Anal. &em, 1993, 65, 2420-2427
2420
Sample Cleanup by Solid-Phase Extraction for the Ultratrace Determination of Polychlorinated Dibenzo-pdioxins and Dibenzofurans in Biological Samples Ruth R. Chang,'tt Walter M. Jarman3 and John A. Henningsg9ll Hazardous Materials Laboratory, Department of Toxic Substances Control, California Environmental Protection Agency, Berkeley, California 94704, Institute of Marine Sciences, University of California, Santa Cruz, California 95064, and California Public Health Foundation, Berkeley, California 94704
A sample cleanup method for the isolation of polychlorinated dibenzo-pdioxins (PCDDs) and dibenzofurans (PCDFs) by solid-phaseextraction is described. A C18 bonded silica cartridge is used for the initial extraction and enrichment of the analytes. Subsequent cleanup is achieved with a dual-cartridgearrangement consisting of a bonded benzenesulfonic acid cartridge in series with a silica cartridge. A Florisil cartridge is employed for the final cleanup step. Sample preparation procedures used for blood plasma and animal tissues are described. The precisionand accuracy of the method is validated by determining recoveries of 2,3,7,8-substitutedPCDD and PCDF congeners spiked at various levels in blood plasma and in animal tissues. The effectiveness of the cleanup method is shown by the analysis of wild bird eggs which are highly contaminated with a variety of pesticides and polychlorinated biphenyls. The application of this cleanup method to human plasma containing PCDDs/PCDFs at parts-per-quadrillionlevels and results of analyses by high-resolution mass spectrometry and low-resolution mass spectrometry are presented. The validity of this method is demonstratedby the comparableresults obtained from this method and from other established methods.
conducted by the California Department of Health Services (DHS). Measuring PCDDs and PCDFs a t levels of low parteper-trillion (ppt) or parte-per-quadrillion (ppq) in biological samples presents problems in many analytical laboratories. Reported procedures*14 for the sample preparation of PCDDs and PCDFs require complicated experimental setups and are time consuming. Lipid extraction, either by liquid-liquid extraction or by solid-liquid extraction, is a common initial step of sample preparation for the analysis of PCDDs and PCDFs. For the liquid-liquid extraction, emulsion formation is a commonly encountered problem. For the solid-liquid extraction,1lJs preparation of column materials and packing of large preparative-scale columns are often laborious and time consuming. In addition, speciallytreated clean glassware is needed for the operation. An automated systeml6 may be employed to reduce the time for sample preparation, but is more costly. A goal of this study was to develop a simple, cost-effective, and fast experimental procedure, capable of generating results with acceptable precision and accuracy. Previously, the Hazardous Materials Laboratory (HML) reported a method employing a C18 bonded silica cartridge to extract PCDDdPCDFs from bird tissues and human blood plasrna.l6 This extraction method was successfully implemented in analyzing a large number bird egg samples for special project studies6 and in analyzing human blood plasma of the 1991 WHO/EURO interlaboratory studies on PCDDs and PCDFs.1' The sample preparation scheme is outlined in Figure 1. In the present study, the conventional cleanup steps after C18 extraction are replaced with three different solidphase extraction (SPE) cartridges. With this method, the complexity of the procedure, the glassware contamination, the amount of solvent used, and the sample preparation time can be significantly reduced. This study was undertaken to assess (1)the ability of this method to determine ultratrace
INTRODUCTION A fire occurred in April 1987 a t a pentachlorophenol wood treatment facility in the area of Oroville, CA.1 Potential food chain biomagnification and concerns for human exposure to polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs)2-8led to several investigations
(7) Firestone, D. Toxicol. Enuiron. Chem. 1991, 74, 376384. (8) Fries, G. F.; Panetenbock, D. J. Toxicol. Enuiron. Health 1990,29, 1-43. (9) Ryan, J. J.; Williame, D. T.; Lau, B. P. Y.; Sakuma, T. Chlorinated
t t
Dioxins and Dibenzofurans in Total Environment; Butterworth Publishers: Boston, 1983; Chapter 7, pp 205-214. (10) Norstrom,R. J.;Sion,M.;Mulvihii,M. J.Int. J.Enuiron.Anal. Chem. 1986,23, 267-287. (11) Smith, L.M.; Stalling, D. L.;J o h o n , J. L.Anal. Chem. 1984,56,
(1)Chang,R.R.;Hayward,D.G.;Flattery,J.J.;Goldman,L.R.;Hamly,
1830-1842. (12) U.S.Environmental Protection Agency Test Methods for Evaluation of Solid Waste, 3rd ed.;GPO Washington, DC,September 1986
California Environmental Protection Agency. University of California. 1 California Public Health Foundation. 11 Present address: Department of Chemistry, Humboldt State University, Arcata, CA 95521.
