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Application of a dual column reaction chromatography system for confirmatory analysis of polychlorinated biphenyl congeners
Chemosphere, Vol.19, Nos.l-6, pp 143-148, Printed in Great Britain
1989
0045-6535/89 $3.00 + .00 Pergamon Press plc
APPLICATION OF A DUAL COLUMNREACTION CHROMATOG~PHY SYSTEM FOR CONFIRMATORY ANALYSIS OF POLYCHLORINATED BIPHENYL CONGENERS D.O. DUEBELBEIS, GENOWEFAPIECZONKA, S. KAPILA, T.E. CLEVENGER, AND A.F. YANDERS J.D. WILSON* Environmental Trace Substances Research Center, University of Missouri, Columbia, MO 65203, and *Monsanto Chemical Co., St. Louis, MO 63167
ABST~CT The present work describes the application of a dual column reaction chromatography system for congener specific analysis of polychlorinated biphenyls in human serum and adipose tissue samples. KEYWORDS Dual Column; polychlorinated biphenyls; specific.
hydrodehalogenation; electron
capture;
congener
INTRODUCTION Because of their widespread use in the past and their persistence in the environment, PCBs are nearly universal contaminants. I t is speculated that most humans are unavoidably exposed to small quantities of these chemicals and various analysis surveys seem to confirm this speculation. Acute and chronic toxic effects of PCBs have been studied in detail. The chronic effects have been monitored in offsprings of exposed populations. Accidental exposure to PCBs and associated PCDFs through cooking oil have resulted in lingering abnormalities in exposed individuals and their children at Yusho, Japan and in Taiwan (Rogan et al., 1988; Rogan, 1982). However, direct correlations between PCB body burden and adverse health effects are d i f f i c u l t to draw due in part to the large number of PCB isomers and the presence of other related contaminants such as polychlorinated dibenzofurans (PCDFs). Thus techniques for isomer specific determination of PCBs are required for making r e a l i s t i c assessment of chronic effects and PCB exposure. Since a l l commercial formulations of PCBs were prepared by direct chlorination of biphenyl, these formulations and PCBs in the environment exist as complex mixtures of 209 possible congeners. However, due to various factors which affect uptake, distribution and elimination of individual PCB isomers, the PCB residues found in the environment rarely resemble the commercial formulations. As a result, PCB residue determination relative to commercial formulation is speculative at best. Studies have shown that in general, higher homologs are metabolized more slowly as a consequence tetrachloro to decachloro congeners and p,p' DDE (a metabolite of p,p' DDT) are the largest contributors to the total chlorinated hydrocarbon body burden in the general i.e. non-occupationally exposed population. Due to i t s excellent s e n s i t i v i t y for polychlorinated organics the electron capture detector continues to be the most commonly used detector for the determinations of PCBs. In all analytical schemes u t i l i z i n g gas chromatography "with electron capture detection (GC-EC) the identification of PCB components is based solely on the retention time and the a b i l i t y of GC columns to separate the congeners. However, complete resolution of all PCB congeners on a single GC column has not been achieved so far (Duinker et al., 1988; Mullin et al., 1984). The problem related to incomplete resolution of congeners with a varying number of chlorines can be addressed by the use of a mass spectrometer as the GC detector (Onuska and Terry, 1986; Pellizzari et al., 1985). However, distinction between coeluting congeners with the 143
144
same number of chlorines is problematic even with this technique. The recently introduced technique of gas chromatography coupled with matrix isolation fourier-transform infra red spectrometry (GC-MI/FTIR) holds considerable potential for congener specific analysis (Bourne et al., 1987; Holloway e t a / . , 1988). However, the technique at present does not meet the s e n s i t i v i t y requirement for the subparts per b i l l i o n determinations. A great degree of success in resolving co-eluting components can be achieved by a two dimensional chromatographic approach, with the combination of non-polar and polar polysiloxane columns (Duebelbeis et a7., 1988; Duinker et at., 1988). Some uncertainty can remain due to a lack of readily available standards. The present report deals with application of a dual column reaction chromatography system for confirmatory analysis of polychlorinated biphenyl congeners in human serum and adipose tissue samples. This system not only yields the improved resolution of the two dimensional approach but also provides added structural information. EXPERIMENTAL The details of the dual column system used in the present study have been given earlier (Duebelbeis e t a / . , 1988). The system was b u i l t around a twelve port, two position valve. The two capillary gas chromatographs were interfaced to a reaction chamber and cryogenic trap through the valve. The i n i t i a l separation of the analyte mixtures was achieved with a 60 m x 0.25 mm i.d. fused s i l i c a column. The stationary phase in the column was "bonded" 95% methyl + 5% phenyl siloxane. The analytes transferred to the second chromatograph were separated with a 60 m x 0.25 mm fused s i l i c a column with bonded cyanopropyl polysiloxane or methyl phenyl polysiloxane stationary phase. All interconnections in the system were made with 0.5 mm o.d. (0.25 mm i . d . ) aluminum clad fused s i l i c a tubing. A reactor consisting of 50 cm X 0.5 mm i.d. nickel tubing was employed throughout this study. The extent of reaction was controlled by varying the residence time of the analyte in the tube reactor and varying the composition of the carrier gas. The product spectra obtained under optimized conditions were used in the confirmation of PCB congeners in human serum and adipose tissue samples. All of the human adipose tissue samples analyzed during the course of the study were obtained through the autopsies performed by Boone County Medical Examiner's office. While the serum samples were obtained from volunteers. The extraction and cleanup procedures used prior to the final analysis are summarized in the flow diagram as follows: 2 9 aliquot adipose tissue
5 ml aliquot serum spike with I.Ss.
