Anal. Chem. 1980, 52, 935-940 (3) Von Lehmden, D. J.; Jungers, R. H.; Lee, R. E. Anal. Chem. 1974, 46, 239-245. (4) Slates, R. V. Dupont Report, DP-1421 (1976). (5) Lyon, W. S. "Trace Element Measurements at the Coal-Fired Stream Plant"; CRC Press: Cleveland, Ohio, 1977. (6) Babu, S. P. "Trace Elements in Fuel"; American Chemical Society; Washington, D.C., 1975. (7) Karr, C. "Analytical Methods for Coal and Coal Products", Vols. I and 11; Academic Press: New York. 1978. (8) Botto, R. I. Jarrell-Ash Plasma News/. 1979, 2(2), 4-8. (9) Ward, A. F . ; Marciello, L. Jane//-AshP k s m News/. 1978, 1(3), 10-13. (10) Botto, R. I. Proc. Int. Conf. Uev. At. Plasma Spectrochem. Anal., Puerto Rlco ( 7980); in press. (11) Hull, R. L. 29th Meeting. Am. Pet. Inst., Uiv. Refining, S f . Louis, Mo., 27-64 11964). (12) S p i e l h o k G.' I.; Diehl, H. Talanta 1966, 13, 991-1002. (13) Capes. C. E.; McIlhinney, A. E.; Russell, D. S.;Sirianni, A. F. Envlron. Sci. Techno/. 1974, 8,1107-1113. (14) Hartstein. A. M.; Freedman, R . W.; Platter, D. W. Anal. Chem. 1973, 45, 61 1-614. (15) Ruch, R. R.; Gluskoter, H. J.; Shimp, N. F. Ill. Stafe Geol. Surv., Environ. Geol. Notes, 1974, 72. (16) Werverka, E. M., Williams, J. M. LA-6835-PR (1978). (17) Guidoboni, R. J. Anal. Chem. 1973, 45, 1275-1277.
935
(18) Pollock, E. N. ACS Uiv. Fuel Chem. Prepr., 1973, 18. 92-105. Schleicher. J. A. Ref. 6, p 35. (19) Dreher, G. 9.; (20) Kometani, T. Y.; Bove, J. L.; Nathanson, B.; Siebenberg, S.; Magyar, M. Environ. Sci. Techno/. 1972, 6 , 617-620. (21) Heinrichs, H. Naturwlss. 1977, 64, 479-481. (22) Santoliquido, P. M. Radlochem. Radioanal. Lett. 1973, 75, 373-377. (23) Gluskoter, H. J . Fuel 1965, 44, 285-291. (24) Lutz, G. J.; Sternple, J.; Rook, H. L. J . Radioanal. Chem. 1977, 39, 277-282. (25) Gleitt. C. E.; Holland, W. D. Anal. Chem. 1962, 34, 1454-1457. (26) O'Gorman, J. V.; Suhr. N. M.; Walker, P. L. Appl. Spectrosc. 1972, 26, 44. (27) Bernas, 9.Anal. Chem. 1968, 40, 1682-1686. (28) Hatch, W. R.; Ott, W. L. Anal. Chem. 1968, 40, 2085-2087. (29) Frant. M. S . ; Ross, J. W. Science, 1966, 154, 1553 (30) Crossley, H. E. J . SOC.Chem. Ind. 1944, 63, 280-342. (31) Thomas, J.; Gluskoter, H . J. Anal. Chem. 1974, 46, 1321-1323.
RECEIVED for review November 29,1979. Accepted February 14, 1980. P a r t of this work was presented a t the 179th National Meeting of the American Chemical Society, Houston, Texas, March 1980.
