Selective Monitoring of Polynuclear Aromatic Hydrocarbons by High Pressure Liquid Chromatography with a Variable Wavelength Detector Ante M. Krstulovic, Douglas M. Rosie, and Phyllis R. Brown* Department of Chemistry, University of Rhode Island, Kingston, R.I. 0288 1
The use of a variable wavelength micro uv detector was investigated for the high pressure liquid chromatographic analysis of polynuclear aromatic hydrocarbons. This detector enhances detection signals by working at optimal wavelengths, thereby increasing the sensitivity of the method and reducing or eliminating interferences. By using a variable wavelength detector in series with a fixed wavelength detector, samples can be monitored simultaneously at two different wavelengths and peak area ratios utilized as a method of identification.
Polynuclear aromatic hydrocarbons ( P A H ) of g r e a t struct u r a l variety occur i n the environment f r o m m a n y different sources. A n u m b e r of these compounds as well as their derivatives are potent carcinogens (1,2)and there is considerable interest i n the development of reliable, sensitive, and r a p i d techniques for the analysis of PAH i n environmental samples. Marly analytical techniques have been used for the analysis of these compounds (3-12). Separations involving high pressure liquid chromatography ( H P L C ) a p p e a r promising because nonvolatile, thermally labile molecules can readily be analyzed without derivatization (13-28). Samples a r e not destroyed in the analysis and i t is possible to collect fractions for further analysis by other methods. In addition, HPLC can utilize the fluorescence or ultraviolet characteristics of PAH which are very sensitive and specific.' This eliminates or reduces interferences from compounds which neither fluoresce nor absorb ultraviolet light. Because the variable wavelength micro uv detector provides a choice of wavelengths, it is possible to select a wavelength to m a t c h the absorption band characteristics of a particular compound. T h u s , sensitivity can be maximized and interferences minimized or eliminated. Furthermore, peak area ratios at two different wavelengths can be utilized as a m e t h o d of
identification of peaks. Therefore, the use of a variable wavelength micro uv detector in combination with a fixed wavelength uv detector was investigated for selective monitoring of PAH by HPLC.
EXPERIMENTAL Apparatus. A DuPont 830 Liquid Chromatograph with a constant pressure pumping system was used. A micro uv detector with fixed wavelength of 254 nm was coupled with a variable wavelength detector, Model SF 770 Spectroflow Monitor (Schoeffel Instrument Corp., Westwood, N.J.) which had a deuterium lamp for the wavelength range of 200 to 400 nm and a tungsten lamp for the range of 350 to 630 nm. The standard unit had dual flow cells with a 1-mm bore and a 10-mm light path between the quartz windows. The column outlet was connected to the detector by means of a Ks-in. steel tubing of minimal length to minimize peak broadening. Column. Preoacked. stainless steel columns. Partisil IO-ODS (Whatman Inc.,AClifton,N.J.) and fi Bondapak CIS (Waters Associates, Milford, Mass.) were used. The Partisil column was 4.6-mm i.d. X 25 cm with the particle size of 10 pm. Octadecyl silane was bonded through Si-0-Si bonds to the silica support. The cI8column which is chemically similar to the Partisil column was 4-mm i.d. X 30 cm in length. Reagents. The model compounds used were as follows: benzene
(Analabs, Inc., North Haven, Conn.); benzo[a]pyrene, chrysene, naphthalene, perylene, and coronene (Aldrich Chemical Company, Inc., Cedar Knolls, N.J.); biphenyl (Pfaltz and Bauer, Inc., Flushing, N.Y.); fluoranthene and pyrene (Calbiochem, Los Angeles, Calif.); phenanthrene (Eastman Organic Chemicals, Rochester, N.Y.). All chemicals were recrystallized from suitable solvents. Solutions were prepared by dissolving these substances in distilled tetrahydrofuran (Mallinckrodt Chemical Works, St. Louis, Mo.), and stored a t 2 "C when not in use. Distilled anhydrous methanol (Mallinckrodt Chemical Works) was used as solvent for gradient elution. Distilled water was filtered using a membrane filter type HA 0.45 pm pore size (Millipore