spike with l.Ss. HOMOGENIZE
Densture serum
Nix with 10 g Na2SO4 (enhyd.)
proteins with 2 mt methanol
Homogenize After
Extract 3X with 5 ml hexane
24 hrs Extract in Column
concentrate
with 150 mt cyc t ohexane: dich I oromethane (1:1)
& S i l i c a gel fr ac ti onati en to separate PC.Be & chlorinated pesticides
Concentrate to 2 ml
+ Fract ionate over F l o r i s i l colulm to remove polar tipids
+ Fractionation with GPC to re~ove other lipids s i l i c a get fractionation to separate PCIB & Pesticides
& Concentrate each fraction to 1 m[ f i n a l volume
Concentrate extract to 0.2 nit f i n a l votume
145
RESULTS AND DISCUSSION The formation of electron capturing products as a result of hydrodehalogenation reactions in a flow-through nickel tube reactor has been demonstrated earlier (Kapila and Aue, 1977; Duebelbeis, 1988). The potential use of these reaction products for the confirmatory analysis of polychlorinated organic was alluded to by Kapila and Duebelbeis (Kapila e t a / . , I987; Duebelbeis, 1988). The dual column system used in the present study could be operated in the classical heart-cutting two dimensional mode where the unresolved congeners are transferred to and resolved in the second column or in the dual reaction chromatography mode, where the unresolved components undergo hydrodechlorination reactions and the products as well as the residual analytes are resolved and analyzed with the second gas chromatograph. The product pattern obtained and the intensity of product peaks can be used in confirming the identify of the congener and determining of its concentration in the sample. The system is thus analogous to mass spectrometry. An example of the distinctive product patterns obtained for two heptachloro congeners 2, 2', 3, 4, 4', S, 6' (182) and 2, 2', 3, 4', 5, 5', 6 (187) are given in Fig. 2. The major products are believed to result from displacement of single chlorine. The assignment of peak is based on retention time data and structural considerations. L
1
4
3
~
4
8
Cl
C1 C1 137
Cl 140
o 400og PCB !182
LIi/~ j
!
,,jI ~, iI
Cl CI
ci [47
![ i
--
ci 146
ci 149
3400g PCB ~181
i ,
'%1
5t.50
52.35
5 3 ~0
5 4 O5
--
5490
~5Z5
5660
RETENTION TIHE
Fig. 2.
58 30
5g15
60
)0
Chromatography of products of coeluting congeners 182 & 187.
These two congeners y i e l d i d e n t i c a l mass d i s t i n g u i s h by mass spectrometry, Fig. 3. '.~8.;) -
324 C1 I:l
57 45
:MINUTES)
-
spectra
and thus are virtually
impossible
324
I~.~C1 C1
Cl
CI 394
Cl
CI Cl
z.ong PCB# I a Z
CI
C1
1.?ng PC8~ 187 CI
C1
C1
4
;53
/
-~"" 260
, "-7~-",'~"1
28g
Fig. 3.
-
SE'~
,~"'1
I',"~'l'"','"[r'",
~2g
34g
F "7
_~0
~-
Sgg
,
4~
~E
il. ............
Mass Spectra of heptachlorobiphenyls 182 & 187.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
......
....
to
146
Distinctive products can be obtained for a l l congeners with four or more chlorines. The intensity of products was found to be d i r e c t l y related to the concentration of the congener in the sample t h u s allowing an accurate determination of congener concentration, as demonstrated in the case of congeners 170 and 190, in Fig. 4-5. The chromatograms show the products obtained from the individual standards and the mixed peak from a human adipose tissue sample. The results based on product peak intensity showed that the congener 170 forms the major portion of the chromatographic peak in the sample, concentration of this congener was determined to be 243 ppb while the concentration of congener #190 was calculated to be 7 ppb. Clearly the system allows the determination of minor components in presence of a major components. ~
co
C1
C1 ~ o
~C1 C1
C]
~ m
co
CI
C1
Is6¢cf
i:@is8
PCB 190 PCB 170
,--4
~A~ I-
I
55 B0 57 00
F
--
f
58 20
55 80
MINUTES
Fig. 4.
~
BS I
I
I
57 O0 5B 20
[-
I-
t-----
55 B0 57 O0 5B 20
MINUTES
MINUTES
Single chlorine loss product chromatograms for: A) 320 pg congener 170. B) 350 pg congener 190. C) congeners 170 & Ig0 combined.
The s e n s i t i v i t y and l i n e a r i t y of the system were found to be sufficient to allow confirmation of congeners down to sub parts per b i l l i o n levels in the serum samples. A number of human adipose and serum samples from individuals with no known occupational exposure were analyzed with the system. The results obtained are summarized in Table I and 2.
g
I
-I------
55 B0 57 O0 5B 20 5q 40 MINUTES
Fig. 5.
