2037
Anal. Chem. 1980, 52, 2037-2044
Determination of Parts-per-Tr iII ion Concentrations of Tetra- , Hexa-, Hepta-, and Octachlorodibenzo-p-dioxins in Human Milk Samples Marsha L. Langhorst' and
L. A.
Shadoff
Analytical Laboratory, 574 Building, Dow Chemical U.S.A., Midland, Michigan 48640
A method has been developed and validated for the determination of parts-per-trillion levels of polychlorlnated dlbenzspdloxh In human breast milk samples. The procedure Involves acidic sample dlgestlon, extraction wlth an organic solvent, multiple cleanups wlth adsorbents and chemically modlfled adsorbents, two high-performance llquld chromatographic cleanups, and flnally detection by multipie-ionmode gas chromatography/mass spectrometry. The procedure fractionates 2,3,7,8-tetrachlorodlbenzo-p-dioxin (2378-TCDD) for separate Isomer-speclflc determlnatlon and specifles conditions for determining 18 other TCDD Isomers. Hexachlorodlbenzo-p-dioxin isomers (HCDD), heptachlorodibenzo-pdloxln isomers (H,CDD), and octachlorodlbenro-pdloxln (OCDD) are also determined. I3C-labeled dioxins were used as Internal standards and carrlers. The procedure has been valldated at levels between 1 and 12 ppt (parts per trllllon) for 2,3,7,8-TCDD and at higher parts-per-trillion levels for HCDD, H7CDD, and OCDD.
A method is described for the determination of tetrachlorodibenzo-p-dioxins (TCDDs), hexachlorodibenzo-p-dioxins (HCDDs), heptachlorodibenzo-p-dioxins(H7CDDs),and octachlorodibenzo-p-dioxin(OCDD) in human milk samples with concentrations in the low parts-per-trillion range. The procedure is an extension and modification of the methodology for dioxins in fish and particulates previously reported (2, 12, 13). This methodology differs by eliminating the use of a caustic digestion which has been shown to affect the stability of higher chlorinated dioxins-HCDD, HTCDD and OCDD (12). One of the most important features of this technique is t h a t it employs a multistep clean-up procedure which eliminates interferences from 106-fold excess concentrations of commonly observed environmental containinants. In addition, by the application of three different forms of chromatography, whose mechanisms for achieving separation involve significantly different parameters, 2,3,7,8TCDD can be determined isomer specifically from the other 21 TCDD isomers (16).
T h e formation, occurrence, persistence, and transport properties of polychlorinated dibenzo-p-dioxins (CDDs) are of interest. For investigation of the bioaccumulation of CDDs in biological and environmental matrices, analytical methodology was developed to determine dioxin residues a t picogram levels. Much of the work reported in this area prior t o 1974 is summarized in ref 1. More recently, a number of methods utilizing gas chromatography/mass spectrometry (GC/MS) have been developed and applied to the determination of dioxins a t parts-per-trillion (ppt, lo-'* g/g) levels in environmental and biological samples (2-10). O'Keefe, Meselson, and Baughman (11) have described a neutral clean-up procedure for the determination of 2,3,7&tetrachlorodibenzop-dioxin (2,3,7,8-TCDD) in bovine fat and milk. These procedures are all limited t o samples not containing highly lipophilic matrices and some depend heavily upon the resolving capabilities of GC/MS instrumentation. A different approach has been to utilize designed multistep chromatographic cleanups t o achieve improved dioxin specificity even with the use of low-resolution GC/MS (12, 13). Recent claims have charged t h a t 2,4,5-T, or its trace contaminant 2,3,7,8-TCDD, is responsible for an abnormally high incidence rate of miscarrages in women residing near coniferous forests in the Pacific northwest where the herbicide is used. This has spawned an interest in determining whether dioxins are accumulating in humans. The most convenient samples of human origin are urine, blood, and breast milk. Because of the high lipid content of mother's milk, a medium likely t o accumulate any dioxins if present, human milk samples are being studied. A recent paper reviews the results of previous studies conducted to determine 2,3,7,8-TCDD in human milk samples ( 1 4 ) . Most recently an Environmental Protection Agency study was completed which found no detedable residues of 2,3,7,8-TCDD in 103 samples of milk from nursing mothers in three western states (15).