M.;Stephens, R. D. Chemosphere 1989,19,481-486. (method 8290). (2) Stephens,R.D.;Harnly,M.;Hayward,D.G.;Chang,R.R.;Flattery, (13) Patterson, D. G., Jr.; Turner, W. E.; Alexander, L.E.; Iseaacs, S.; J. J.; Petreas, M.X.; Goldman, L. R. Chemosphere 1990,20,1091-1096. Needham, L. L. Chemosphere 1989,18,875-882. (3) Goldman, L. R.; Hayward, D. G.; Flattery, J. J.; Harnly, M.; (14) Prlpke, 0.;Liz,2.A.; Scheunert, K. Chemosphere 1989,19,941Patterson, D. G.; Needham, L.L.;Siegal, D.; Chang, R. R.; Stephens, R. D.; Kizer, K. Chemosphere 1989,19, 841-848. (4) Hayward, D. G.; Charles, J. M.;Bettancourt, V. D.; Stephens, R. D.; Papanek, P.; Lane, L.; Ward, C. Chemosphere 1989,18,455-468. (5) Petreas, M. X.; Goldman, L. R.; Hayward, D. G.; Chang, R. R.; Flattery, J. J.; Wiesmijller, T.; Stephens, R. D. Stephens; Fry, D. M.; Rappe, C. Chemosphere 1991, 1731-1741. (6) Fiedlev, F.; Hutzinger, 0.:Timms, C. W. Toxicol. Enuiron. Chem. 1990,29,157-234. 0003-2700/93/0385-2420$04.00/0
948. (15) Turner, W. E.; Isaaca, S. G.; Patterson, D. G., Jr.; Chemosphere 1992,25,805-810. (16)Chang, R. R.; Jarman, W. M.;King, C. C.; Esperanza, C. C.; Stephens, R. D. Chemosphere 1990,20,881-886. (17) Stephens, R. D.; Rappe, C.; Hayward, D. G.; Nygren, M.; Startin, J.; Esbprll, A.; CarlB, J.; Yjbheikki, E. J. Anal. Chem. 1992,64, 31093117.
0 IS93 Amerjcan Chemlcal Society
ANALYTICAL CHEMISTRY, VOL. 85, NO. 18, SEPTEMBER 15, 1993
1
1. homo enlra In acetonltrlle 2. S p l b C12- PCDDdPCDFs 3. shake
1. splb"C12- PCDDslFCDFs 2. add lormlc a d d 3. degassing
f!
Preclpltata (discard)
Supernatant
I
Precipitate
1. add dalonized H 2 0 2. add lormlc acld
w I
Bonded C18 Slllca
C18 Cartridge
2421
1. homo en120 In awtonltrlie 2. aplkeq3CI2-FCDDslPCDFs
1. splka "C 1 2 - PCDDsIPCDFs 2. add lormlcacld
3. ahake
3. degassing
Supernatant
1. add delonlzed H p 12. add formic acld
(discard)
\c3/ C18 Csrtrldpo
Bonded C18 Slllca
1. drying 2. hexane
I. drying 2. hexane
Sample Extract
Sample Exiract
Potassium Sillca
hexane
50% CH, C12 /Cyclohexane SI cartridge
18% Carbopack CICallte
Carbon Column Florisil
toluene
I
I
FI c a y g e
I
4% CH,CI, I hexane
1. hexane 2. losb CH, CI,/ hexane
Fractlon I (discard)
I
50% CH2C121Hexane
Fractlon I
1
Flgurr 1. Method A: sample preparation for the analysis of PCDDs and PCDFs.
levels of PCDDs and PCDFs in animal tissues and in blood plasma, (2) the effectiveness of this simplified method to remove interferences, (3) the precision and accuracy of the analysis, and (4) the capability to produce results comparable to those from other established methods.
EXPERIMENTAL SECTION Chemicals. All solvents (methanol, acetonitrile, hexane, dichloromethane, acetone) were Resi-analyzed reagent grade (J. T. Baker Chemical Co.). Native and laClz-1abeled PCDD and PCDF standards were purchased from Cambridge Isotope Laboratories, Woburn, MA. Apparatus and Materials. All glassware and apparatus in contact with samples should be inert to common organic solvents and strong acids and bases. The glassware was washed with detergent and rinsed with distilled water, acetone, and hexane. Containers having areas of difficult access were sonicated in toluene for 5 min. The cleaned glassware was then heated to 300 OC for 3 h. All glassware openings were covered with aluminum foil. Prior to use, the treated glasswarewas rinsed with the solvent with which it would subsequently be in contact. Allbiological specimens were stored at -40 OC in hexane-rinsed aluminum foil. Human blood plasma was obtained from Irwin Memorial Blood Bank, San Francisco, CA. Bovine calf serum was obtained from Hyclone, Logan, UT. Chicken eggs were obtained from a local supermarket in Berkeley, CA. A tissue homogenizer (Polytron) with a 20-mm-diameter generator and adjustable speed control (Brinkman Instrument, Inc., Westburg, NY) was used. The solid-phase extraction device (Supelco,Inc. Bellfonte, PA) with a vacuum manifold, vacuum gauge, bleed valves, and Teflon solvent guides was employed. A Visidry drying unit was attached to the manifold for concentrating samples by evaporation with nitrogen. The solid-phase extraction cartridges used (Varian Association,Harbor City, CA) included octadecylbond elute (C18, 1 g), benzene-sulfonylpropyl bond elute (SCX, 1 g), silica (Si, 1 g), and Florisil (Fl, 1 g) cartridges. Procedure. A. Sample Preparation. (1)Blood Plasma. A 100-mLsample of human plasma or bovine serum was spiked with a standard mixture containing 17 W12-labeled 2,3,7,8-
Fractlon II (PCDDSIPCDFS)
(discard)
Fraction I (PCDDsIPCDFs) HRGCILRMSINCI or HRGCIHRMSIEI
CH2Cln
I
HRGCILRMSINCI
or HRGCIHRMSIEI
Figure 2. Method B: sample preparation for the analysis of PCDDs
and PCDFs. Table I. Percent Recoveries of Labeled Congeners from Spiked Human Plasma Samples. spiking level (na/kg) 0.05 0.1 0.5 86 f 19 77 f 14 86f 10 76f8 62 f 10 58 f 14 41 f 1 113 f 13 75 f 19 72 f 9 95 f 22 88 f 18 69f 17 62 f 27 68 f 9 96 f 15 73 f 18
89 f 13 96 f 10 108 f 21 107 f 9 91 f 8 86f 11 75 f 18 95 f 24 83 f 20 90 f 22 98 f 17 96 f 18 87 f 13 87 f 20 87 i 13 91 f 10 74 f 13
69 f 16 84f13 87 f 22 88f21 79 f 19 88f37 81 f 21 84f 17 79 f 12 78 f 13 9 0 f 16 87 f 17 81 f 14 64 f 21 89f 21 90 f 36 82 f 19
a Samples were analyzed by HRMS-EI.Values are means f standard deviation (n = 3).
