Anal. Chem. 1981, 53, 1213-1217
The fact that other metals give separate peaks suggests that the general method proposed in this work will ultimately lead to a very attractive method of multielement analysis. In the present work only the determination of copper has been optimized and equivalent studies, on the other elements, will be required before a highly efficient method for multielement determinations becomes possible.
Determination of Capper in Water Samples by LCEC and Atomic Absorption Spectrometry. All of the above data suggest that a relatively interference free method for the determination of copper should be possible with LCEC. Determination of real samples over the concentration range lo* to 5 x 10" M was undertaken with tap water in contact with copper pipes and results compared with those obtained by atomic absorption spectrometry (AAS). Excellent agreement, for example, (2.20 f 0.05) X lo* M and (14.2 f 0.3) X lo4 M (LCEC) vs. (2.20 f 0.05) X lo4 M and (14.3 f 0.3) X lo4 M (AAS), suggests total copper is being determined. Certainly this should be true with AAS, and in view of the very strong complex formation of Cu(dtc)2,this is also probably not surprising with the LCEC method. Applications to a wide range of industrial effluents and other samples were equally successful, so the method is believed to be relatively specific. For determination of copper, AAS would generally be the preferred technique except when only small volumes of samples are available. The detection limits of the two techniques are similar (flameless AAS methods would be considerably more sensitive) and can be used with small volumes but potential for extensive multielement determination on very small volumes of sample could provide a distinct use for LCEC in trace metal determinations.
ACKNOWLEDGMENT Extensive discussions with Ian Russell in the early stages of this work are gratefully acknowledged.
LITERATURE CITED (1) Kotthoff, I.M.; Lingane, J. J. I n "Polarography", 2nd ed.; Interscience: New York, 1952. (2) Bond, A. M. I n "Modern Polarographic Methods In Analytical Chemistry"; Marcel Dekker: New York, 1980. (3) Flato, J. B. Anal. Chem. 1972, 44 ( l l ) , 75A-87A. (4) Barendrecht, E. In "Electroanalytical Chemistry"; Bard, A. J., Ed., Marcel Dekker: New York, 1967, Vol. 2, pp 53-103.
1213
(5) Copeland, T. R.; Osteryoung, R. A.; Skogerboe, R. K. Anal. Chem. 1974, 46, 2093-2097. (6) Kissinger, P. T. Anal. Chem. 1977, 49, 447A-456A. (7) Felice, L. J.; Kissinger, P. T. Anal. Chem. 1976, 48, 794-796. (8) Swartzfager, D. G. Anal. Chem. 1976, 48, 2189-2192. (9) Fleet, B.; Little, C. J. J. Chromatogr. Sci. 1974, 12, 747-752. (10) Kissinger, P. T.; Refshauge, C.; Dreiling, R.; Adams, R. N. Anal. Lett. 1973, 6, 465-477. (11) Magee, R. J. Rev. Anal. Chem. 1973, 1, 335-377. (12) Delepine, M. Bull. SOC. Chim. h. 1908, 3 , 652-654. (13) Hulanicki, A. Talanta 1967, 14, 1371-1392. (14) Koirtyohann, S. R.; Wen, J. W. Anal. Chem. 1973, 45, 1986-1989 and references cited therein. (15) Ai-Mahdi, A. K.; Wilson, C. Mlkorchem Ver. Mikrochim. Acta 1951, 36/37, 218-223. (16) Smith, E.; Hayes, J. R. Anal. Chem. 1959, 31, 898-902. (17) O'Laughiin, J. W.; O'Brien, T. P. Anal. Lett. 1978, 1 1 , 829-844. (18) Liska, 0.; Lehotay, J.; Brandsteterova, E.; Guichon, G.; Colin, H. J. Chromatogr. 