Reagents. A modified sweep codistillationtechnique (17)using methylene chloride saturated nitrogen is used to effectively remove trace contaminants from adsorbents. The preparation of 44% sulfuric acid on silica, 10% silver nitrate on silica, and basic alumina and the purification of nitrogen for use in the evaporation of solvenh for trace analysis have been previously described (12). For identification purposes, this purified nitrogen has been termed FEMTOGAS. Silica. Prepare this reagent from chromatographic grade silicic acid purified and activated as described for the preparation of 44% sulfuric acid on silica (12). 22% Sulfuric Acid on Silica. Prepare and store the same as 44% sulfuric acid on silica, except adjust the weight of the concentrated sulfuric acid to obtain a material with an acid concentration of 22% based upon the total weight. 33% 1 M Sodium Hydroxide on Silica. Prepare the same as 44% sulfuric acid on silica, except substitute 1M aqueous sodium hydroxide for concentrated sulfuric acid and prepare material to be 33% by weight 1 M NaOH. This reagent is not stored in a desiccator. Solvents and Chemicals. All solvents are Burdick and Jackson, distilled-in-glass quality, which have been tested through the procedure to verify the absence of contamination. Laboratory chemicals--H2SO4,NaOH, HC1, and AgN0,were ACS reagent grade (J. T. Baker) and are also checked for contamination. Dioxin Standards. The primary standard of 2378-TCDD was prepared by W. W. Muelder (Dow Chemical Co.) and its structure was confirmed by single-crystal X-ray diffraction techniques. Purity was assessed at 98% by mass spectrometry. Standards of other TCDD isomers were synthesized and isolated aa previously described (16). Primary standards of 1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin (1234678-H,CDD) and OCDD were synthesized by H. G. Fravel and W. W. Muelder (Dow Chemical Co.). A standard containing two HCDD isomers was synthesized by Aniline (18). Standards of 1234679-H7CDDand the 10 HCDD isomers were synthesized and isolated in a manner similar to that reported for TCDDs (16). Isotope-enriched 13C-2378-TCDDand I3C-123478-HCDDwere synthesized by A. S. Kende (University of Rochester, Rochester, NY). Mass spectrometric analysis indicated these standards to be 86 atom % 12Cand 43 atom % 13C,
EXPERIMENTAL SECTION
0003-2700/80/0352-2037$01.00/00 1980 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 52, NO. 13, NOVEMBER 1980
respectively. Perchlorination of the 13C-2378-TCDDprovided 13C-OCDD. Apparatus. Reverse-Phase HPLC. The reverse-phase liquid chromatographic clean-up system consists of an Altex Model 110 pump, Rheodyne Model 7120 injector valve with 50-yL loop, two 250 mm X 6.2 mm (0.d.) DuPont Zorbax ODS columns in series, and a Perkin-Elmer LC-65T variable wavelength detector operated at 235 nm. Use methanol as the mobile phase at 1.9 mL/min and operate the LC column oven at 50 "C. Sensitivity is 0.01 absorbance units full scale (AUFS). Silica HPLC. The silica liquid chromatographic clean-up system consists of an Altex Model 110 pump, Rheodyne Model 7120 injector valve with 100-yL loop, two 250 mm X 6.2 mm (0.d.) DuPont Zorbax SIL columns in series, and a Perkin-Elmer LC-65T variable wavelength detector operated a t 235 nm. Use hexane as the mobile phase at 1.7 mL/min and operate the LC column oven at 31 "C. Sensitivity is 0.008 AUFS. Gas Chromatography/Mass Spectrometry. The GC/MS determinations were performed on a Kratos MS-30 GC/MS with multiple-ion-monitoring capabilities. The instrument was modified by replacing the membrane separator with an all-glass single-stagejet separator (SGE). The GC column was a 2 m X 2 mm i.d. glass column packed with 0.60% OV-17 silicone/0.40% Poly S-179 on a specially activiated Chromosorb W-AW, 80/100 mesh support (a recent development of The Dow Chemical Co. which has been licensed to HNU Systems, Inc.). A flow rate of 30 cm/min helium carrier gas was used. Column operating temperatures were 230 "C isothermal for TCDD analyses, 260 "C isothermal for HCDD, and 275 "C for H&DD and OCDD. Operating parameters for the Kratos MS-30 mass spectrometer are as follows: trap current = 300 yA; multiplier gain = 90% of full; electron energy = 34 eV; mass spectrometer reaction = 1OOO. The analysis of TCDD is achieved by monitoring the ions m/e 320 and m/e 322 for the molecular ion cluster of native TCDD and m/e 335 from the molecular ion cluster of 13C-2,3,7,8-TCDD. For higher chlorinated dioxins, the followng ions were monitored: native HCDD, m/e 388, 390; W-HCDD, m/e 396, 397; native H,CDD, m/e 422,424; native OCDD, m/e 458,460; 13C-OCDD, m/e 472. Procedure. Sample Preparation and Initial Cleanups. The sample preparation procedure consists of (a) sample digestion and extraction to remove the bulk of the sample matrix and to transfer the dioxins residue fraction into a suitable solvent, (b) removal of lipids through reaction with oxidizing reagents (c) separation of dioxins from common chemical interferences (PCBs, DDE), and (d) two