substituted PCDDS/PCDFs. After spiking, the concentration which of each congener was 0.3 ng/kg (ppt), except [13Cl~10CDD, was 0.6 ng/kg. Before extraction, the spiked sample was equilibrated by gently swirling for 20 min. The spiked sample was then mixed with an equal volume of formic acid and was degassed for 15 min under vacuum. (2) Animal Tissues. The biological specimens were thawed at room temperture. For the animal tissues, connective tissues were removed prior to the processing. For egg samples, yolks were separated from the albumin. Bird samples were prepared by homogenizing the whole carcass, after the beak and primary feathers were removed. Aliquots of the homogenate were taken for analysis. Ten grams of tissue was homogenized in 100 mL of acetonitrile. A mixture of l3C12-labeled 2,3,7,8-substituted PCDDs/PCDFs containing one congener of each chloro homolog group was added to the tissue homogenate. For the tetra through hepta congeners 100pg was added, and for octa 300pg was added.
ANALYTICAL CHEMISTRY, VOL. 65, NO. 18, SEPTEMBER 15, 1993
2422
Native 2378 - TCDD Isotopic ratio : 0.79
m/z: 3 19.8965
318 132 305
245 115 33 1
50
-,
2h?
#Et02 16.257
I
Q) . I I
m/z: 333.9335 4 100393 175 390
50-
I
0 15:30
16: 30
Retention Time
17:OO
-
17:30
Flgure 3. Extracted ion chromatogram of TCDD channel of human plasma determined by HRMS-EI. m/z:303.9816
L ioe
398
Native 2378
- TCDF
lsotoplc ratio : 0.85
50
a, u E cd
w E a
m/zr385.8988
.
SO
9 Q)
-3 Y
Et02 a47
L l
L lee
m/z:315.9418
cd
I
2
50
L le0 50 0 15:30
16:00
16: 30
17100
17130
Retention Time Flgure 4. Extracted ion chromatogram of TCDF channel of human plasma determined by HRMS-EI.
The spiked tissue homogenate was extracted as described in our previous publication.16 The acetonitrile extract was concentrated to about 50 mL and mixed with an equal volume of formic acid and an equal volume of deionized water. The sample mixture was degassed for 15 min by vacuum. B. Sample Enrichment and Cleanup. (1) Extraction by Octadecyl Bonded Cartridge. Prior to the sample extraction, the C18 cartridge was activated with two 6-mL portions of methanol followed by two 6-mL portions of deionized water. The sample extract was loaded into a 75-mL reservoir and applied to an activated C18 cartridge with suction at a flow rate of 6-7 mL/min. The cartridge was dried thoroughly with a stream of nitrogen using a Visidry attachment or by suction through a vacuum manifold. The polar interferences and lipids were not retained by the column. The column was then eluted with two
3-mL portions of hexane. The eluate was concentrated to 1mL and was subjected to the following SPE cleanup steps. ( 2 ) Dual-Cartridge Cleanup. Before use, a bonded SCX cartridge and a Si cartridge were activated as follows: the SCX cartridge was washing with two 6-mL portions of methanol, 6 mL of acetone, and two 6-mL portions of hexane, and the Si cartridge with two 6-mL portions of hexane. The SCX cartridge was connected to the top of the silica cartridge. This dual-cartridge unit was attached to the vacuum manifold. The concentrated sample eluate in hexane was applied to the dual cartridge and three 1-mLhexane rinses from the sample vial were subsequently added. The dual cartridge was eluted with 6 mL of hexane. The upper cartridge was removed. An additional 3 mL of hexane was applied to the silica cartridge. The eluent, which contained PCDDs and PCDFs, was evaporated to 1 mL under nitrogen
ANALYTICAL CHEMISTRY, VOL. 65, NO. 18, SEPTEMBER 15, 1993
2423
