Anal. Chem. 1999, 71, 1448-1453
Automated Toxaphene Quantitation by GC/MS Susan T. Glassmeyer, Kathryn E. Shanks, and Ronald A. Hites*
School of Public and Environmental Affairs and Department of Chemistry, Indiana University, Bloomington, Indiana 47405
Toxaphene is a complex mixture of at least 600 hexa- to decachlorinated bornanes and bornenes, which was used as an insecticide from the late 1950s to the early 1980s. Like PCBs and other environmentally persistent organochlorine pesticides, toxaphene is ubiquitous in the environment. Toxaphene’s complex composition makes its accurate quantitation difficult. We report here an automatic, gas chromatographic mass spectrometry method (using electron capture negative ionization) that is precise and fast. This method is implemented by a small QBasic program that compares peak area ratios to the predicted chlorine isotopic ion ratios. This method decreases the time required for analysis while maintaining precise quantitation. The method is verified with standard and unknown samples contaminated with various amounts of other organochlorine pesticide interferents. From 1972 until the early 1980s, toxaphene was a popular replacement for DDT. Toxaphene was, in turn, banned in 1982, when the U.S. Environmental Protection Agency canceled the registrations for most of its uses.1 By 1982, toxaphene was primarily used as a broad-spectrum insecticide on cotton; therefore, its application was concentrated in the southeastern United States, cotton-growing region. Some minor amounts of toxaphene (less than 1% of the total annual use2 were used in the Midwest, where it was applied to soybeans, corn, and sunflowers. The Hercules Chemical Co. first synthesized toxaphene in 1947. Their production scheme had three steps: (a) the extraction of R-pinene from pine stumps, (b) the isomerization of this compound to camphene, and (c) the photochlorination of camphene to produce toxaphene.2,3 The carbon skeleton of toxaphene consisted of both bornanes and bornenes; thus, a generalized structure of toxaphene is
Note that the double bond is present in some toxaphene * E-mail address:
[email protected]. (1) Sergeant, D. B.; Onuska, F. I. In Analysis of Trace Organics in the Aquatic Environment; Afghan, B. K., Chau, A. S. Y., Eds.; CRC Press: Boca Raton, FL, 1989; pp 69-118.
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congeners but absent in others and that the level of chlorination ranges between 6 and 10. There are 10 different carbon atoms that can be substituted with 1 or 2 chlorines each leading to a very large number of isomers, many of which are chiral. As a result, the composition of toxaphene is very complex, consisting of over 32 000 possible congeners.4 Using high-resolution gas chromatography, ∼600 of these congeners have been found in commercial toxaphene mixtures,5 but many of these GC peaks represent several congeners. Less than 30 toxaphene congeners have been isolated and structurally identified.6 Because of it complexity, the quantitation of toxaphene in environmental samples is difficult. Most analysts have used one of three quantitation protocols: selected peak quantitation, total mass chromatogram analysis, or mass spectral isotope ratio comparison. In selected peak quantitation, the total area of 15-20 selected peaks in a gas chromatogram of a reference standard of toxaphene are used to calculate a relative response factor (RRF). Peaks in the sample gas chromatograms with retention times that correspond to the selected peaks in the standard are then used to determine the concentration.7,8 The simplicity of this method allows quantitation to be performed using only a gas chromatograph with an electron capture detector, but this method has some inherent problems. Because this method uses GC only, it is not highly specific for toxaphene. In addition, this method assumes that all of the selected GC peaks will be present in both the standard and the sample chromatograms. Lack of peak correspondence between the chromatograms can result in a significant error in the toxaphene concentration. Furthermore, since the peaks are selected somewhat randomly, they may not give the true homologue composition of the sample. The total mass chromatogram analysis method goes to the opposite extreme. This method uses gas chromatographic mass spectrometry (preferably in the electron capture, negative ionization, ECNI, mode). Selected ion monitoring is used with m/z values picked to represent the different levels of toxaphene’s chlorination and unsaturation. After the GC/MS experiment, the (2) Von Rumker, R. A.; Lawless, W.; Neiners, A. F.; Lawrence, K. A.; Kelso, G. C.; Horaz, F. A Case Study of the Efficiency of the Use of Pesticides on Agriculture. US EPA 540/9-75-025, 1975. (3) Saleh, M. A. Rev. Environ. Contam. Toxicol. 