high-performance liquid chromatographic (HPLC) cleanups to provide additional removal of contaminants (PCBs, DDE, phthalates) and to remove compounds very similar to dioxins (chlorinated benzyl phenyl ethers) (16, 19). Weigh a 30-g portion of human breast milk (shaken to homogenize) into a clean glass bottle and spike with isotopically labeled internal standards. Add 5 ng of [ 13C]-2,3,7,8-tetrachlorodibenzo-p-dioxin and 1ng of [ 13C]~ta~hlorodibem~p-dioxin. Add 200 mL of concentrated HC1 and shake with a mechanical shaker for 1h. Add 100 mL of hexane and return to the shaker overnight (approximately 20 h) at ambient temperature. The next day, remove from shaker and let stand -5 min or until layers have separated. Occasionally there will be a foam between the layers. Addition of several drops of distilled deionized water will break the foam and allow clean separation of the layers. Pipette/transfer the hexane layer to an 8-oz bottle. Add 70 mL of hexane to the digestate and return to the shaker for an additional 2 h. Pack the first set of gravity-flow liquid chromatographic clean-up columns consisting of (1) a 2 x 22 cm glass column packed with 5 g of 22% HzS04on silica under 1 g of silica gel and (2) a 2 X 22 cm glass column packed with 1g of silica under 2 g of 33% 1 N NaOH on silica under 1g of silica under 4 g of 44% H2S04on silica under 1.5 g of silica (see Figure 1). The freshly packed columns are prewashed with 30 mL of hexane and the effluent is discarded. The hexane extract, -100 mL, is passed through the column and the effluent run directly into the second column. When hexane has drained to the bed levels pipette the second hexane extract (-70 mL) from the digestion bottle onto column 1. Discard the acid digestate. When the extract has again drained to bed level, rinse the columns with an additional 15 mL of hexane and 2 times 10 mL of hexane washes. Collect the total
20mm
2Omm
-A
-B
Flgure 1. Glass chromatography columns for sample cleanups: A = silica. B = 22% suifuic acid on silica, C = 44% sulfurlc add on sfflca, D = 33% 1 M sodium hydroxide on silica, E = 10% AgNO:, on siHca.
and F = basic alumina.
column effluent (-205 mL) in a 250-mL flat-bottomed flask. Add boiling stones and evaporate sample volume to -20 mL using a six-stage Snyder column and heating mantle. The H$304/silica columns remove the bulk of the lipids and other oxidizable components from the hexane extract. The 22% H2S04/silica column is a less effective reagent than the 44% H2S04/silicabut is less prone to plugging or reduced flow. The combination of these reagents is quite successful. The NaOH/silica reagent neutralizes the acid to prevent charring during solvent evaporation in the flask. Pack the second set of gravity-flow liquid chromatographic clean-up columns consisting of (1)a 1 x 10 cm glans column packed with 1.5g of 10% &NO3 on silica and (2) a 6 X 35 cm glass column packed with 5.0 g of basic alumina (see Figure 1). The freshly packed AgN03/silica column is prewashed with 15 mL of hexane and the effluent discarded. Add the 20mL hexane sample extract to the top column and allow this to drain to bed level. Wash the flask and columns with 2 times 5mL of hexane, allowing each to drain to the bed of the top column. Add 30 mL of hexane through these columns. Let all the effluent flow into a 150-mL beaker. Next discard the top column. Elute the basic alumina column with 50 mL of 50% (v/v) carbon tetrachloride in hexane into the waste beaker. Discard the waste solvent. Finally, elute the dioxins from the basic alumina column with 22 mL of 50% (v/v) methylene chloride in hexane, collecting the eluate in a small vial. This column system separates dioxins from common environmental residues such as DDE, chlorinated aliphatic hydrocarbons, sullides, and PCBs (12). The final 22 mL of CH2Clz/hexane fraction contains all 22 TCDD isomers, all 10 HCDD isomers, both H F D D isomers, and OCDD if they are present. Evaporate the methylene chloride/hexane eluate to dryness and transfer the residue (generally not visible) to a 300-yL conical vial with a small volume of 40% methylene chloride in hexane. Again evaporate to dryness. HPLC Cleanups. The next step in the clean-up scheme is a reversed-phase HPLC separation. This is a clean-up step and
ANALYTICAL CHEMISTRY, VOL. 52, NO. 13, NOVEMBER 1980
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1
12.3.7.3- I S O r i
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COLLECTION ZONE CALIBRATION STANDARD
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2 3.7.8-TCDD
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8
10
12
14
16
1 . 2 . 3 . 6 . 7 9 - o r 1 2.3.6.89-hexaCDD
18
20
22
24
26
30
28
B.
M I L K SAMPLE
MILK SAMPLE 2378 1
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26
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28
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Figure 3. Silica HPLC chromatograms: A = collection zone calibration standard; B = typical human milk sample. Brackets represent dioxin collection zones.