I
I 55 BO
- I
-I
5 7 O0 5 8 20 MINUTES
I---59 40
Single chlorine loss product chromatograms for the tentatively i d e n t i f i e d congeners 170 and 190: A) Adipose sample from a 65 yr old male. B) Adipose sample from a 27 yrs old male.
147
Table I.
Concentration of polychlorinated biphenyls in human adipose tissue.
PCB
Donor AQe/Sex
Cen~ener 15 mo
23 N
27 N
052
19.0
24.0
060
5.5
14.0
15.0
101
7.0
22.0
26.0
28 F
35 F
37 14
44 N
44 14
48 N
57 N
63 F
65 M
69 N
73 F
23.0
5.0
5.4
14.0
10.0
4.7
16.0
15.0
24.0
8.5
8.7
12.0
7.4
8.2
2.2
5.2
7.3
8.1
15.0
15.0
17.0
13.0
13.0
9.2
13.0
5.3
6.4
11.0
14.0
7.3
17.0
11.0
23.0
151
2.0
4.1
12.0
15.0
2.7
11.0
2.2
4.7
9.1
13.0
1.7
15.0
9.1
21.0
118
36.0
27.0
69.0
141.0
52.0
46.0
11.0
24.0
101.0
46.0
332.0
190.0
435.0
124.0
114
1.4
15.0
5.6
7.6
12.0
5.5
11.0
9.5
6.9
23.0
18.0
31,0
10.0
153
47.0
50.2
3]3.0
231.0
203.0
113.0
16.0
177.0
379.0
220.0
472.0
406.0
1200.0
213.0
105
11.0
11.0
22.0
34.0
19.0
18.0
7.5
16.0
37.0
20.0
70.8
48.0
120.0
44.0
141
1.2
4.6
7.7
19.0
3.9
7.9
3.2
3.3
7.1
10.0
4.0
6.9
8.4
16.0
137
2.0
3.6
16.0
18.0
15.0
7.9
7.1
11.0
17.0
11.0
21.0
14.0
77,0
11.0
138
27.0
27.0
175.0
137.0
86.0
25.0
129.0
257.0
147.0
239.0
191.0
988.0
136.0
129
6.3
16.0
27.0
14.0
15.0
12.0
16.0
20.0
26.0
22.0
27.0
32.0
54.0
23.0
182, 187
16.0
18.0
80.0
87.0
40.0
42.0
13.0
55.0
120.0
80.0
90.0
131.0
230.0
74.0
183
11.0
5.9
50.0
47.0
18.0
22.0
3.0
18.0
52.0
31.0
38.0
50.0
93.0
35.0
8.3
23.0
13.0
13.0
4.6
9.4
20.0
13.0
24.0
21.0
48.0
18.0
128 185
4.4
2.9
7.5
3.3
4.7
4.0
3.2
171, 202
3.2
11.0
14.0
15.0
12.0
11.0
17.0
18.0
26.0
31.0
156
3.7
8.3
13.0
8.0
15.0
9.3
5.6
13.0
18.3
173, 200
5.8
7.8
8.7
6.8
9.9
10.0
3.6
7.0
14.0
23.0
80.0
165.0
165.0
129.0
123.0
56.0
207.0
]01.0
250.0
180
0.86
0.27
0.92
1.6
2.0
4.6
67.0
0.84
45.0
56.0
19.0
16.0
29.0
20.0
61.0
12.0
12.0
20.0
15.0
36.0
8.6
340,0
396.0
712.0
178.0
191
1.3
5.7
3.3
2.1
4.4
3.9
2.5
3.5
7.0
5.3
7.3
6.9
12.0
4.1
170
9.0
26.0
74.0
51.0
52.0
53.0
21.0
70.0
104.0
92.0
107.0
110.0
243.0
67.0
201
6.8
26.0
36.0
43.0
38.0
41.0
43.0
65.0
95.0
111.0
163.0
152.0
193.0
5.0
196, 203
5.4
12.0
32.0
38.0
32.0
36.0
10.0
49.0
79.0
80.0
117.0
128.0
167.0
48.0
189
3.8
1.5
1.7
1.8
3.9
6.0
3.0
6.9
4.3
5.2
7.0
5.3
11.0
2.6
2O8
0.63
2.1
2.2
2.9
4.2
3.0
7.4
9.5
18.0
18.0
24.0
7.0
195
1.9
2.4
8.7
7.9
7.7
13.0
16.0
14.0
16.0
16.0
21.0
30.0
11.0
3.7
6.7
3.0
5.4
194
4.3
27.0
28.0
38.0
205
3.4
7.3
31.0
14.0
17.0
18.0
8.4
8.1
207
2O6
3.54
15.0
209
5.2
9.8
12.0
7.6
5.9
9.2
20.0
9.9
43.0
3.3
54.0
64.0
63.0
64.0
77.0
129.0
38.0
15.0
6.6
12.0
9.0
11.0
7.4
4.5
48.0
51.0
55.0
92.0
147.0
99.0
137.0
35.0
26.0
20.0
39.0
61.0
238.0
59.0
112.0
32.0
All concentrations are expressed as perts pen b i l l i o n nenogrm/g).
