Ion Trap GC-MS Analysis of Tissue Samples for Chlorinated

Chapter 18

Ion Trap GC-MS Analysis of Tissue Samples for Chlorinated Compounds Downloaded by UNIV MASSACHUSETTS AMHERST on September 17, 2012 | http://pubs.acs.org Publication Date: November 1, 2000 | doi: 10.1021/bk-2001-0771.ch018

Pamela P. Hamlett, Gary L. Steinmetz, and David M. Klein Texas Parks and Wildlife Environmental Contaminants Laboratory, 505 Staples Road, San Marcos,TX78666 (phone 512-353-3486; email [email protected])

Currently, ion traps mass spectrometers are used in the Texas Parks and Wildlife (TPW) Lab to obtain full scan mass spectra of compounds at a detection limit comparable to selected ion monitoring (SIM) data from bench top quadrapole instruments. This combined with the ion trap's chemical ionization capability, tandem mass spectrometry, and simultaneous full scan with tandem mass spectrometry greatly enhances the quality of tissue analysis data. Ion trap identification/verification of both false-positive and false-negative results from electron capture detectors (ECD) and SIM are also discussed.

Introduction The Texas state legislature has given Texas Parks and Wildlife (TPW) trustee responsibility for all public waterways, wildlife and both fresh and coastal water fishes of Texas. The TPW maintains a chemistry laboratory to: 1. provide information about fish and wildlife kills 2. support criminal investigations of polluters 3. obtain background information about contaminant levels in Texas 4. provide analysis in support of related projects for local, state and federal government entities The majority of the samples submitted to the laboratory are fish and wildlife tissues although water, soil, and sediment samples are also received. Many of these samples are analyzed for chlorinated organic residues including pesticides, fungicides, herbicides and poly-chlorinated biphenyls (PCBs). Classical methods of sample

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© 2001 American Chemical Society In Pesticides and Wildlife; Johnston, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 2000.

243 preparation include Soxhlet extraction and Kuderna-Danish solvent evaporation. Both of these techniques are too time consuming and have been replaced by validated methods using static solvent extraction and automated nitrogen evaporation of solvents. The sample preparation scheme used by TPW is shown in Figure 1.

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Experimental Samples were frozen at -20° C. The samples were thawed and homogenized with a Robot Coupe RSI 6V. 10 g of sample was weighed out and mixed with 100 g of anhydrous sodium sulfate. This mixture was frozen overnight to remove all traces of moisture from the sample. Extraction was with three portions of dichloromethane. Evaporation was done with a Zymark Turbovap. Samples were defatted on a Waters HPLC system set up with a gel permeation column. Samples that were analyzed by electron capture were further purified by liquid chromatography using a mixed alumina and silica column.(3) Gas chromatography with electron-capture detection was performed on DB 5 and DB 1701 30 m X 0.25mm X 0.25 urn column in a Hewlett-Packard 5890 gas chromatograph. SIM were recorded on a Hewlett-Packard 5970 mass selective detector. Ion trap mass spectral detection was performed on a Thermoquest GCQ. For PCB analysis a 60 m X 0.25 mm X 0.25 urn DB-5 column.

Safety Considerations Chemical analysis poses hazardous situation and appropriate safety precautions should be taken. No one should work in a chemistry lab without specific education on methods, techniques and the hazards of the chemicals that must be handled. Therefore, any laboratory doing analysis of hazardous substances must have a working chemical hygiene plan with a designated and trained chemical hygiene officer. This is not a recommendation, as it is required by law under the 29 CFR 1910.1450 which states, 'This section shall apply to all employers engaged in the laboratory use of hazardous chemicals. Employers shall have developed and implemented a written Chemical Hygiene Plan no later than January 21, 1991." A specific note of caution concerns the hazards of working with pesticide extracts. Extracting tissue involves concentrating contaminants from a large quantity of sample into a small volume of solvent. The concentrated extract poses a special risk in handling and proper personal protection is required. Latex gloves are NOT appropriate for this work. Only nitrile gloves or those rated to resist organic solvents should be used. Finally, anyone working with concentrated dioxin extracts and standards should obtain specific training before working with concentrates of these extremely toxic compounds.

In Pesticides and Wildlife; Johnston, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 2000.