30
Figure 2. RPLC chromatograms: A = collection zone calibration standard; B = typical human milk sample. Brackets represent dioxin collection zones.
is not used to quantitate dioxins even though a UV detector is used to monitor the column effluent. It is generally not sensitive enough to detect dioxins in milk samples but can detect the 5 ng of isotopically labeled 13C-2,3,7,8-TCDDused in the internal standard. The residue is quantitatively injected into the RPLC in the following manner. Place 16 pL of chloroform into the conical vial and allow to stand about 10 min. Using a 50-pL syringe, draw in 4 pL of methanol and a 2-pL air bubble plug. Then draw the residue solution carefully into the sytringe until an air bubble appears in the barrel. Place a second 16-pL aliquot of chloroform into the vial, rotate quickly for rinsing and draw into the syringe. Inject the total volume into the liquid chromatograph. Collect chlorinated dioxins fractions from the HPLC according to previously determined zones from a calibration standard. The calibration standard contains 10-20 ng of each dioxin to be determined, injected in no more than 30yL of chloroform. Collect four fractions: (a) 13.8 -15.0 min for 2,3,7,8-TCDD; (b) 12.6-13.8 min and 15.0-17.2 min for the other TCDD isomers; (c) 18.4-22.6 min for the HCDD isomers; (d) 23.0-25.8 rnin for the two H&DD isomers and 28.0-30.1 min for OCDD (See Figure 2). To collect fractions, allow the methanol effluent to drain directly into a 25-mL volumetric flask which was previously charged with approximately 1 mL of hexane. After collection, mix the solvents by swirling and add sufficient aqueous 2% sodium bicarbonate to raise the organic layer into the neck of the volumetric flask. Transfer the hexane layer to a small vial. Extract the remaining aqueous layer three more times with approximately 1ml of hexane each, shaking vigorously. Combine the extracts and evaporate to dryness under prepurified nitrogen. The final step is a silica HPLC separation to provide further isomer resolution of 2,3,7,8-TCDD. This step is only used for the 2,3,7,8-TCDD fraction collected off the RPLC. Transfer the residue drom the RPLC 2,3,7,8-TCDD fraction to a 3WpL conical vial with a small volume of 40% methylene chloride in hexane and evaporate to dryness. Calibrate the silica LC system using approximately 10 ng of 2,3,7,8-TCDD to establish the correct collection zone. The 2,3,7,8-TCDDretention time will drift slightly to shorter retention
times as small amounts of water present in the hexane eluent changes the activity of the silica. The absolute retention time for 13C-2,3,7,&TCDDby silica HPLC should be between 12.5 and 17.0 min to use the following fraction collection scheme (16). Inject a blank solvent to clean the valve. Then analyze the reagent blank sample, followed by the set of milk samples. To do this, dissolve and inject the entire residue sample as described for the RPLC cleanup except use 40 pL of hexane plus 30 pL of hexane. Collect two fractions of effluent from the silica LC in small vials: (a) for 2,3,7,8-TCCD collect from 0.90 to 1.05 times the absolute retention time of 2,3,7,&TCDD; (b) for other TCDD isomers, collect from 1.05 to 1.45 times the absoluate retention time for 2,3,7,&TCDD (see Figure 3). Evaporate these fractions to dryness under prepurified nitrogen. Combine the TCDD isomer fractions (“iso” fractions) from the RPLC cleanup and the silica HPLC cleanup. Transfer all four fractions (a) 2,3,7,8-TCDD, (b) TCDD isomers (contains quantitatively 18 other TCDD isomers, not 2,3,7,8-TCDD), (c) HCDD isomers, and (d) H7CDD isomers and OCDD to 300-pL conical vials. Dissolve the residues in 10 pL of isooctane approximately 10 min prior to analysis by GC/MS. Additional details of method design and development are comprehensively described (12). The HPLC steps have been designed by using Zorbax ODS and Zorbax SIL columns. Any column must be calibrated with the dioxin standards to be determined to verify collection zones and chromatographic conditions. The two HPLC steps are effective in removing the potential interferences (12) and in providing isomer specificity for 2378-TCDD (16). Sample Determination and Calculations. After dissolving the residue and rinsing the vial walls, inject 5 pL into the GC/MS. For 2,3,7,8-TCDD and TCDD isomer (“iso”) fractions, monitor m / e 320, 322, and 335 (see Figure 4). For HCDD fractions, monitor m l e 388, 390, 396, and 397 (see Figure 5). For the H,CDD/OCDD fraction, monitor m / e 422 and m / e 424 until H7CDD elutes. Then adjust the multiple-ion-monitor to follow m / e 458, 460, and 472 until the OCDD elutes (see Figure 6). Calibrate the instrument by injecting standards of native dioxins (10 ppb each). To calibrate the recovery determinations, use 13C-labeleddioxin standards at concentrations equal to the levels added to samples. Calculate the apparent dioxin concentrations by using eq 1, dioxin concn (ppt) =