The relative concentration of a l l congeners in most non-occupationally exposed individuals was found to be generally quite similar with congeners 118, 138, 153, 170, 180 and 187 being the most predominant ones. Congeners 105, 114, 118, and 156 were found to be the predominant of the toxic congeners. In general a rough correlation between the PCB concentration and the age of the individual was observed. A similar trend was observed in case of PCB congener concentration in serum of a non-occupationally exposed individual. The results showing the
148
concentrations of major congeners are summarized in Table 2. The results indicate that the congeners with adjacent unsubstituted carbons are less persistent in human serum and adipose tissues. Table 2. Samlpte
SE_.XX
PCB Congener Concentration in Serum (ppb) AGE
SER23 N 20 SER19 N 23 SER17 F 24 SER11 F 26 SER4 F 28 SER16 F 29 SERIO N 30 SER27 f 30 ~R20 M 32 ~R7 F 32 ~R14 N 35 SER8 F 38 SER15 N 41 SER3 M 51 SER22 N 53 SER12 14 57 ~R20 M 52 SER~ M 67 SER9 F 76 SER30* N 25 ~R31" I,I 21 SER32* N 27 *Occqpationatty exposed individuals.
****************'***********"*****CONGENER * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 11.~8 15:3 138 183 15~6 180 0.052 O. 16 0.52 0.22 0.29 0.042 0.096 0.42 0.13 0.15 0.11 0.19 0.11 1. 1 0.28 0.62 O. 13 O.R'q 1.3 3.0 4.8 12.0
0.17 0.29 0.58 0.42 0.21 0.11 0.30 0.50 0.34. 0.28 0.36 0.19 0.17 1.3 0.54 0.82 0.38 0.76 1.5 2.9 12.0 11.0
0.096 O. 17 0.46 0.26 O. 17 0.054 O. 17 0.38 0.22 0.20 0.22 0.15 0.11 0.95 0.36 0.70 0.24 0.54 1.1 2.6 11.0 12.0
0.034 0.048 0.042 0.052 0.039 0.028 0.038 0.044 0.054 0.~3 0.048 0.030 0.024 O. 16 0.086 0.098 0.068 0.068 0.21 0.34 2.0 1.4
0.012 0.031 O. 11 0.070 0.029 0.004 0.048 O.092 0.0/*2 0.039 0.060 0.020 0.016 0.25 0.062 0.16 0.030 0.16 0.23 0.38 0.88 0.92
0.14 0.21 0.20 0.38 O. 16 0.11 0.34 O. 24 0.32 0.24 0.32 0.11 0.092 1.1 0.42 0.62 0.30 0.78 1.2 1.6 11.0 6.2
170 0.038 0.072 0.076 0.16 0.065 0.028 O. 13 O. 10 0.12 0.11 0.10 0.049 0.026 0.45 0.14 0.24 O. 10 0.34 0.51 0.72 4.2 2.6
The data clearly demonstrate that the dual column system allows unambiguous detection of individual congeners down to sub parts per billion levels with relatively small sample. ACKNOWLEDGEMENTS Partial support for this work was provided by a grant from Monsanto Chemical Co., St. Louis, Missouri. The assistance of Dr. Jay Dix in obtaining the adipose tissue samples is also acknowledged. REFERENCES Bourne, S., G.T. Reedy and P.T. Cunningham (1978). J. ChromatoaraDhic Scj .... ]_Z, 460-463. Duebelbeis, D.O. A dual column reaction gas chromatographic system for the structural confirmation of chlorinated organics, 1988, Ph.D. Dissertation, University of Missouri, Columbia. Duebelbeis, D.O., S. Kapila, T. Clevenger, A.F. Yanders and S.E. Manahan (1988). Chemosphere, in press ]988. Duinker, J.C., D.E. Schulz and G. Petrick (]988). Anal. Chem., 6_00(6), 478-482. Holloway, T.T., B.J. Fairless, C.E. Freidline, H.E. Kimball, R.D. Kloepfer, C.J. Wurrey, L.A. Jonooby and H.G. Palmer (]988). Appl. Spectrosc., 42(2), 359-369. Kapila, S. and W.A. Aue (]977). J. Chromatoqraphic Sci., ]__55,569-572. Kapila, S., D. Duebelbeis, R. Malhotra, A.F. Yanders and S.E. Manahan (1987). In: Pesticide Science and Biotechnoloqy (R. Greenhalgh and T.R. Roberts, eds.) pp 325-328, B1ackwell Scientific Publications. Mullin, M.D., C.M. Pachini, S. McCrindle, M. Romkes, S.H. Safe and L.M. Safe (1984). Environ. Sci. Technol., 18(6), 468-476. Onuska, F.I. and K.A. Terry (1986). HRC CC, J__~.Hiqh Resolut. Chromatoqr. Chromatoqr. Commun. 9, 67]-675. Pellizzari, E.D., M.A. Morley and S.D. Cooper (1985). J_,. Chromatoqr., 339, 277-314. Rogan, W.J. (1982). Teratology, 26, 26. Rogan, W.J., B.C. Gladen, K-L. Hung, S-L. Koong, L-Y. Shih, J.S. Taylor, Y-C. Wu, D. Yang, N.B. Ragan, C-C. Hsu (1988). Science, 24__!, 334-336.