Table 11. Levels of PCDDs and PCDFs Found in the Egg Samples. spiking level (ng/kg) 0 2,3,7,&TCDD 1,2,3,7,&PeCDD 1,2,3,4,7,8-H~CDD 1,2,3,6,7,8-H~CDD 1,2,3,7,8,9-H~CDD 1,2,3,4,6,7&HpCDD OCDD 2,3,7,8-TCSF 1,2,3,7,8-PeCDF 2,3,4,7,8-PeCDF 1,2,3,4,7,8-H~CDF 1,2,3,6,7,8-H~CDF 2,3,7,8,9-H~CDF 1,2,3,7,8,9-H~CDF 1,2,3,4,6,7&HpCDF 12 3 4 7 8 9-HpCDF ddDF' ' '
2.5
5.0
10
20
NAb
NA
NA
NA
NA
NA
NA
NA
0.04' 0.05 Bd 0.11 B 0.05' 0.59 B 6.3 0.22 0.04 B 0.08 B 0.09 B 0.05 B 0.04 B 0.03 0.12 B 0.04 B 0.26 B
0.02' 0.01' 0.02' 0.03' 0.22 10 3.1 B 0.23 0.01' 0.02' 0.05' 0.01' 0.01' 0.01c 0.08 B 0.02c 0.16 B
2.0 2.1 2.0 1.7 3.5 11 3.1 2.6 2.8 3.1 2.5 2.5 2.5 3.0 2.4 8.6
2.1 2.0 1.9 1.7 3.5 10 2.6 2.2 3.1 3.0 2.5 2.9 2.5 2.6 2.1 8.2
4.9 4.5 4.3 4.1 7.4 19 6.0 6.5 6.7 6.2 5.0 6.3 6.2 6.3 5.9 13
NA
NA
5.2 5.3 4.0 4.9 6.9 19 6.1 4.5 5.4 6.3 5.0 6.2 5.3 5.6 4.9 16
9.5 9.3 8.6 8.0
11 10 10 9.8 12 39 11 13 12 13 11 12 12 12 11 36
18 20 19 18 22 65 21 20 21 29 23 23 22 25 23 68
21 20 19 20 22 65 22 21 21 23 20 24 24 22 21 64
11
33 12 8.7 10 10 9.7 11
10 10 9.6 30
Aliquota (10 g) of a pooled egg yolk were spiked with tetra through hepta congeners at the level of 2.5,5,10, and 20 ng/kg, except octa congeners,which were at 3 times higher levels. NA, not analyzed. TCDD is too low to be detected in LRMS-NCI. MDL. d B, indicates value between MDL and PQL. e I, interferences present. Table 111. Percent Recoveries of the lBclz Homologs from Spiked Egg _ _ Samdes. spiking level (ng/ka) 2.5 ["CizITCDD [13C12]-PeCDD ["CI~I-HXCDD ["CIZI-H~CDD ["C121-OCDD ["CEJ-TCDF [13C12]-PeCDF ["C~~I-HXCDF ["C~~I-HPCDF [13C12]-OCDF
5.0
NAb
NA
98 f 10 98 f 1 70 17 51 f 7 69 f 4 71 f 4 64 f 3 66 f 2 42 f 1
100 f 34 96 f 46 76 f 25 65 112 94 113 go* 1 81 116 75 f 13 51 f 13
10
NA 97f2 100f4 9 1 1 14 74f8 94f 13 83f2 86*6 83111 6117
20
NA 9Of 15 87f46 72f15 50f9 78f 16 7515 78f8 77f8 41f7
a Samples were analyzed by LRMS-NCI. Average of two analyses and percent of difference. b NA, not analyzed.
before the final step of cleanup on a Florisil cartridge. (3) Florisil Cartridge Cleanup. A Florisil cartridge was prewashed with two 6-mL portions of dichloromethane, 6 mL of 4% dichloromethane in hexane, and 6 mL of hexane. The concentrated sample eluate recovered from the dual cartridge was applied to the Florisil cartridge. Three 1-mL hexane rinses from the sample vial were added. Interferences, such as polychlorinated biphenyls (PCBs), chlorinated benzenes, and chlorinated diphenyl ethers,ls were separated from PCDDs and PCDFs by eluting the cartridge with 6 mL of 4 % dichloromethane in hexane (fraction I). PCDDs and PCDFs were then eluted with 20 mL of dichloromethane (fraction 11). The dichloromethane extract was concentrated and transferred to a reactive vial containing 2 pL of tetradecane as a keeper. After dichloromethane was evaporated, additional tetradecane was added to the extract to make the final volume of 5 pL for the blood plasma and 10 p L for the animal tissue. A sample preparation scheme for the PCDD and PCDF analysis is presented in Figure 2. C. Lipid Determination. Totallipid was determined by the method of Ryan et al.9 Fifty grams of blood plasma or 10 g of tissue homogenate was extracted initially with 100mL of hexane/ acetone mixture (1:2) and subsequently with 50 mL of hexane. The extracted lipids were determined gravimetrically. D. Instrumental Analysis. A Finnigan Model 4500 lowresolution mass spectrometer (LRMS) and a Finnigan MAT 90 high-resolution mass spectrometry (HRMS) were employed for these analyses. A 60-mDB-5 fused-silica capillary column with 0.25-rm film thickness (J&W Scientific) was directly connected (18)Ryan,J. J.; Lizottre, R.; Newsome, W. H. J. Chromatogr. 1984, 303,351-360.
