Anal. Chem. 1983, 5 5 , 677-681 (IO) Adams, F.; Bloch, P.; Natusch, D. F. S.; Surkyn, P. Proceedlngs of the International Conference on Environmental Pollution, Thessalonikl, Greece, Anagnostopoulos, A., Ed.; Thessaloniki University, 1982; pp 122- 142. (11) Surkyn, P.; De Waeie, J.; Adams, F. Envlron. Anal. Chem., in press. (12) Denoyer, E.; Van Grieken, R.; Adams, F.; Natusch, D. F. S. Anal. Chem. 1982, 54, 26A-4lA. (13) Spurny. K. R.; Schormann, J.; Kaufmann, R. Fresenlus’ 2. Anal. Chem. 1981, 308, 274-279. (14) De Waele, J.; Van Espen, P.; Vansant, E.; Adams, F. “Proceedlngs of the 17th Annual Conference on Microbeam Analysis, Washlngton, DC, Aug 9-13, 1982; Heinrich, K. F. J., Ed.; San Francisco Press. Inc.: San Francisco, CA, 1982; pp 371-377. (15) Kaufmann, R.; Hillenkamp, H.; Wechsung, R. Med. Prog. Technol. 1979, 6 , 109-120. (16) Vogt, H.; Heinen, M. J.; Meier, S.; Wechsung, R. Fresenlus’ Z . Anal. Chem. 1981, 308, 195-200. (17) Helnen, M. J.; Meler, S., Vogt, H., Wechsung, R. Fresenlus’ Z . Anal. Chem. 1981, 308, 290-296. (18) Busch, K. L.; Unger, S. E.; Vlncze, A,; Cooks, R. 0.; Keough, T. J. Am. Chem. SOC.1982, 104, 1507-1511. (19) Balasanrnugam, K.; Fuan Anh Dang, R. J.; Hercules, D. M. Anal. Chem. 1981, 53, 2296-2298. (20) Timbrell, V.; Rendall, R. E. G. Powder Technol. 1971/1972, 5 , 279-287. (21) Tirnbrell, V. Pneumoconiosis, Proceedings of the Internatlonal Conference, Johannesburg, 1969; pp 28-36. (22) Tlmbrell, V.; Gilbson, J. C.; Webster, I. Inf. J. Cancer 1968, 3 , 406-408. (23) Tlmbrell, V. “Biological Effects of Mineral Fibers”; Wagner, J. C., Ed.;
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IARC Sclentlflc Publications: Lyon, 1980; No. 30, Voi. 1, pp 127-142. (24) Korfmacher, W. A.; Miguel, A. M.; Mamamtov, G.; Wehry, E. L.; Natusch, D. F. S. Envlron. Scl. Technol. 1878, 13, 1224. (25) Commins, B. T.; Glbbs, G. W. Br. J. Cancer 1969, 23, 358-362. (26) Hibborn, J.; Thomas, R. S.; Lao, R. C. Sci. TotalEnviron. 1974, 3 , 129-140. (27) Lahav, N., Razlel, S. I s r . J. Chem. 1871, 9 , 683. (28) Furukawa, T.; Brindley, G. W. Clays C/ay Miner. 1973, 21, 279. (29) Yariv, S.; Lahav, N.; Lacher, N. C/ays Clay Miner. 1976, 24, 51. (30) Markham, M. C.; Wosczyna, K. Environ. Sci. Technol. 1978, 10, 930-931. (31) Chowdhury, S. J. Appl. Chem. Protechnol. 1975, 25, 347-353. (32) Vansant, E. F.; Yariv, S. J. Chem. SOC.,Faraday Trans. 1 1877, 73, 1815-1824. (33) Kruger, D.; Oberiies, F. Nafurwlssenschaffen1943, 31, 92. (34) Noller, C. R. “ Textbook of Organlc Chemlstry”, 3rd ed.; Saunders: Philadelphia, PA, 1966. (35) Feigl, F. “Spot Tests In Organlc Anaiysls”, 7th ed.; Eisevier: Amsterdam, 1966.
