Methods of Analysis of Picogram Quantities of Organic Substances in Real Samples Michael L. Gross University of Nebraska, Lincoln. NE 68588 Certain toxic chemicals resist degradation and, as a result, they may accumulate in the environment and ultimately pose dadgers-to human health. If the materials are highlytoxic, accumulation in environmental, food, or human tissue samples, even at levels as low as parts-per-trillion, is a cause for concern because the chemicals may cause long-term adverse health effects such as cancer. Consequently, there exists aneed to develop analytical methods for these compounds at trace levels. The analysis must he both highly sensitive and specific. That is, the method must be not only capable of detecting small numbers of molecules but also of insuring the identity of the molecules detected. In this article. we describe sensitive and s~ecificmethods that haw heen used to monitor tetrachlor~~dihenzodioxins (TCDDI and tetrachlorodihen7ofur~ns(TCDF,: examr)lesare compounds I and 11.
"m;;=; C1
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The procedures are suitable for detecting and quantitating these substances a t the ~arts-ner-trillionlevels in comnlex environmental and biological samples. The 2,3,7,X-isomers (shown as I and 111are members of H complrx isomer families. There are 22' 'I'CDD isomers and more than ;It) TCIW isomers whirh differ in the oositions of chlorint: suhjtitution. Thus. there are two aspecis of the question of specificity for these compounds. First, a reliable method must be capable of distinguishing TCDD or TCDF from other closely related chlorinated materials such as PCB's or DDE. Second, it may he necessary to quantitate a specific isomer, such as the 2,3,7,8-TCDD, in a mixture which contains other TCDD's. Even if other isomers are not present, it may he necessary to insure that the detected compound is the 2,3,7,8-TCDD or 2,3,7,8-TCDF because these isomers are the most toxic members of the isomer families. Chlorinated dioxins occur as contaminants in the commercial production of various chlorinated phenols. For example, 2,4,5-trichlorophenol can condense to form 2,3,7,8TCDD (eqn. (1)). ~
~
The contaminated phenol is subsequently used in the nianukrture of the herbicides 2.4.5-T. 111. and Silvex, IV, and the antiseptic hexachloruphene, V.
The final products contain the 2,3,7,8-TCDD a t concentrations as high as parts-per-billion (ppb). Both TCDD and TCDF can also be produced in the combustion of materials
containing suitable precursors (e.g., municipal waste) and enter the environment adhered to fine particulate emissions from the stack of the combustor. The concentration of TCDD and TCDF on the particulates is also in the ppb range. The latter source is much more complex chemically than an industrial synthesis, and, as a result, many of the TCDD and TCDF isomers are to be expected. The materials will he diluted as they enter the environment leading to lower contamination levels. Therefore, the analytical procedures must be sensitive to concentrations at least as low as one part per trillion (ppt). Gas ChromatographylMass Spectrometry (GCIMS) The GCiMS combination is suitable for detecting picogram levels (ppt in 1to 10 g samples) provided the GC, MS, or both are operated in a high resolution or highly specific mode ( I ) . The basis for the high sensitivity is the electron multiplier detector of the mass spectrometer. The capability of detecting a single ion using the electron multiplier has been described in other papers of this series. However, the high sensitivity of the detector does not permit counting single or even thousands of molecules in a real samnle. In fact. hundreds of millions of molecules are required for a specific detection using state-of-the-art methods in mass soectrometrv. It is im~ortant to understand the reasons for this.1n the fol~~wing paragraphs we discuss the reasons in terms of the actual procedure used in a part-per-trillion analysis of 2,3,7,8-TCDD. Sample Preparation The first step in the analysis of a real sample is to remove the substance of interest from the sample matrix. This can be done by simple or continuous extraction of soil, fly ash, or water samples using a solvent such as hexane (2-4). A hiological sample such as adipose tissue must he digested first usine ethanolic ootassium hvdroxide. This treatment converts fatty acid estrrs and pn~teininto water soluble materials. The dieisst is then extracted with hexanr to remove ihc TCDI). other