EPA 560/6-8-007
ANALYSIS OF POLYCHLORIMATED BIPHENYL (PCB)
IN HUMAN BLOOD SERUM SAMPLES
OCTOBER 1977
ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF TOXIC SUBSTANCES
WASHINGTON, D.C.
-------
DISCLAIMER
This report has been reviewed by the Office of
Toxic Substances, U.S. Environmental Protection
Agency, and approved for publication. Mention of
trade names or commercial products does not con-
stitute endorsement or recommendation for use.
-------
ANALYSIS OF POLYCHLORINATED BIPHENYL (PCB)
IN HUMAN BLOOD SERUM SAMPLES
Final Report
Research Request No. 2
Contract 68-01-3248
Prepared By:
Charles L. Stratton and Paul C. Geiszler
ENVIRONMENTAL SCIENCE AND ENGINEERING, INC,
P. 0. Box 13454, University Station
Gainesville, Florida 32604
For:
OFFICE OF TOXIC SUBSTANCES
ENVIRONMENTAL PROTECTION AGENCY
Washington, D.C. 20460
October, 1977
-------
CONTENTS
Figures
Tables
1. Introduction 1
2. Analytical Procedure 2
Quantitation 2
Confirmation 9
References 16
Appendices
A. Analytical data results
B. Analytical procedure
C. Sample calculation
D. Data for PCB pool samples
£. Perchlorination procedure
-------
FIGURES
Number Page
1 Typical Chromatogram of a Serum Extract 3
2 Chromatogram of Aroclor 1242 4
3 Chromatogram of Aroclor 1254 5
11
-------
TABLES
Number Page
1 Inter-Run Statistics for PCB Pool Samples 7
2 Inter-Laboratory Comparison of PCB Pool
Sample Results 8
3 Results of Confirmatory Analysis for PCB 10
4 Reagent Blank Statistics 14
5 Comparison of Pattern versus Perchlorination
Results for PCB Pool B 15
111
-------
INTRODUCTION
A total of 208 human blood serum samples and two mother's milk samples
were analyzed for polychlorinated biphenyl (PCB). The samples were
supplied to Environmental Science and Engineering, Inc. (ESE), in frozen
condition by the Department of Health, Education, and Welfare, Center
for Disease Control, Atlanta, GA (CDC). This report includes the
analytical results for these samples and an assessment of the degree of
uncertainty involved in the analysis. The analytical results, expressed
as Aroclor® 1242 and Aroclor® 1254, are tabulated in Appendix A.
-------
ANALYTICAL PROCEDURE
The analytical procedure used is described, in detail, in Appendix B.
This procedure, which is based on the procedure used by the Michigan
Department of Health, was developed by the cooperation of CDC and ESE.
Quality control was maintained during each sample run by:
1. Establishing and documenting chromatogram resolution by
analysis of a chlorinated hydrocarbon pesticide mixture;
2. The analysis of blind duplicate samples;
3. The analysis of reagent blank samples;
4. The analysis of a chlorinated hydrocarbon pesticide mixture
carried through the procedure;
5. The analysis of two serum pool samples.
QUANTITATION
Each chromatogram was quantitated as Aroclor 1242 and Aroclor 1254 as
described in Appendix B. Figure 1 is a typical chromatogram of a serum
sample extract. Figure 2 is a standard Aroclor 1242 chromatogram at the
same gas chromatographic conditions. Figure 3 is a standard Aroclor
1254 chromatogram. Figure 1 shows the baseline used for peak height
measurement. The same convention was used for the construction of the
baseline on all samples. The peak heights were summed for all matching
peaks (within a 4 percent retention time window) for the respective
Aroclor pattern. Appendix C shows the steps of typical sample
quantitation.
-------
Figure 1. Typical Chromatogram of a Serum Extract
-------
Figure 2. Chromatogram of Aroclor 1242
-------
Ul
Figure 3. Chromatogram of Aroclor 1254
-------
Each analytical run contained a PCB Pool A and a PCS Pool B sample.
These pool samples consisted of serum prepared and provided by CDC.
Aroclor 1242 was spiked into PCB Pool B. Appendix D is a summary of CDC
analytical results for these two pool samples.
Table 1 is a summary of the inter-run statistics for the PCB pool
samples as analyzed by ESE. PCB Pool A contained a mean Aroclor 1242
concentration of 6.2 ng/ml and a mean Aroclor 1254 concentration of 7.4
ng/ral using the quantitation convention described above. These low PCB
levels are very near the analytical detection limit of the method. At
these levels, the analytical relative standard deviation is approxi-
mately 36 percent. PCB Pool B was spiked by CDC with Aroclor 1242 to a
calculated concentration of 81.1 ng/ml. The mean Aroclor 1242 concen-
tration reported for the 26 analytical runs is 80.5 ng/ml with a
relative standard deviation of 10.7 percent from run to run. The back-
ground level of Aroclor 1254 in PCB Pool B is 17.7 ng/ml with about a 30
percent relative standard deviation.
Table 2 summarizes inter-laboratory statistics for the PCB pool samples.
The correspondence between CDC and ESE results for Aroclor 1242 in these
pool samples is good. It should be noted in Table 2 the standard
deviation of the ESE analyses reflects inter-run variability, while the
standard deviation for the CDC analyses reflects intra-run variability.
For PCB Pool B, the ESE mean result is 99 +_ 10 percent of the Aroclor
1242 spiked amount and 92 +_ 10 percent of the mean CDC result.