to the ion source. The LRMS was operated in negative ion chemical ionization mode (NCI) with methane as reagent gas. The ionizer pressure was 0.4 Torr with a source temperature of 100 "C. One microliterof sample extract was injected in a splitleas mode. Helium, with a flow rate of 1.1mL/min, was usedas carrier gas. The temperature program consisted of an initial temperature of 180 OC for 2 min, programmed to 260 OC at 20 OC/min and then 1OC/min to 300 OC. The injector temperature was 250 OC. The HRMS was operated in electron impact ionization mode (EI). The ionizer pressure was 1V Torr with a source temperature of 250 OC. The temperature program consisted of an initial temperature of 220 OC, programmed to 260 OC at 5 "C/min and then 1 OC/min to 300 "C. A SPI injector with initial temperature of 200 OC was programmed to 300 OC at 180 "C/min and held for 40 min. Selected ion monitoring (SIM) was employed to acquire pairs of specific ions for PCDDs and PCDFs. SIM of M and (M + 2) ions for the TCDD/TCDF through to the HpCDD/HpCDF were used for identification. The (M + 2) and the (M+ 4) ions were monitored for the OCDD/OCDF. A mixture containing 5 pg each of the native and Wlz-labeled PCDDs/PCDFs was injected daily to calibrate the response factors and to validate the instrument performance. Criteria for positive identification of PCDDs and PCDFs are (1)an isotopic ratio of *lo% of the theoretical value, (2) two specificions maximize within f 2 scans, (3) signal-to-noiseratio greater than 1 0 1 , and (4) elution of the analyte within the proper relative retention window as compared to a standard run. Safety Considerations. PCDDs and PCDFs are considered probably carcinogenic and teratogenic to human. All chemical components involved in the analysis and human tissues should be treated as potential health hazards and handled with the appropriate procedures in the laboratory.19
RESULTS AND DISCUSSION One of the advantages of this solid-phase extraction method is that PCDDs and PCDFs can be simply extracted from complex biological matrices. This method eliminates the elaborate procedures required by the traditional methods for total lipid extraction, followed by various treatments to remove lipids. In this method, the PCDDs and PCDFs are extracted from the animal tissue with acetonitrile rather than other solvents (e.g., hexane, ether, and chlorinated solvents) commonly used to extract lipids. Results from our previous study16 and from other investigatorsm show that although ~
(19)Rappaport, S.M.; Campbell, E. E. Am. Znd. Hyg. Assoc. J. 1976, 37,690-696. (20) Norh K.; Sjevall, J. J. Chromtogr. 1989,422, 103-105.
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ANALYTICAL CHEMISTRY, VOL. 65, NO.
18, SEPTEMBER 15, 1993
Table IV. Comparison of PCDDPCDF Levels Found in Plasma and Serum Samples As Determined by HRMS and LRMS concn (pu/ku of plasma) unexposed subjectb exposed subject bovine serum HRMS LRMS HRMS LRMS HRMS LRMS 2,3,7,8-TCDD 1,2,3,7,&PeCDD 1,2,3,4,7,&HxCDD 1,2,3,6,7,8-H~CDD 1,2,3,7,8,9-H~CDD 1,2.3.4.6.7,&H~CDD OCDD.. 2,3,7,8-TCDF 1,2,3,7,&PeCDF
1;2;3;4;6;7,&HpCDF 12 3 4 7 8 9-HpCDF
ddDi ' '
TCDD equivalent DHS" TCDD equivalent ITEFn f
17 f 9.2 44 f 13 32 f 10 270 f 67 62 f 21 480f 100 3300 f 720 18 f 10
2oc
42 f 12 42 f 13 39 f 13 23' 23 f 7.7 130 f 39 25' 4oc 170 f 75 120 f 54
NAu 62 f 18 29 f 66 280 f 66 50 f 14 500 f 116 3200 f 1188 18 f 9.7 25' 45 f 13 43 f 16 37 f 13 2oc 10f 2
120 f 41 25' 65 f 52 200 f 57 120 f 35
37 Bd 120 I' 36' lo00 170 B 770 go00 15' 9.5' 110 I 120 B 110 B 30' 37 ' 300
NA 140 88 1100 140 840 9600 43 I 5.2 100 120 97 5.2'
3.8' 6.3' 13' 27 I 15' 79 B 240 75 B 6.1 I 4.3'
4.2'
42'
11' 27'
57c 372 333
372 303
0.9' 3.2' 26 2.4' 80 260 16 0.7' 6.1
11'
10
7.6' 13' 62 B 5.7 I 85 B 35
6.8 2.8 B 0.7' 62 5.1 B 53 31
22
12
11'
220
NA
0 NA, not analyzed. b Values are the mean and standard deviation of six analyses of pooled human plasma from a California blood bank. Detection limit. d B, between detection limit and PQL. e I, interference present.
acetonitrile extracts only 10% of the total lipid it effectively extracts PCDDs and PCDFs from animal tissues. In addition, acetonitrile is a protein precipitant. Formic acid is added to the tissue extract and to the plasma sample to ensure the analytes are fully released from the sample matrices. Thus, both the acetonitrile and reduced pH due to formic acid inhibit protein binding and increase the extraction efficiency of the analyte by the C l 8 sorbent. The acetonitrile extract is miscible with water and forms an aqueous medium that also facilitates the retention of nonpolar analytes on the C18 sorbent. The total lipids, depending on the sample matrix, can be determined either by a gravimetric method9 or by a sensitive colorimetric method.21 When the sample is limited, the colormetric method is recommended due to the small sample size needed (27 p L ) to determine serum lipids. The lipids extracted from hexane/acetone extraction was 0.4 f 0.06% in the human plasma and 28 f 2% in the egg yolk. In this study, a C18 cartridge was employed to extract PCDDs/PCDFs from the sample extract. The C18 sorbent is an octadecyl hydrocarbon covalently bond to a silicate substrate. It has a nonpolar character to retain PCDDsl PCDFs and other nonpolar compounds. It also has a size exclusion function to eliminate macromolecular, biogenic interferences (such as plasma proteins) in biological extracts. Both analyte enrichment and sample cleanup can be simultaneously accomplished by using a C18 cartridge. In cases when a large sample volume or a dirty sample is needed for analysis, the sample flow rate through the C18 cartridge may reduce due to accumulation of impurities on the column. This disadvantage can be overcome by an automatic systern,Z2 which with the aid of a pressure regulator allows automatically adjustment of column back pressure. The sample can thus be passed through the C18 cartridge at a constant flow rate, and the column drying and elution can be carried without personal attention. The use of SCX and Si cartridges for the analysis of PCDDs/ PCDFs and other chlorinated aromatic hydrocarbons was (21)Christopher, S.; Frings, W.T. D u n , R. T. Queen, C. A. Clin. Chem. 1972,18,673-674. (22) Jordan, L.; Dowlina, S. D. Natl. Meet.-Am. Chem. SOC.,Diu. Environ. Chem. 1992,32,846.