Corporation, Bedford, Mass.). Preparation of Ehvironmental Samples. An aluminum sampling probe containing an organic-free glass fiber filter, 10.16-cmdiameter, GF/B type (Whatman, Inc.) was used for collecting the airborne particulate matter. Before every collection, the sample holder was rinsed with tetrahydrofuran. A Cadillac pump, Model HP33 (Clements Mfg. Co., Chicago, Ill.) was used and the air flow monitored with a flow meter. Environmental samples were collected for 11h. The air flow rate was 6 cubic feet per minute, and the total volume of air passed through the filter was 3960 cubic feet. The filters were extracted with 300 ml of tetrahydrofuran for 1h in a Soxlet apparatus and the extract was flash-evaporated. The final volume was adjusted to 1 ml with tetrahydrofuran. HPLC Operating Conditions. A 34-min linear gradient from 60/40 CH30H/HnO (v/v) to a 100%CH30H was used. The separations of the standard compounds and the environmental samples were performed under the same conditions at ambient temperature. Column pressure was 650 psi. The standard compounds and the environmental samples were monitored at the following wavelengths: 210, 220,240,254,280,290,300,340,and 360 nm. Identification of Peaks. Chromatographic peaks of the environmental samples were identified by three methods. First, tentative identification was made on the basis of the retention times and comparison with standards. Second, standards were added to the samples and the mixture was re-chromatographed. A quantitative increase in the peak area was taken as further characterization. Finally, the ratios of peak areas at two wavelengths of peaks in chromatograms of the environmental samples were compared to those of known standards. Quantitation of Peaks. Chromatographic peaks were quantitated electronically by means of a Hewlett-Packard 3380 A Model Electronic Integrator, which marks the retention time of every peak and its respective area.
RESULTS Reproducibility. The retention times, peak areas, a n d peak shapes were found t o be highly reproducible. Since the liquid chromatograph used has a constant pressure pumping system, the flow rates change during t h e course of the gradient. Therefore, retention times rather than volumes were used for t h e capacity factors which were calculated using the following formula:
where t l is t h e retention time of t h e peak of interest a n d t o the
retention time of unretained components~Table I shows the reproducibility of t h e k' values for t h e s t a n d a r d s r u n i n triplicate at 254 *m. T h e capacity factors were calculated at 95% confidence limits, using the following formula: ANALYTICAL CHEMISTRY, VOL. 48, NO. 9, AUGUST 1976
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2 4 6 nm
3 0 0 nm 7 I
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Figure 1. Detection of PAH at different wavelengths, order of elution: (1) benzene (37.9 pg), (2) naphthalene (1.55 pg), (3) biphenyl (1.50 pg), (4) phenanthrene (0.65 pg), (5) fluoranthene (1.00 wg), (6) pyrene (0.65 1s). (7) chrysene (0.65 pg), (8) perylene (1.00 pg),(9) benzo[a]pyrene (0.35 pg), and (10) coronene (0.91 pg)
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Table I. k' Values a t 254 nm
2
0.38 f 0.04 0.89 f 0.15 1.29 f 0.23 1.76 f 0.26 2.24 f 0.24 2.35 f 0.23 2.89 f 0.20 3.35 i 0.20 3.50 f 0.18 4.50 f 0.26
Benzene Naphthalene Biphenyl Phenanthrene Fluoranthene Pyrene
Chrysene Perylene Benzo [a]pyrene Coronene
1
Table 11. Identification of Area Ratios Standard Naphthalene (254 nm/280 nm) Phenanthrene (254 nm/240 nm) Fluoranthene (254 nm/240 nm) Benzo[a]pyrene (290 nm/240 nm)
Environmental sample
1.56
1.55
0.64
0.63
0.66
0.66
1.87
1.87