nonpolar chemical substances are also removed by the ~rocedure.Therefore, the hexane extract must he "cleaned interfering suhstances. For the Lp" to remove andvsis of TCDD, the hexane is extracted with concentrated su1t;ric acid whirh removes weakly hasic sut~sunces(ketonrs, aldehydes, aromatics, etc.1. After cmcentrating the htxane. a iew etayes of short column liquid chron~atogr~iphy (gravity tlowl are used to complete the cleanup. At this point, 11 relativclv clean extract is obtained which is suitable tor GC M S analisis. No matter how carefully this extraction and cleanup have been executed, a small fraction (10-50%) of the TCDD will be lost, and the sensitivity reduced. GCIMS Analysis: Sample lonlzation The second stage of the procedure is the actual analysis of the cleaned-up extract using GC/MS. It is necessary to use both a GC and MS in tandem because the extraction and clean-up do not remove all interiering substances. Furthermore, the retention time of a chemical substance on the (;C column is a strong indicator of the identity of the substance. If there is no question about isomers, a packed column is Volume 59 Number 11 November 1982
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sufficient. This would be the case in carefully controlled monitoring studies such as analysis of animal tissue in a feeding study in which the identitv of the isomer administered is known. Since a packed column will separate only a small fraction of the 22 TCDD isomers, a higher resolution cadllarv . . column is preferred for the analysis bf an environmental or biological sample for which the source of TCDD is unknown. For example, 2,3,7,8-TCDD would be detected as a consequence of herbicide use whereas a mixture of isomers would result from contamination bv incineration. Two other advantages acme to the use of capillary column GCIMS. First, no interface between the GC and MS is necessary. The full flow of helium carrier gas (-- 1ml/min) can be admitted to the MS without deleterious effects to the ionization source and vacuum pumps. The separator interface used for packed column GCIMS removes most of the helium, but it also takes a small fraction of the substance being analyzed. Second, the instantaneous concentration of TCDD in the mass spectrometer ionization source is higher because of the narrow elution profile (- 5 see) from a capillary column compared to a packed column (= 30-45 sec). Let us next consider the source of the mass spectrometer. Often electron ionization is used for analysis of trace substances. The source chamber is not a closed system. Sample molecules continuallv flow in from the GC column..~ and thev are continuously pumped out so that the source pressure can be maintained a t torr. Consequently, the molecules spend only a brief amount of time in the ionizing beam of electrons, and onlv one of 1000-10.000 molecules is converted into a gas-phase ion. Recycling the sample molecules in a combined GCIMS is not a possibilitv if there are ~ o t e n t i a l interfering compounds whGh elute kfter the compound of interest. Thus, we see an inherent limitation of the sensitivity of the method of GC/MS; that is, most of the sample molecules are never ionized and consequently never detected. In addition to ionization, the electron beam activates some of the molecule ions to decompose giving a mass spectrum or fingerprint of the molecule. If the entire mass spectrum can be acquired for sample molecules emerging from the GC, high specificity will be obtained. Unfortunately, scanning the spectrum takes valuable time during which many of the intense ions are never focused onto the detector. As a result, specificity is traded for sensitivity. The sensitivity can be remined bv focusine the mass soedrometer on a limited number of ions (ealled mktiple ion ietection) but a t a loss of specificitv. Furthermore, the decom~ositionsdisoerse the ionization-among fragment ions causing an additional loss of sensitivitv if the molecular ion is to be monitored. other methods of ionization are available for mass spectrometry analysis. Chemical ionization charges the molecules by way of an ion-molecule reaction. A reagent gas such as methane or water is admitted to a ti~htlv'constructedionization chamber a t a pressure of 1torr, and the gas is ionized by electron bombardment. Because of the higher pressure, the initially-formed ions interact with the background gas and are converted to CH5+ and C2H5+in the case of methane and Hs0+ in the case of water. These