Table V. Comparison of the Methods for the Analysis of Western Sandpiper (Calidris mauri? Whole Body Homogenates. concn (ng/kg of dry wt) method A method B method C 2,3,7,8-TCDD 1,2,3,4,7,8-PeCDD 1;2;3;4;7;8-HxCDD 1,2,3,6,7,&HxCDD 1,2,3,7,8,9-H~CDD 1,2,3,4,6,7,8-HpCDD OCDD 2,3,7,8-TCDF 1,2,3,7,8-PeCDF 2,3,4,7,8-PeCDF 1,2,3,4,7,8-H~CDF 1,2,3,6,7,8-HxCDF 2,3,4,6,7&HxCDF 1,2,3,7,8,9-H~CDF 1,2,3,4,7,8,9-HpCDF 1 2 3 4 7 8 9-HpCDF
ddD< ' '
TCDD equivalent DHSB TCDD equivalent ITEF27
NAb
1.8 1.6 3.4 0.05' 5.7 56 0.5 0.08
0.58 0.47 0.28 0.33 0.02' 0.59 0.02' 3.5 3.4 2.0
NA 1.7 1.2
NA 1.7
2.3 0.74 7.5 190
2.5 0.02'
1.2
0.02' 0.43 0.44 0.19 0.34 0.09 0.60 0.45 5.8 3.4 2.0
1.1
11
200 0.51 0.08 0.50 0.30 0.16 0.34 0.02' 0.83 0.02' 2.0 3.3 1.9
Method A a C18 cartridge;potassium silica/acidicsilica column; Carbopack C and basic alumina columns. Method B: a C18 cartridge; benzenesulfonicacid/silicacartridges and Florid cartridge. Method C: concentrated sulfuricacid treatment; potassium silicajacidic silica column; Carbopack C and basic alumina columns. Samples were analyzed by LRMS-NCI. b NA, not analyzed. Detection limit. reported by Kleinveld et aLZ3 SCX has a benzenesulfonylpropyl functional group covalently bonded to a silicate substrate. SCX is a strong cation exchanger. Due to the presence of the benzene ring on its surface, SCX also has high potential for nonpolar interactions. Acidic silica, containing 40% concentrated sulfuric acid, is commonly used for PCDD/ PCDF sample cleanup.1216 In this strong acidic environment the silica may undergo protonation, dehydration, and subsequent sulfonation. The sulfonyl group present in the SCX and in the acidic silica seems to provide a similar function for the removal of basic interferences. The dual SCX/Si column (23)Kleinveld, A. H.; Verhoeve, P.; Nielen, M. W. F. Chemosphere
1989,18, 1401-1412.
ANALYTICAL CHEMISTRY, VOL. 65, NO. 18, SEPTEMBER 15, 1993
Table VI. Mean Levels of Organochlorine Compounds in Clapper Rail (Rallus Longirostris levioes) Eggs from California concn concn (wg/kg of (rglkg of compound wet wt) compound wet wt)
H2O (%) lipid (%) p,p’-DDE p,p’-DDT p,p’-DDD HCB mirex wHCH @HCH Y-HCH oxychlordane heptachlor epoxide trans-nonachlor cis-nonachlor MC-2 MC-5 cis-chlordane
77 11
3000 200 64 4.2 0.41 0.86 5.4 0.56 250 9.6 45 4.1 64 57 2.5
tram-chlordane PCB congeners 99 119 146 153 138 187 180 170/190 199 1961203 105 194 206 of congeners
1.8 20 140 24 210 53 30 73 30 5.6 11
2.8 9.4 1.8
610
Table VII. Comparison of the Methods for the Analysis of Clapper Rail (Rallus longirostris levioes) Eggs. concn (ng/kg of wet wt) method A method B
2,3,7,8-TCDD 1,2,3,7,8-PeCDD lt2,3,4,7,8-HxCDD 1,2,3,6,7,8-H~CDD lY2,3,7,8,9-HxCDD 1 2 3 4 6 7 8-HpCDD
ddDD ’ ’
2,3,7,8-TCDF
1,2,3,7,8-PeCDF 2,3,4,7,8-PeCDF 1,2,3,4,7,8-H~CDF 1,2,3,6,7&HxCDF 2,3,4,6,7$-HxCDF 1,2,3,7,8,9-HxCDF 1,2,3,4,6,7,8-HpCDF 1 2 3 4 7 8 9-HpCDF ddDi ’ ’ TCDD equivalent DHSB TCDD equivalent ITEFZ7
1-16
0.96
4.0 1.5 4.2 1.3 3.7 12 1.3 IC 1.4b 0.9 3.2 2.1
3.6
1.2
0.79 0.46 2.5 0.34 0.7 11
6.1
1.1
3.6 0.80
3.2 4.9 1.1I‘ 1.2* 0.6 2.9 1.1 0.85
0.59 0.24 0.89 0.2 0.81 9.4 5.1
a Method A a C18 cartridge; potassium silica/sulfuric acid silica column;Carbopack C and basic alumina columns. Method B: a C18 cartridge; benzenesulfonic acidhilica cartridges and a Florisil cartridge. Data were generated by HRMS-EI. ‘1, interference.