Micromethods for Toxic Residue Screening by Negative Chemical Ionization Mass Spectrometry Douglas W. Kuehl* U.S. Environmental Protection Agency, Environmental Research Laboratory- Duluth, 620 7 Congdon Boulevard, Duluth, Minnesota 55804
Michael J. Whitaker and Ralph C. Dougherty Department of Chemistry, Florida State University, Tallahassee, Florida 32306
Methods were developed for the analysis of polychlorinated chemical residues found in milligram quantities of biological samples. Sample preparation by micro continuous liquid-liquid extraction steam distlllation or by micro gel-permeation chromatography gave sufficiently clean residue extracts for negative chemical Ionization analysis. WHh these techniques, chemicals such as chlorophenols and chloroblphenyls have been confirmed in human adipose samples as small as 12.5 mg. These methods make it possible to screen k
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7
250
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Figure 3. Typical NCI mass spectrum for steam distillation of 2.0 mL of human seminal plasma. Only "C negative mass defect ions are shown
for polychlorinated chemicals a n d Kiwus (8) for polychlorinated biphenyls (PCBs) and l,l-dichloro-2,2-bis(p-chlorophenyl)ethylene @,p '-DDE) from fish tissue. Two experiments were conducted to evaluate the utility of the microsteam distillation as a clean-up procedure for NCI screening for persistent toxic residues. First, we examined eight 250-mg samples for human adipose tissue and fourteen 2.0-mL samples of human seminal plasma t o determine the different types of chemicals that may be isolated and identified in real samples by these techniques. Second, we analyzed smaller and smaller quantities of t h e same adipose tissue to determine t h e minimum quantity of tissue required for detection of t h e major toxic chemicals in these samples. T h e
predominant contaminants identified in the adipose tissue survey were components of tech-chlordane, PCB isomers, l,l,l-trichloro-2,2-bis(p-chlorophenyl)ethane (p,p'-DDT) and l-chloro-2,2-bis(p-chlorophenyl)ethylene ( p , p'-DIIMU) (Table I). The predominant contaminants identified in human seminal plasma were PCB isomers and pentachlorophenol (Table 11). The mass assignments in Tables I and I1 are pseudomolecular ions that were formed by reactions 1-5 above. Compound identifications are based upon a comparison with NCI spectra of standards. In each sample an ion a t m / z 463 (CL7) was observed; however, this compound remains unidentified. Typical NCI mass spectra for steam distillation of 250 mg of adipose tissue and 2.0 mL of human seminal
938
ANALYTICAL CHEMISTRY, VOL. 52, NO. 6, MAY 1980
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ANALYTICAL CHEMISTRY, VOL. 52, NO. 6, MAY 1980 n
that observed in the smallest samples. Pentachlorobiphenyl was therefore reported t o be present only if it was 10 times greater t h a n t h a t observed in the blank. Additional samples of 100, 50, and 25 mg of a human adipose tissue (2-20-2-MW-I, Table I) were analyzed. Although absolute peak intensity for rdentificat ion decreased with decreasing sample size, all identifications made on t h e 250-mg sample were also possible on t h e 25-mg sample. An estimate of detectability of polychlorinated chemicals by NCI was established by observing t h e pseudomolecular ion of trans-chlordane, pentachlorophenol, and octachlorostyrene, which had been spiked into t h e residue of 500 mg of steam distilled cleaned up oyster tissue a t 10 ppb. In each case t h e pseudomolecular ions were readily identifiable. We have examined GPC as an alternate clean-up procedure. The system described by Kuehl and Leonard (7) was capable of isolating both polar and nonpolar toxic chemicals from fatty tissue; however, the system described was used for chromatography of 0.5-1.0 g of oil, which represented 5-10 g of a fish of 10% fat content. A new column 500 mm X 10 mm packed with Bio-Rad SX-8 beads and eluted a t 1.5 rnL/min with cyclohexane/methylenechloride provided separation of corn oil, PCBs, and pentachlorophenol comparable to t h a t of t h e large columns (7). In addition, Arochlor 1254 (1 