RECEIVED for review September 21,1982. Accepted December 15, 1982. This research was funded by the EEC through research Grant No. ENV-486-BCN and by the Interministrial Commission for Science Policy, Belgium, through research grant 80-85/10, J.K.D.W. is indebted to the “Instituut tot Aanmoediging van het Wetenschappelijk Onderzoek in Landbouw en Nijverheid (IWONL)” for financial support.
Determination of Chlorinated Pesticides in Urine by Molecular Neutron Activation Analysis Laura R. Opelanio and Edward P. Rack’ Department of Chemistry, University of Nebraska -Lincoln, Llncoln, Nebraska 68588-0304
Alan J. Blotcky Medical Research, Veterans Administration Medical Center, Omaha, Nebraska 68 105
Frank W. Crow Midwest Center for Mass Spectrometry, University of Nebraska -Lincoln,
A molecular neutron actlvatlon analysis (MoNAA) procedure employing solvent extraction and high-performance liquid chromatography separation techniques with Subsequent irradiation of the eluted fractions and radioassay for 38CIactivity Is applied to the determlnation of trace quantities of DDT and Its metabolites DDA [bis(p -chlorophenyl)acetlc acld], DDD [l,l-dlchloro-2,2-bis(p-chlorophenyl)ethane], and DDE [l,ldlchloro-2,2-bls(pthlorophenyl)ethylene] In urine. Data and confirmation analyses using mass spectrometry are presented.
The toxic nature and unknown long term effects of chlorinated pesticides to living organisms necessitate the need to evaluate continually their presence in the environment. While the trichlorobis(pchloropheny1)ethane family of compounds (DDT) have been banned from use in many countries, they are still widely used in many Third World countries, and thus their continued use can constitute a serious problem for the global population (1). The urinary excretion of bis(pchloropheny1)acetic acid (DDA), a metabolite of DDT, can be used as an indicator of exposure to DDT (2-5).
Lincoln, Nebraska 68588-0304
The current Environmental Protection Agency (EPA) procedure available for the determination of DDA involves chemical derivatization and gas chromatographic separation employing a 63Ni electron capture detector (6, 7). Unlike previous procedures, where chemical derivatization is employed prior to GC separation, this study develops a method of separating out, and then determining DDT and its metabolites, DDA, DDD, and DDE by means of instrumental neutron activation analysis-simultaneously with minimum interference from other chlorinated hydrocarbons. Separation is performed by high-performance liquid chromatography (HPLC) with subsequent neutron activation of the collected eluent and radioassay for 3sCl activity. It is important to realize that if one is analyzing urine for trace DDA by neutron activation analysis, all chemical separations must be performed prior to neutron irradiation rather than after activation. By virtue of the (n,y) reaction on 37Cl,the emission of the prompt y-rays creates an energetic ”Cl recoil atom which breaks greater than 99% of the original chlorine bonds. The recoil 38Cl atom undergoes new chemical reactions with its environment; thus it finds itself in a different chemical form than originally present in the urine sample. Our confirmation analysis is not an automated LC/MS approach (8),but an
0003-2700/83/0355-0877$01.50/00 1983 American Chemical Soclety
678
ANALYTICAL CHEMISTRY, VOL. 55, NO. 4, APRIL 1983
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off-line LC/MS technique involving an independent collection of HPLC fractions followed by mass spectrometric analysis. This approach has a distinct advantage of giving those workers in the field choices in undertaking their DDA determinations. DDT has an affinity for lipid tissues in animals since it is fat soluble. Ingestion, even in trace amount, resulb in storage and concentration in fatty tissues, where it is slowly dehydrochlorinated to DDE and/or DDD and ultimately excreted as DDA in urine. Figure 1illustrates the structures of DDT and its metabolites. Urine is a complex matrix for analysis, regardless of the technique employed, because of the number of ions and molecular compounds present. The presence of radioactivable sodium and chloride ions in urine presents a severe problem in neutron activation analysis. In a recent study we developed a procedure for trace level determination of iodoamino acids and hormonal iodine in a urine matrix employing a combined HPLC/NAA technique which is a form of what we term "molecular neutron activation analysis" (MoNAA) (9). Our procedure requires that the compounds of interest contain radioactivable atoms that have high neutron cross sections and that form radionuclides of suitable half-lives for radioassay. It is also important that these compounds be in a form amenable to chemical separation without prior irradiation. No acid digestion procedure can be employed, since this can change the identity of the trace compounds. Since chlorinated compounds, such as plasticizers and pesticides, are ubiquitous in nature, their potential interference can make the analytical task for specific identification, especially a t the trace level, very difficult. For this reason, analysts have employed various cleanup techniques, such as column adsorption, prior to a gas or liquid chromatographic step (3, IO).