-------
Table 1. Inter-Run Statistics for PCB Pool Samples
Aroclor 1242
Aroclor 1254
PCB Pool A
X = 6.2 ng/ral
n = 26 runs
a = _+ 2.4 ng/ml
a% = + 38.8%
X =
n -
7.4 ng/ml
26 runs
cr = +_ 2.5 ng/ml
a% = +• 33.9%
PCB Pool B
X =
n =
0 =
a% =
80.5 ng/ml
26 runs
4-8.5 ng/ml
+ 10.7%
X = 17.7 ng/ml
n = 26 runs
0 = _+ 5.4 ng/ml
a% = + 30.3%
-------
Table 2. Inter-Laboratory Comparison of PCB Pool Sample Results
Mean Cone.
Aroclor 1242 No. of Standard
(ng/ml) Analyses Deviation
PCB Pool A
CDC Analysis,
ESE Analysis,
PCB Pool B
Aroclor 1242*
Aroclor 1242
Spiked Aroclor 1242*
CDC Analysis,
ESE Analysis,
Aroclor 1242*
Aroclor 1242
6.9
6.2
81.1
87.5
80.5
5
26
NA
5
26
+_ 0.6
± 2'4
NA
+_ 5.8
_+ 8.5
* Data from Virlyn W. Burse memo dated May 5, 1977 (Appendix D)
8
-------
CONFIRMATION
Confirmation of PCS was accomplished by perchlorination to decachloro-
biphenyl (DCS) using antimony pentachloride as the derivatizing reagent
as described by Armour (1973). Thirteen percent of the total number of
samples were subjected to perchlorination. The perchlorination proce-
dure is described in detail in Appendix E. Tests have shown (ESE, 1977)
that this perchlorination procedure yields 100 _+ 4.2 percent recovery of
Aroclor standard solutions over the range of 103 ng to 10 ug.
The results of the confirmatory analyses along with the pattern matching
results are tabulated in Table 3. This table shows the pattern analyzed
values for Aroclor 1242 and Aroclor 1254, the calculated DCB concentra-
tion based on the pattern matching results, the analyzed DCB concentra-
tion after perchlorination of the sample, and the percent correspondence
between the actual DCB value and the calculated DCB value. Conversions
between Aroclor and DCB concentrations were calculated, using the
factors of Armour (1973). For example, the calculation of DCB
concentration in sample A0102 is as follows:
15 ng/ml Aroclor 1242 . 2 ng/ml Aroclor 1254 _ 32 _„/_-, „_ DrR
0.52 0.65 S
Implicit in the use of these conversion factors is the assumption that
Aroclor 1242 and Aroclor 1254 are present in the serum sample in unmodi-
fied form. This is definitely not the case and, hence, the assumption
is a very weak point in comparison of the pattern matching results with
the perchlorination results.
-------
Table 3.
CDC No. Name/Sample
Analyzed
A0102
A0103
B0103
40-01
01-01
11-01
46-01
84-03
14-03
.16-01
PCB pool B
PCB pool B
Reagent blank
PCB pool B
Reagent: blank
PCB pool B
Reagent blank
PCB pool B
Run No.
1
1
1
2
3
3
3
4
4
4
5
7
8
8
8
8
9
10
Sequence
No.
9
2
13
1
12
9
4
9
3
4
4
11
1
3
9
11
10
6
Sample
Vol. (ml)
2.5
4.3
3.6
2.5
5.0
2.5
4.6
5.0
2.5
4.2
3.2
5.0
5.0
5.0
5.0
4.4
3.5
3.2
Aroclor 1242
(ng/ml)
95
15
11
96
0
83
16
4
98
12
9
19
7
0
82
7
11
14
Aroclor 1254
(ng/ml)
21
2
5
17
0
20
8
1
9
9
12
23
37
0
17
24
1
15
DCB
(ng/ml)
215
32
29
211
0
191
43
10
202
37
36
72
70
0
184
50
23
50
DCB
(ng/ml)
166
18
15
133
11
150
30
7
132
26
23
63
50
6
134
50
18
17
• X. J.VJU
DCB Calculated
%
77.2
56.3
51.7
63.0
—
78.5
69.8
—
65.3
70.2
65.1
87.7
71.4
—
72.8
100.0
78.3
34.0
-------
Analyzed
Calculated Analyzed 11CB Analyzed
Cue No. Name/Sample
-sss
17-01
81-01
53-01
79-04
Nanograde hexane
20-03
23-02
PCB pool B
78-01
Reagent blank
PCB pool B
67-01
86-01
82-02
80-01
57-02
74-01
76-01
Run No.
11
12
13
13
—
14
15
16
16
16
17
17
18
19
20
21
22
23
Sequence
No.
1
11
3
9
—
5
6
3
7
13
1
9
8
8
10
12
5
6
Sample
Vol. (ml)
5.0
3.8
5.0
4.8
5.0
5.0
4.2
5.0
3.8
5.0
4.8
5.0
5.0
5.0
5.0
5.0
3.1
4.7
Aroclor 1242
(ng/ml)
11
20
12
15
0
34
20
81
58
2
71
14
14
10
14
15
33
161
Aroclor 1254
(ng/ml)
9
24
12
15
0
29
26
25
40
0
19
10
17
15
27
12
23
92
DCB
(ng/ml)
35
75
41
52
0
110
78
194
173
4
166
42
53
42
68
47
99
451
DCB
(ng/ml)
14
25
7
19
0
37
16
108
127
13
119
17
36
24
52
27
35
167
DCB Calculated"
%
40.0
33.3
17.1
36.5
—
33.6
20.5
55.6
73.4
—
71.5
40.5
68.3
57.1
76.5
57.4
35.4
37.0
-------
Analyzed Calculated Analyzed OCR Analyzed
CUC No.
13-06
—
—
021337
—
—
64-01
—
021338
-Name/Sample
DCB
PCB pool B
Aroclor 1254
PCB pool B
Reagent blank
Run No.
24
—
25
25
—
26
26
26
26
Sequence
No.