1989
0045-6535/89 $3.00 + .00 Pergamon Press plc
APPLICATION OF A DUAL COLUMNREACTION CHROMATOG~PHY SYSTEM FOR CONFIRMATORY ANALYSIS OF POLYCHLORINATED BIPHENYL CONGENERS D.O. DUEBELBEIS, GENOWEFAPIECZONKA, S. KAPILA, T.E. CLEVENGER, AND A.F. YANDERS J.D. WILSON* Environmental Trace Substances Research Center, University of Missouri, Columbia, MO 65203, and *Monsanto Chemical Co., St. Louis, MO 63167
ABST~CT The present work describes the application of a dual column reaction chromatography system for congener specific analysis of polychlorinated biphenyls in human serum and adipose tissue samples. KEYWORDS Dual Column; polychlorinated biphenyls; specific.
hydrodehalogenation; electron
capture;
congener
INTRODUCTION Because of their widespread use in the past and their persistence in the environment, PCBs are nearly universal contaminants. I t is speculated that most humans are unavoidably exposed to small quantities of these chemicals and various analysis surveys seem to confirm this speculation. Acute and chronic toxic effects of PCBs have been studied in detail. The chronic effects have been monitored in offsprings of exposed populations. Accidental exposure to PCBs and associated PCDFs through cooking oil have resulted in lingering abnormalities in exposed individuals and their children at Yusho, Japan and in Taiwan (Rogan et al., 1988; Rogan, 1982). However, direct correlations between PCB body burden and adverse health effects are d i f f i c u l t to draw due in part to the large number of PCB isomers and the presence of other related contaminants such as polychlorinated dibenzofurans (PCDFs). Thus techniques for isomer specific determination of PCBs are required for making r e a l i s t i c assessment of chronic effects and PCB exposure. Since a l l commercial formulations of PCBs were prepared by direct chlorination of biphenyl, these formulations and PCBs in the environment exist as complex mixtures of 209 possible congeners. However, due to various factors which affect uptake, distribution and elimination of individual PCB isomers, the PCB residues found in the environment rarely resemble the commercial formulations. As a result, PCB residue determination relative to commercial formulation is speculative at best. Studies have shown that in general, higher homologs are metabolized more slowly as a consequence tetrachloro to decachloro congeners and p,p' DDE (a metabolite of p,p' DDT) are the largest contributors to the total chlorinated hydrocarbon body burden in the general i.e. non-occupationally exposed population. Due to i t s excellent s e n s i t i v i t y for polychlorinated organics the electron capture detector continues to be the most commonly used detector for the determinations of PCBs. In all analytical schemes u t i l i z i n g gas chromatography "with electron capture detection (GC-EC) the identification of PCB components is based solely on the retention time and the a b i l i t y of GC columns to separate the congeners. However, complete resolution of all PCB congeners on a single GC column has not been achieved so far (Duinker et al., 1988; Mullin et al., 1984). The problem related to incomplete resolution of congeners with a varying number of chlorines can be addressed by the use of a mass spectrometer as the GC detector (Onuska and Terry, 1986; Pellizzari et al., 1985). However, distinction between coeluting congeners with the 143
144
same number of chlorines is problematic even with this technique. The recently introduced technique of gas chromatography coupled with matrix isolation fourier-transform infra red spectrometry (GC-MI/FTIR) holds considerable potential for congener specific analysis (Bourne et al., 1987; Holloway e t a / . , 1988). However, the technique at present does not meet the s e n s i t i v i t y requirement for the subparts per b i l l i o n determinations. A great degree of success in resolving co-eluting components can be achieved by a two dimensional chromatographic approach, with the combination of non-polar and polar polysiloxane columns (Duebelbeis et a7., 1988; Duinker et at., 1988). Some uncertainty can remain due to a lack of readily available standards. The present report deals with application of a dual column reaction chromatography system for confirmatory analysis of polychlorinated biphenyl congeners in human serum and adipose tissue samples. This system not only yields the improved resolution of the two dimensional approach but also provides added structural information. EXPERIMENTAL The details of the dual column system used in the present study have been given earlier (Duebelbeis e t a / . , 1988). The system was b u i l t around a twelve port, two position valve. The two capillary gas chromatographs were interfaced to a reaction chamber and cryogenic trap through the valve. The i n i t i a l separation of the analyte mixtures was achieved with a 60 m x 0.25 mm i.d. fused s i l i c a column. The stationary phase in the column was "bonded" 95% methyl + 5% phenyl siloxane. The analytes transferred to the second chromatograph were separated with a 60 m x 0.25 mm fused s i l i c a column with bonded cyanopropyl polysiloxane or methyl phenyl polysiloxane stationary phase. All interconnections in the system were made with 0.5 mm o.d. (0.25 mm i . d . ) aluminum clad fused s i l i c a tubing. A reactor consisting of 50 cm X 0.5 mm i.d. nickel tubing was employed throughout this study. The extent of reaction was controlled by varying the residence time of the analyte in the tube reactor and varying the composition of the carrier gas. The product spectra obtained under optimized conditions were used in the confirmation of PCB congeners in human serum and adipose tissue samples. All of the human adipose tissue samples analyzed during the course of the study were obtained through the autopsies performed by Boone County Medical Examiner's office. While the serum samples were obtained from volunteers. The extraction and cleanup procedures used prior to the final analysis are summarized in the flow diagram as follows: 2 9 aliquot adipose tissue
5 ml aliquot serum spike with I.Ss.