EXPERIMENTAL SECTION Reagents and Solvents. Burdick and Jackson Laboratories, Inc. (Muskegon, MI), HPLC-grade hexane and methanol were used without further purification. ACS reagent-grade glacial acetic acid from Fisher Scientific, Inc. (Fairlawn, NJ), was used as received. Solvent mixtures were degassed under vacuum prior to use. For preparation of spiked urine, l,l,l-trichloro-2,2-bis(pchloropheny1)ethane (or p-p'DDT), l,l-dichloro-2,2-bis(pchloropheny1)ethylene (or p-p'DDE), l,l-dichloro-2,2-bis(pchloropheny1)ethane (or p-p'DDD), and bis(p-chloropheny1)acetic acid (or p-p'DDA) from Aldrich Chemical Co., Inc. (Milwaukee, WI), were used. Triply distilled water, showing negligible activity after a 140-min reactor irradiation, was used in all phases of the procedure. Pre-HPLC Sample Preparation. The solubility of DDT and its metabolites in organic solvents allows their extraction from urine, using 2% glacial acetic acid in hexane. For each determination, one urine blank and one urine sample spiked with a known amount of DDA, DDD, DDT, and DDE were used, employing the same extraction procedure for both. Twenty milliliters of urine (raw or spiked) was diluted with 20 mL of distilled deionized water and extracted twice with 10 mL of 2% glacial acetic acid in hexane for a maximum extraction
yield. It was necessary to acidify the urine during solvent extraction to keep the DDA in the acid form and hence to obtain a maximum extraction recovery. Although urine contains a large amount of radioactivable sodium and chlorine, removal of these contaminants was afforded by the solvent extraction procedure. The chlorinated compounds of interest are insoluble in the aqueous phase, while inorganic salts, such as those of sodium and chlorine, are highly soluble. The extracts were combined and centrifuged at 3300 rpm for 45 min to break the emulsion formed by shaking the sample. The hexane layer was evaporated to dryness with nitrogen gas, to allow a change of solvent into the reversed-phase elution solution-consisting of a 60 + 20 + 20 (v/v) methanol-water-acetic acid mixture ready for injection into the liquid chromatograph. HPLC Instrumentation, Columns, and Separation. An ISCO Model 1440 liquid chromatograph equipped with a Model 314 metering pump and a Model UA-5 absorbance monitor with built-in recorder was used. The eluent was collected employing an ISCO Model 328 fraction collector (collecting 1 mL volume in polystyrene Autotechnicon vials). Two reversed-phase Whatman columns were evaluated 6.35 X 250 mm Partisil5-ODS and Partisil 10/C8. Both columns were protected with a 7 cm x 2.1 mm i.d. guard column dry-packed with Whatman Co:Pell ODS (25-37 m). The Partisil5-ODS column afforded the better separation for DDT and its metabolites, as seen in Table I. The solvent reservoir, guard column, and the column itself were operated at ambient temperature. The mobile phase consisted of 60 + 20 + 20 (v/v) methanol-water-glacial acetic acid mixture was maintained at a flow rate of 1mL/min and a pressure of 2100 psi. The mobile-phase solvent was degassed prior to use, and isocratic elution was employed. The ultraviolet detection was set at a wavelength of 254 nm. Two separate HPLC runs were made, one of the spiked urine and the other of the blank urine sample. The fractions in which the chlorinated compounds of interest elute were combined and subjected to neutron activation and radioassay. Samples