BW[(DE)/ (FG)I
x 103
(1)
2040
ANALYTICAL CHEMISTRY, VOL. 52, NO. 13, NOVEMBER 1980
Table I. Dioxin Determinations in Control Milk Zomogenate 2,3,7,8-TCDD iso-TCDD HCDD H,CDD concn, pptbse OCDD % recov- concn,b,e concn/'e % recov- 1,2,3,4,6,7,8- 1,2?3,4,6,8,9- concn/ie % concn,a,e PPt recovery isomer isomer no. PPt ery PPt PPt eryC 1.2 (0.5) 68 nd (0.2) 1.3 (0.2) 2.5 (0.3) 90 31 nd (0.2) 1 0.5 (0.5)d 1.1(0.5) 76 0.6 ( 0 . 2 ) 1.4 (0.2) 1 5 (0.9) 29 2 0.6 (0.5) 37 nd (0.3) nd (0.2) 1.0 (0.2) 5.3 (0.4) 57 1.4 (0.4) 82 3 0.5 (0.4) 41 nd (0.2) 1.3 (0.2) 7.7 (0.5) 50 0.6 (0.2) 106 0.4 (0.2) 4 0.6 (0.4) 45 nd(0.2) 1.1 (0.4) 74 0.2 (0.2) 0.9 (0.2) 5.6 (0.5) 42 5 0.7 (0.7) 24 nd(0.3) 1.0 (0.3) 101 nd (0.2) 0.9 (0.2) 5.2 (0.4) 54 6 0.7 (0.6) 34 nd(0.3) 0.6 (0.4) 1.0 (0.8) 3.5 ( 2 ) 83 75 nd (1.2) 7 0.4 (0.4) 40 1.2 (0.5) 7.7 (1.1) 42 1.3 (0.5) 60 nd (0.5) 8 0.3 (0.2) 37 nd (1.9) 34 nd (0.7) 1.2 (0.3) 64 nd (0.3) 1.6 (0.3) 6.5 (0.7) 50 9 0.4 ( 0 . 2 ) av 0.5 (0.4) 36 nd (0.3) 1.0 (0.4) 78 1.2 (0.3) 6.0 (0.7) 55 std dev k O . 1 +1 k0.3 * 16 c 0.4 k3.0 k 20 a Corrected for internal standard recovery. Absolute concentration of dioxin observed, not corrected for internal standNumbers in parentheses are the LOD. ard recoveries, % recovery refers to recovery of 13C-labeled internal standards. e Numbers are not corrected for dioxin concentrations found in reagent blanks (see Table IV).
5 el 10 ppb hexaCDD STANDARD
I
h
-
xl
X l
xl
REAGENl BLANK
322
X l
REAGENT BLANK
320
4 2
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2.3.7.8-TCDD 4
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388
C o / ~ ~ ~ I T E
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CONTROL
322
4
Xl
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Flgure 4. GC/MS monitoring of native 2,3,7,8-TCDD at m / e 320 and 322 and 13C-2,3,7,8-TCDDat m l e 335.
where A = the peak height of native CDD in the sample, B = the peak height of native CDD in the standard, C = the concentration of the standard, ng/mL, V = final volume of sample extract, mL, W = initial sample weight, g, D = peak height of the internal standard in the sample, E = the concentration of internal standard in the standard solution, ng/mL, F = the peak height of the internal standard in the standard solution, and G = the weight of the internal standard added to the sample divided by the final volume of the sample, ng/mL. The term enclosed in brackets [(DE)/(FG)] X 100% is the recovery of the internal standard that had been added to the sample before workup. Calculate the instrumental limit of detection (LOD) by using the equation shown above, except replace the value for A with 2.5N, where N is the peak-to-peak instrument noise. Calculate the dioxin concentrations for each ion separately and report the average of the two values for each component.
CONTROL COMPOSITE MILK
-
1.2.3.6.7.8- hexaCDD 4
3
2
1
I
0
Flgure 5. GC/MSmonitoring of native HCDD at m l e 388 and 390 and I3C-HCDD at m / e 396 and 397.
RESULTS AND DISCUSSION Validation Data. T h e method was validated by determining the precision and accuracy on human milk samples fortified with known concentrations of TCDD, HCDD, H&DD, and OCDD, and the accuracy was studied as a function of concentration. T h e validation range was determined by analyzing nine portions of composited control milk homogenate. T h e milk samples were obtained on a voluntary basis from nursing mothers. Table I shows the results of replicate analyses of the control composite milk. Notice that concentrations and LODs for 2,3,7,8-TCDD, HCDD, and OCDD are corrected for
ANALYTICAL CHEMISTRY, VOL. 52, NO. 13, NOVEMBER 1980 mie 472
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424
422
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A X1 Xl
424
Xl
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REAGEYl BLANK
Amount Added ippt!