1991, 118, 1-85. (4) Vetter, W. Chemosphere 1993, 26, 1079-1084. (5) Jansson, B.; Wideqvist, U. Int. J. Environ. Anal. Chem. 1983, 13, 309-321. (6) Hainzl, D.; Burhenne, J.; Parlar, H. Chemosphere 1994, 28, 245-252. (7) Muir, D. C. G.; Ford, C. A.; Rosenberg, B.; Norstrom, R. J.; Simon, M.; Be´land, P. Environ. Pollut. 1996, 93, 219-234. (8) Henry, K. S.; Kannan, K.; Nagy, B. W.; Kevern, N. R.; Zabik, M. J.; Giesy, J. P. Arch. Environ. Contam. Toxicol. 1998, 34, 81-86. 10.1021/ac980906b CCC: $18.00
© 1999 American Chemical Society Published on Web 03/04/1999
areas of all of the peaks in the mass chromatograms for each chlorination and unsaturation level are summed. A response factor is then determined from the calibration standard and applied to the unknown sample.9-11 While this method is quick, it requires a careful examination of the data to make sure that all of the peaks in a sample are, in fact, from toxaphene. The third method of quantitation, mass spectral isotope ratio comparison, is the most conservative of the three methods. It too uses selected ion monitoring in the ECNI mode with m/z values picked to cover the range of 6-10 chlorine atoms and 0-1 double bonds. However, in this case, every peak in every mass chromatogram is examined to determine whether it is or is not toxaphene based on the correct chlorine isotope ratios.12,13 This method makes sure that only toxaphene peaks will be included in the quantitation calculation, but it too has its difficulties. For example, a GC/MS experiment may give over 300 peaks, each of which needs to be examined by an experienced analyst; as a result, each data file can take 1-2 h to examine. In addition, because so many isotope ratios are being examined, there is a strong potential for human error, particularly if several data files are being analyzed at once. Our laboratory has developed an automated form of this mass spectral isotope ratio comparison method, which combines the benefits of the original method with increased speed and reproducibility. This paper explains this automated method and demonstrates its applicability to standard and unknown samples. EXPERIMENTAL SECTION Sample Preparation. To test the proficiency of the quantitation program, several standards as well as environmental samples were analyzed. A 22-congener toxaphene standard mixture developed by H. Parlar (from Dr. Ehrenstorfer GmbH, Augsburg, Germany) was used to determine the ability of the program to accurately classify a peak as a toxaphene congener. Mixtures of Hercules toxaphene and technical chlordane were used to verify the chlordane interference removal portion of the program. Finally, a fish sample was analyzed using the three quantitation methods discussed above to determine the comparability of the automated method to the other techniques. For all samples, [13C1]chlordane (Cambridge Isotope Laboratories, Andover, MA) was used as the internal standard. As purchased, this standard contains both the R and γ isomers, but we standardize on the GC peak corresponding to the latter compound because of its better response under our ionization conditions. The archival fish samples were obtained from the U.S. EPA and the U.S. National Biological Survey (NBS) though the Great Lakes Fish Contaminants Monitoring Program. At the NBS, the fish were composited (five whole fish per sample), homogenized, and stored frozen at less than -30 °C. Approximately 10 g of (9) Pearson, R. F.; Swackhamer, D. L.; Eisenreich, S. J.; Long, D. T. Environ. Sci. Technol. 1997, 31, 3523-3529. (10) Jantunen, L.; Harner, T.; Bidleman, T.; Wideman, J.; Parkhurst, W. Organohalogen Compd. 1997, 33, 285-289. (11) Onuska, F. I.; Maguire, J. In Selected Topics and Mass Spectrometry in the Biomedical Sciences; Caprioli, R. M., et al., Eds.; Kluwer Academic Publishers: Dordrecht, The Netherlands, 1997; pp 533-558. (12) Glassmeyer, S. T.; DeVault, D. S.; Myers, T. M.; Hites, R. A. Environ. Sci. Technol. 1997, 31, 84-88. (13) Swackhamer, D. L.; Charles, M. J.; Hites, R. A. Anal. Chem. 1987, 59, 913917.