for confirmation analysis by mass spectrometry were subjected to exactly the same solvent extraction and liquid chromatographic procedures. The HPLC fractions containing the chlorinated compounds were evaporated to dryness with dry nitrogen gas and brought up to 1-mL volume with methanol for DDA, and with hexane for the fractions containing DDD, DDT, and DDE. Neutron Irradiation. Glassware, polystyrene, and polyethylene collection vials were all acid-washed with nitric acid and rinsed with distilled deionized water to remove possible radioactivable contaminants. Irradiation was done in the Omaha VA Medical Center TRIGA Mark I Reactor operating at a thermal-neutron flux of 1X 10l1neutrons cm-2s-'. The samples were irradiated in the rotary specimen rack of the facility for 140 min and allowed to decay for 5 min before conventional radioassay. Radioassay. y-Ray spectrometry was conducted with a 60-cm3 coaxial lithium-drifted germanium detector (Princeton Gamma Tech) and a Nuclear Data ND 600 4096-channel analyzer, or two each 3 in. X 3 in. NaI(T1) detectors in parallel and an ND 2400 2096-channel analyzer. The Ge(Li) system had a resolution of 2.0 kV (fwhm), a peak-to-Compton ratio of 37:1, and a relative peak efficiency of 11.3% for the 1.332 MeV y of 6oCo. Mass Spectrometric Conditions and Instrumentation. Solid probe analysis of DDA was performed on a Kratos MS-50 (Manchester, England) double-focusing high-resolution mass spectrometer. The instrument was operated in the electron-impact mode, with a source temperature of 250 "C and an electron energy of 70 eV. Samples were introduced via solids inlet probe and volatilized by heating the probe. The molecular ion peaks at m/z 280 and m/z 282 were monitored via a multiple ion monitor peak switch unit (11). The instrument was operated at a resolution of 20 000 to avoid interference peaks resulting from sample contamination. Data for these two ions were stored in a Nicolet (Madison, WI) signal averager and processed by a computerized quantitative analysis program written to operate in the Kratos DS-55 data system. DDD, DDT, and DDE were determined employing a Kratos MS-80medium-resolutionmass spectrometer (ultimateresolution 20 000) equipped with a five-channel multiple peak monitoring (MPM) device. The instrument was directly coupled with a Carlo Erba gas chromatograph operating with a SE-54 fused silica
ANALYTICAL CHEMISTRY, VOL. 55, NO. 4, APRIL 1983
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capillary column (0.25mm X 30 m). The gas chromatograph (GC) was operated employing a helium carrier gas with a linear velocity of -35 m/s, an injector temperature of 250 "C, and a detector temperature of 275 "C. The oven temperature was programmed from 200 "C to 280 "C at 5 "C/min during the analysis. A split injection technique was used. The mass spectrometer was operated in the electron-impact mode, with a source temperature of 250 "C and an electron energy of 70 eV at a resolution of 3000. The peak profiles were acquired at an amplifier bandwidth of 30 000 Hz. MPM was used to monitor the following ions: m / z 317.9352 (molecular ion + 2) for DDE, m/z 237.0052 (fragment ion) for DDD and for DDT as well. The instrument was tuned by using m/z 280.9824 of perfluorokerosene (PFK), which was also used as a check mass during the analysis. The output was recorded on a Linear Model 595 three-pen strip chart recorder.