A
Flgure 7.
I
Statistical treatment
of
validation data
for 2,3,7,8-TCDD.
HEXACHLDRODIBENZD-p-DlOXlN
r
/
CONTROL COMPOSITE MILK
424
422
Xl
I 7
I 6
I 5
I 4
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I 0
monitoring of native H CDD at m l e 422 and 424, at m l e 458 and 460 and ‘%-OCDD at m l e 472.
Flgure 0. GClMS
native
OCDD
internal standard recoveries and levels for iso-TCDDs and H7CDDs are not corrected. Because detectable (but unconfirmed) amounts of 2,3,7,8-TCDD were found in the control milk homogenate composited for the validation study, the lower end of the validation range was limited. For 2,3,7,8TCDD a reasonable lower limit for validation was calculated by the following equation: control concentration + (2.5 X standard deviation of the control concentration). That is, 0.5 ppt + (2.5 X 0.1 ppt) = 0.8 ppt. Likewise, the lower validation limit for HCDD, H7CDD, and OCDD were calculated as 1.9 ppt, 2.4 ppt, and 15 ppt, respectively. For determination of accuracy and precision, portions of the same milk homogenate were spiked with native 2,3,7,8TCDD, 1,2,3,6,7,8HCDD, and 1,2,3,6,7,9-or 1,2,3,6,8,9-HCDD, 1,2,3,4,6,7,8-H7CDD,and OCDD a t various levels and were analyzed acccording to the procedure described. The results obtained from recovery experiments are shown in Table 11. Percent recovery is the recovery of the isotoptically labeled internal standard. For iso-TCDD and H7CDD, where 13Clabeled internal standards were not available, percent recovery is the recovery of the spiked native dioxin not corrected for internal standard recovery. Percent accountability refers to the amount of observed native dioxin corrected for recovery of internal standard compared to the amount of the native dioxin that was added. The precision of the method was demonstrated by analyzing eight spiked portions of composite milk homogenate a t approximately 2.5 times the lower validation limit. These results are also incorporated into Table I1 (no. 8-15). Validation data obtained for other TCDD isomers are shown in Table 111. These data show the difficulty in trying to analyze a t the limit of detection and demonstrate an improved precision a t concentrations much higher than the limit of detection. I t can be noted t h a t the percent accountability of the native dioxin when corrected for internal standard recovery was 78-94% of the fortificaiton level.
Y 0
I
20
10
33
I
I
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40
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Amount Added (ppt!
Flgure 8.
Statistical treatment of validation data
for HCDD.
OCTACH LORODIBEUZO-P-DIOXIN
0
50
100
150
200
250
Amount Added ippt!
Figure 9.
Statistical treatment of validation data for
OCDD.
A statistical treatment of the validation data is shown in Figures 7-9. The heavy solid line is the actual level of native dioxin spiked. The dashed line represents the least-squares fitted line for the level of dioxin found. The shaded area represents the total uncertainty of the determination including the error associated with the least-squares fitted line and the error associated with final recovery of the dioxin from the vial for GC/MS. For 2,3,7,&TCDD, the standard deviation of the least-squares fitted line is h0.45 ppt. The error associated with vial recovery (423%) is superimposed upon the plot as
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ANALYTICAL CHEMISTRY, VOL. 52, NO. 13, NOVEMBER 1980
n n u 0
C C r,
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e 5:
n n ?
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9-
8E
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ANALYTICAL CHEMISTRY, VOL. 52, NO. 13, NOVEMBER 1980
RPLC 11.0 (Minuter)
I
12.0
13.0
16.0 2378-TCDD COLLECTION ZONE
"IS0"TCDD COLLECTION
SILICA HPLC
1379
1378
1
t
1237/1238
1
0.90 1.m Relative T o 2,3,7,8-TCDD L2.3.7.8-TCDD Retention Time = COLLECTION ZONE 0.01 1.00
1247/1248
Packed Column GC Relative To 13c-2.3.7.8
1
t
"IS0"-TCDO
1I
1
1,3,7,8-TCDD
1 2 3
3.3 3.3 3.3
1.6 1.7
II
I
I
-I
1.10
1.20
1.30
HCDDs, H7CDDs, and OCDD. By using multicolumn, multiadsorbent procedures, it is possible to fractionate chlorinated dibenzo-p-dioxins into groups according to their degree of chlorination, separate isomers within a group, and simultaneously separate the CDDs from a wide variety of other species (i.e., polychlorinated dibenzofurans, polychlorinated biphenyls, polychlorinated terphenyls, polybrominated biphenyls, DDTs, and phthalate esters) (12, 26). By use of RPLC, silica LC, and gas chromatography-three different forms of chromatography whose mechanisms for separation involve significantly different molecular parameters-the separation of all 22 TCDD isomers has been documented (26). The procedure applied here separates and recovers 2,3,7,8-TCDD in a fraction by itself. The "iso" fraction, recovers 18 other TCDD isomers. T h e remaining three isomers of TCDD are sacrificed to ensure complete collection of 2378-TCDD. 1,3,6,9-, 1,4,7,8-, and 1,2,6,8- or 1,2,7,9- are lost during the LC collection process (16). The fractionation of the 22 isomers of TCDD by RPLC, silica LC, and GC are diagramed in Figure 10. The RPLC fractionates
1,3,7,9-TCDD
1.3
concn, ppt recovery added found
recovery
3.3 3.3 3.3
39 45 48
%
1.1 1.5
1.6
Absolute concentration of dioxin observed, rected for internal standard recovery. a
not
12361 1239
1.00
70
48 52 39
I 1.50
I
m
no.