arrangement removes both basic and acidic polar interferences. Dioxins and other nonpolar related compounds pass through the two cartridges in the hexane eluent. The separation of PCDDs and PCDFs from other structurally related compounds is achieved in the last cleanup step on the Florisil cartridge. Monoortho and nonortho PCBs and other chlorinated aromatic compounds are found in fraction I (4 % dichloromethane in hexane), and PCDDs and PCDFs in fraction I1 (dichloromethane). A. Precision a n d Accuracy Studies. To evaluate the precision and accuracy of this analytical procedure, the following quality control samples were analyzed. (1) Blood Plasma. Human blood plasma, a composite sample obtained from a local California blood bank, was used for this study. Seventeen l3C12-labeled 2,3,7,8-substituted congeners were spiked into 100-mL aliquots of the plasma at levels of 0.05,0.1, and 0.5 ng/kg, except [13C12]OCDD,which was spiked 2 times higher. The levels of the native and labeled PCDDs and PCDFs were determined by HRMS with electron
2425
impact ionization. Triplicate analyses were made at each spike level. The overall recoveries range from 70% to 100% , with the exception of [13C12lOCDD with a recovery of 41 % when spiked a t the lowest level (Table I). Our present and previous studies16 have shown that recoveries from blood plasma with a less complex extraction procedure are generally higher and more consistent than those from animal tissues. At the spiked level of 0.05 ng/kg, the isotopic ratios of the M+ and (M 2)+ ions for 13C12-labeledTCDD and TCDF were 0.79 and 0.81, respectively (Figures 3 and 4). The ratios for the identified native TCDD and TCDF in composite blood were 0.79 and 0.85, respectively. The theoretical value is 0.78. Concentrations of 2,3,7,8-TCDD and 2,3,7,8-TCDF found in this blood sample were 0.017 and 0.018 ng/kg, respectively. At the spiked level of 0.05 ng/kg, signal-tonoise ratios for [13C1+2,3,7,&TCDD and [W12]-2,3,7,8-TCDF were 80 and 150, respectively. On the basis of the criteria generally accepted by the laboratory community24and EPA method 8290,26 HML has defined a method detection limit (MDL) as three standard deviations of the background of the sample. The practical quantitation limit (PQL) is lostandard deviations of the background of the sample (3.3 times the MDL). Based on these criteria, the estimated MDL for tetrachlorinated PCDDs and PCDFs was below 0.005 ng/kg. (2) Animal Tissues. Two dozen chicken egg yolks were combined and aliquots were fortified with a mixture containing 17 native 2,3,7,8-substituted congeners. Based on our preliminary study1 on the contaminated levels of PCDD and PCDF found in the Oroville area, the following spiking levels were validated. The spiked levels were 2.5,5,10, and 20ng/kg for eachcomponent, except OCDD and OCDF, which were 3 times higher. In addition, one [13C1~1-2,3,7,8-substituted congener from each chloro homolog group was added as internal standards and to monitor recoveries. These samples were only analyzed by LRMS-NCI. When this phase of the study was conducted, a HRMS was not available in the laboratory. Duplicate analyses were made a t each spiked level. Concentrations of the spiked 2,3,7,8-substituted congeners and percent recoveries of l3C12 congeners are shown in Tables I1 and 111. Percent recoveries ranged from 64 % to 103%,with the exception of octa congeners at 41-74%. The relative percent differences for these duplicate analyses were predominately within *15%. Due to the low response of TCDD in NCI mode, 2,3,7,8-TCDD was not detected at these low levels. B. Comparison of HRMS-E1 a n d LRMS-NCI Results. A comparison of results obtained from HRMS using E1 and LRMS using NCI was made. Blood samples from an exposed subject and from unexposed subjects were treated by the described cleanup method. A commercial bovine serum, which was found to be relatively uncontaminated with PCDDs and PCDFs, was also concurrently processed as a matrix blank. The same blood extract was analyzed for PCDDs and PCDFs by both HRMS-E1 and LRMS-NCI (Table IV). In the NCI mode, the response of PCDD is generally lower than that of PCDF. The response of TCDD in NCI is 2-3 orders of magnitude lower than other congeners and is too low to allow the detection and analysis of TCDD in blood samples. The
+
(24) Tuner, W. E.; Patterson, D. G., Jr.; Isaacs, S. G.; Alexander, L. R. Chemosphere 1992,25, 793-804. (25) Tondeur,Y.; Beckert, W. F.; Billets, S.; Mitchum, R. K. Chemo-
sphere 1989, 18, 119-131. (26) Health effectsof2,3,7,8-tetrachlorodibenro-p-diorinand related compounds; Report to the California Air Resources Board, California
Department of Health Services: Berkeley, CA, 1985. (27) NATO. Committee on the Challenges to Modern Society. Pilot study on international information exchange on dioxins and related compounds. InternationalToxicityEquivalencyFactor. Method of Risk Assessment of Complex Mixtures of Dioxins and Related Compounds, 1988;Report No. 176.
2426
ANALYTICAL CHEMISTRY, VOL. 05, NO. 18, SEPTEMBER 15, 1993 1967 244261.
Native 2,3 7,8-TCD F Isotopic ratio : 1.4 I
A
21340.
f
304.891 0.m
.
I19089. 745337.