ppm), hexachlorobenzene (500 ppb), and pentachlorophenol (500 ppb) spiked into corn oil and eluted on this system were recovered a t greater than 95%. A series of loo-, 50-, 25-, and 12.5-mg samples of t h e same adipose tissue (2-12-2-MW-I, Table I) was then used t o evaluate the GPC performance to determine if the same toxic chemicals could be found by G P C / N C I as were found by steam distillation/NCI. Figure 4 shows UV detector traces of the four tissue samples. As can be seen, maximum resolution and sensitivity occur with t h e 25-mg sample. Apparently a maximum optimum loading for this column was about 35 mg of oil. Negative chemical ionization analysis of each sample showed t h e same series of toxic compounds as was observed in the steam distillation/NCI experiment. In addition d compounds observed in the LOO-mg sample were also observed in the 12.5-mg sample. We applied the micro-GPC/NCI screening system to t h e analysis of a sample of Lake Ontario lake trout. A Soxhlet extract of 50 mg of trout tissue was cleaned u p by GPC and analyzed by "21. Polychlorinated biphenyls, hexachloro-
v
T
I
I Elution
Volume
(mi)
Figure 4. Gel-permeation chromatographic traces of UV detector response vs. elution volume for human adipose tissue samples of (A) 10 mg, (B) 50 mg, (C) 25 mg, and (D) 12.5 mg
plasma are shown in Figures 2 and 3, respectively. In addition
to the toxic substances listed in Tables I and 11, low molecular weight free fatty acids readily steam distill. Since free fatty acids have a high sensitivity for chloride attachment (9),they are t h e most consistent interference in NCI screening of environmental samples. Polychlorinated chemicals are therefore identified in t h e mass spectra by their negative mass defects as well as t h e characteristic isotopic ratios of the chlorine isotope clusters. In both the steam distillation and GPC procedures, a pentachlorobiphenyl was observed in the blanks a t a level approximately an order of magnitude lower than
2 \
250
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MASS
939
0-
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Figure 5. Negative chemical ionization mass spectrum of 50 mg of Lake Ontario lake trout cleaned u p by GPC. Only "C negative mass defect ions are shown for polychlorinated chemicals
Anal. Chem. 1980, 52, 940-942
940
benzene, polychlorinated styrenes ( I O ) , chlordane, and nonachlor are readily identified in this sample (Figure 5). Although the use of micromethods limits the possibilities for contamination of the sample, the amount of adventitious material t h a t will appear as a positive is also smaller for microtechniques. Adequate procedural blanks must be carried through the entire procedure. We have used both water and corn oil for procedural blanks. Realistic tissue sample blanks would be desirable, b u t we are not aware of sources of sufficiently contaminant-free tissue. T h e steam distillation system may be the best for micromethods because smaller amounts of solvent and fewer pieces of glassware are required, so it is generally easier to obtain clean blanks with this system.
LITERATURE CITED (1) Dougherty, R. C.; Piotrowska, K. Proc. Natl Acad. S o USA 1976, 73, 1777- 178 1,
(21 Dougherty, R. C.: Piotrowska. K. J . Assoc. Off. Ana;. Chem. 1976, 59, i. n x ~. i n. .w (3) Tindle, R. C.; Stalling. D. L. Anal. Chem. 1972, 4 4 , 1768-1773. (41 Stalling, D. L.; Tindle, R. C.; Johnson, J. L. J . Assoc. Off. Anal. Chem. 1972. 55. 32-38. ! 5 ) Veith, G. D.; Kuehl, D. W.; Rosenthall, J. J , Assoc. Off. Anal. Chem. 1975, 58, 1-5. (6) Kuehl, D W.; Glass, G E : Pugllsi, F. A Anal. Chem 1974, 96, 804-805. (7) Kuehl, D. W.: Leonard, E. N. Anal. Chem. 1978, 5 0 , 182-185. !81 Veith, G. D : Kiwus, L. M. Bull. Environ. Contam. Toxicol. 1977, 17, 631-636. (9)Tannenbaum. H. P.: Roberts, J. D.: Dougherty. R. C. Anal. Chem. 1975,
- -- - -
47 49-54 - 1
(10) Kuehl, D. W.; Kopperman. H. L.; Veith. G. D : Glass, G. E. Bull. Environ. Contam. Toxicol. 1976, 16. 127-132
RECE :IVED for review October 15, 1979. Accepted January 28, 1580. This work was supported by grants from the National and the u.s. Environnlental Protection Institute Agency.