3
—
1
3
—
6
7
1Q
13
Sample
Vol. (ml)
2.4
1.0
5.0
3.1
0.5
5.0
4.3
5.0
5.0
Aroclor 1242
(nR/ml)
5
0
70
10
0
72
18
0
220
Aroclor 1254
(ng/ml)
12
0
18
10
480
11
8
1
63
DCB
(ng/ml)
28
2140
162
35
738
155
47
2
520
DCB
(ng/ml)
25
2100
133
22
666
96
14
2
65
DCB Calculated
7,
89.3
98.1
82.1
62.9
90.2
61.7
30.8
—
12.5
-------
Reagent blank analyses were conducted during each sample run. Statisti-
cal analysis of the reported reagent blank data is presented in Table 4
for both the pattern quantitation and perchlorination data. Reagent
blank values are uniformly low.
Table 5 summarizes all analytical results for PCS Pool B. The
perchlorination results are 83 +_ 13 percent of the CDC reported Aroclor
1242 spike, and 71 _+ 12 percent of the ESE reported total Aroclor
concentration.
For the 26 serum samples analyzed by both pattern matching and
perchlorination, the mean analyzed DCB value is 51.4 percent of the mean
calculated DCB value. The correlation coefficient between the analyzed
and calculated DCB values is 0.89. Linear regression analysis yields
the following empirical relationship.
analyzed = nT**«/analyzed Aroclor 1242 + analyzed Aroclor 1254V g^Q
DCB (ng/ml) ' \ 0.52 0.65 )
13
-------
Table 4. Reagent Blank Statistics
Pattern Quantitation
Aroclor 1242 Aroclor 1254
n = 36 n = 35
I = 0.6 ng/ml X = 0.3 ng/ml
a = jf 1.0 ng/ml cr = +_ 0.7 ng/ml
Perchlorination
DCB
n = 5
"X = 7.8 ng/ml as DCB
a = + 4.3 ng/ml as DCB
14
-------
Table 5. Comparison of Pattern versus Perchlorination Results for PCB
Pool B
PCB (ng/ml) DCB (ng/ml)
Spiked Aroclor 1242* 81.1 156 (c)
Pattern Analyzed Aroclor 1242 (n=26) 80.5 _+ 8.5 155 +_ 16 (c)
Pattern Analyzed Aroclor 1254 (n=26) 17.7 +_ 5.4 27 +_ 8 (c)
Perchlorination Analyzed PCB Pool B (n=9) NA 130 _+ 21 (a)
* Data from Virlyn W. Burse memo dated May 5, 1977 (Appendix D)
(a) Analytical result
(c) Calculated DCB concentration using conversion factors of Armour
(1973)
15
-------
REFERENCES
Armour, J. A. 1973. Quantitative Perchlorination of Polychlorinated
Biphenyls as a Method for Confirmatory Residue Measurement and
Identification. Journal of the Association of Official Analytical
Chemists, 56(4):987-993.
Environmental Science and Engineering, Inc. 1977. Unpublished data.
16
-------
APPENDIX A
ANALYTICAL RESULTS
-------
CDC
Number
A0101
A0102
A0105
A0106
A0107
A0108
A0111
A0112
A0113
A0114
A0115
A0116
A0117
A0118
A0121
A0122
A0124
A0125
B0101
B0102
B0103
B0204
B0405
B0406
39-02
40-01
90-01
85-01
01-02
01-02
01-06
PPM
Birthdate 1242
0.009
0.015
0.008
0.006
0.011
0.005
0.005
0.009
0.011
0.021
0.011
0.015
0.007
0.008
0.002
0.003
0.007
0.005
0.007
0.013
0.016
0.020
0.011
0.014
7/03/38 0.003
5/16/42 0.006
1/02/50 0.012
11/16/39 0.016
12/22/55 0.011
7/16/57 0.020
' 8/19/33 * 0.011
12/29/60 0.008
PPM
1254
0.002
0.002
0.002
0.004
0.007
0.004
0.007
0.006
0.005
0.002
0.005
0.003
0.002
0.003
0.003
0.004
0.003
0.003
0.001
0.005
0.008
0.005
0.017
0.009
0.003
0.008
0.009
0.005
0.006
0.010
0.008
0.009
-------
CDC
Number
01-05
01-04
01-01
91-01
39-03
05-02
04-01
76-03
02-02
05-01
02-01
07-01
06-01
88-02
08-06
08-01
08-02
08-04
08-07
10-01
50-01
10-02
11-01
79-05
46-01
46-02
11-02
50-02
51-01
46-03
47-02
84-03
Birthdate
8/26/64
8/14/38
8/26/33
2/26/45
3/20/67
10/10/15
5/13/07
8/11/72
8/03/43
7/04/19
10/11/41
4/05/12
2/15/34
4/17/30
8/25/71
8/02/34
6/10/40
3/28/63
6/07/64
10/30/51
12/02/25
11/22/47
5/01/25
9/30/71
1/21/42
1/02/63
11/20/21
3/06/24
10/12/55
10/31/61
2/24/45
1/03/72
PPM
1242
0.011
0.019
0.009
0.011
0.005
0.005
0.010
0.008
0.015
0.004
0.051
0.005
0.006
0.042
0.004
0.010
0.007
0.003
0.002
0.002
0.011
0.004
0.019
0.018
0.007
0.005
0.010
0.031
0.016
0.020
%
0.015
0.007
PPM
1254
0.005
0.002
0.012
0.006
0.003
0.009
0.007
0.005
0.008
0.014
0.020
0.005
0.006
