1774
Anal. Chem. 1980, 52, 1774-1777
with a preconcentration procedure should be sufficient for most clinical and pharmacological purposes. Applications for analysis of illicit or pharmaceutical preparations of PCP could be carried out without preconcentration. The very low cost and ease of operation of potentiometric instrumentation make potentiometric analyses of PCP+ a highly desirable alternative and worthy of further investigation.
Gupta, R. C.; Lu, I.; Oei, G.; Lundberg, G. D. Clin. Toxicol. 1975, 8.611. Marshman, J. A.; Ramsay, M. P.; Sellers, E. M. Toxicol. Appl. Pharmacol. 1978, 35, 129. Saferstein, R.; Manura, J. J.; Brettell, T. A,; De, P. K. J. Anal. Toxicol. 1978, 2 , 245. Lin, D. C. K.; Fentiman, A. F.; Foltz, R. L.; Forney, R. D. Sunshine, I. Biomed. Mass. Spectrom. 1975, 2 , 206. Wilson, A. E.; Domino, E. F. Biomed. Mass Spectrom. 1978, 5 , 112. Baker, J. K.; SkeRon, R. E.; Ma, C. J. Chromatogr. 1979, 168, 417. Rosenberg, L. S.;Vunakis, H. V. Res. Commun. Chem. fafhol. Pharmacol. 1979, 25, 547. Done, A. K.; Aronow, R.; Miceli. J. N.; Lin, D. C. K. "Management of the Poisoned Patient", Rumack. 8. H., Temple, A. R., Eds.; Princeton Scientific Press: Princeton, N.J., 1977: pp 72-102. Lin, D. C. K.; Foltz, R . L.; Done, A. K.; Aronow, R.; Arcinue, E.; Miceli, J. N. "Quantitative Mass Spectrometry in Life Sciences", DeLeenheer, A. P.; Roncucci, R. R., Eds.; Elsevier: Amsterdam; pp 121-129. Wong, L. K.; Beiman, K. Clin. Toxicol. 1976, 9 , 583. Martin, C. R.; Freiser, H. Anal. Chem. 1980, 52, 562. Rosenberg, L. Farmacia(8ucharest) 1978, 26, 141. Goina, T.; Habai, S.; Higuchi, T.; Illian, C.; Tossounian, J. Anal. Chem. 1970, 42, 1674. Hassan, S . S.M.; Elsayes, M. 6. Anal. Chem. 1979, 57, 1651. Martin. C.R.; Freiser. H. Anal. Chem. 1979, 51, 803. Martin, C.R.; Freiser, H. J. Chem. Educ.. in press. James, H. J.; Carmack, G. P.; Freiser, H. Anal. Chem. 1972, 4 4 , 853. Gustavii, K. Acta Pharm. Suec. 1967, 4 , 233. Marinsky, J.; Marcus, Y. "Ion Exchange and Solvent Extraction", Vol. 6; Marcel Dekker: New York, 1974; p 5.
ACKNOWLEDGMENT We greatfully acknowledge helpful discussions with Paul Consroe of the University of Arizona. LITERATURE CITED Greifenstein. F. E.; DeVauR. M.; Yashitake, J.; Gajewski. J. E. Anaesfh. Analg. Curr. Res. 1958, 3 7 , 283. Davies, 8. M.; Beech, H. L. J. Mental Sci. 1960, 706,912. Garey, R. E.; Weisberg, L. A.; Heath, R. G. J. Psychedelic Drugs 1977, 9 , 280. National Institute on Drug Abuse, Res. Monogr.; National Institute on Drug Abuse: Rockville, Md.; 1978, Vol. 21. Done, A. K.; Aronow, R.; Miceli, J. M. National Institute on Drug Abuse Res. Monogr.; National Institute on Drug Abuse: Rockviile, Md.. 1978; VOl. 21, p 210. Misra, A. L.; Pontani, R. 6.; Bartolomeo, J. Res. Commun. Chem. fafhol. Pharmacol. 1979, 24, 431. Chait, L. D.; Bolster, R. L. Commun. Psychopharmacol. 1978, 2 , 351. Pierce, W. 0.; Lamoreaux, T. C.; Urry, F. M.; Kopjak, L.; Finkle, B. S. J . Anal. Toxicol. 1978, 2 , 26.