Simultaneous Titrimetric Determination of Bismuth Ion and Free Nitric Acid Concentrations David E. Hughes" and Mario J. Cardone Pharmaceutical Research Division, Norwich-€aton Pharmaceuticals, Box 19 1, Norwich, New York
A tandem titrimetric system has been developed in which a complexometric assay for bismuth(II1) ion is coupled with a simultaneous alkalimetric determination. The metallochromic indicator pyrocatecholsulfonephthaleinis used to determine the end points of both titrations. The procedure has been developed in such a way as to produce four distinct end points from the indicator such that proper pH control and avoidance of bismuth(111) nitrate hydrolysis have been accomplished.
Complexometric titration of bismuth ion has remained the assay of choice even in a time when titrimetric analysis has been supplanted by newer methods. A need exists to assay for bismuth(II1) ion in the presence of nitric acid and to determine the nitric acid concentration in some systems. During the manufacturing of several antidiarrheal formulations ( I ) , for example, bismuth(II1) appears concomitantly with nitric acid. The importance of the following titrimetric system is t h a t it allows the tandem determination of both species. T h e stability of the bismuth-EDTA complex (the log of the stability constant is 28.0 f 0.3 (2, 3)j dictates that only metal ions that form very stable complexes can successfully compete with bismuth for EDTA chelation. Thus, Th4+(41,Fe3+,H$+, In3+,Zr4+, and HP+ interfere in any bismuth complexometric titration and must be absent, removed, or reduced to ensure the selectivity of the assay ( 5 ) . Trivalent iron and mercury(I1) ion, for example, are reduced by the addition of ascorbic acid. Complexometric titrations of bismuth with EDTA have employed a host of indicators. Alizarin chrome black R, stilbazo, thorin (6, 7); picramineazo compounds (8, 9);the yellow Bi14- complex ( I O ) , the bismuth thiourea complex (51, azoimidazole ( 1I ) , 2-2(2-pyridylazo)-4-methylphenol. (PAC) (12), thiazolyazonaphthol (13),4-(4-nitro-2-thiazolyazo) resorcinol and 2-methyl-4-(4-nitro-2-thiazolyazo) resorcinol (141, semixylenol orange and xylenol orange (151, 1 2-naphtho0003-2700/80/0352-G940$@1 .CO/O
138 75
quinone-4-sulfonic acid-2-thiosemicarbazone (16),and pyrocatecholsulfonephthalein ( I 7) have been employed as indicators. For reasons t h a t will become evident later, pyrocatecholsulfonephthalein was chosen as the indicator for this investigation. Pyrocatechol violet, catechol violet, and brenzcakchin violet are all trivial names for pyrocatecholsulfonephthalein (18). Although pyrocatecholsulfonephthalein undergoes air oxidation in alkaline solutions, it is stable in neutral solution for several months and is suitable for use as a bench reagent (19). Analyses of bismuth in the presence of other metal ions, including pharmaceutical formulations, have been performed (20). The structure of py~ocatecholsulfonephthaleirlreveals that wo
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t.he indicator has two hydroxy groups meta to the central carbonium ion which are available for complexation. T h e protons on the hydroxy groups para to the central carbon atom are acidic and auxochromic; successive deprotonation results in a transition from a red aqueous solution (pH el) to yellow (pH 1--2) to blue (pH -8). T h e sulfonate group serves the function that it does in other benzaurin-like dyestuffs, namely, to sterically hinder the triphenylmethane structure from becoming a colorless carbinol by the addition of a hydroxyl group to the central carbon atom, The sulfonate group does not protonate even in very ;pH