where 8 is the sample mean, m is the estimated population mean, s is the standard deviation, and t is the t-table value at the stated confidence level. Linearity of Response. Calibration plots for the compounds in the standard mixture were made at all the wavelengths used. They showed good linearity of response over a wide concentration range. The repression coefficientsfor these plots were between 0.998 and 1.000. Detection of PAH at Different Wavelengths. As can be seen in Figure 1,0.91pg of coronene (peak 10) can barely be detected at 240,254, and 340 nm. This compound has a low absorbance at 300 nm and practically no absorbance at 360 nm. However, at 290 nm, coronene exhibits maximum absorbance. At 290 nm benzo[a]pyrene (peak 9) also has maximum absorbance and can be monitored almost completely free of interference from perylene (peak 8) which has minimum absorbance at this wavelength, but has maximum absorbance at 240 nm. On the other hand chrysene (peak 7 )has maximum absorbance at 254 nm. Although pyrene (peak 6) has maximum absorbance at 254 nm, the optimal wavelength for monitoring this compound in the presence of fluoranthene (peak 5) is 360 nm a t which it is detected with some loss in sensitivity, but completely free from the interference of fluoranthene. To monitor the fluoranthene selectivity, either 240 or 340 nm can be optimal. Phenanthrene (peak 4) has maximum absorbance at 240 nm. Its absorbance decreases with increasing wavelength and a t 300 nm, 0.65 kg of phenanthrene can not be detected. At 240 nm, biphenyl (peak 3) also has maximum absorbance which decreases with increasing wavelength. However, the same amount of biphenyl can no longer be detected at 290 nm. For benzene (peak 1)and naphthalene (peak 2), 254 nm appears to be the optimal wavelength for selective detection. Applications. The applicability of the described detection technique was tested in the HPLC analysis of PAH in atmospheric particulate matter. Environmental samples were collected at the University of Rhode Island in Kingston, R.I. This location can be classified as a relatively nonpolluted area and has been chosen as an intermediate area between the urban and rural collection locations which will be used in further studies.
I
0
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30
TIME ( m i d Figure 2. PAH in the atmospheric particulate matter (Kingston, R.I.). (1) Naphthalene; (2) phenanthrene; (3) fluoranthene; (4) benzo[a] pyrene
The environmental samples were chromatographed under the same conditions as the standards. Several major peaks were tentatively identified by retention times. Standards were then added to the sample for further identification. Finally, the ratios of peak areas at two different wavelengths were determined and ,compared with the ratios for the standards. Area ratios for the peaks of interest are listed in Table 11. Four peaks, naphthalene, phenanthrene, fluoranthene and benzo[a]pyrene (Figure 2) were identified using retention times, co-injection with standards and peak area ratios. CONCLUSIONS The use of a variable wavelength micro uv detector in combination with a fixed wavelength detector shows promise for routine analysis of selected PAH in environmental samples by HPLC. This technique has the advantages of high sensitivity, selectivity, and specificity and does not require expensive instrumentation. However, it should be noted that although the use of a variable wavelength can be useful in characterizing selected peaks of environmental samples by peak area ratios, absolute identification of all the peaks in a chromatogram of a complex mixture can be done only by a combination of several methods. Since the sample preparation and extraction are fundamental in the analysis of environmental samples, new collection systems and extraction procedures as well as those presently in use are being investigated and critically evaluated. ACKNOWLEDGMENT The authors thank the DuPont Company for the use of the DuPont 830 Liquid Chromatograph; the SchoeffelInstrument Corporation for the 770 SF Spectroflow Monitor; Whatman Inc., for the columns and glass filters; Richard A. Hartwick for reading this manuscript and his helpful discussions; Charles Olney for the use o$ the collection system; Paul Walsh for the help in collecting environmental samples; and Roberta Caldwell for help in preparation of the manuscript. LITERATURE CITED (1) Committee on Biological Effects of Atmospheric Pollutants, "Particulate Polycyclic Organic Matter", National Academy of Sciences, Washington, D.C. (1972). (2) Survey of Compounds Which Have Been Tested for Carcinogenic Activity, Public Health Service Publication, No. 149. (3) 0.Hutzinger, S.Safe, and M. Zanders, "Polycyclic Aromatic Hydrocarbons", Analabs, Inc., Res. Notes, 13, 3 (Dec 1973).