1979, 172, 384-387. (19) Toropova, V. F.; Budnikov, R. G. K.; Ulakovich, N. A. Talanta 1977, 25, 263-267. (20) Chant, R.; Hendrickson, A. R. Martin, R. C.; Rohde, N. J. Aust. J . Chem. 1973, 26, 2533-2536. (21) Wheeler, S. H.; Mattson, B. M.; Mlessler, G. L.; Pignolet, L. H. Inorg. Chem. 1978. 17, 340-350. (22) Dix, A. H.; Diesveid, J. W.; Van der Linden, J. G. M. Inorg. Chim. Acta 1977, 24, L51-L52. (23) Hendrickson, A. R.; Martin, R. L.; Rohde, N. M. Inorg. Chem. 1975, 14, 2980-2985. (24) Hendrickson, A. R.; Martin, R. L.; Rohde, N. M. Inorg. Chem. 1974, 13, 1933-1939. (25) Hendrickson, A. R.; Martin, R. L.; Rohde, N. M. Inorg. Chem. 1976, 15, 2115-2119. (26) Budnlkov, G. K.; Toropova, V. F.; Ulakhovich, N. A,; Viter, I.P. Zh. Anal. Khim. 1975, 30, 2120-2124. (27) Kitamura, H.; Ichimura A.; Kitigawa, T. Nippon Kagaku Kaishi 1979, 354-358 and references cited therein. (28) Uiakovich, N. A.; Budnikov, G. K.; Fomina, L. G. Zh. Anal. Khlm. 1979, 34, 241-244. (29) Tindall, G. W.; Bruckensteln, S. J . Nectroanal. Chem. 1969, 22, 367-373. (30) Cathro, K. J.; Walkley, A. J. J. Polarogr. SOC. 1958, 2 , 36-40. (31) . . Voael. A. "A Textbook of Quantitative lnoraanic Analvsis"; Lonamans: Loridon, 1968; p 869. (32) O'Donnell, J. F.; Ayres, J. T.; Mann, C. K. Anal. Chem. 1965, 37, 1161-1164. . , - . . . - .. (33) Cauquis, G.; Lachenal, D. J . Electroanal. Chem. 1973, 43, 205-213. (34) Scrimage, C.; Dehayes, L. J. Inorg. Nucl. Chem. Lett. 1978, 14, 125-1 33. (35) Bigley, I.E. Ph.D. Thesis, University of Massachusetts, Amherst, MA, 1978. (36) Maitoza, P.;Johnson, D. C. Anal. Chim. Acta 1980, 118, 233-241.
RECEIVED for review August 15, 1980. Accepted March 17, 1981.
Liquid Chromatographic Determination of Benzo[ a Ipyrene in Natural, Synthetic, and Refined Crudes Bruce A. Tomkins," Roberta R. Reagan, John E. Caton, and Wayne H. Griest Analytical Chemistry Divislon, Oak Ridge National Laboratory, P.O. Box X, Oak Ridge, Tennessee 37830
Benzo[a]pyrene (BaP) is isolated and quantitated by using a sequential high-pressure llquid chromatographlc (HPLC) procedure. The sample is first injected onto a semipreparative HPLC column containing a bonded polar aminocyano (PAC) packlng material, from which a BaP-enriched fraction is obtained. This Isolate is then reinjected onto a Zorbaw ODS reversed-phase analytical-scale column, and fluorescence detection is used to quantitate BaP. This procedure Is applicable to samples with BsP concentrations ranging from 0.02 to 500 pg/g. The precision is nominally f6% (relative standard deviation), and the accuracy compares favorably with that dlsplayed by more tedious methods. Recoveries, as determined by counting a radioactive BaP tracer, usually exceed 95 %. Two samples may be processed per personday.