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Extraction and Analysis Scheme 11 0 g F i s h + 1 0 0 g S o d i u m

[

Sulfate)

Extract 3x with 1 0 0 ml_ D i c h l o r o m e t h a n e C o n c e n t r a t e & Filter

GPC Separate Fats from C o n c e n t r a t e to 1 m L Analyze for Pesticides & PCBs by G C - M S

Analytes

C o n c e n t r a t e to 5 m L Solvent Exchange to H e x a n e Place on C o l u m η of A l u m i n a & Silica

Elute with 3 0 m L Hexane C o n c e n t r a t e to 1 m L Analyze for P C B s by G C - E C D

Elute with 30 m L II A c e t o n e : H e x a n e (50:50)1 C o n c e n t r a t e to 1 m I Analyze for Pesticides by G C - E C D

Figure 1. Sample Preparation Scheme


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Electron Capture Detectors Electron-capture detectors have been the standard for gas chromatographic analysis of chlorinated pesticides and PCBs (1,2). While these detectors are very sensitive, they suffer from a lack of selectivity. Confirmation requires a second column and detector. Due to overlapping retention time of many compounds, a time consuming liquid chromatography step is routinely used to separate the non-polar PCBs from more polar chlorinated pesticides (Figure 1) (3). Current analytical requests seek part per billion (ppb) or lower detection limits. Concentrating samples to provide this "ultratrace" level of detection produces gas chromatograms where interfering compounds from the tissue matrix can equal or exceed those of the chlorinated residues.

Selected Ion Monitoring Due to the complex backgrounds of tissue samples, the gas chromatograph-mass spectrometer (GC-MS) has been used to provide data with less false positive identifications. Full scan mass spectra from bench top quadrapole instruments lack the sensitivity required so the technique of selective ion monitoring (SIM) GC-MS is used to obtain a lower detection limit (4). In SIM only two or three ions are detected for each compound. The presence and ratio of the ions are used to identify analytes. However, there are some limitations inherent to SIM analysis. First, it is can only be used for known analytes. Ions of interest must be assigned to retention time windows and only compounds which are in a time windows will be detected. Therefore, unknown compounds will give no signal with this system. While this technique is useful at eliminating background, it provides no information about unknown compounds which may be important for environmental evaluations. Another area that presents difficulty is the ion ratio(s) used to confirm an analyte. This ratio has a large window of acceptance. In EPA methods, the ion ration can vary by 20 - 30% depending on the method followed (e.g.. EPA 625 or 8270 C). This practice can give rise to a false positive detection if the background includes ions close to the m/e of the secondary confirmation ion(s). If there is an interfering background ion it may cause false negative reports by obscuring the correct ratio of a confirmation ion. False negatives can also arise if the retention time of the analyte changes and the ions are no longer in the correct retention time window. SIM has given rise to both false positive and negative identifications in the TPW when samples were re-analyzed by more sensitive ion-trap instruments.

Analyte Quantitation Quantitation by SIM is usually performed from calculations on the most abundant ion. Often the confirmation ions fall below a 5:1 signal to noise ratio yet this problem is



246 rarely addressed in SIM data. With greater sensitivity than full scan mass spectra, SIM provides analyte concentrations with lower detection limits. Because of this, confirmation of suspected false positive or false negative reports is not possible without re-analyzing the sample on a more sensitive instrument. There are two instruments that have significantly higher sensitivity than quadrapole instruments operated in the SIM mode. One is the ion trap and the other is a magnetic sector mass spectrometer. Sector instruments are extremely sensitive and selective, however, the expense and difficulty of use have kept these mass spectrometers beyond the realm of most analytical laboratories.

Ion Trap Detectors Ion trap detectors (ITD), are relatively inexpensive and readily available from several manufacturers. The ITD is robust enough for everyday use and though an ITD is more expensive than an electron capture detector, the quality of the data is far superior. While a selective detector provides only a retention time and some information about a compound the ion trap can provide a full mass spectrum of a compound. Early ion traps had a limitation because samples with a complex matrix, a so-called "dirty sample," would not give "classical" mass spectra. This was resolved with Automatic Gain Control was incorporated into the second generation of ion traps.(5) External ionization now available from Thermoquest is another technique available to further enhance the quality of the mass spectra obtained from an ion trap. These innovations along with (-) chemical ionization make the ion trap a tool worth considering for the analysis of chlorinated compounds in wildlife tissue samples.