spike with l.Ss. HOMOGENIZE
Densture serum
Nix with 10 g Na2SO4 (enhyd.)
proteins with 2 mt methanol
Homogenize After
Extract 3X with 5 ml hexane
24 hrs Extract in Column
concentrate
with 150 mt cyc t ohexane: dich I oromethane (1:1)
& S i l i c a gel fr ac ti onati en to separate PC.Be & chlorinated pesticides
Concentrate to 2 ml
+ Fract ionate over F l o r i s i l colulm to remove polar tipids
+ Fractionation with GPC to re~ove other lipids s i l i c a get fractionation to separate PCIB & Pesticides
& Concentrate each fraction to 1 m[ f i n a l volume
Concentrate extract to 0.2 nit f i n a l votume
145
RESULTS AND DISCUSSION The formation of electron capturing products as a result of hydrodehalogenation reactions in a flow-through nickel tube reactor has been demonstrated earlier (Kapila and Aue, 1977; Duebelbeis, 1988). The potential use of these reaction products for the confirmatory analysis of polychlorinated organic was alluded to by Kapila and Duebelbeis (Kapila e t a / . , I987; Duebelbeis, 1988). The dual column system used in the present study could be operated in the classical heart-cutting two dimensional mode where the unresolved congeners are transferred to and resolved in the second column or in the dual reaction chromatography mode, where the unresolved components undergo hydrodechlorination reactions and the products as well as the residual analytes are resolved and analyzed with the second gas chromatograph. The product pattern obtained and the intensity of product peaks can be used in confirming the identify of the congener and determining of its concentration in the sample. The system is thus analogous to mass spectrometry. An example of the distinctive product patterns obtained for two heptachloro congeners 2, 2', 3, 4, 4', S, 6' (182) and 2, 2', 3, 4', 5, 5', 6 (187) are given in Fig. 2. The major products are believed to result from displacement of single chlorine. The assignment of peak is based on retention time data and structural considerations. L
1
4
3
~
4
8
Cl
C1 C1 137
Cl 140
o 400og PCB !182
LIi/~ j
!
,,jI ~, iI
Cl CI
ci [47
![ i
--
ci 146
ci 149
3400g PCB ~181
i ,
'%1
5t.50
52.35
5 3 ~0
5 4 O5
--
5490
~5Z5
5660
RETENTION TIHE
Fig. 2.
58 30
5g15
60
)0
Chromatography of products of coeluting congeners 182 & 187.
These two congeners y i e l d i d e n t i c a l mass d i s t i n g u i s h by mass spectrometry, Fig. 3. '.~8.;) -
324 C1 I:l
57 45
:MINUTES)
-
spectra
and thus are virtually
impossible
324
I~.~C1 C1
Cl
CI 394
Cl
CI Cl
z.ong PCB# I a Z
CI
C1
1.?ng PC8~ 187 CI
C1
C1
4
;53
/
-~"" 260
, "-7~-",'~"1
28g
Fig. 3.
-
SE'~
,~"'1
I',"~'l'"','"[r'",
~2g
34g
F "7
_~0
~-
Sgg
,
4~
~E
il. ............
Mass Spectra of heptachlorobiphenyls 182 & 187.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
......
....
to
146
Distinctive products can be obtained for a l l congeners with four or more chlorines. The intensity of products was found to be d i r e c t l y related to the concentration of the congener in the sample t h u s allowing an accurate determination of congener concentration, as demonstrated in the case of congeners 170 and 190, in Fig. 4-5. The chromatograms show the products obtained from the individual standards and the mixed peak from a human adipose tissue sample. The results based on product peak intensity showed that the congener 170 forms the major portion of the chromatographic peak in the sample, concentration of this congener was determined to be 243 ppb while the concentration of congener #190 was calculated to be 7 ppb. Clearly the system allows the determination of minor components in presence of a major components. ~
co
C1
C1 ~ o
~C1 C1
C]
~ m
co
CI
C1
Is6¢cf
i:@is8
PCB 190 PCB 170
,--4
~A~ I-
I
55 B0 57 00
F
--
f
58 20
55 80
MINUTES
Fig. 4.
~
BS I
I
I
57 O0 5B 20
[-
I-
t-----
55 B0 57 O0 5B 20
MINUTES
MINUTES
Single chlorine loss product chromatograms for: A) 320 pg congener 170. B) 350 pg congener 190. C) congeners 170 & Ig0 combined.
The s e n s i t i v i t y and l i n e a r i t y of the system were found to be sufficient to allow confirmation of congeners down to sub parts per b i l l i o n levels in the serum samples. A number of human adipose and serum samples from individuals with no known occupational exposure were analyzed with the system. The results obtained are summarized in Table I and 2.
g
I
-I------
55 B0 57 O0 5B 20 5q 40 MINUTES
Fig. 5.