The current interest in synthetic fuels, derived from either shale oil or coal, has been matched only by the desire that these materials present a minimal hazard to health and to the environment. For this reason, there is a substantial interest in identifying and quantitating any biologically harmful component present in these new fuels, Benzo[a]pyrene (BaP) is a case in point. BaP is a wellknown carcinogen ( I ) which is usually present in fossil fuels and has been used for years as an indicator of the polycyclic aromatic hydrocarbon content of fuels. As a result, there has been an extensive effort to develop rapid, convenient, accurate, and precise methods for determining BaP in natural, synthetic, and refined crudes. BaP has been determined by using direct-injection gas
0003-2700/81/0353-1213$01.25/0 0 1981 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 53, NO. 8, JULY 1981
chromatography-mass spectrometry (Z), anodic differential pulse voltammetry (31, and matrix isolation fluorescence spectroscopy (4). Recently, we published (5) two procedures for determining BaP in crudes in which conventional chemical isolation procedures were used with standard solution fluorescence spectroscopy, gas chromatography, and highpressure liquid chromatography (HPLC). While the isolation procedures described in ref 5 are highly effective, they are also extremely time-consuming and require a high degree of technical skill to yield accurate, reproducible results. We attempted to design a procedure which would retain the accuracy and precision of our earlier procedures, yet would be simpler and more convenient to use. For this purpose, HPLC appeared to be the technique of choice. Several authors have used HPLC for class fractionation of crudes (6-10). Two of these procedures (8,9)employed general UV detection at 254 nm, while the others employed either a more selective UV wavelength for BaP (7) or selective fluorescence detection (6, 10). The work done at the National Bureau of Standards (10-12) was particularly noteworthy because it ultimately led to the quantitation and certification of BaP in a reference shale oil. This procedure, which employed a bonded aminosilane-HPLC column in the isolation step, has also been applied to virgin, used, and recycled lubricating oils (12). Previous HPLC fractionation procedures (10-12) have not addressed the following problems: (a) isolation of BaP if the activity of the isolation column changes slightly; (b) overall yield of BaP for each aliquot of sample analyzed; and (c) thorough cleaning of the isolation column and restoration to its original activity. In this paper, we have modified and somewhat simplified the National Bureau of Standards procedure (10-12) to overcome these three constraints. The resulting method has been successively applied to the analysis of BaP in a wide range of fossil fuel material types consisting of crude and refiied coal liquids, shale, and petroleum crudes as well as semisolid tars. Isolation and quantitation are achieved in not more than 3 h, and the relative standard devation is typically 6%.
EXPERIMENTAL SECTION Solvents and Chemicals. Pentane, methylene chloride, and acetonitrile were purchased from Burdick and Jackson Laboratories (Muskegon, MI) and used without further purification. Hexane was purchased as ACS reagent grade from Fisher Scientific, Inc. (Fairlawn, NJ), and redistilled before use. Solvent mixtures were degassed with stirring and under vacuum for at least 15 min prior to use. BaP was obtained from the Chemical Repository at the Illinois Institute of Technology Research Institute (IITRI; Chicago, IL; lot no. ET-9-63-1). Its purity was established as described previously (5). Naphthalene and pyrene were purchased from the Aldrich Chemical Co. (Milwaukee,WI) and were used as received. The radiolabeled BaP tracer, 7,10-14C-BaP(239 pCi/mg), in benzene solution, was acquired from the Amersham Corp. (Arlington Heights, IL) and diluted to 1OOooO dpm/mL in 50% (v/v) methylene chloride/hexane. Samples. All samples except the SRC-I1coal oil, gasifier tar, and standard shale oil were obtained through the US. Environmental Protection Agency/Department of Energy Fossil Fuel Research Materials Facility (13, 14). The latter included three Comparative Research Materials (CRMs) which are available (14) for biological assay and analytical methods research and development. The SRC-I1 coal oil and Wilmington Petroleum Crude were obtained from the National Bureau of Standards (Washington, DC) through the joint DOE/NBS-sponsored Analytical Characterization Group. The gasifier tar, which was collected at the low Btu coal gasifier at the University of Minnesota at Duluth, was supplied by B. R. Clark, Oak Ridge National Laboratory. SRM 1580 (certified shale oil standard) was purchased from the Office of Standard Reference Materials, National Bureau of Standards (Washington, DC). Equipment. Isolation System. The isolation system was
1-
t SOLVENT RESERVOIRS
-________ I
2
C T-UNION
VALVE
t SOLVENT FILTER
t PUMP AND GAUGE
C GUARD
COLUMN
t PAC COLUMN
COLLECT
WASTE
Flgure 1. General schematic of the HPLC system.