0.010
0.006
0.013
0.009
0.005
0.006
0.007
0.011
0.007
0.023
0.025
0.024
0.019
0.018
0.032
0.023
0.014
0.010
0.024
-------
CDC
Number
12-02
47-01
12-01
13-03
13-07
13-02
14-03
87-01
13-01
13-04
14-01
16-01
16-02
15-02
15-01
79-03
17-01
17-02
52-02
63-01
55-01
56-01
18-01
88-03
18-02
19-01
59-02
19-03
48-01
48-04
81-01
48-02
Birthdate
1/30/31
3/03/42
6/19/29
6/25/61
10/03/64
4/18/40
2/23/60
7/31/48
1/02/39
2/16/63
9/02/36
2/23/54
8/23/53
4/11/32
12/11/28
7/13/68
1/02/49
1/14/50
11/30/47
6/12/41
2/29/29
8/14/46
7/12/45
8/01/56
2/16/45
10/04/47
5/09/28
9/09/66
6/12/39
6/26/68
11/18/40
4/28/43
PPM
1242
0.007
0.000
0.000
0.018
0.007
0.003
0.011
0.002
0.008
0.015
0.022
0.014
0.018
0.047
0.034
0.044
0.020
0.019
0.007
0.024
0.003
0.004
0.012
0.011
0.023
0.009
0.012
0.009
0.015
0.002
0.020
0.008
PPM
1254
0.021
0.009
0.010
0.010
0.001
0.002
0.001
0.004
0.002
0.004
0.006
0.015
0.009
0.014
0.011
0.023
0.008
O.OOS
0.006
0.009
0.026
0.006
0.005
0.010
0.006
0.011
0.016
0.004
0.010
0.011
0.024
0.013
-------
CDC
Number
48-03
53-01
59-01
53-02
65-01
79-04
80-02
49-02
61-01
49-01
20-03
92-01
20-04
20-01
88-04
77-01
22-01
21-01
23-01
23-02
24-02
26-02
87-02
83-01
45-01
25-02
27-01
29-01
78-01
29-02
28-02
79-01
Birthdate
11/10/63
4/10/31
1/11/28
5/28/21
8/07/40
1/18/70
6/30/03
11/06/15
6/01/05
9/28/24
3/05/40
11/18/46
7/21/71
8/17/40
9/06/60
1/14/22
1/28/39
12/02/48
1/16/42
4/07/44
4/16/27
9/14/13
5/08/51
4/07/36
9/11/34
9/22/16
9/08/46
2/22/19
3/18/31
9/28/15
9/07/65
1/03/46
PPM
1242
0.012
0.012
0.041
0.004
0.013
0.015
0.020
0.032
0.019
0.014
0.034
0.010
0.009
0.001
0.011
0.009
0.006
0.010
0.011
0.020
0.014
0.040
0.007
0.026
0.009
0.006
0.002
0.006
0.058
0.002
0.010
0.013
PPM
1254
0.008
0.012
0.025
0.011
0.007
0.015
0.022
0.053
0.014
0.011
0.029
0.010
0.008
0.006
0.020
0.014
0.017
0.012
0.012
0.026
0.015
0.016
0.006
0.011
0.041
0.009
0.004
0.013
0.040
0.006
0.014
0.024
-------
CDC
Number
_«.
73-01
71-01
66-01
61-02
67-01
78-02
88-01
24-01
26-01
30-01
30-02
86-01
30-01
54-01
76-02
28-01
42-01
62-01
01-07
82-02
84-01
74-01
34-01
52-01
33-02
75-01
33-01
35-01
80-01
58-01
Birthdate
8/09/15
7/25/29
5/03/40
9/27/53
5/29/33
3/23/32
11/21/34
9/24/28
6/22/24
8/25/08
6/27/34
4/28/34
5/23/26
11/24/57
11/04/19
6/01/49
1/20/29
6/23/34
9/23/02
10/27/59
10/19/51
3/22/50
12/15/37
8/25/52
5/17/51
10/22/51
9/23/18
9/07/53
8/18/—
12/11/89
5/31/49
PPM
1242
0.008
0.011
0.016
0.017
0.004
0.014
0.008
0.015
0.004
0.025
0.003
0.011
0.014
0.012
0.006
0.011
0.004
0.008
0.011
0.009
0.010
0.008
0.007
0.015
0.003
0.005
0.014
0.007
0.004
0.014
0.005
PPM
1254
0.015
0.015
0.009
0.014
0.005
0.010
0.018
0.010
0.012
0.018
0.014
0.017
0.017
0.014
0.011
0.017
0.018
0.014
0.012
0.012
0.015
0.012
0.011
0.009
0.007
0.012
0.024
0.018
0.010
0.027
0.017
-------
CDC
Number
38-03
44-01
70-01
41-01
36-01
84-02
57-01
57-02
38-02
38-01
74-01
68-04
68-02
79-02
68-01
68-03
69-01
43-01
76-01
41-05
60-05
02-03
13-05
13-06
14-05
16-03
17-03
19-04
22-02
22-03
23-03
Birthdate
8/22/59
10/01/26
6/27/26
8/22/45
10/25/58
7/16/50
9/04/15
4/14/20
9/10/35
5/07/50
2/12/43
8/25/48
4/18/24
2/24/21
2/26/47
9/14/51
3/07/45
5/07/21
10/10/51
9/27/48
PPM
1242
0.004
0.002
0.004
0.009
0.008
0.010
0.010
0.015
0.013
0.007
0.012
0.033
0.012
0.014
0.020
0.015
0.003
0.169
0.084
0.161
0.004
0.004
0.005
0.002
0.005
0.003
0.004
0.004
0.004
0.003
0.003
0.003
PPM
1254
0.008
0.010
0.007
0.010
0.011
0.014
0.026
0.012
0.018
0.012
0.011
0.023
0.007
0.032
0.022
0.012
0.009
0.103
0.032
0.092
0.009
0.025
0.004
0.006
0.012
0.006
0.003
0.005
0.008
0.020
0.013
0.007
-------
CDC
Number Birthdate
23-04
38-04
38-06
41-02
41-03
41-04
64-01
60-02
60-03
4/30/62
in/19/ss
8/17/40
021336
021338
B7
B8
B9
021337 1/02/50
PPM
1242
0.003
0.004
0.002
0.003
0.006
0.000
0.018
0.006
0.008
0.003
Onn?