I
I 55 BO
- I
-I
5 7 O0 5 8 20 MINUTES
I---59 40
Single chlorine loss product chromatograms for the tentatively i d e n t i f i e d congeners 170 and 190: A) Adipose sample from a 65 yr old male. B) Adipose sample from a 27 yrs old male.
147
Table I.
Concentration of polychlorinated biphenyls in human adipose tissue.
PCB
Donor AQe/Sex
Cen~ener 15 mo
23 N
27 N
052
19.0
24.0
060
5.5
14.0
15.0
101
7.0
22.0
26.0
28 F
35 F
37 14
44 N
44 14
48 N
57 N
63 F
65 M
69 N
73 F
23.0
5.0
5.4
14.0
10.0
4.7
16.0
15.0
24.0
8.5
8.7
12.0
7.4
8.2
2.2
5.2
7.3
8.1
15.0
15.0
17.0
13.0
13.0
9.2
13.0
5.3
6.4
11.0
14.0
7.3
17.0
11.0
23.0
151
2.0
4.1
12.0
15.0
2.7
11.0
2.2
4.7
9.1
13.0
1.7
15.0
9.1
21.0
118
36.0
27.0
69.0
141.0
52.0
46.0
11.0
24.0
101.0
46.0
332.0
190.0
435.0
124.0
114
1.4
15.0
5.6
7.6
12.0
5.5
11.0
9.5
6.9
23.0
18.0
31,0
10.0
153
47.0
50.2
3]3.0
231.0
203.0
113.0
16.0
177.0
379.0
220.0
472.0
406.0
1200.0
213.0
105
11.0
11.0
22.0
34.0
19.0
18.0
7.5
16.0
37.0
20.0
70.8
48.0
120.0
44.0
141
1.2
4.6
7.7
19.0
3.9
7.9
3.2
3.3
7.1
10.0
4.0
6.9
8.4
16.0
137
2.0
3.6
16.0
18.0
15.0
7.9
7.1
11.0
17.0
11.0
21.0
14.0
77,0
11.0
138
27.0
27.0
175.0
137.0
86.0
25.0
129.0
257.0
147.0
239.0
191.0
988.0
136.0
129
6.3
16.0
27.0
14.0
15.0
12.0
16.0
20.0
26.0
22.0
27.0
32.0
54.0
23.0
182, 187
16.0
18.0
80.0
87.0
40.0
42.0
13.0
55.0
120.0
80.0
90.0
131.0
230.0
74.0
183
11.0
5.9
50.0
47.0
18.0
22.0
3.0
18.0
52.0
31.0
38.0
50.0
93.0
35.0
8.3
23.0
13.0
13.0
4.6
9.4
20.0
13.0
24.0
21.0
48.0
18.0
128 185
4.4
2.9
7.5
3.3
4.7
4.0
3.2
171, 202
3.2
11.0
14.0
15.0
12.0
11.0
17.0
18.0
26.0
31.0
156
3.7
8.3
13.0
8.0
15.0
9.3
5.6
13.0
18.3
173, 200
5.8
7.8
8.7
6.8
9.9
10.0
3.6
7.0
14.0
23.0
80.0
165.0
165.0
129.0
123.0
56.0
207.0
]01.0
250.0
180
0.86
0.27
0.92
1.6
2.0
4.6
67.0
0.84
45.0
56.0
19.0
16.0
29.0
20.0
61.0
12.0
12.0
20.0
15.0
36.0
8.6
340,0
396.0
712.0
178.0
191
1.3
5.7
3.3
2.1
4.4
3.9
2.5
3.5
7.0
5.3
7.3
6.9
12.0
4.1
170
9.0
26.0
74.0
51.0
52.0
53.0
21.0
70.0
104.0
92.0
107.0
110.0
243.0
67.0
201
6.8
26.0
36.0
43.0
38.0
41.0
43.0
65.0
95.0
111.0
163.0
152.0
193.0
5.0
196, 203
5.4
12.0
32.0
38.0
32.0
36.0
10.0
49.0
79.0
80.0
117.0
128.0
167.0
48.0
189
3.8
1.5
1.7
1.8
3.9
6.0
3.0
6.9
4.3
5.2
7.0
5.3
11.0
2.6
2O8
0.63
2.1
2.2
2.9
4.2
3.0
7.4
9.5
18.0
18.0
24.0
7.0
195
1.9
2.4
8.7
7.9
7.7
13.0
16.0
14.0
16.0
16.0
21.0
30.0
11.0
3.7
6.7
3.0
5.4
194
4.3
27.0
28.0
38.0
205
3.4
7.3
31.0
14.0
17.0
18.0
8.4
8.1
207
2O6
3.54
15.0
209
5.2
9.8
12.0
7.6
5.9
9.2
20.0
9.9
43.0
3.3
54.0
64.0
63.0
64.0
77.0
129.0
38.0
15.0
6.6
12.0
9.0
11.0
7.4
4.5
48.0
51.0
55.0
92.0
147.0
99.0
137.0
35.0
26.0
20.0
39.0
61.0
238.0
59.0
112.0
32.0
All concentrations are expressed as perts pen b i l l i o n nenogrm/g).