Application of Modern Ion Traps The current ion traps have significant advantages over SIM and selective detectors for residue analyses. The current state-of-the-art ITD is capable of multiple mass spectra or M S . This includes tandem mass spectroscopy (MS-MS or MS ) which is especially useful when analyzing dirty matrices and in cases where an unambiguous confirmation is required. Tandem mass spectroscopy isolates a single parent ion in the trap while all other ions are ejected. The isolated ion then undergoes a collisionally induced dissociation (CID) giving rise to ions known as daughter or secondary ions. This is known as "tandem in time." "Tandem in space" can also be done with a triple quadrapole instrument but the cost is significantly higher and the instruments are not capable of simultaneous full scan - tandem mass spectroscopy. n

2

2

Guidelines for the use of M S for quantitation require at least two daughter ions be detected, each with a signal to noise ration of not less than 5:1 (6). Tandem mass spectroscopy is demonstrated in Figure 2. This technique has been successfully


247 applied to several pesticide residue problems. One group is using MS-MS extensively for the determination of residues in fruits and vegetables (7). Another interesting application has been in the investigation of polychlorinated dibenzodioxins (8). Several of these compounds are extremely toxic with the level of interest in the part per trillion range. Until the use of MS-MS with ion traps, the only EPA approved method of analysis for these compounds was with a magnetic sector instrument in what is known as High Resolution Gas Chromatography-High Resolution Mass Spectrometry (HRGC-HRMS) (9). With MS , detection limits in the range of 25 ppt have been obtained, depending on the isomer (8). While this is still more than an order of magnitude greater than that of HRGC-HRMS, the ion trap is more readily available and significantly reduces the cost of these analyses. This places analysis of dioxins within the realm of possibility for many labs which would never have the funds or expertise to have a magnetic sector mass spectrometer on site.


2

Application of GC-Ion Trap MS in the TPW Laboratory At the TPW Lab the goal is not to "err on the side of caution" but to provide reliable analytical data. To meet that goal several samples have been analyzed by ECD, SIM, and ion trap detectors. There is not sufficient space to review all of the results but some selected examples will help to illustrate the advantages obtained from the use of the ion trap detector. Dieldrin is a chlorinated pesticide* prepared from epoxidation of aldrin. This epoxide compound has a low LC 50 in fish (0.037 mg/L) (10). ECD analysis indicated that this compound was present in a fish tissue sample at 40 ppb. Further analysis by SIM also confirmed that dieldrin was present. However, when the sample was re-analyzed by ion trap GC-MS full scan, no dieldrin was detected. To ensure the ion trap was not giving a false negative, the extract was fortified with dieldrin to give an equivalent of 5 ng/g tissue level. Dieldrin was then readily detected (Figure 3). Fish kills are a frequent occurrence during the hot summer months in southern states. Most of these events can be traced to elevated temperature and low dissolved oxygen. In some cases information is obtained about chemical use that leads to an investigation of a specific compound. One such case involved the reported application of the fungicide chlorothalonil. Extracts from dead fish tissues were analyzed by GC-MS-MS and chlorothalonil was identified at a concentration of 7 ng/g. (Figure 4). To further confirm this identification, negative chemical ionization was done followed by MS-MS to unambiguously identify the fungicide.

Simultaneous Full Scan and Tandem Mass Spectrometry One of the most interesting applications of modern ion traps is the recent technological innovation which allows the instrument to perform full scan mass



EI Single Stage Mass Spectrum

EI MS-MS Mass Spectrum



Figure 2. E.I mass spectrum where the m/e 373 ion is isolated after a CID gives rise to the MS-MS Spectrum


V0


250

Figure 3. Dieldrin Spiked in Fish Tissue at 5 ng/g

Figure 4. EI MS-MS of Sample with chlorothalonil detected



251

2

Figure 5. Chromatogram showing alternating full scan and MS for simultaneous analysis across peak.