• UU J
0.002
Oflfi9
. \J\Jf.
0.160
0.220
0.012
0.015
0.030
0.010
PPM
1254
0.006
0.013
0.009
0.007
0.008
0.008
0.008
0.007
0.011
0.011
n 01 9
u . u J. £.
0.004
Onns
. uu_?
0.066
0.063
0.007
0.011
0.017
0.010
-------
APPENDIX B
ANALYTICAL PROCEDURE
-------
PROCEDURE FOR
ANALYSES OF POLYCHLORINATED BIPHENYL (PCB) IN BLOOD SERUM
Prepared by:
Toxicology Branch
Clinical Chemistry4Division
Bureau of Laboratories
Center for Disease Control
Atlanta, GA
and
ENVIRONMENTAL SCIENCE AND ENGINEERING, INC,
P. 0. Box 13454, University Station
Gainesville, FL 32604
-------
I. ANALYTICAL PROCEDURE
A. Extraction
1. Pipet 5 ml of serum into a 12.5 cm length culture tube to
which was added 4 ml of methanol (all solvents should be of
pesticide quality and stored in glass or teflon containers),
attach a teflon-lined screw cap.
2. Mix the contents of the tube on a rotary mixer (50-55 rpm)
for 4 minutes.
3. Add 5 ml of hexane-ethyl ether (1:1, V/V) .
4. Attach the teflon-lined screw cap and place on a rotary
mixer (50-55 rpm) for 15 minutes.
5. Centrifuge at 2000 rpm for 2-5 minutes.
6. Transfer the upper solvent layer by pipette to a 25 ml
graduated Kuderna-Danish concentrator tube.
7. Carry out steps 3-6 twice more with fresh hexane-ethyl ether
solutions; the extractions are combined in the concentrator
tube.
8. Concentrate the extract to approximately 0.5 ml under a slow
stream of dry organic-free nitrogen.
B. Saponification
1. Add 2 ml of a 2% (V/W methanolic potassium hydroxide
solution, add an ebbulator.
2. Attach a micro-snyder column.
3. Using a. Kontes Tube Heater, allow the contents to gently
boil, reducing the volume to 0.3 ml.
-------
Note: If a precipitate has formed, add a few drops of 2%
methanolic KOH and warm gently in steam with swirling until
the precipitate dissolves.
4. After the solution has cooled slightly, add about 2 ml of
methanol-water (1:1).
5. Allow solution to reach room temperature and add 2 ml hexane
into tube.
6. Stopper tube and shake vigorously.
7. Pipet out top layer.
8. Repeat steps 7-9 two additional times.
C. Column Preparation and Elution
1. Activate in a 200°C oven for 24 hours Woelm silica gel
activity Grade I and granular sodium sulfate (Mallinckrodt
#8024). Store in air tight flasks.
2. Deactivate the silica gel with 3% water (W/V) and allow to
equilibrate at least 5 hours before use.
3. Pack a small mat of glass wool into the bottom of a 7 mm
i.d. x 200 mm chromatography column, which contains a 50 ml
reservoir and a teflon stopcock.
4. Mix 3g of silica gel with approximately 50 ml of hexane and
pour the slurry into the column.
5. Allow the silica gel to settle and then top with a 5-7 g
layer of sodium sulfate.
-------
6. Elute the columns with 20 ml of hexane and as the last of
the hexane enters the sodium sulfate layer, add the
concentrated, saponified extract. Place graduated 25 ml
receiver under column. Open stopcock and allow extract to
just enter the sodium sulfate layer before closing stopcock.
7. Rinse the concentrator tube with 1 ml of hexane and add to
the column. Open stopcock and allow rinse to just enter the
sodium sulfate layer before closing stopcock.
8. Rinse the micro-Snyder-concentrator tube assembly with 2 ml
of hexane and add to the column. Open stopcock and allow
rinse to just enter sodium sulfate layer before closing
stopcock. Note: The sodium sulfate layer should never
become "dry".
9. Carefully add 25 ml hexane to the column (without disturbing
the sulfate layer). Open stopcock and allow column to elute
until the 25 ml receiver is filled.
10. Concentrate eluant under a slow stream of dry, organic-free
nitrogen to approximately 1.0 ml.
11. Rinse the assembly by allowing 1-2 ml of hexane to run down
the sides of the column.
12. The solution is then reduced to exactly 1 ml under nitrogen
and is now ready for GLC analysis. Note: After analyzing
by GLC it may be necessary to further concentrate or dilute
this solution in order to re-analyze.
-------
D. GLC Analysis
All analyses will be on a Varian 2700 GLC with electron capture
detector. The flow rate which gives at least a 10% peak height
to valley resolution between p,p'-DDE and dieldrin on the 6 ft x
1/8 in. column containing 1.5% OV-17-1.95%QF-1 should be used.
The injector and detector temperature must be higher than the
column which is run isothermally at 200°C. Also the operational
range for the electrometer should be 10~10 and the attentua-
tion should be x!6 or more. All analyses must be run at the
same electrometer setting; therefore, it might be necessary to
dilute the sample.
E. Confirmation
A minimum of five percent of the samples will be confirmed by
perchlorination to decachlorobiphenyl to insure that PCB is
indeed being analyzed. The samples of the highest concentration
will be selected by ESE for this analysis.
II. DESCRIPTION OF A "RUN"
A. Unknown samples and controls—to be put through the entire
analytical procedure
Eight unknown samples (exception: the 27 samples from
Mississippi will be in 3 runs of 9 samples each).
2. Two controls (made from human serum at CDC and designated as
PCB Pool A and PCB Pool B).