ground fish tissue were blended with 80 g of sodium sulfate and loaded into a glass wool-plugged glass thimble, placed in a Soxhlet extractor, spiked with the internal standard, and extracted for 24 h with 50% acetone in hexane. The lipid concentrations in the fish extracts were determined through gravimetric measurement. The majority of the lipids were removed using gel permeation chromatography. The solvent was 60% cyclohexane in dichloromethane. The toxaphene fraction was subjected to further chromatographic cleanup on 1% water deactivated silica. Four solvent fractions were collected: hexane, 10% dichloromethane in hexane, dichloromethane, and methanol. The first three fractions were combined, solvent was exchanged into hexane though rotary evaporation, and the sample reduced to 200 µL under a steady stream of nitrogen and analyzed by electron capture, negative ionization, gas chromatographic mass spectrometry (ECNI GC/MS). Analysis by Electron Capture Gas Chromatographic Mass Spectrometry. A Hewlett-Packard 5989A mass spectrometer was used to analyze the samples. The samples were injected into a Hewlett-Packard 5890 Series II gas chromatograph containing a 30-m DB-5MS column (250 µm i.d., film thickness 0.25 µm, J&W Scientific, Folsom, CA). Helium was used as the carrier gas. The 1-µL injections were made in the splitless mode, with a vent time of 1.9 min. The injection port temperature was maintained at 285 °C to ensure complete volatilization of the sample. The temperature program for the column began with a 1-min hold at 40 °C, followed by a 10 °C/min ramp up to 200 °C, a 1.5 °C/min ramp up to 230 °C, and a 10 °C/min ramp to 300 °C, which was held for 5 min. After eluting from the GC column, the samples were carried through a 300 °C transfer line into the ion source of the mass spectrometer, which was held at 125 °C. Methane was used as the ECNI reagent gas; its pressure was maintained at 0.43 Torr. The electron capture negative ionization GCMS analysis procedure was developed by Swackhamer et al.13 and modified by Myers,14 except that the internal standard has been changed from a octachlorinated PCB to [13C1]chlordane and the hexachlorinated toxaphene homologues are monitored at 2 Da higher. The M- or (M - Cl)- ions of the hexa- to decachlorinated bornanes and bornenes were monitored in the selected ion monitoring (SIM) mode. Four time windows, each monitoring a subset of the ions, were used to increase sensitivity relative to monitoring all of the ions through the entire run. To set the windows, a toxaphene standard was run in full-scan mode. The individual ion chromatographs were examined to determine the beginning and ending times for the windows. Table 1 gives the m/z values of the ions monitored for toxaphene. For each chlorine homologue, two ions are monitored: a quantitation ion and a confirmation ion. Thus for each ion selected to quantitate toxaphene, another ion (always 2 Da lower) is used to confirm that toxaphene is, in fact, being measured. Both ions are in the same ion cluster, and both are due (with the exception of the hexachlorinated homologue) to the loss of one chlorine from the molecular anion. The last column of Table 1 gives the theoretical ratio of the quantitation ion to the confirmation ion. The automatic inspection of the ratio of these two ions is the focus of this paper. (14) Myers, T. M. Masters Thesis, Indiana University, Bloomington, IN, 1994.
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Table 1. Homologue Groups and SIM Program Parameters empirical formula
mol wt
ion compn
unsatn
quant ion (m/z)
confirmn ion (m/z)
Q/C ion
C10H10Cl6 C10H12Cl6 C10H9Cl7 C10H11Cl7 C10H8Cl8 C10H10Cl8 C10H7Cl9 C10H9Cl9 C10H6Cl10 C10H8Cl10
340 342 374 376 408 410 442 444 476 478
M M M - Cl M - Cl M - Cl M - Cl M - Cl M - Cl M - Cl M - Cl
1 0 1 0 1 0 1 0 1 0
344 344 343 343 377 377 413 413 449 449
342 342 341 341 375 375 411 411 447 447
0.80 1.92 0.80 1.92 0.96 2.24 0.64 1.12 0.48 0.75
Table 2. Ions Monitored for Toxaphene Quantitationa window 1
window 2
window 3
window 4
336 I 338 I 341 C 342 I, C 343 I, Q 344 Q 406 I 408 I 409 IS 410 I 411 IS
336 I 338 I 341 C 342 I, C 343 I, Q 344 Q 371 I 373 I 375 C 376 I 377 Q
336 I 338 I 341 C 342 I, C 343 I, Q 344 Q 371 I 373 I 375 C 376 I 377 Q 411 C 412 I 413 Q 447 C 448 I 449 Q
411 C 412 I 413 Q 447 C 448 I 449 Q
a I, interference ion; C, confirmation ion; Q, quantitation ion; IS, internal standard ion.