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Several methods have been developed for the routine determination of DDA excretion levels in urine. Cueto et al. (7) employed a colorimetric procedure after DDA recovery from urine by using an ion exchange resin. The method which has been adopted by the Environniental Protection Agency involves derivatization with boron trifluoride in methanol after a solvent extraction step, ultimately followed by a gas chromatographic separation utilizing 63Ni electron-capture detection. No attempt has been made, prior to the present work, to approach the problem via a liquid chromatographic route. While an electron capture detector may be a thousand times as sensitive as the conventional HPLC ultraviolet detector, neutron activation analysis can be employed as a specific HPLC detector for compounds having radioactivable atoms in their structures. It was our main intent to develop a combined HPLC/NAA procedure for the quantitation of DDA in a urine matrix in the presence of the other metabolites and of DDT itself. The presence of radioactivable chlorine atoms in the structures of DDT and its metabolites makes them eligible for determination by neutron activation. Solvent Extraction Procedure. Solvent extraction is the technique of choice for preparing pesticide samples, prior to chromatographic separation, because of the major advantages it offers, such as concentration of the sample, removal of aqueous radioactivable contaminants (such as sodium and chlorine), and its inherent simplicity. Since our MoNAA procedure involves a radioassay of W 1 activity, it is important that all the reagents and solvents to be used be evaluated for their chlorine content and for other possible radioactivable interferences. Jacobs et al. (12)conducted a study on the trace element composition of some commonly used organic solvents and concluded that HPLC-grade solvents do not necessarily have lower trace element contents than reagent grades. We used instrumental neutron activation analysis as employed by Jacobs e t al. to determine the possible radioactivable elements in all our solvents and reagents. A low but significant 38Clactivity was observed in methanol, thus causing an increase in the detection limit of our MoNAA technique. We did not see such contribution from hexane, glacial acetic acid, and distilled water when subjected to the same analysis. Because it is highly possible for DDA to be excreted in urine as conjugates of amino acids (IO),we incorporated a digestion step into our solvent extraction procedure to evaluate this possibility. We added 9 M sulfuric acid into the raw or spiked urine and performed a digestion, at ambient temperature prior to the solvent extraction step, by shaking the mixture in an automatic shaker for 30 min. However, the sulfuric acid digestion resulted in a gross enhancement in the DDA recovery, with concomitant decrease in the yields of DDT, DDD, and DDE. Apparently the sulfuric acid digestion facilitates the conversion of DDT and lower metabolites to DDA. Since we were using a reversed-phase HPLC, it was necessary to evaporate the hexane layer to dryness, using nitrogen gas, and
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Table 11. Efficacy of the Method amt expected? MoNAA result, MS result,b d m L Pg/mL CI g/mL DDA 0.57 0.51 (88.8%)' 0.52 (91.0%)' DDD 4.10 3.76 (91.6%) 3.77 (92.0%) DDT 1.49 1.29 (86.7%) 1.20 (80.5%) DDE 1.78 1.70 (95.4%) 1.68 (94.4%) a The number of micrograms of compound per milliliter of urine. The mass spectrometry result for DDA was' obtained by using the solid probe technique, while results for DDD, DDT, and DDE were obtained by GC/MS. Mass spectrometric conditions are given in the Experimental Section. ' The recoveries have been found to be reproducible within 5% variation and independent of the trace molecular concentrations in the range studied. Yields can be determined by employing spiked samples. then dissolve and adjust the residue to volume with the HPLC elution solution so that it was ready for injection into the liquid chromatograph. Extraction recovery of the chlorinated compounds was investigated by doing solvent extraction on urine spiked with DDT and its metabolites in the sub-parts-per-million range. The combined extracts (hexane layer from the fiist and second extractions) were irradiated with neutrons and radioassayed for 38Clactivity. Extraction recoveries obtained for all the compounds were nearly 100%.