-------
3. One chlorinated hydrocarbon control containing 1 ml of the
following: Y and 3 hexachlorocyclohexane;
heptachlorepoxide; o,p'DDE; dieldrin; p,p'-DDE; o.p'DDT,
p,p'-DDD and P,p'-DDT with a range of 50-100 ng/ml. This
control will be prepared by ESE. When running the unknown
samples, any peaks that have the same retention times as the
control-should not.be included for quantitation.
4. Two reagent blanks, the second blank should be composed of
solvents that will be used in the subsequent run to validate
purity of solvents (especially useful when new solvent
bottles or lot numbers are used in the next run).
B. Standards—to be analyzed by gas liquid chromatography
1. Three Aroclor 1242 standards of varying concentrations. The
upper concentration should be chosen so that the tallest
peak is approximately 80% full scale. Then without
adjusting the attenuation, the next two concentrations
should be chosen to encompass the working range.
2. Three Aroclor 1254 standards of one concentration. This
concentration should be chosen such that it falls in the
middle of the 1242 working concentration range.
-------
III. INJECTION ORDER
Std 1242 (High) Std 1254 II
Sample Sample
Sample Sample
Std 1254 I Std 1242 (Medium)
Sample Sample
Sample Sample
Std 1242 (low) Std 1254 III
Sample Sample
Sample Sample
IV. QUANTITATION
A. As 1242
Baselines for the chromatograms of the standards and the
samples will be drawn by following down the sloping solvent
tail and continuing out until a stable baseline is
established. The retention times of all the peaks in the
three 1242 standards will be measured from the solvent front
to the nearest tenth of a millimeter. The three retention
time values for each specific peak in the 1242 standard (i.e.
from the three separate injections) are averaged and a four
percent retention time window is then generated from this
average retention time value. The four percent retention time
window (i.e. plus and minus two percent of the retention time
distance of each particular peak of the 1242 standard) is then
applied for matching of sample peaks to standards.
-------
Samples that have peaks within these windows that are offscale
will be repeated to obtain all quantifiable peaks on scale.
The chlorinated hydrocarbon control is gas chromatographed in
the same range as the samples (i.e. if all or nearly all
samples are in 1.0 ml and 5 microliters are being injected
then this will be followed for the chlorinated hydrocarbon
control). Peaks in the chlorinated hydrocarbon control that
are within the retention time windows of the standard and are
offscale in the control will be disregarded and these particu-
lar peaks will not be quantified in the standards, blanks, or
in the samples since they may be attributable to pesticides in
the samples and not specific PCB's. Peaks in the blank, which
is analyzed in the same range as the samples, that fall in the
retention time windows are measured and summed. This value is
used as zero nanograms injected in the generation of a
standard curve. All peaks in each of the tree Aroclor 1242
standards that have not been discarded are measured and summed
and, together with the blank value, are used to generate the
k
standard curve. The curve is established by plotting the
total peak height as the Y axis versus nanograms of Aroclor
1242 injected on the X axis. The height in millimeters of all
sample peaks that have not be discarded and are in the
specific windows are measured and summed and the total peak
height value is then plotted on the Y axis of the standard
curve to .give nanograms of Aroclor 1242 injected for each
sample. All sample analysis points on this curve must be
-------
between the high standard and the origin. Calculations are
then carried out to give micrograms Aroclor 1242 per
milliliter sample.
B. As 1254
The retention times of all the peaks in the Aroclor 1254
standards will be measured from the solvent front to the
nearest tenth of a millimeter. The three retention time
values for each specific peak in the Aroclor 1254 standards
(i.e. one for each injection) are averaged and a four percent
window is then generated from this value. Peaks in the
chlorinated hydrocarbon control that are within the retention
time windows of the Aroclor 1254 standards and are offscale in
the control will be disregarded as was done in the Aroclor
1242 quantitation. All peaks in each of the three Aroclor
1254 standards that have not been discarded are measured and
summed. The total peak height of the three separate Aroclor
1254 standards must agree within +_ 10%. An average total peak
height response factor is then calculated, i.e. total peak
height per nanogram Aroclor 1254 injected. Sample peaks that
fall within the retention time windows of the Aroclor 1254
standard, and have not been discarded must be onscale and are
then measured and summed to give a total peak height of
Aroclor 1254 in the samples. This value is then divided by
the Aroclor 1254 response factor to give nanograms of Aroclor
1254 injected for each sample. Calculations are then carried
out to give micrograms Aroolor 1254 per millimeter sample.
-------
c. REPORTING OF RESULTS
The chromatograms will include the following information: initials
of operator; date; run number; chart speed; temperature of
injector; detector; and column; carrier gas and flow rate; standing
current; percent full scale deflection at range of 10~° and
attenuation of 32; operating electrometer settings; volume of
sample injected; final sample volume; and volume of serum sample.
The chromatograms of the standards (1242 and 1254) will include
concentration and volume injected.
VI. STORAGE OF SAMPLES
All serum samples will be stored frozen until analysis. Any unused
serum sample will be stored for at least 2 months after notifica-
tion of results.
VII. PREPARATION AND STORAGE OF STANDARD PCB SOLUTIONS
Aroclor stock solutions will be prepared using the Aroclor obtained
directly from Monsanto Company, St. Louis, Missouri, from the
following lots:
Aroclor 1016 Lot KC10-7008
Aroclor 1242 Lot KC09-415
Aroclor 1254 Lot KD05-613
Approximately 0.05 ml of the Aroclor is placed directly into a tare
weighed 50 ml volumetric flask. The flask with Aroclor is then
weighed and the mass of Aroclor is calculated by difference. The
flask is filled to volume with nanograde 2,2,4 trimethylpentane at
-------
20°C. This solution is shaken for a period of 4 hours and allowed
to stand at least one full day to assure complete solution. This
stock solution is stored at 4°C in the dark. Dilutions of this
stock solution are used as Aroclor standards. All standards are
stored in the dark at 4°C when not in immediate use. They are
brought to room temperature and mixed before each use. Working
standards should be prepared from the stock solution at least once
per week.