Other ions must be monitored to account for the internal standard and for potential interferences. The internal standard, [13C1]chlordane, has a molecular ion at m/z 407 with a eightchlorine isotopic cluster; m/z 409 and 411 are monitored for this compound. Chlordane and its isomers and homologues are the main interferents with our toxaphene analysis scheme.13 Technical chlordane is a complex mixture containing over 100 compounds.15 Chlordane itself has a molecular weight of 406 with a eightchlorine isotopic cluster, but it and its homologues have molecular and fragment ions due to the loss of one or two chlorines at m/z 371 and 336, respectively, with seven- and six-chlorine isotopic clusters, respectively. Therefore, we monitor m/z 406, 408, 410, 371, 373, 336, and 338 to account for these interferences. There is another type of interference due to 13C contributions to a toxaphene quantitation ion. The problem ions at m/z 342, 343, 376, 412, and 448 are 1 Da lower than the respective quantitation ions and are also monitored. All of the toxaphene quantitation and confirmation ions and the interference ions are summarized in Table 2, which gives the ions monitored in the various retention time windows. At the beginning of each day of analysis and after every four to five sample injections, a standard sample containing known quantities of Hercules toxaphene and [13C1]chlordane was injected (15) Dearth, M. A.; Hites, R. A. Environ. Sci. Technol. 1991, 25, 245-254.
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Figure 1. Theoretical isotope ratio pattern for the quantitation and confirmation ions (see Table 1) of a heptachlorinated toxaphene congeners showing the m/z values for both the bornane (A) and the bornene (B).
into the GC/MS system. Once the automated program (see below) determined the areas for each homologue group, the group areas were summed, and the total toxaphene response factor, relative to the internal standard, was calculated. This factor and the known amount of the internal standard that had been added to the sample were then used to quantitate the toxaphene in it. The contribution from each homologue group was determined by dividing the area of the ion for that homologue by the sum of the areas.
RESULTS AND DISCUSSION Because of the 3.08:1 natural-abundance ratio of chlorine-35 to chlorine-37, chlorinated compounds exhibit distinctive isotope ratio patterns that depend on the number of chlorine atoms in a given ion. One of these patterns is illustrated for the ions used to measure the heptachlorinated bornane congeners; see Figure 1A. Because this is an ion due to the loss of a chlorine atom from the molecular ion, the elemental composition of the lightest ion in this cluster is C10H11Cl6 at m/z 341, and the isotopic pattern indicates the presence of six chlorine atoms. The quantitation ion is m/z 343, which is the second ion in this isotopic cluster, and the confirmation ion is m/z 341, which is the first ion in this cluster. Given that there are exactly six chlorine atoms in this ion, the ratio of these two ions is known to be precisely 1.92. Thus, we can look at the area ratio of m/z 343-341 for each peak in each selected ion-monitoring record and determine whether the ratio is 1.92 plus or minus some specified error. If the ratio is not 1.92, then the peak cannot be due to a heptachlorinated bornane, and its area should not be included in the quantitation of toxaphene. If one were dealing with a potential peak from a heptachlorinated bornene, then the same argument tells us that m/z 343 (the quantitation ion) is the third ion in this six-chlorine cluster and m/z 341 (the confirmation ion) is the second; see Figure 1B. In this case, the expected ratio is 0.80. A similar
calculation gives the expected quantitation to confirmation ion ratios listed in the last column of Table 1. These ratios are the heart of our automated toxaphene quantitation scheme, which has been implemented by a small computer program, written in QBasic, that examines each GC/MS selected ion peak to make sure the peak shows the correct chlorine isotope ratio. Automated Toxaphene Quantitation. The QBasic program (see the Supporting Information) consists of several sections of code including variable declaration, test array creation, toxaphene analysis calculation, and interference identification and correction. This program calculates the chlorine isotope ratios between the measured quantitation and confirmation ion peak areas and compares these experimental values with the theoretical ratios. Currently, the program is designed to run with HewlettPackard instrumentation and software. The background-subtracted data files