ANALYTICAL CHEMISTRY, VOL. 48, NO. 9, AUGUST 1976
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(4) P. K. Mueller and E . L. Kothny, Anal. Chem., 45, (5),1R (1973). (5) R. C. Lao, R. S.Thomas, and J. L. Monkman, J. Chromatogr., 112, 681 (1975). (6) R. C. Lao, R. S.Thomas, H. Oja, and L. Dubois, Anal. Chem.,45,906 (1973). (7) D. A. Lane, H. K. Moe, and M. Katz, Anal. Chem., 45, 1776 (1973). (6)K. D. Bartle, M. L. Lee, and M. Novotny, lnt. J. Environ. Anal. Chem., 3,349 (1973). (9) W. Giger and M. Blumer, Anal. Chem., 46, 1663 (1974). (10) I. P. Fisher and A. Johnson, Anal. Chem., 47, 59 (1975). , (11) M. L. Lee, K. D. Bartle, and M. V. Novotny, Anal. Chem., 47, 540
(1975). (12) G. M. Janini, K. Johnvton, and W. L. Zielinskl, Jr., Anal. Chem., 47, 670 (1975). (13) M. A. Fox and S.W. Staley, "Analysis of Polycyclic Organic Compounds
in Atmospheric Particulate Matter by HPLC with Fluorescence Detection", 170th National Meeting, Am. Chem. Soc.,Chicago, Ill., August 25-29, 1975. (14) . , J. A. Schmit, R. A. Henry, R. C. Williams, and J. F. Dieckman, J. Chromatogr. Sci., 9, 645 (1971). (15) S.Soedigdo, W. W. Angus, and J. W. Flesher, Anal. Biochem., 67, 664 (197.51 . - . -,. (16) C. G. Vaughan, B. B. Wheals, and M. J. Whitehouse, J. Chromatogr., 78, ~
203 (1973).
(17) J. C. Suatoni and F. R. E. Swab, J. Chromatogr. Sci., 13, 361 (1975). (18) J. C. Suatoni, H. R. Farber, and B. E. Davis, J. Chromatogr. Sci., 13, 367 (1975). (19) M. Novotny, M. L. Lee, and K. D.Bartle, J. Chromatogr. Sci., 12,606 (1974). (20) R. Gladen, Chromatographia, 5, 237 (1972). (21) J. L. Monkman, J. Chromatogr., 26, 456 (1967). (22) M. Dong, D. C. Locke, and E. Ferrand, Anal. Chem., 48, 368 (1976). (23) B. Wheals, C. G. Vaughan, and M. J. Whitehouse, J. Chromatogr, 106, 109 (1975). (24) H. J. Klimisch, J. Chromatogr.,23, 11 (1973): Anal. Chem., 45, 11 (1973). (25) R. E. Jentoft and T. H. Gouw, Anal. Chem., 40, 178 (1968). (26) B. L. Karger, M. Martin, J. Loheac, and G. Guiochon, Anal. Chem., 45,496 (1973). (27) R. Vivilecchia, M. Thiebaud, and R. W. Frei, J. Chromatogr. Sci., 10,411 (1972). (28) C. H. Lochmuller and C. W. Amoss, J. Chromatogr., 108, 85 (1975).
RECEIVEDfor review February 23,1976. Accepted May 19, 1976. Work supported by Grant No. CA 1760301 from the National Cancer Institute.