-------
APPENDIX C
SAMPLE CALCULATION
-------
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-------
APPENDIX D
DATA FOR PCB POOL SAMPLES
-------
May ^, j.977
E:IORAKDUK TO THE RECGE:
XCM: Virlya-W.-Burse,-jieaearch-Chemist, ..Toxicology Branch^ CC,_BL
U3J: "Characterization" of the Third ESE Pool
he purpose of this mer.orandui?. is to report data obtained by this laboratory
hen an attempt was made to "characterize" the third set of pools prepared
or the ESE PCB project. These pools are to be used as internal bench controls
y ESE once the analysis of unknowns is commenced.
a the process of deterrining. residue levels of AR 1242, in each of the two
ools, the analyst adhered to (as much as possible) those aspects of the
rotocol proposed for ESE (See JAL's correspondence to Charles Strattcn
fESE, April 20, 1977), i.e. I.D., II.B., Ill and IV.A.
he values for the two pools are shown in Table 1, since the3r are to be
iewed as two separate pools no correction was made for background of AR 1242
Ethe spiked pool.
he standard curve used for calculations is attached.
Virlyn W. Burse
Attachment
Dr. Liddle «"
.Dr. Ki mb r ough
Dr. Needh=r".
-------
"CHARACTERIZATION OF THIRD ESE PCB POOL
•P""^
Pool I.D. Aroclor Sample I.D.
ESE Pro j eel- 1242 A5
PCD I'ool A
" ; " A4
.
11 | " Al
" i i " -A3
" » A2
ESE Project " B2
PCB Pool- B
" " Bl
i
' " » B4
11 ' " B5
11 , " B3
"Datd generated ur. 'up, proposed protocol approach
i
i
i
!
1 ; '
1 ' ,
1
Level of PCB
in ppm
0.0068
0.0072
0.0068
0.0061
0.0077
0.0948
0.0858
010921
0.0816
0.0831
PCB Added Prelim.
in ppm Stats
None x = 0.0069
G.D. - O.COOu
(Jl)% UCL = 0.0001
Nona gg?0 UCL = O.OOB7
95% LCL = 0.0057
None gg% LCL = 0.0051
None '
None
0.0811 x = 0.0875
S.D. = 0.0050
95% UCL = 0.0991
" 99% UCL = 0.1049
95% LCL = 0.0759
11 99% LCL = 0.0701
ii
it
-------
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1 .C.
-------
APPENDIX E
PERCHLORINATION PROCEDURE
-------
PERCHLORINATION PROCEDURE FOR CONFIRMATION OF PCB
I. Principal. Perchlorination of a PCB mixture using antimony penta-
chloride (SbCl^) as the active reagent results in conversion of
all PCB present to DCB which is a single isomeric, fully-substituted
species. This species has a high electron capture (EC) detector
response. It can also be more readily separated from other compounds
by GC techniques than can the complex PCB mixture. Quantitation can
be accomplished by measurement of a single peak.
II. Sample Preparation. The sample extract (1.0 ml) prepared in the
method described above is quantitatively transferred to a Kuderna-
Danish apparatus with micro-Snyder column.
III. Removal of Solvent. The extracted PCB must be quantitatively
exchanged from the hexane solvent to chloroform. All residual hexane
must be removed from the extract prior to perchlorination. Even small
amounts of residual hexane will result in the formation of a black
solid residue upon the addition of SbCl,.. This severely reduces
PCB recovery. The hexane is removed by azeotrophic evaporation from
the hexane/chloroform mixture.
-------
il Add 3 ml of nanograde chloroform to the Kuderna-Danish receiver con-
taining the sample extract in hexane and concentrate, using a micro-
Synder column, by slow boiling in a tube heater or water bath to about
0.2 ml. Do not allow to evaporate to dryness.
.2 Repeat step III.l three additional times in order to remove all resi-
dual hexane. Rinse the micro-Snyder apparatus with a minimum amount
of chloroform. Final volume should be approximately 1.0 ml.
.3 Quantitatively transfer to a reaction vial (Figure 1) using three
chloroform rinses (Total rinse volume about 2 ml).
.4 Add two micro-Hengar boiling chips and immerse reaction vial upright
in a 70°C water bath to a depth of 6 _+ 2 cm.
.5 Increase water bath temperature slowly until the solvent begins to
boil. Boiling temperature should be 72-76°C.
.6 Concentrate slowly to a volume of approximately 0.1 ml. Under no cir-
cumstances should the water bath temperature be permitted to exceed
76°C or the solvent be evaporated to dryness. If either of these hap-
pen, PCB will be lost by volatilization and consequent recoveries will
be low. The final volume (0.1 ml) may be determined with sufficient
accuracy by comparison of solvent level with another reaction vial
containing 0.1 ml of chloroform.
* V
.7 When a volume of 0.1 ml is achieved, cap the reaction vial immediately
and allow to cool.
-------
Teflon-lined cap
Figure 1. Perchlorination reaction vial.
-------
IV Sample Perchlorination.
.1 To the concentrated sample extract in the reaction vial add 0.2 ml of
SbCl5 and immediately re-seal the vial tightly
with the Teflon-lined screw cap.
.2 Place the reaction vial into a preheated (160 _+ 3°C) aluminum block
heater for a period of 15 hours.
.3 After the reaction period, remove the reaction vial from the aluminum
block heater and allow to cool to room temperature. Then cool to 0°C
in an ice water bath.