High-Performance Liquid Chromatography Separation Procedure. Table I shows the retention times of DDT and its metabolites, as well as the mobile phases and flow rates employed. Both normal and reversed-phase elution modes were considered and evaluated. The Partisil 10/C8 column afforded poor resolution for the polychlorinated compounds. For all compounds and conditions, the chloride ion eluted almost immediately and was separated before the appearance of any of the polychlorinated compounds. The Partisil5-ODS column gave the best separation for DDT and its metabolites with the solvent system consisting of 60 + 20 + 20 (v/v) methanol-water-acetic acid. DDA contains a carboxyl group and undergoes a normal chemical equilibrium based on ionization. Separations of ionizable species in conventional reversed-phase HPLC would ordinarily result in poor retention and peak tailing. However, by suppressing the ionization, that is, by having a mobile phase with a low pH of about 2 to 3, retention would be increased and peak symmetry improved (12-15). The acetic acid (pK, = 4.76) in the solvent mixture served this purpose. The reproducibility of the separation was also enhanced by the presence of acetic acid in the mobile phase. Because of the multiplicity of the polychlorinated biphenyls (PCBs) and other related pesticides, such as 2,4D, the mere separation of the DDT and its metabolites may not be sufficient as a criterion for their identification. Polychlorinated biphenyl mixtures, Arochlor 1254 and Arochlor 1242, interfere sufficiently with the HPLC separation of DDT, DDE, and DDD but not of DDA. However, 2,4D or any other substituted acetic acid analogue will interfere with the determination of DDA without any previous derivatization or separation employing Florisil. An ultraviolet detector (UV) with a fixed wavelength at 254 was used on all chromatographic runs. It can be seen from Figure 2 (upper figure), which illustrates the UV detector response at 254 nm with respect to the elution volume containing urine from the solvent extraction procedure discussed in the previous section, that human urine contains compounds, probably amino acids, which would interfere in the determination of DDA. It would then be a difficult task to distinguish between these compounds and DDA just by relying on the UV detector response; hence, it becomes necessary to use neutron activation analysis as a detection device in lieu of the built-in UV detector. DDT
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I I Flgure 3. Selected ion high-resolution (R = 20 000) mass spectra for 100 ng of DDA. Each scan window is 300 ppm wide. and its metabolites contain chlorine (24.23% chlorine-37), which when irradiated with neutrons in the nuclear reactor produces 38Clwith a half-life = 37.3 min) and radioemanations suitable for y-ray spectrometry. Neutron activation of HPLC fractions where DDT and its metabolites elute is highly specific and sensitive in analyzing for the %C1activity. Illustrated in Figure 2 (upper figure) is a typical HPLC chromatogram of 1000 ppb spiked DDT, DDD, DDE, and DDA in an urine matrix. The dotted lines represent the chromatogram of a blank urine sample, showing a peak which is not DDA but probably due to amino acids in urine. The radioassay responses of the chlorinated compounds are shown in Figure 2 (lower figure). Mass Spectrometry. Confirmation analysis for DDA was performed employing a double-focusing high-resolution mass spectrometer monitoring molecular ion peaks at m/z 280 and m / z 282 (Figure 3) at 20000 resolving power. Solid probe analysis may also be performed on DDE, which gives intense molecular ion peaks at m/z 318 and m/z 320. However, DDT is expected to interfere because it gives small fragment ions in the region of m / z 320. DDD and DDT have very similar electron-impact fragmentation patterns, both giving significant fragment ions at m / z 235 and no molecular ion. Therefore, the distinction between DDD and DDT could not be made by using the solid probe technique. However, these three chlorinated compounds, DDD, DDT, and DDE, could be distinguished based on their GC retention data. By employing gas chromatographic mass spectrometry, the molecular ion peaks of DDE could be monitored without any interference from DDT. Also, DDD and DDT could now be monitored at m/z 235. The gas chromatograms of DDD, DDT, and DDE are shown in Figure 4. Detection Limits. Detection limits afforded by the MoNAA procedure are quite dependent on the neutron flux of
ANALYTICAL CHEMISTRY, VOL. 55, NO. 4, APRIL 1983
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Table IV. Limits of Detection by Mass Spectrometry limit,O w/mL of urine SINb resolution DDA 0.35 2:l 20 000 (solid probe technique) 0.003 2:l 3 000 DDD (GC/MS) DDT (GC/MS) 0.013 2:l 3 000 0.001 2:l 3 000 DDE (GC/MS) a For this limit of detection, 20 mL of urine is extracted into the n-hexane solvent according to the procedure described in the Experimental Section. b Signal to noise ratio.
Figure 4. GClMS selected ion chromatograms of the chlorinated compounds DDD, DDT, and DDE using a three-pen strip chart recorder. Peaks shown correspond to DDE, (1) and (2); DDD, (3); and DDT, (4) and (5). The masses monitored were mlz 317.9352 for DDE and mlz 237.0052 for DDD and DDT.