1
I
1.40
0.90
Table 111. Validation Data for Other TCDD Isomersa concn, ppt added found
If
2.3.7.8TCDD
1378
0.80
0.70
1.30 136911478
*
1379
124611249
I
1.20
I
"IS0"-TCDD COLLECTION
1268112
1 1
11
1.10
l7.0
2043
cor-
an error cone and the total uncertainty is represented by the shaded area. At higher dioxin concentrations, the vial recovery errors are most significant. In addition, reagent blanks were analyzed simultaneously with each set of samples to check laboratory contamination. These data are detailed in Table IV. Analytical Considerations. This work demonstrates the ability t o separate and determine any or all of the TCDDs,
Table IV. Validation Data for Reagent Blanks to Check for Laboratory Contamination 2,3,7,8-TCDD
no. 1
2 3 4 5 6 7 8
9 10
av std dev
concn," PPt nd ( 0 . 5 ) c nd (0.6) nd ( 0 . 5 ) nd (0.1) nd (0.1) 0.1 (0.1) 0.1 (0.1) 0 . 0 9 (0.08) 0.2 (0.2) nd ( 0 . 4 ) nd ( 0 . 3 ) i0.3
HCDD
SO-TCDD
concqb ppt
concn,a ppt
33 31 32
nd(0.3) nd ( 0 . 2 ) nd (0.2)
26
0 . 3 (0.1) 0.07 (0.1)
0.3 (0.3) nd ( 0 . 3 ) nd ( 0 . 2 ) nd ( 0 . 3 ) nd ( 0 . 3 ) nd ( 0 . 3 ) nd ( 0 . 3 )
%
recovery
33 41 41
60 39 37 34 i13
nd(0.09)
nd (0.1) nd(0.9) nd(O.l) nd ( 0 . 2 ) nd (0.2) ~0.1
nd(0.4) 0.2(0.2)
nd(0.4) nd ( 0 . 3 ) kO.1
H,CDD 7%
recovery 69 59 84 85 93 61
77 120 95 103 85 *19
OCDD
early isomer
late isomer
nd(0.2)
nd ( 0 . 2 )
nd(0.4) nd ( 1 . 2 ) 0.7 ( 0 . 2 ) 0.4 (0.2) 0 . 3 (0.2) 0.2 (0.2)
nd(0.8) nd ( 0 . 4 ) nd (0.6) nd ( 0 . 4 ) +0.2
nd ( 0 . 4 ) nd ( 1 . 2 ) 0.7 ( 0 . 2 ) 0.5 ( 0 . 2 ) 0.4 (0.2) 0 . 3 (0.2) nd ( 0 . 8 ) 0.5 ( 0 . 4 ) 0.7 (0.6) nd ( 0 . 4 ) + 0.3
concn," PPt
recovery
2.0 (0.9) 4.3 ( 2 . 4 ) nd ( 5 ) 14 (24)
33 31 39 15
6 (0.9)
34
5.5 ( 2 ) 3.9 ( 2 ) 4.1 ( 1 . 4 ) 9.3 ( 5 ) 5.5 ( 2 ) +4
43 35 29 31 +9
%
16
a Corrected for internal standard recoveries. Absolute concentration of dioxin observed, not corrected for internal standard recoveries. The numbers in parentheses are the LODs.