.4 Cautiously vent pressure from the vial in a fume hood, directing away
from the analyst. Add 1 ml of 6 If HC1 to the cool reaction vial,
replae the cap tightly and shake for 30 seconds. The HC1 stops the
perchlorination reaction. CAUTION: IF THE REACTION VIAL IS NOT COOL,
THE ADDITION OF HC1 MAY CAUSE DANGEROUS SPLATTERING OF THE REAGENTS
FROM THE CONTAINER.
.5 Add 1 ml hexane, shake vigorously for 30 seconds and carefully draw
off the hexane layer with a disposable pipet.
.6 Place this hexane extract on the top of a 6 mm x 12 cm disposable
pipet packed with 2 g of anhydrous Na2S04. This column is prewashed
with hexane.
.7 Repeat steps IV.5 and IV.6 five times to assure complete
extraction of all DCS from the reaction vial.
-------
.8 Pass two 1 ml portions of fresh hexane through the Na2SC>4 column
and collect all fractions in a 10 ml graduated Kuderna-Danish receiving
vial •
.9 Connect a modified micro-Snyder column to the Kuderna-Danish apparatus
add one Hengar boiling chip, and evaporate in a water
bath (70°C) to less than 0.5 ml. Care must be taken to avoid bumping
and loss of sample.
.10 Cool the apparatus to room temperature and remove the micro-Snyder
column. Rinse the micro-Snyder column with sufficient fresh hexane to
bring the volume up to 1.0 ml as indicated on the graduated Kuderna-
Danish receiving vial. Remove the micro-Snyder column and mix by gentle
swirling.
.11 Transfer the extract immediately (before significant solvent
evaporation) to a properly-labeled Hypo-vial.
.12 Close the Hypo-vial with a Teflon septum and aluminum cap.
All PCB present in the sample has been converted to DCB in
1.0 ml hexane. The sample is now ready for GC analysis.
V. Analysis
.1 Analyze the DCB present in the perchlorinated extract by GC/EC. The
following column conditions have been found to be suitable:
Glass column: length 6 ft., ID 1/8"
1.5% OV-17/1.95% QF-1 liquid phase
Chromosorb W-HP, 80/100 mesh support
-------
N2> 35 psig inlet, approximately 40 ml/min.
Column temperature—220°C
Injection port temperature—240°C
Detector Temperature—200°C
Under these conditions, DCB will elute in approximately 20 minutes,
Figure 2 is a typical chromatogram.
.2 Quantitate as DCB by comparison of the peak area with that of a known
concentration of pure DCB taking into consideration
all concentration factors. Care must be taken to assure the sample
concentration and the standard concentration are near the same value
so the EC detector is operating in the linear range. Peak areas of
the standard and the sample should not differ by more than a factor
of two.
A - B
ng DCB/ml = S x C
-------
I"
bl
I 1
! I
o : .10 :
I minutes
*
Figure 2. Typical Chromatogram Showing PCBs After Perchlorination to DCB.
20
-------
where A = area of the DCS peak of the sample
B - area of the DCB peak of the procedural blank
S = area of DCB standard peak
C = concentration of the DCB standard in ng
V = volume of the sample extract
.3 To convert DCB values to approximate equivalent PCB values, Table 1
may be employed.
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Table 1. Factors to Mathematically Convert Decachlorobiphenyl to an
Equivalent Amount of Aroclor (5)
Aroclor
1221
1232
1242
1016
1248
1254
1260
1262
DCS
Av. No. Cl*
1
2
3
3
4
5
6
7
10
MWt
188.5
223
257.5
257.5
292
326.4
361
395.3
499
X**
0.38
0.45
0.52
0.52
0.59
0.65
0.72
0.79
1.00
* Average whole number of chlorines calculated from percent chlorine
substitution for a specific Aroclor
t Molecular weight of Aroclor based on the average whole number of
chlorines calculated from percent chlorine substitution
** X = molecular wt Aroclor/molecular wt DCS (499). To convert ppm
DCB to ppm of a specific Aroclor, multiply ppm x DCB by X for the
Aroclor.
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TECHNICAL REPORT DATA
(Pleat nod Instructions on the reverse before completing)
\. REPORT NO.
EPA 560/6-8-007
3. RECIPIENT'S ACCESSION NO.
4. TITLE AND SUBTITLE
Analysis of Polychlorinated Biphenyl (PCB) in
Human Blood Serum Samples
8. REPORT DATE
October 1, 1977
9. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Charles L. Stratton and Paul C. Geiszler
«. PERFORMING ORGANIZATION REPORT NO
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Environmental Science and Engineering, Inc.
P.O. Box 13454
Gainesville, Florida 32604
10. PROGRAM ELEMENT NO.
11. CONTRACT/GRANT NO.
68-01-3248
12. SPONSORING AGENCY NAME AND ADDRESS
Environmental Protection Agency
Office of Toxic Substances
401 M Street, SW
Washington, D.C. 20460
13. TYPE OF REPORT AND PERIOD COVERED
Final 1/77-10/77
14. SPONSORING AGENCY CODE
EPA-OTS
15. SUPPLEMENTARY NOTES
16. ABSTRACT
A total of 208 human blood serum samples and two mother's milk samples were
analyzed for polychlorinated biphenyl (PCB). The samples were supplied to
Environmental Science and Engineering, Inc. (ESE) in frozen condition by the
Department of Health, Education, and Welfare, Center for Disease Control,
Atlanta, Georgia. This report includes the analytical results for these
samples and an assessment of the degree of uncertainty involved in the analysis.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lOENTIFIERS/OPEN ENDED TERMS C. COSATI Field/Group
Polychlorinated biphenyls, PCB, blood
serum, analytical methods
IS. DISTRIBUTION STATEMENT
19. SECURITY CLASS (TTia At port/
unclassified
21. NO. OP PAGES
59
Unlimited—release to public
j_
20. SECURITY CLASS IThii pay/if
unclassified
22. PRICE
CPA Form 222O-1 (S-73)
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