arranged as shown in Figure 1. The solvent was pumped through in. 0.d. Teflon tubing to a three-way T-union valve with 90° plug porting (Hamilton Co., Reno, NV, part no. 86633). Solvent passed from the union valve to an inline solvent filter in. connecting tubing, 2-p pore size filter, Alltech Associates, Arlington Heights, IL), which in turn was connected to the loop injector. Exactly 250 pL of sample solution was injected onto an MPLC Guard Column equipped with an “Amino” cartridge (Brownlee Labs, Inc., Santa Clara, CA) using a six-port Valco loop injector tested to 7000 psig (Valco Instrument Co., Houston, TX, part no. CV-6-UHD-a-NGO). A loop filler port (Rheodyne, Inc., Cotati, CA, part no, 7011) connected to the loop injector permitted the sample loop to be filled by a conventional 1-mL syringe. The solvent mixture was pumped by using a Model 396 Simplex minipump (Milton Roy Co., Laboratory Data Control Division, Riviera Beach, FL) adjusted to deliver 2.6 mL/min at 500-1000 psig. The Partisil M9 10/25 PAC (polar aminocyano phase) column, 25 cm X 9.4 nun id., was purchased from Whatman, Inc. (Clifton, NJ). The chromatographic peaks were detected by using a Model 153 UV detector, obtained from Altex Scientific, Inc. (Berkeley, CA), which was equipped with an 8-pL flow cell and a monitoring wavelength of 254 nm. The eluant could be either collected or shunted to waste. Analytical System. The analytical system was a simplified version of the HPLC unit depicted in Figure 1. A single reservoir was used, and the solvent was filtered with a low-pressure solvent filter (Universal Scientific, Inc., Atlanta, GA, part no. 02-237). The solvent was pumped by a Model 2396 Duplex minipump (Milton Roy Co., Laboratory Data Control Division,Riviera Beach, FL). A pulse dampening unit (Laboratory Data Control Division) was used to minimize flow rate fluctuations by the pumping system. The sample was injected onto the analytical column using a Rheodyne 7125 sample injector with a 20-pL sampling loop (Rheodyne, Inc., Cotati, CA), which was also equipped with a Rheodyne 7011 loop filler port. The column itself was a 25 cm X 4.6 mm i.d. Zorbax ODS (octadecylsi1ane)-BP 7 p particle size unit supplied by Alltech Associates. The column was preceded by an MPLC Guard column (Brownlee Labs, Inc.) equipped with a reversed-phase (RP-18) cartridge. The emerging peaks were detected by using a fluorescence detector, Model 420-E/420-C, purchased from Waters Associates, Inc. (Milford, MA). The filters used were 360 nm (excitation) and 425 nm (emission). Connections in both the isolation and analytical systems were made with either 1/16 in. 0.d. type 316 stainless steel, l/16 in. 0.d. Teflon, or in, 0.d. Teflon tubing. The latter was used solely to transfer solvent from a reservoir to the pump. Recovery Measurement. A 50-pL aliquot of each isolate was mixed with 100 mg of powdered cellulose and 100 p L of Combustaid (Packard Instrument Co., Inc., Downers Grove, IL) in a paper cone. Both cone and contents were completely combusted
ANALYTICAL CHEMISTRY, VOL. 53, NO. 8, JULY 1981
9% PENTANE/MeC12 I
__
--
MeC12
~
MeC12-
2h MeCN/MeC12
-
'%
-
1215
5 g / g , the tracer was omitted from the sample, and y was assumed to be 100%.
PENTANE/MeCIZ
RESULTS AND DISCUSSION W '
2 v) 0
4
L - .
0
I 20
_ _ - _-__ 40
60
80
L - I (00 120
L -2
140
160
(80
TIME h n l
Flgure 2. Profile of a typical isolation chromatogram: (---) is a standard of naphthalene, pyrene, and benzo[a]pyrene; (-) is the chromatogram of coal oil A.