from the mass spectrometer are opened with MS ChemStation, a proprietary Hewlett-Packard program. A preliminary macro integrates the SIM peaks for each of the ions listed in Table 2 and extracts the retention times, widths, areas, and start and end times for each peak found. This output is written to a text file, which is then used as the input file for the QBasic program. After reading the peak retention times and the corresponding peak areas, the Qbasic program checks to make sure that the retention times are between 1 and 120 min and that the peak areas are between 0 and 107 area counts. Next the appropriate quantitation, confirmation, and interference m/z values are read in (from DATA statements) along with the correct bornane and bornene ratios. Then for each homologue group, the following tests are performed: (a) The quantitation ion peak is found, and the first confirmation ion peak is identified. (b) The difference between the quantitation peak and confirmation peak retention times is calculated and compared to an acceptable value (usually (0.025 min). (c) If the difference in retention times is acceptable, the quantitation to confirmation area ratio is calculated and compared to the correct bornane and bornene ratio, and the difference between the calculated and the correct ratio is checked against the allowable error (usually (20%). (d) If the ratio is within the acceptable range, the data are corrected for interferences (see below) and written to the output file. (e) Finally, if for any reason, the test ion pair fails to meet the criteria (retention times too far apart or the ratio too far from the theoretical value), the confirmation ion peak and its corresponding information are discarded and the next available confirmation ion peak is tested. This process continues until all confirmation ion peaks have been tested. The program then progresses to the next homologue set and the next quantitation ion. Once a peak is determined to be potentially toxaphene, interferences are removed. For the carbon-13 interferences, the program goes through the output described above peak by peak and searches for corresponding ((0.05 min) peaks 1 Da lower than the quantitation ion. The area of the lower peak is multiplied by 11% (based on 10 carbon atoms in the bornane carbon skeleton and a 1.1% C-13 isotopic abundance), and that value is subtracted from the area of the quantitation ion. This correction is very small (on average less than 10%). The chlordane check is slightly more
Figure 2. Example of the quantitation of three potential octachlorinated toxaphene peaks. The top panel is the quantitation ion at m/z 377, the middle is the confirmation ion at m/z ) 375, and a chlordane interference ion at m/z 373. Eight peaks are labeled with their retention times (min) and the areas (in counts).
complex. The program again looks for interference peaks (see Table 2) that have retention times corresponding to the peaks found to be toxaphene. If the area of these peaks is greater than 25% of the area of the quantitation peak, the interference is deemed significant, and the area of the quantitation peak is set to zero. By setting these values to zero, instead of completely removing them from the output, the analyst can monitor the relative amount of peaks being removed due to interferences. A specific example may help to make this process clear. Figure 2 shows a small section of the SIM records for m/z 377 and 375 (the quantitation and confirmation ions for octachlorinated toxaphene) and m/z 373 (an interference ion). Let’s just focus on three of the peaks that had matching retention times at 33.08, 34.23, and 36.16 min. For octachlorinated compounds, the expected ratio of these quantitation to confirmation ions for a bornane is 2.24, while that for a bornene is 0.96 (see Table 1). The ratio of the m/z 377 to 375 areas for the peak at 33.08 min is 2.22, which is 0.9% lower than theoretically expected. Since it falls within the allowed error, it is retained as toxaphene. Similarly, the peak at 36.16 min (ratio 0.81, which is 16% lower than the expected value) also passes the first test. However, the peak at 34.23 min is discarded at this point, because the difference of its ratio (0.76) from the expected is 21%, which is greater than the allowed error. Analytical Chemistry, Vol. 71, No. 7, April 1, 1999
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Table 3. Results of Hercules Toxaphene and Technical Chlordane Study
Figure 3. A total ion chromatogram for the 22-congener Parlar standard. The numbers are the Parlar numbers assigned to the individual isolated congeners; the congeners indicated by parentheses were not detected.