2044
ANALYTICAL CHEMISTRY, VOL. 52, NO. 13, NOVEMBER 1980
the TCDD isomers into two fractions. One fraction contains 2,3,7,8-TCDD and nine other TCDD isomers and the combined “iso” fraction contains the remaining TCDDs. The RPLC 2,3,7,&TCDD fraction is further fractionated by silica HPLC to obtain an isomer-specific fraction containing only 2,3,7,8-TCDD. Even if the RPLC collection zone is not precise enough to eliminate all of the 1378 and 1379 isomers, the packed column GC separation still guarantees isomer specificity for 2,3,7,8-TCDD as demonstrated in Figure 10. The HCDD, H,CDD, andd OCDD fractions contain all the isomers of these higher chlorinated dioxins (13). The procedure provides excellent removal of potential interferences by the various chromatographic steps. The removal efficiencies of the basic alumina and RPLC steps for polychlorinated biphenyls, l,l-dichloro-2,2-bis(chloropheny1)ethylene (DDE), and polychlorinated benzylphenyl ethers from fish samples has been documented (12). In all cases, it is expected that a 106-foldexcess of these compounds will not interfere with the dioxin determination. The use of isotopically labeled internal standards is important to monitor the recovery of dioxins through the steps of the procedure and, more importantly, to act as a “carrier” for the trace levels of native dioxin. As a residue becomes cleaner and cleaner the possibility for loss of picogram dioxin quantities increases. This work displays the value of a well-designed validation. T h e examination of a t least seven controls, seven replicate samples, and seven spiked samples allows a fairly good evaluation of the method performance and the determination of LOD. In this work, the instrumental LOD was defined as the concentration a t which the measured signal is 2.5 times the noise. The actual limits of the procedure are defined by the dioxin level in control milk and by the statistical treatment of the validation data. Using this multiple step procedure requiring approximately 3 days to complete, it is possible to determine reliable 2,3,7,8TCDD concentrations down to 1ppt. Finally, this methodology has also been applied for the determination of dioxins in a wide variety of other environmental and biological samples. Following the initial digestion
and/or extraction, the clean-up procedures are appropriate for a wide variety of sample types.
ACKNOWLEDGMENT The authors wish to express their appreciation to T. J. Nestrick and L. L. Lamparski for adding definition to this dioxin methodology and to W. B. Crummett for his initiation and continued support for this research. The human milk samples were obtained through the gracious cooperation of M. David Sutton, MD, and his staff.
LITERATURE CITED Lucier, G.. Ed. EHP, Environ. Hsaffh Perspect. 1973, No. 5 . Bumb, R. R.; Crummett, W. B.; CutiQ, S. S.; Gledhiii, R. R.; Hummei, R. H.; Kagei, R. 0.; Lamparski, L. L.; Luoma, E. V.; Miller, D. L.; Nestrkk. T. J.; Shadoff, L. A.; Stehi, R. H.; Woods, J. S. Sclence in press. Hummei, R. A. J. Agrc. FoodChem. 1977, 2 5 , 1049-1053. Pfeiffer, C. D. J. Chromatogr. Sci. 1978, 14, 386-391. Pfeiffer, C. D.; Nestrick, T. J.; Kocher, C. W. Anal. Chem. 1978, 50. 800-804. Shadoff, L. A.; Hummel, R. A. Blomed. Mass Spectrom. 1978, 5 , 7-13. Mahle, N. H.; Higgins, H. S.; Getzandaner, M. E. Bull. Envlron. Contam. Toxicol. 1977, 18, 123-130. Stehi, R. H.: Lamparski, L. L. Sclence 1977, 197, 1008-1009. Lamparski, L. L.; Mahie, N. H.; Shadoff, L. A. J. Agric. Food Chem. 1978, 26, 1113-1116. Eicemen. 0. A., Clement, R. E.; Karasek, F. W. Anal. Chem. 1979, 51, 2343-2350. OKeefe, P. W.; Meseison, M. S.; Baughman, R. W. J. ASSOC.Off.Anal. Chem. 1978, 61. 621-626. Lamparski, L. L.; Nestrick. T. J.; Stehi, R. H. Anal. Chem., in press. Lamparski, L. L.; Nestrick, T. J., Anal. Chem.. in press. Shadoff, L. A. “Abstracts of Papers”, 178th National Meeting of the American Chemical Society, Washington, DC, Sept 1979; American Chemical Society: Washington, DC, 1974. PEST 052. Testimony of M. L. Gross, In re: The Dow Chemical Company, et el.. FIFRA Docket No. 415. et ai., EPA Exhibit No. 223, pp 27-29, 1980. Environmental Protection Agency, Hearing Clerk’s Offlce, 401 N. Street S.W., Washington, DC 20460. Nestrick, T. J.; Lamparski, L. L.; Stehi, R . H. Anal. Chem. 1979, 5 1 , 2273-2281. Storherr, R. W.; Watts, R . R. Assoc. Off. Anal. Chem. 1085, 48, 1154-1 158. Aniline, 0. Adv. Chem. Ser. 1973, No. 120, 126-135. Shadoff, L. A.; Blaser, W. W.; Kocher, C. W.; Fravei, H. G. Anel. Chem. 1978, 50, 1586-1588.
RECEIVED for review March 10, 1980. Accepted August 11, 1980.