Table 111. Limits of Detection by Molecular Neutron Activation Analysis limit, ng/mL of urine“ Ge(Li) NaI(T1) detectorb detector 1 x 10l2thermal-neutrons cm-’ s-’ DDA 10.6 2.1 DDD 7.0 1.4 DDT 6.0 1.2 DDE 6.4 1.3 1 X 1013thermal-neutrons cm-a s-’ DDA 1.1 0.2 DDD 0.7 0.1 DDT, DDE 0.6 0.1 1 x 1014 thermal-neutrons cm-2 s-l DDA 0.11 0.02 DDD 0.07 0.01 DDT, DDE 0.06 0.01 a Detection limit-minimum detectable concentrations that will give a signal exceeding critical level 95% of the time. In comparing the efficiencies of the two detectors, it was found that the Ge(Li) detector is 19.98% as efficient as the NaI(T1) crystal for T 1 activity. the nuclear reactor. Presented in Table I11 are the limits of detection for the reactor procedure employing both Ge(Li) and NaI(T1) detectors. It is quite apparent from inspection of Table I11 that for the determination of chlorinated pesticides in urine in the parts-per-billion range a nuclear reactor with a neutron flux of at least 1 X 10l2neutrons cm-2 s-l must be employed. By employing urine samples that have been spiked with sub-parts-per-million concentrations of the chlorinated compounds, we found, in general, relative standard deviations of about 2~5%.This study demonstrates the feasibility of nuclear activation as a specific HPLC detector for compounds having radioactivable atoms in complex biological samples such as urine or serum. Presented in Table IV are the limits of detection of the chlorinated pesticides employing mass spectrometry. The
same procedure leading up to neutron activation and radioassay was employed in preparing samples for mass spectrometry. As can be seen in Table IV, the limits of detection are sufficiently low to allow routine analysis for any of the chlorinated pesticides by the described procedure. It appears that the mass spectrometry procedure would allow routine analysis of urine samples processed by the solvent extraction-HPLC steps.
ACKNOWLEDGMENT The assistance of N. C. A. Weerasinghe with the GC/MS analysis is greatly appreciated. Registry No. DDT, 50-29-3; DDA, 83-05-6; DDD, 72-54-8; DDE, 72-55-9.
LITERATURE CITED (1) Buslness Week June 12, 1978, p 152. (2) Florentlna. D. H.; De Graeve, J.; Grogna, M.; De Wiest, F. J. Chromaf o p . 1978, 157, 421. (3) Durham, W. F.; Armstrong, J. F.; Quinby, G. E. Arch. Environ. Health 1965, I f , 76. (4) Laws, F. R.; Curiey, A.; Biros, F. J. Arch. Envlron. Health 1967, 15. 766. (5) Wolfe, H. R.; Durham, W. F.; Armstrong, J. F. Arch. Envlron. Health 1970, 27, 711. (6) Cranmer, M. F.; Carroll, J. J.; Copeiand. M. F. Bull. Envlron. Confarn. Toxlcol. 1969, 4 , 214. (7) Cueto, C.; Barnes, A. G.; Mattson, A. M. J. Agrlc. FoodChern. 1956. 4 . 943. (8) Guiochon, G.; Arpino, P. J. Anal. Chem. 1979, 5 1 , 682A. (9) Firouzbakht, M. L.; Garmestani, S.K.; Rack, E. P.; Biotcky, A. J. Anal. Chem. 1981, 5 3 , 1746. (IO) Enos, E. F.; Biros, F. S.;Gardner, D. T.; Wood, J. P. Presented at the 154th National Meeting of the American Chemical Society, Chicago, IL, Sept 10-15, 1967, Ag. and Food Section. (11) Crow, F. W.; Giblin, D. E. Presented at the 29th Annual Conference on Mass Spectrometry and Allied Topics, ASMS, Minneapolis, MN, May 24-29, 1981. (12) Jacobs, F. S.;Ekambaram, V.; Filby, R. H. Anal. Chern. 1962, 54, 1240. (13) Horvath, C.; Melander, W. J. Chrornafogr. Sci. 1977, 9, 393. (14) St. Claire, R. L., 111; Ansarl, G. A. S.;Abell, C. W. Anal. Chern. 1982, 54, 186. (15) Majors, R. E. “High Performance Llquld Chromatography, Advances and Perspectives”; Horvath, C., Ed.; Academic Press: New York, 1980; Vol. I, p 95.
RECEIVED for review November 10, 1982. Accepted January 3, 1983. This research was supported by the Division of Chemical Sciences of the U.S.Department of Energy (Contract No. DE-A502-76ER01617.OAOO4), Medical Research Service of the Veterans Administration, and a University of Nebraska Research Council NIH Biomedical Research Support Grant.