in an oxygen-rich atmosphere using a Packard Tricarb Model B 306 Sample Oxidizer (Packard Instrument Co.). Burn time was 45 s. The liberated 14C02was collected in 7 mL of Carbosorb carbon dioxide absorber and mixed with 12 mL of Permafluor V scintillation cocktail in the instrument (products of the Packard Instrument Co.). The subsequent liquid scintillation counting of the "COz liberated from the combusted aliquot was performed for 4 rnin at room temperature using the carbon-14 counting channel of a Tri-Carb Liquid Scintillation Counter, Model (2-2425 (Packard Instrument Co.). All sample counts were corrected for scintillation quenching with the Automatic External Standard option of the instrument. Procedure. Isolation of the BaP-Bearing Fraction. A standard containing 100 pg/mL each of naphthalene, pyrene, and BaP, dissolved in 50% (v/v) methylene chloride in hexane, was eluted with 6% methylene chloride in pentane through the isolation system. The peak maximum time of BaP was noted, and the volume of solvent eluting between h10 min of this time taken as the BaP isolate. One gram of liquid sample was dissolved in 5 mL of 50% (v/v) methylene chloride in hexane solution which contained approximately 1 X lo5 dpm/mL, I4C-BaP and was diluted to a total volume of 10 mL with hexane. One gram of solid sample was dissolved as thoroughly as possible in the spiked methylene chloride in hexane solution and later centrifuged to remove solids. A 250-pL portion of the sample supernatant was then injected into the isolation system. The BaP fraction was taken to dryness by using both vacuum and dry, flowing nitrogen and later redissolved in exactly 1 mL of acetonitrile. After the BaP fraction was isolated, the column was washed for 30 min with methylene chloride and for 30 min with a 66% (v/v) solution of acetonitrile in methylene chloride. It was then flushed for 30 rnin with methylene chloride and for a final 30 min with 6% methylene chloride in pentane solution before injecting another sample. The absorption profile of a typical isolation run (at 254 nm) is shown in Figure 2 and indicates not only the column washing/flushing sequence but also the timing of the BaP fraction collection. Analysis of BaP. The HPLC mobile phase, 25% (v/v) water in acetonitrile, was degassed for at least 0.5 h before use. A series of five standards ranging between 0.1 and 2.0 pg/mL BaP in acetonitrile were analyzed daily, preceding any samples. Under the usual analytical conditions employed (1000 psig pressure, 1.7 mL/min flow rate) BaP normally eluted in 25 min. Calculations. The peak heights for the five BaP standards were fitted to a standard linear regression equation, thus yielding f(h), the BaP concentration in pg/mL as a function of peak height. [The correlation coefficient of f(h) typically exceeded 0.995.1 The concentration of BaP is calculated by using the following equation: BaP' pg'g
f(h) wg/mL X 1 mL = w , g/10 mL X 0.250 mL
1.07 pg X y y w,g 4 0 f ( h ) / ~X y - 1.07 Y / W X
where w is the mass of the sample in grams, y is the overall recovery, and 1.07 is the mass of the radioactive tracer added in pg. In cases where the native BaP concentration was less than
Work performed at the National Bureau of Standards (10, 11) clearly demonstrated the utility of HPLC for accurately and rapidly determining BaP in a sample of crude shale oil and new, used, and recycled oils (12). These procedures, however, did not address the problems of (a) recovery of the analyte, (b) cleaning the isolation (aminosilane) column, and (c) long-term stability of the peak maximum time for BaP. In addition, it was not clear how these two procedures would handle samples such as coal gasifier tars. Private communications (15) indicated that the aminosilane column is cleaned by back-flushing with methylene chloride. I t is not possible to reactivate the PAC or aminosilane isolation column conveniently with a single polar solvent such as either 2-propanol or acetonitrile. While either