RECEIVED for review February 29,1980. Accepted May 2,1980. This work was conducted with financial assistance from the Office of Naval Research.
Determination of Trace Amounts of Chlorinated Phenols in Human Urine by Gas Chromatography Thomas R. Edgerton," Robert F.
Moseman,'
Emile M.
Lores,
and Lynn
H. Wright
Environmental Toxicology Division. Health Effects Research Laboratory, U S . Environmental Protection Agency, Research Triangle Park, North Carolina 2771 1
T h e most frequently employed analytical procedures for measuring low levels of chlorinated phenols in urine involve solvent extraction and either derivatization (often with toxic derivatizing reagents) to a more chromatographible moiety (1-7) or determination of the free phenol on various polyester gas chromatographic columns (8-12). Recently, reports of analytical methods which use porous polymer resins as the sorbing agents for the removal of phenols from water (13-15) and organic contaminants from aqueous biological media (16, 17) have been published. The most widely used resin has been Amberlite XAD-2. The recovery efficiency of pesticides (14,18-20) and phenols (13, 14) from water using XAD-2 or the chemically identical XAD-4 has been reported. Because these results are for water only, a n analytical method was developed employing XAD-4 and using both double support-bonded diethylene glycol succinate (DSB-DEGS) and support-bonded butane-1,4-diol succinate (SB-BDS) columns (21,22) for chromatography of the isolated free phenols. T h e analytical procedure involves extraction of the chlorinated phenols by passing hydrolyzed urine through a column of clean XAD-4 resin. The chlorinated phenols are then eluted from the column with 2-propanol in hexane. This eluate is concentrated and analyzed by electron capture-gas chromatography (EC-GC). EXPERIMENTAL Apparatus. A Tracor 222 gas chromatograph, equipped with a nickel-63 electron capture detector, was operated in the pulsed
Present address: Radian Corporation, Austin, Texas 78766.
linearized mode. Borosilicate glass columns (1.8 m X 4 mm i.d. or 0.6 m X 4 mm id.) were packed with double support bonded diethylene glycol succinate or support-bonded butane- 1,4-diol succinate on 80/100 mesh acid washed Chromosorb W. The columns were operated at 18G210 "C with a 5% methane in argon carrier gas flow rate of 60-80 mL/min. Other temperatures were: detector, 300 "C; inlet 225 "C; transfer line, 220 "C. The following glassware was used. A Chromaflex column, 250 x 10.5 mm i.d., with a Teflon stopcock (Kontes Catalogue No. K-420280);a Kuderna-Danish concentrator assembly (K-570000); 25-mL graduated tubes, size 2525 (K-570050);and a 15 x 125 mm screw-cap culture tube. Reagents and Materials. Chlorinated phenol reference standards were obtained from Aldrich Chemical Co. (Milwaukee, Wis.). For fortification purposes, the phenols were converted to their respective sodium salts prior to addition to urine. All solvents were pesticide quality or equivalent. Reagent materials (3 N HC1,O.l N NaOH, NaHS03, deionized water) were extracted with hexane and toluene prior to use. The macroreticular resin, XAD-4, was obtained from Rohm and Haas (Philadelphia,Pa.). The Ties were removed by slurrying in methanol and decanting (14). The remaining beads were purified by Soxhlet extraction with 3 N HCl for 18 h followed by neutralization with water in a Buchner funnel. The neutralized resin was washed with six 50-mL volumes of 0.1 N NaOH. The resin was again neutralized with water and allowed to dry. The dried resin was then sequentially extracted with methanol, acetonitrile, acetone, and hexane in a Soxhlet extractor for 8 h per solvent ( 1 4 , 2 3 ) . The purified resin was stored under methanol in a glass stoppered bottle. Preparation of XAD-4 Column. A small plug of hexaneextracted glass wool was placed in a 250 X 10.5 mm i.d. Chromaflex column. Approximately 6 cm (1.5-2 g dry weight) XAD-4 resin in methanol was added to the column. The flow of methanol was