Undiluted Sediment Sample 100 90

ζ

80

ζ

70

ζ

p

c

B

1 3 8

n o t o n

^

m

41.83

φ Ζ g 60 -

«40 Φ

ΓΓ 30 ~ 20 10 20.79 0 20

Figure 6. PCB and DDE in simultaneous mode


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spectrometry simultaneously with MS-MS. The instrument does this by alternating a full scan spectrum with an MS-MS scan. There are four to five scans per second. The software separates the scans and provides two separate chromatograms for the sample. (Figure 5) This technique was used to investigate a challenging problem: how to identify trace amounts of pesticides in the presence of significant amounts of PCBs. Historically, the only method for this analysis was to use liquid chromatography to separate the compounds followed by separate gas chromatographies. As stated previously, this is a time consuming step which greatly increases waste solvents and adds additional sample transfers with the potential of sample loss at each transfer. With the advent of simultaneous full scan and MS-MS it is possible to analyze extracts for PCBs and DDE with only a gel permeation clean up to remove fats as illustrated in Figure 6. In this chromatogram, ρ,ρ'- DDE is seen by MS-MS as less than baseline in the presence of ppm levels of various PCB isomers. In this case the ion trap is functioning as a chromatograph to separate and isolate the DDE parent ion. The ion trap then performs MS-MS for identification and confirmation of DDE in this sample at the retention time of 34.82.

Conclusion Ion trap detectors have proven to be sensitive, selective, and reliable for investigation of residues in tissue extracts. At ultra-trace levels of interest there are too many interferences from the matrix to provide acceptable residue confirmation by electron capture or selected ion monitoring detection methods. An ion trap is more expensive than an electron capture detector but the data obtained is significantly more reliable. With the advent of simultaneous full scan and tandem mass spectrometry, ultra trace levels of residues can be detected without giving up the ability to look for large amounts of unknown or unexpected residues as is the case in selected ion monitoring and without giving up any sample through-put.

Acknowledgement We would like to thank the U.S. Fish and Wildlife Service and the Sport Fish Restoration Fund for financial support of this work.

References 1.

Official Methods of Analysis of the Association of Official Analytical Chemists; Helrich, K. Ed;, Fifteenth Edition; Association of Official Analytical Chemists, Gaithersburg, MA, 1990, Vol. 1, pp 283-284.


253 2.


3.

4.

5. 6. 7. 8. 9.

10.

Methods for Organic Chemical Analysis of Municipal and Industrial Wastewater; Longbottom, J.E. and Lichtenberg, J.J.: Eds.; Organochlorine Pesticides and PCBs - Method 608, U.S. Environmental Protection Agency: Cincinnati, OH, 1982; pp 608.1 - 608.11. Methods of Analysis by the U.S. Geological Survey National Water Quality Laboratory—Determination of Chlorinated Pesticides in Aquatic Tissue by Capillary-Column Gas Chromatography with Electron-Capture Detection; Leiker, T.J.; Madsen, J.E.; Deacon, J.R.; Foreman, W.T., Eds., U.S. Geological Survey, Denver, CO, 1995; pp. 11-12. Methods for Organic Chemical Analysis of Municipal and Industrial Wastewater; Longbottom, J.E. and Lichtenberg, J.J.: Eds.; Organochlorine Pesticides and PCBs - Method 625, US Environmental Protection Agency: Cincinnati, OH, 1982; pp 625.1 - 625.17. Huston, C.K.; J. Chromatogr. 1992, 606, 203-209. Goodenowe, D.; Duffy, M . ; Establishing LC-MS Confirmation Criteria; 47 American Society for Mass Spectrometry Conference, Dallas, TX, Sheridan, R.S.; Meola, J.R. J. AOAC Int. 1999, Vol. 82, 00. Hayward, D.G.; Hooper, K.; Andrzejewski, D. Anal. Chem. 1999, 7, 212-220. Guidance for Assessing Chemical Contaminant Data for Use in Fish Advisories, Bigler, J. and Greene, A. Eds; Volume 1: Fish Sampling and Analysis; Office of Science and Technology, Office of Water, U.S. Environmental Protection Agency, Washington, DC, 1993, ρ 8.6. Farm Chemicals Handbook, R.T., Ed., Meister Publishing Company: Willobughby, OH, 1997; p. C 128 th


Ion Trap GC-MS Analysis of Tissue Samples for Chlorinated

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