I 11232.
m.891
306
0.590
f
1600 1320
1s:m
14: 10
19w
ISISB
m/z:303.9816
B
nee
'2108 1?:3
16140
Native 2,3,7,8-TCDF Isotopic ratio :0.72
ie:m
19: 18
24"
ma
;;:: 1?29
88 60
40 20
I
r.'z:30S.d9d7 1084 2524
iEt03 2.611
I
m/x: 3 17.9389
'3C
122 3 ,7 ,8-TC D F 42%'7' Spiked at 10 ppt 211?e5
% 198.
e e6e.
333 6330
619 684 1736 894 92?7 ,49119
!,,A 95??
20.
e?
413
.
;? 18:89
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.
l A . ~ - ,
763
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652 27?7
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20199
I
I
I
..
rhr-
24: 00
2.52s
1171 696 3889
m'ie
I
28: BO
Retention Time
Flgure 5. Extracted ion chromatogram of TCDF channel of clapper rail eggs. (A) LRMSNCI. (B) HRMS-EI.
relative percent differences of results generated from these two analytical systems are mostly within &lo%. Due to the extreme low concentration of 1,2,3,7,8-PeCDD, 1,2,3,4,7,8HxCDD, and 1,2,3,7,8,9-HxCDFin unexposed subjects, the variations of analytical results of these congeners are larger than other positively identified congeners. The congener pattern of the unexposed blood plasma is similar to that commonly found in human plasma. C. Validation of Cleanup Efficiency. To validate cleanup efficiency, several contaminated environmental samples were treated by three methods and results were compared. Method A is our previously reported procedure16 (Figure 1). Method B is the procedure presented in this study (Figure 2). Method C is one of the classical methods9 commonly used
for biological samples. Samples were compositesof migratory western sandpipers (Caulidrismauri),collected in California (1985). Although these samples are contaminated with PCDDs/PCDFs at low levels, the results obtained from these three methods are very close (Table V). The level of contamination in the method blank for the animal tissue ranged from 0.5 to 5 ng/kg for OCDD and 0.1 ng/kg for hepta PCDDs and PCDFs and that in the method blank for the blood plasma was about 0.01 ng/kg for human plasma. A second environmental sample with high contamination was treated by methods A and B. This sample was whole eggs from clapper rail (Rallus longirostris levipes), collected a t Ventura, CA (1985). A preliminary study from the University of California a t Santa Cruz, indicated the presence
ANALYTICAL CHEMISTRY, VOL. 65, NO. 18, SEPTEMBER 15, 1993
of very high levels of PCBs and other chlorinated pesticides (Table VI). Thus, this sample was chosen to evaluate the efficiency of the SPE procedure for cleanup of a highly contaminated sample. Results obtained from methods A and B are consistent (Table VII). The data were generated by LRMS-NCI. The extracted ion chromatograms of the TCDF channel, as determined by LRMS-NCI, are shown in Figure 5A. Many interferences, possibly hexachlorinated naphthalenes, are present in the TCDF channel. The isotopic ratio for 2,3,7,8-TCDF is 1.4, which is higher than the theoretical value 0.78. The same egg extract was reanalyzed by HRMSE1 for the TCDD and TCDF. As shown in Figure 5B, many interferences found in the chromatogram generated by LRMSNCI were not observed. With high-resolution exact mass monitoring, the isotopic ratio for 2,3,7,8-TCDF is now 0.72. This result suggests that interferences are mainly present at mlz 304 rather than at the quantitative ion mlz 306. Therefore, in Table VI1 there is no significant difference in reported 2,3,7,8-TCDF values determined by LRMS or by HRMS. There were only minor interferences observed in the penta and hexa PCDD and PCDF channels, when the analytes were determined by LRMS-NCI. No apparent interferences were observed in the hepta and octa channels. However, for the tetra channels, as also shown in other studies,24 a mass spectrometer with a resolution of a t least 8000 is required to eliminate interferences from TCDD and TCDF.
CONCLUSIONS The cleanup procedure for the analysis of PCDDs and PCDFs by solid-phaseextraction provides an effective means to generate results in response to emergencies or for monitoring a large number of samples. In addition to the described matrices, this cleanup method was successfully used in HML
2427
for the analyses of PCDDs and PCDFs in liver, muscle, and molasses. A carbon column, which is widely used for the sample cleanup for the analysis of PCDDs and PCDFs, is eliminated in this method. The time required for the sample preparation can be significantly reduced by using commercially available prepacked SPE cartridges. Analysis of six samples could be completed in 1(plasma) to 2 (animal tissues) days by a single chemist with manual operation. The inexpensiveand simple SPE apparatus makes it affordable for most laboratories. Cross contamination from high-level samples and glassware is also minimized by using disposable cartridges. Plasticizers, contributed by the synthetic housing of SPE cartridges, are not a problem when selected ion monitoring is used. Moreover, this cleanup method requires relatively small quantities of expensive and hazardous chlorinated solvents. With the combination of using a selective solvent for tissue extraction, removal of interferences by various phases of SPE cartridges, and detection of analytes by HRGCIHRMS with SIM, PCDDs and PCDFs can be satisfactorily identified. The analytical results generated by LRMS-NCI compared to HRMS-E1were within f10% for most congeners.
ACKNOWLEDGMENT The authors acknowledge Drs. Robert D. Stephens, Gunilla Lindstrljm, and Theodore Belsky for their critical review and comments on the manuscript. We also thank Wilson Wong and Mandy Mok for their technical assistance and David Ledig from US. Fish and Wildlife Services for providing bird samples to complete this study. Data were presented in part at the annual meeting of Dioxin '91, North Carolina, September 1991. RECEIVED for review December 1, 1992. Accepted June 2, 1993.