ions can react in turn with a sample molecule, S, to ionize it by proton transfer forming SH+. If the reaction Deriod is sufficientlv lone. a ereater fraction of the sample molecules will be converted Lto &arged species than bv electron ionization. Unfortunatelv. .. the ion exit H i made smalier to permit a greater buildup of pressure, and many of the ions are not transmitted to the mass analvzer. Thus, the sensitivity is about comparable to electron ionization even though a greater fraction of sample molecules can be ionized in the C~source. Chemical ionization procedures have also been employed for the analysis of TCDD. One logical strategy is to convert the TCDD molecules into negative ions taking advantage of the favorable electron affinity of a chlorine-substituted mol-
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922
Journal of Chemical Education
ecule. Using a l torr source, the sensitivity of the method was found to he less than electron ionization to form positively charzed molecules ( 5 ) .More recentlv. ,. an atmosnheric nressure suurcr has been used ior ihe rmulysis of 'I'CI)U. loni~stion ocrurs bveltctrun rautureand hvreactiun with O,;tG~.l'his - . . proceduie yields detkction limits comparable to those obtained by electron ionization. Mass Analysis After ionization, the molecule ions and fragment ions are accelerated from the source and submitted to mass analvsis. The mass analyzer is set to transmit only molecular ions so that high sensitivity can be achieved. As was mentioned previously, this is done at the expense of specificity. Uncertainty about identification will occur if other interfering compounds produce signals of the same mass as TCDD and emerge from the GC during the elution time window for TCDD. Two exa m ~ l e of s possible interferences are DDE (a metabolite or dec&nposition product of DDT) and a fragment ion of PCB. Their formula, exact masses. and mass resolution needed to separate them' from TCDD are given in the Table. The information provided in the Table eives a clue for increasing the specificity of the method. 1f tKe mass spectrometer can be tuned to a mass resolution of 10.000, and peak profiles acquired during the elution, the interferences can be separated from TCDD. An example of a high resolution (double focusing) mass spectrometer output for such a procedure is given in Figure 1. The mass spectrometer is programmed to switch between two of the molecular ion peaks of TCDD and an internal standard mass (a 2,3,7,8-TCDD for
--
Possible Interferlna Sianals at mle 322 Formula
Exact Mass
C I ~ H & ' ~ C I ~ ~ ~ C I 321.8936 CIIH~~~CP'CI~ 321.9289 C,2H335C15 321.8677
Origin
Re~olution~
-
TCDD MC DOE Mt Chlorinated Biphenyl Fraomant
9,100 12.500
ResolYtion necewry to separate TCDD signal and me imerterence.
I
I
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~
m.m%
m P U 4
Figure 1. Three peak monitoringof 2.3.7,aTCDD eluting from a packed column gas chromatograph. The upper *ace is the raw data. The lower trace has been smoothed using a 16poinl moving average procedure. Masses 319.8965 and 321.8936 are moiecular ions of the 2.3.7.8-TCDD: m l r 333.9344 is the molecular ion of the internal standard. The mass resolution is approximately 10,000.
coverv of the entire nrocedure. Second, it can be emnloved as tthirh all of the rarbo~tatcms have been replaced with WJ. a standard for quantitation. This is based on the reasonable 7'CDD has a nnmher oi molecular ions. M'e haw chosen to assumption that any losses of endogeneons TCDD present monitor two them: m z 319.8965, C,,3HI0.~''CI.,,and initially in the sample will be the same as for exogeneous in321.09:%, CI:H40:. T l r S - ( ' l which owur in the abundance ternal standard. Thus, accurate quantitation can be achieved ratio or 0.7: to 1.t1Uchased on the im that chlorine U(UIIS in by comparing the signals for unknown amounts of TCDD with nature as "Cl and 37C1 in a 3:l ratio). that of the known amount of internal standard. Third, i t Snecificitv is eained in three wavs. The most obvious is that serves as a standard for assigning the retention time of he high r&lutFo"m
Mitchum,R.K.,Moler,G.F.,and Korfmacher, W.A.,Anol. Chem.,52,2278(19801. 17) Hsrles. R. L.,Oswald,E.0..Wilkinson. M . K.,Dupuy, &,A. E.. McDaniel. D. D.,snd Tai. H..Anal.Chrm.,52,1239(19801. 181 Busor, H. R. and Rsppe, C.. A n d Chem., 52.2251 119801. 191 Lamwnki. L.L. and Nesviek,T. J.. A n d Chem.. 52,2M5(198Q)and referenc~jdted (6)
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