toxaphene in sample (µg)
chlordane in sample (µg)
toxaphene recovered (µg)
diff (%) (from 10 µg)
10 10 10 10 0
0 1 2 4 2
10.5 11.2 8.5 9.7 0.003
+5 +12 -15 -3
Table 4. Comparison of Toxaphene Concentration Determined by the Four Quantitation Methods Discussed in the Text
homologue
Because the peaks shown in Figure 2 are from octachlorinated compounds, the program examines the peaks in the m/z 373 SIM record (see Figure 2, bottom) for peaks corresponding to those found to be potentially toxaphene. There was no corresponding peak at m/z 373 for the 33.08-min peak. But there were corresponding peaks at 34.23 and 36.16 min. The areas of these peaks are 80 and 79% of the quantitation ion, respectively. Both of these ratios are far above the maximum of 25%, so these peaks are rejected as toxaphene peaks and not used in the quantitation. Note that the peak at 34.23 min failed both the isotopic ratio test and the interference check. In this time window, the only peak to be retained as toxaphene was the one at 33.08 min. The QBasic program offers several advantages over the manual mass spectral isotope ratio comparison method. It is reproducible and impartial, because it removes the “human visual inspection” factor. The program also provides significant savings in analysis time. Analysis of a single file by hand took well over 1 h, whereas the program can complete the same analysis in less than 10 min, which includes running all macros and making all the file transfers. Verification of the Program. Our first test of the program was to determine whether it could find all of the compounds in a standard mixture containing 22 congeners synthesized by Parlar and colleagues.16 Five injections were made and the data analyzed by the program. In all cases, 20 of the 22 congeners were verified as toxaphene in the sample. Approximately 30 additional, minor peaks were also called toxaphene. A labeled total ion chromatogram from one of these experiments is shown in Figure 3. The 30 small, extraneous peaks were impurities in the internal standard and in the 22-congener mixture. The two congeners that were not found, 31 [2,2,3-exo-trichloro-6-(E)-chloromethylene-5,5-bis(dichloromethyl)-8,9,10-trichloronorbornane] and 38 (2,2,5,5,9b, 9c,10a,10b-octachlorobornane), were eliminated during the chlordane interference removal portion of the program. As mentioned above, when the chlordane interference ions exceed 25% of the quantitation ion, the peak area is set to zero. We contemplated increasing this threshold but eventually decided not to. Because the overall design of this program is to provide a conservative quantitation platform, it is best to err on the side of removing toxaphene rather than augment the potential of including nontoxaphene compounds. (16) Parlar, H.; Angerhoefer, D.; Coelhan, M.; Kimmel, L. Organohalogen Compd. 1995, 26, 357-362.
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selected peak
total toxaphene, ppma
19
hexab hepta octa nona deca
0.2 26 55 19 0
total mass chromatogram
mass spectral ratio
automated toxaphene quant
4.0
3.9
3.4
23 29 41 7.4 0.1
1.5 25 62 12 0
2.6 35 48 14 0.1
a Microgram per gram lipid in all cases. b Homologue composition in percent of total.
To further test the chlordane removal section, four Hercules toxaphene samples (50 ng/µL) were prepared and combined with varying amounts of technical chlordane (0, 5, 10, and 20 ng/µL). If the chlordane were not being efficiently discriminated against during the quantitation of the toxaphene, one would expect to see increasing measured toxaphene concentrations with increasing chlordane concentrations. Conversely, if the technical chlordane peaks were being unnecessarily removed, one would expect the measured toxaphene concentration to decrease with increasing chlordane concentrations. One additional sample containing no toxaphene, but with 10 ng/µL technical chlordane, was used as a secondary check to make sure that chlordane did not register falsely as toxaphene. Aliquots (200 µL) of the five test solutions were pipetted into five separate autosampler vials and spiked with the internal standard, [13C1]chlordane. Table 3 lists the amount of toxaphene and technical chlordane in each vial, the amount of toxaphene recovered, and the percent difference from 10 µg. Clearly, the varying amounts of technical chlordane had no systematic effect on the measured toxaphene levels. The quantitation of the sample that had no toxaphene added to it showed 0.003 µg, which falls within the range for blank samples. We believe that these tests demonstrate that the automated toxaphene quantitation program we have developed can accurately detect toxaphene and remove problematic chlordane interferences. Comparison of Methods. To further explore the differences between the four quantitation methods, a fish tissue sample was analyzed using the quartet of protocols; these results are shown in Table 4. Clearly, the selected peak quantitation method is overestimating the concentration at 19 ppm. The other three methods gave about the same total toxaphene concentration but different homologue ratios. The mass spectral isotope ratio
method and the automated toxaphene quantitation method give similar homologue ratios (as they should), but the total mass chromatogram method shows elevated hexachlorinated contributions. The complex nature of toxaphene makes quantitation difficult, but the automated toxaphene quantitation program described here provides quick and reproducible results in less time than it took to evaluate every peak visually. ACKNOWLEDGMENT We thank the U.S. EPA and the NBS for the fish samples used to verify the program. We thank the Great Lakes National Program
Office for supporting this work through Cooperative Agreement X985360. SUPPORTING INFORMATION AVAILABLE Details of the QBasic toxaphene analysis program. This material is available free of charge via the Internet at http:// pubs.acs.org.
Received for review August 12, 1998. Accepted November 18, 1998. AC980906B
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