solvent is excellent for merely cleaning the aminosilane or PAC column, these solvents are strongly retained by the column and cannot be removed easily. The rather extensive cleaning procedure reported in this paper, in which methylene chloride, acetonitrile/methylene chloride, and methylene chloride solvents are used, not only cleans the column but also restores it to its original activity. The BaP retention time therefore is reproducible even over long periods of routine use, allowing reproducible isolations to be achieved ewily. During the course of this work, we have prepared nearly 75 isolates a t the rate of 2 per day, yet the peak maximum time for BaP has remained within f 5 min of a nominal average of 50 min. The other major source of variation is the slight difference in the composition of the 6% methylene chloride in pentane solutions, possibly caused by the degassing procedure. Chemical species elute from the PAC column in order of increasing polarity. For this reason, we suspect that aliphatic hydrocarbons, aromatic hydrocarbons, ketones, phenols, and amines elute in roughly that order as the eluant is changed from methylene chloride/pentane to neat methylene chloride and finally to acetonitrile/methylene chloride. The procedure used by Brown et al. (12) enables a BaP isolation to occur somewhat faster than in the method described herein and uses a very narrow cut to obtain a BaP fraction. In our experience, permitting BaP to elute over a slightly longer period of time offers an advantage in that there is no particular difficulty if the column activity changes slightly with use. The rather large cut used for the BaP isolate (20 rnin in a 60-min run) enables a recovery exceeding 95% in spite of possible minor changes in the activity of the PAC column. Recovery corrections were achieved through the use of the radiolabeled BaP spike. However, direct liquid scintillation counting of the tracer in aliquots of the BaP isolate proved unsatisfactory because certain fluorescent materials present in the sample significantly augmented the true number of counts detected by the phototube. The Automatic External Standard option provided in the instrument did not completely correct this problem. Combusting an aliquot in an oxygen-rich atmosphere and counting the 14C02colleded from the escaping gas provided an unbiased, accurate means of recovery determination. The analytical HPLC system, employing the Zorbax column and fluorescence detector, was optimized so that BaP and a minimum number of interferents would be detected. Nevertheless, compounds such as noncarcinogenic benzo[e]pyrene, benzo[b, j , or klfluoranthenes, and perylene are known to interfere with many BaP analytical methods. Solution of these materials were injected into the analytical system to test for a significant interference. Perylene, benzo[ b and klfluoranthene, and BeP eluted as a single peak at 22 min, while
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ANALYTICAL CHEMISTRY, VOL. 53, NO. 8, JULY 1981
Table I. Accuracy and Precision Evaluation of Sequential HPLC Procedure repository sample no.
sample description
CRM-1
I4C-BaP BaP concn, pg/g recovery, %
coal oil A
97
162 154 146 140 182 177 174 180 12
100 100 100
NBS coal liquid oil (SRC-11) CRM-2
98 95 100 93 99 100 97 10;
crude shale oil A
CRM-3
SRM 1580 (certified shale oil)
10 3.3 2.4 2.4 35 31 31 30 1.3
97 93 92
...7b6 ...
NBS Wilmington petroleum crude A
RSD, %
BaP concn by reference procedure, pg/g
151 t 9.5
6
179 t 4
2
134 * 7a1d
11 t 0.8
7
13.2 i. 0.7a
10 11
... ... ...
petroleum crude A
av BaP concn,a
2.7
t
0.5
20
31.5 i: 2.4
8
1.2 f 0.2
13
3.5
* 0.4a
21 i: 6 c
2.0
?
0.5a
1.0
...
1.2
a Mean ?r standard deviation, No 14C-BaPadded. 100% recovery assumed. Mean with 95% confidence limits; value certified by the National Bureau of Standards. Value provided by the joint DOE/NBS Analytical Characterization Group.
Table 11. BaP Concentrations for Various Shale and Coal Oil Products repository sample no.
