7.5 mg) with arrow indicating the expected retentlon time for EDB,(D)papaya extract (equivalent to 7.5 mg), (E)papaya extract diluted 1 In 10 (analysis indicated 200 ppb); recording conditions, 150 cm X 2 mm 10% OV-17, 40 mL/min argonlmethane at 90 OC.
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Figure 5. Grapefruit extract examined by GUMS on 10% OV-17 using chemical ionization with methane as reagent gas: (A) combined m l z 107 and m l z 109; (B) mass chromatograms for m l z 107 and m l z 109. Arrows indicate the expected retention time for EDB.
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Flgure 3. Hall electrolytic conductivity detector chromatograms: (A) an EDB standard representing 10 ppb, (B) papaya extract (equivalent to 70 ppb), (C)papaya extract diluted 1 in 10; recording conditions, 125 cm X 2 mm 20% Carbowax, 60 mLlmln hydrogen at 90 'C. Reconstructed Ion Chromatogram
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Flgure 6. Grapefruit extract examined by GUMS on OV-17 using chemical ionization with methane as reagent gas: (A) combined m l r 107 and m l z 109; (B) separate mass chromatograms for m l z 107 and m l z 109 to demonstrate the peak ratio observed.
Flgure 4. Grapefruit extract examined by GClMS on 10% OV-17 uslng chemical ionization with methane as reagent gas: (A) m l z 107 and 109 combined; (B)separate mass chromatograms for r n l z 107 and r n l z 109 to illustrate the interference observed in the m l z 107 profile. Arrows indicate the expected retention time for EDB.
of a number of different extracts were carried out. In the case of an orange extract using OV-225 as the stationary phase, severe interference in both mass chromatograms was observed. In addition to these interferences a t the exact RRT for EDB, other extremely large components containing both of these ions were also evident in the mass chromatograms. Closer examination of these peaks in the total ion chromatogram mode of operation revealed such entities to be hydroxymonoterpenes, i.e., coextractable flavor components from the orange. The use of OV-225 as a stationary phase was therefore abandoned. To illustrate the potential power of the mass chromatogram as a mode of data manipulation, a grapefruit extract was fi st
examined on OV-17 by the total ion chromatogram technique (TIC) (Figure 4A). As can be seen the RRT for EDB is on the solvent decline after injection. However, the resultant mass chromatograms (Figure 4B) from this data base clearly indicate no interferences for the detection of EDB using m / z 107/109. For these reasons, the adoption of OV-17 using MID was established for routine confirmation of EDB. While mass chromatograms can indicate the strong possibility of noninterference, the extract was reanalyzed with MID (Figure 5). A slight interference was observed at m / z 107 only which might lead to an nonideal ratio measurement for m/z 107/109. This grapefruit extract examined above was determined to be negative for EDB by EC. A positive determination by EC was then examined by MS and is illustrated in Figure 6. In this case, the two ions at m / z 107/109 are clearly visible at the correct RRT. However, the measured ratio was 1:0.82, i.e., m / z 107 more intense than expected. Presumably this shift in the ratio is due to the previously demonstrated interference in the m / z 107 measurement. These ions represented 30 ppb EDB. In the mass spectral examination of wheat extracts (Figure 7) by E1 using MID techniques, the confirmation of EDB at
ANALYTICAL CHEMISTRY, VOL. 56, NO. 12, OCTOBER 1984
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Mass Chromatograms
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Flgure 7. Multiple ion detection chromatograms by E1 for acetonewater extracts: (A) wheat flour at the 1.9 ppb level of EDB, (B) wheat flour at the 269 ppb level of EDB; recording conditions, electron impact GC/MS using 3% OV-17 at 70 OC.
1.9 and 269 ppb, respectively (Figure 7), was complicated by the appearance of an adjacent peak at a higher retention time. Clearly ethyl acetate, hexane, and acetone-water extracts can present analytical difficulties in the confirmation procedure. However, the ratio of m / z 107/109 was within the experimental range observed with reference material (1.05 to 0.85). This experimental range is about 10% on either side of the theoretical value (0.98) expected for one bromine atom. Measurement of isotope abundance ratios via quadrupole mass spectrometers at the picogram level employed in this study obviously cannot achieve greater precision due to instrumental and procedural difficulties. As an extension to the confirmation procedure discussed above, some samples were also examined by negative chemical ionization (NCI) using methane as reagent gas (Figure 8). The only abundant ions observed in this particular mode of detection were m / z 79 and m / z 81 representing Br-. While the detection of bromine is not absolute proof of the presence of EDB, the evidence obtained via this method can be regarded as additional support for the confirmation by positive ion CI or EI. Figure 8A illustrates the NCI mass chromatograms obtained for a flour sample previously confirmed by E1 to contain EDB at 1.9 ppb. The ion ratios are as expected for one bromine. While the S I N for these data are about 4:1, there can be no doubt that one bromine is present particularly in light of the previous E1 results. In the case of negative findings by E1 or positive ion CI (Figure 8B),the observed ratio of m / z 79/81 under NCI (1.05 to 0.9) supported the previous conclusion.
/’
Figure 8. Flour extract examined by GUMS using negative chemical ionization techniques with methane as reagent gas: (A) a confirmed positive identification for EDB at scan no. 125 at 1.9 ppb level: (B) an example of a negative identification for EDB.
CONCLUSIONS There can be no doubt that EC detection for EDB and a whole host of organohalogens is of primary use in screening large numbers of food sample extracts. This method of detection has both specificity and sensitivity (parts per billion or less). However, during EDB confirmation by MS the observance of interferences from the choice of solvent and coextractables is commonplace and troublesome. Intelligent use of a stationary phase to avoid most of these pitfalls can result in a reliable confirmation technique by MS. Admittedly only two major ions are available for this measurement (mlz 107 and m / z 109). However, the observance of the correct intensity ratio for a carbonium ion containing one bromine is paramount to an unambiguous identification when dealing with such a relatively small molecule. In this way positive identification of EDB in a wide variety of samples has been confirmed by MS to ensure analytical integrity of the preliminary reported incidences of EDB. Registry No. Ethylene dibromide, 106-93-4. LITERATURE CITED (1) Borman, S. A. Anal. Chem. 1984, 5 6 , 573-575A. (2) Ciower, M. J. Assoc. Off. Anal. Chem. 1980, 6 3 , 539. (3) Rains, D. M.; Holder, J. W. J. Assoc. Off. Anal. Chem. 1981, 6 4 , 1252. (4) Ciower, M. “Laboratory Information Bulletin”; U.S.Food and Drug Administration, July 1980; 23380. (5) Luke, M. A.; Froberg, J. E.; Doose, G. M.; Masumoto, H. T. J. Assoc. Off. Anal. Chem. 1981, 6 4 , 1187. (6) Cairns, T.; Siegmund, E. G.; Jacobson, R. A.; Barry, T.; Petzinger, G.; Morris, W.; Heikes, D. Biomed. Mass Spectrom. 1983, 1 0 , 301. (7) Daft, J. L. J. ASSOC.Off. Anal. Chem. 1983, 6 6 , 228. (8) Cairns, T.; Siegmund, E. G. Org. Mass Spectrom., in press.
RECEIVED for review April 17, 1984. Accepted May 14, 1984.