The relative concentration of a l l congeners in most non-occupationally exposed individuals was found to be generally quite similar with congeners 118, 138, 153, 170, 180 and 187 being the most predominant ones. Congeners 105, 114, 118, and 156 were found to be the predominant of the toxic congeners. In general a rough correlation between the PCB concentration and the age of the individual was observed. A similar trend was observed in case of PCB congener concentration in serum of a non-occupationally exposed individual. The results showing the
148
concentrations of major congeners are summarized in Table 2. The results indicate that the congeners with adjacent unsubstituted carbons are less persistent in human serum and adipose tissues. Table 2. Samlpte
SE_.XX
PCB Congener Concentration in Serum (ppb) AGE
SER23 N 20 SER19 N 23 SER17 F 24 SER11 F 26 SER4 F 28 SER16 F 29 SERIO N 30 SER27 f 30 ~R20 M 32 ~R7 F 32 ~R14 N 35 SER8 F 38 SER15 N 41 SER3 M 51 SER22 N 53 SER12 14 57 ~R20 M 52 SER~ M 67 SER9 F 76 SER30* N 25 ~R31" I,I 21 SER32* N 27 *Occqpationatty exposed individuals.
****************'***********"*****CONGENER * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 11.~8 15:3 138 183 15~6 180 0.052 O. 16 0.52 0.22 0.29 0.042 0.096 0.42 0.13 0.15 0.11 0.19 0.11 1. 1 0.28 0.62 O. 13 O.R'q 1.3 3.0 4.8 12.0
0.17 0.29 0.58 0.42 0.21 0.11 0.30 0.50 0.34. 0.28 0.36 0.19 0.17 1.3 0.54 0.82 0.38 0.76 1.5 2.9 12.0 11.0
0.096 O. 17 0.46 0.26 O. 17 0.054 O. 17 0.38 0.22 0.20 0.22 0.15 0.11 0.95 0.36 0.70 0.24 0.54 1.1 2.6 11.0 12.0
0.034 0.048 0.042 0.052 0.039 0.028 0.038 0.044 0.054 0.~3 0.048 0.030 0.024 O. 16 0.086 0.098 0.068 0.068 0.21 0.34 2.0 1.4
0.012 0.031 O. 11 0.070 0.029 0.004 0.048 O.092 0.0/*2 0.039 0.060 0.020 0.016 0.25 0.062 0.16 0.030 0.16 0.23 0.38 0.88 0.92
0.14 0.21 0.20 0.38 O. 16 0.11 0.34 O. 24 0.32 0.24 0.32 0.11 0.092 1.1 0.42 0.62 0.30 0.78 1.2 1.6 11.0 6.2
170 0.038 0.072 0.076 0.16 0.065 0.028 O. 13 O. 10 0.12 0.11 0.10 0.049 0.026 0.45 0.14 0.24 O. 10 0.34 0.51 0.72 4.2 2.6
The data clearly demonstrate that the dual column system allows unambiguous detection of individual congeners down to sub parts per billion levels with relatively small sample. ACKNOWLEDGEMENTS Partial support for this work was provided by a grant from Monsanto Chemical Co., St. Louis, Missouri. The assistance of Dr. Jay Dix in obtaining the adipose tissue samples is also acknowledged. REFERENCES Bourne, S., G.T. Reedy and P.T. Cunningham (1978). J. ChromatoaraDhic Scj .... ]_Z, 460-463. Duebelbeis, D.O. A dual column reaction gas chromatographic system for the structural confirmation of chlorinated organics, 1988, Ph.D. Dissertation, University of Missouri, Columbia. Duebelbeis, D.O., S. Kapila, T. Clevenger, A.F. Yanders and S.E. Manahan (1988). Chemosphere, in press ]988. Duinker, J.C., D.E. Schulz and G. Petrick (]988). Anal. Chem., 6_00(6), 478-482. Holloway, T.T., B.J. Fairless, C.E. Freidline, H.E. Kimball, R.D. Kloepfer, C.J. Wurrey, L.A. Jonooby and H.G. Palmer (]988). Appl. Spectrosc., 42(2), 359-369. Kapila, S. and W.A. Aue (]977). J. Chromatoqraphic Sci., ]__55,569-572. Kapila, S., D. Duebelbeis, R. Malhotra, A.F. Yanders and S.E. Manahan (1987). In: Pesticide Science and Biotechnoloqy (R. Greenhalgh and T.R. Roberts, eds.) pp 325-328, B1ackwell Scientific Publications. Mullin, M.D., C.M. Pachini, S. McCrindle, M. Romkes, S.H. Safe and L.M. Safe (1984). Environ. Sci. Technol., 18(6), 468-476. Onuska, F.I. and K.A. Terry (1986). HRC CC, J__~.Hiqh Resolut. Chromatoqr. Chromatoqr. Commun. 9, 67]-675. Pellizzari, E.D., M.A. Morley and S.D. Cooper (1985). J_,. Chromatoqr., 339, 277-314. Rogan, W.J. (1982). Teratology, 26, 26. Rogan, W.J., B.C. Gladen, K-L. Hung, S-L. Koong, L-Y. Shih, J.S. Taylor, Y-C. Wu, D. Yang, N.B. Ragan, C-C. Hsu (1988). Science, 24__!, 334-336.