sample description
no. of replicates; av % 14C-BaP recovery
Coal-Derived Materials atmospheric still overhead 2; b atmospheric still bottom 4; 98 vacuum still overhead 2; 100 vacuum still bottom 4; 95 atmospheric still overhead 2; b atmospheric still bottom 2; 100 vacuum separator over2; 97 head vacuum still bottom 2; 100 coal gasifier tar 2; 100
1308 1309 1310 1311 1312 1313 1314 1315
concn,= pg/g
ref procedure value, pg/g
0.3 115 t 8 451 233 * 14 5 37 260
0.19 * 0.03 144 * 5.0 456 * 18 162 t 8.4 1.5 t 0.5 43 f 3.3 296 f 20
142 61
118 f 4.0
73 t 2.7
Shale Oil-Derived Materials 11 ?r 2 2; 100 8 crude shale oil 10 t 1.6 hydrotreated shale oil 2; 95 9 2; 100 16 16 t 4.5 hydrotreated residue 2; b 0.02 0.03 * 0.005 diesel fuel marine refined from shale oil N o radioactive Values represent averages of two runs, or mean t standard deviation of data if more than two runs. tracer added; 100% recovery assumed. 4601 4602 4607 4610
0
10
20
30
4C
50
TIME l r n ~ n l
Flgure 3. Analytical chromatogram of the final BaP isolate using the fluorescence detector (conditions listed in the text). benzou]fluoranthene eluted a t 21 min. BaP, which eluted at 25 min, was chromatographically resolved from these in-
terferents. Furthermore, a gas chromatogram of an isolate (sample 1311) confirmed the presence of these species, as well as that of benzo[ghi]perylene and anthanthrene. In every sample examined, BaP always appeared as a clean, sharp peak, well-resolved from neighboring peaks and containing no obvious shoulders. An example of the HPLC chromatogram of the BaP fraction isolated from a coal oil is shown in Figure 3. Oxygen, which is present in the acetonitrile/water mixture, can cause fluorescence quenching and therefore significantly change the apparent concentration of BaP in a given isolate. Our practice is to degas the solvent mixture for 30 min under vacuum and to run standards to calibrate the instrument daily before determining BaP in the sample isolates. The accuracy and reproducibility of the BaP procedure described in this paper was evaluated by application to several very complex fossil fuels samples consisting of two coal oils, two crude shale oils, and two petroleum crudes, as shown in
Anal. Chem. 1981,53,
Table I. The data indicate quantitative recovery of BaP (>95%), a nominal relative standard deviation of 6 % , and a working range of at least 2 orders of magnitude. The agreement between our HPLC procedure and our reference procedure (5) was fairly good, considering the complexity of the crude samples. The omission of tracer from the petroleum crude is justified because of the low native BaP concentration and the high recovery expected, based on the other samples. Under normal operating conditions, the detection limit of the procedure (calculated from twice the noise level of the analytical HPLC) was 2.5 pg of BaP/g of crude sample. Improvements in this limit were achieved by (a) direct injection of the sample for isolation without prior dilution, (b) increasing the size of the sampling loop, or (c) concentrating the final BaP extract to a known volume less than 1 mL. The HPLC procedure for BaP has been applied to a variety of materials derived from several coal conversion and shale oil refining processes to explore the limits of its utility. These samples included materials such as a coal gasifier tar residue, two solid residues from coal liquefaction, a finished coal oil product, and a highly refined shale oil product. The results of duplicate analyses are presented in Table I1 and are compared with existing data generated from a ref 5 procedure. No spike was added to any sample for which the expected BaP concentration was less than 5 pgfg. Furthermore, in two samples (repository numbers 4610 and 1308) the BaP concentration was so low that the samples were injected onto the PAC column without the usual dilution. The isolation of BaP from sample 4610 was further modified by replacing the 250-pL loop with a 1000-pL injection loop. The results in Tables I and I1 demonstrate that the HPLC procedure is a viable alternative to our longer, more tedious reference procedure. In every sample except 1311, the HPLC procedure yielded values with a precision and accuracy comparable to those of the reference procedure, regardless of the matrix. When the data generated by the two methods were plotted against each other, a straight line with an intercept of 0.214, a slope of 0.987, and a correlation coefficient of 0.984 was obtained. This result indicates that our improved HPLC
1217
1217-1222
procedure can be applied with confidence to the measurement of BaP in a wide variety of natural, synthetic, and refined crudes.
ACKNOWLEDGMENT The authors express their thanks to Gary M. Henderson for determining the overall yield of BaP by combustion of the sample and to Hisashi Kubota for providing BaP data on the samples reported using the longer, more rigorous isolation procedure indicated in this paper.