This article not subject to U.S. Copyright. Published 1980 by the American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 52, NO. 11, SEPTEMBER 1980
Table I. Retention Data for Chlorinated Phenols on a Double Support-Bonded DEGS Columna compound 2,6-dichlorophenol 2,4-dichlorophenol 2,5-dichlorophenol 2,3-dichlorophenol 2,4,6-trichlorophenol 2,3,5-trichlorophenol 2,3,6-trichlorophenol 2,4,5-trichlorophenol 2,3,4-trichlorophenol 2,3,5,6-tetrachlorophenol 2,3,4,6-tetrachlorophenol 3,5-dichlorophenol 3,4,5-trichlorophenol 2,3,4,5-tetrachlorophenol 3,4-dichlorophenol pentachlorophenol
minutes RRT2,4,5-TCP 2.95 0.35 3.19 0.37 3.19 0.37 3.39 0.40 5.47 0.64 6.81 0.80 6.85 0.80 8.54 1.00 8.94 1.05 13.15 1.54 14.84 1.74 19.29 2.26 20.47 2.40 21.18 2.48 25.83 3.02 35.20 4.12
Double support-bonded DEGS, 1.8 m x 4 mm; 170 OC, 60 mL/min. Table 11. Retention Data for Chlorinated Phenols on a Support-Bonded BDS Columno compound 2,6-dichlorophenol 2,4-dichlorophenol 2,5-dichlorophenol 2,3-dichlorophenol 2.4.6-trichloro~henol 2;3;5-trichlorophenol 2,3,4-trichlorophenol 2,4,5-trichlorophenol 2,3,6-trichlorophenol 3,5-dichlorophenol 3,4-dichlorophenol 2,3,4,5-tetrachlorophenol 2,3,4,6-tetrachlorophenol 3,4,5-trichlorophenol 2,3,5,6-tetrachlorophenol DentachloroDhenol
minutes RRT 2,4,5-TCP 0.98 0.30 0.98 0.30 1.02 0.31 1.02 0.31 2.83 0.86 2.83 0.86 0.95 3.15 1.00 3.31 1.29 4.25 2.02 6.69 2.48 8.19 3.36 11.10 5.31 17.56 7.26 24.62 8.39 27.76
1775
Preparation of Urine. Four milliliters of urine was placed in a 15 X 125 mm screw-cap culture tube. To this were added 100 mg of sodium bisulfite and 1 mL of concentrated HCl. The tube was sealed with a Teflon lined screw cap and placed in a boiling water bath for 1h with periodic shaking. The sample was removed and cooled to room temperature. Column Elution and Regeneration. The XAD-4 column was rinsed with 10 mL of deionized water. When the water level reached the top of the resin bed, 15 mL of 3 N HCl was added. After approximately 5 mL of acid eluted through the column, the flow was halted and the column allowed to equilibrate for 5 min. After equilibration, the flow of acid was continued and a phenol standard or hydrolyzed urine sample was added to the column when the level of acid reached the top of the resin bed. Quantitative transfer of the sample was accomplished with two 1-mL rinsings with 3 N HC1. The column was eluted with an additional 25 mL of 3 N HC1 followed by 25 mL of deionized water. Both acid and water eluates were discarded. When the water level reached the top of the resin bed, a Kuderna-Danish (K-D) concentrator assembly was placed under the column and the phenols were eluted from the column with 100 mL of 10% 2-propanol in hexane (v/v). After the first 5 mL were eluted, the flow was halted and the column was allowed to equilibrate for 5 min. After equilibration, the flow was continued and the eluate collected. The K-D assembly was removed and the column was regenerated by washing with approximately 25 mL of methanol which was discarded. A volume of methanol was kept in the column until further use. Concentration. The K-D assembly was placed on a steam bath and the sample was concentrated to a final volume of 1+15 mL (two phases). The sample was cooled and the hexane layer (upper) transferred to a 15-mL centrifuge tube. The extract was then further concentrated to a volume of 1-2 mL for analysis by EC-GC.