Gas Chromatographic and Mass Spectral Properties of Sulfonylurea N-MethyI-"-perf Iuoroacyl Derivatives W. E. Braselton, Jr.,* E. D.'Bransome, Jr., and H. C. Ashline Deparfment of Medicine, Medical College of Georgia, Augusta,
J.
Ga.30902
1.Stewart and 1. L. Honigberg
School of Pharmacy, University of Georgia, Athens, Ga. 30602
N-Methyl and N-methyltrlfluoroacetyl, N-methylpentafluoropropionyl, and N-methylheptafluorobutyryl derlvatlves of the sulfonylureas tolbutamide, hydroxyltolbutamide, carboxytolbutamide, chlorpropamide, and tolazamide were prepared. The derlvatlzed compounds were thermally stable as shown by mass spectrometry and exhibited excellent gas chromatographic properties on the liquid phases 3% OV-I, 3% OV-17, and 3% SP-2401.The derivatives showed high sensitivity to electron-capture detection: tolbutamlde-N-methyltrlfluoroacetate sensitivlty = 2.8 X mol s-' at 3:l signal to noise. The mass spectral properties of the methyl-perfluoroacyi derlvatlves of chlorpropamide, tolbutamide, and tolbutamide metabolites were characterized by rearrangemenf involving loss of SO1, In contrast to the toiazamide derivative which did not lose SO2.
The recently renewed controversy over the University Group Diabetes Program (UGDP) report concerning increased cardiovascular mortality from treatment with tolbutamide, an antidiabetic sulfonylurea (1-5),highlights the need for a rapid and precise method of measurement of this class of drugs and their metabolites in body fluids. Although sulfonylureas have been used in the treatment of maturityonset diabetes for more than 20 years, inadequate quantitative methods have resulted in a paucity of reliable information on the pharmacokinetics and mechanism of action of this class of drug. Quantitative procedures employing colorimetric and spectrophotometric methods to measure sulfonylureas and their metabolites in body fluids have suffered from lack of sensitivity and specificity unless involved solvent extraction procedures were used (6, 7). High pressure liquid chroma1386
ANALYTICAL CHEMISTRY, VOL. 48, NO. 9, AUGUST 1976
tography (HPLC) has been used to analyze pharmaceutical preparations (8), but present HPLC methodology is not sensitive enough for measurements of therapeutic levels in blood. Matin and Knight (9) recently reported a sensitive and specific chemical ionization mass spectrometry method although the requirements of expensive specialized equipment limit its use as a routine clinical procedure. GLC methods of quantitation have been described (6,10-12)but, for the most part, these have been unsuccessful because of the thermal instability of the sulfonylureas and their N-methyl derivatives. We have recently found that the N-methyl-N'-perfluoroacyl derivatives of the sulfonylurea drugs I, IV, V, and some sulfonylurea metabolites 11, I11 (Figure l),are stable to conditions of gas chromatography and thus provide the basis for a simple and accurate quantitative procedure employing flame ionization GLC (13). This paper describes in detail the mass spectral (electron impact) and GLC characteristics of the N-methyl-N'-trifluoroacetyl, pentafluoropropionyl, and heptafluorobutyryl derivatives. This work has led to development of a more sensitive electron capture GLC method of quantitation. A forthcoming paper will describe methodology for routine extraction, derivatization. and measurement of sulfonylureas in 50-pl serum samples.
EXPERIMENTAL Apparatus. Compounds were analyzed o n a Finnigan 1015D gas chromatograph-mass spectrometer interfaced w i t h a Systems Industries (Sunnyvale, Calif.) System 150 data acquisition a n d control system. T h e injector and separator temperatures were 240 OC; spectra were taken a t 70 eV. Columns were 1.5 m X 2 mm i.d. glass, a n d H e carrier gas flow was approximately 30 ml/min. Compounds were chromatographed o n 3% OV-1 (on SO/lOO Supelcoport, Supelco, Inc.),