LITERATURE CITED (1) Dipple, A. In ”Chemical Carcinogens”; Searle, C. E., Ed.; American Chemical Society: Washington, DC, 1976; pp 245-314. (2) Fructer, J. S.; Laul, J. C.; Petersen, M. R.; Ryan, P. W.; Turner, M. E. Adv. Chem. Ser. 1978, No. 770, 255-261. (3) Coetzee, J. F.; Kazi, G. H.; Spurgeon, J. C. Anal. Chem. 1978, 48, 2170-2174. (4) Wehry, E. L.; Mamantov, G. Anal. Chem. 1979, 57, 643A-656A. (5) Tomkins, 8. A.; Kubota, H.; Griest, W. H.; Caton, J. E.; Clark, B. R.; Guerin, M. R. Anal. Chem. 1980, 52, 1331-1334. (6) McKay, J. F.; Latham, D. R. Anal. Chem. 1973, 45, 1050-1055. (7) Boden, H. J . Chromatogr. Scl. 1978, 74, 391-395. (8) Dark, W. A.; McGough, R. R. J . Chromatogr. Sci. 1978, 16, 6 10-615. (9) Matsunaga, A.; Yagl, M. Anal. Chem. 1878, 50, 753-756. (10) Wise, S. A.; Cheder, S. N.; Hertz, H. S.; Hilpert, L. R.; May, W. E. Anal. Chem. 1977, 49, 2306-2310. (11) Hertz, H. S.; Brown, J. M.; Cheder, S. N.; Guenther, F. R.; Hilpert, L. R.; May, W. E.; Parris, R. M.; Wlse, S. A. Anal. Chem. 1980, 52, 1650-1657. (12) Brown, J. R.; Wise, S. A.; May, W.E. J . Environ. Scl. Health, Part A 1980, A75, 613-623. (13) Coffin, D. L.; Guerin, M. R.; Griest, W. H. Proceedings of the Symposium on the Potential Health and Environmental Effects of Fossil Fuel Technologies, Sept 1978; Oak Ridge National Laboratory: Oak RMge, TN, 1979; CONF-780903, p 153. (14) Griest, W. H.; Coffin, D. L.; Guerin, M. R. Fossil Fuels Research Matrix Program, ORNLITM-7346; Oak Ridge National Laboratory, Oak Ridge, TN, June 1980. (Referenceable document, avallable from National Technical Information Service, Springfield, VA 22161.) (15) May, W. E., National Bureau of Standards, washington, DC, private communication.
RECEIVED for review December 29, 1980.
Accepted April 6, 1981. This work was funded by the U.S. Department of Energy, Office of Health and Environmental Research, Contract No. W-7405-eng-26,with the Union Carbide Corp.
Quantitative Determination of Conjugated and Esterified Estrogens by Gas ChromatographyKhemical Ionization Mass Spectrometry .
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Thomas Cairns,” Emil G. Slegmund, and Bobby Rader Department of Health and Human Services, Food and Drug Administration, Office of the Executive Director of Regional Operations, 152 1 West Pic0 Boulevard, Los Angeles, California 900 15
A quantltatlve gas chromatographic method has been developed by uslng trlfluoroacetyi (TFA) derlvatives of the constituent estrogenlc compounds derlved from pregnant mares’ urine found In tablet preparations of conjugated and esterifled estrogens. This assay procedure has permitted measurement and verification of the criteria established for such products by the current USP requirements. Possible interfering related estrogenlc compounds have been quantltaled by GCMS to provide a more detailed chemical profile of the total contents of these formulations.
Conjugated and esterified estrogens are used for the
treatment of menopausal syndrome and other conditions where there may be an estrogen deficiency. Commercial products are formulated from extracts derived from pregnant mares’ urine (1)which contains a whole host of closely related estrogenic compounds: estrone (I),equilin (11),equilenin (111), 17-a-estradiol (IVa), 17-&estradiol(IVb), 17-a-dihydroequilin (Va), 17-P-dihydroequilin(Vb),17-a-dihydroequilenin (VIa), and 17-P-dihydroequilenin(VIb). The principal constituent members of this particular class of equine steroids in commercial products are sodium estrone sulfate and sodium equilin sulfate. Dependent upon the collection time of the urine from the pregnant mares, the relative amounts of these principal estrogens and related compounds can and do vary. For this reason the USP (2) has described the criterion to
This article not subject to US. Copyright. Published 1981 by the American Chemical Society