RESULTS AND DISCUSSION
a Support-bonded BDS, 0.6 m x 4 mm; 190 'C, 60 m L / min.
halted when the level of solvent reached the top of the resin bed. A second plug of glass wool was placed on top of the resin bed.
Relative retention times of the chlorinated phenols under investigation are presented in Tables I and 11. T h e difference in elution patterns of SB-BDS from that of DSB-DEGS allows for the confirmation of chlorinated phenols in urine. A 0.6-m DSB-DEGS column was used for the rapid elution of pentachlorophenol. Work is continuing in our laboratory on the use of high performance liquid chromatography (HPLC) for the seDaration and confirmation of chlorinated Dhenols. These results will be presented in a later publication. As shown in Table 111, recoveries of chlorinated phenols from fortified urine averaged better than 80%. Method " sensitivity is dependent upon chlorine substitution and elution time of the phenol through the gas chromatographic column.
Table 111. Recoveries of Chlorinated Phenols from Fortified Urine ppm addeda
0.05
0.01
compound 2,6-dichlorophenol 2,4-dichlorophenol 2,3-dichlorophenol 2,5-dichlorophenol 3,4-dichlorophenol 3,5-dichlorophenol 2,3,4-trichlorophenol 2,3,5-trichlorophenol 2,3,6-trichlorophenol 2,4,5-trichlorophenol 2,4,6-trichlorophenol 3,4,5-trichlorophenol 2,3,5,6-tetrachlorophenol 2,3,4,6-tetrachlorophenol 2,3,4,5-tetrachlorophenol
pentachlorophenol a
av % rec 83 86 86 80 85 80 88
87 85 87 87 84 79
% SD
av % rec 96 89
i
7.7 12.3 3.4
i
1.3
83 86 82 87
i i
i i i i i
4.6 2.8 7.7 5.0 15.0
i 5.3 I3.6 i 6.8 i
5.6
Av. 5 determinations each fortification level.
83
5% SD
* c * i ? ?
i
81
i
92
i
89
i i
98 82 91 94 92 86
0.10
i
*
f
i
*
5.6 5.3 4.2 3.0 6.3 3.2 6.3 5.4 7.5 6.6 8.9 4.3 9.2 7.7 10.3 4.9
av % rec 90 89 87 89
87 81 88 85 90 81
93 86 88 92 82 85
%SD
9.0 i 7.4 i 3.2 i 4.0 i 4.0 i 1.9 i 4.4 + 4.2 i 7.3 i 3.8 i 5.7 i 4.6 i 7.2 + 5.3 I7.1 i 4.9 i
0.50 av % rec % SD
___-
83 84 89 84 87 81 89 87 90 86 84 84 87
i i i i
88
91
i i
88
i
Range for all fortification levels.
2.5 2.8 3.8 2.4 i 4.5 i 2.3 i 7.6 i 7.5 i 5.6 i 3.1 i 2.9 i 2.8 + 6.7 5.1
6.7 5.2
-
1.00
av % rec 84 86 88 86
86 82 88 86 83 86 88
86 92 94 97 93
7% SD
rangeb
5.3 * 4.1 ? 3.8 i 4.1 i 4.9 * 1.6 i 4.0 I3.8 ? 6.7 i 4.9 I7.5 f 5.6 i 7.6 * 6.0 i 4.7 5 5.1
75-104 70-103 78-92 79-92 80-96 77-88 80-101
I
74-100
79-102 76-94 70-110 78-92 75-106 81-102 74-102 70-101
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ANALYTICAL CHEMISTRY, VOL. 52, NO. 11, SEPTEMBER 1980
Table IV. Chlorinated Phenol Residues from General Human Population Samples co mDoun ds a 2,5- and/or
sample no.
2,6-dichlorophenol
1 2
3 4 5 6 7 8 9 10 11
12 a
2,4-dichlorophenol 21 13 20 11
