mathematical treatment of the data and the use of the GC patterns simplifies the comparison of chromatograms and lends itself t o computer assisted data retrieval. T o date, the technique has been applied solely to the indentification of crude and bunker oils; however, it should also be applicable to other petroleum products.
LITERATURE CITED (1) C. A.
Wilson, E. P. Ferrero, and H. J. Coleman, Am. Chem. Soc., Div. Pet.
Chern.. Preor.. 20. 613 119751.
(2) M. E. Garzaand J.~Muth,'€nvi;on. Sci. Techno/., 8, 249 (1974). (3)E. R. Adlard, L. F. Creaser, and P. H. Matthews, Anal. Chern., 44, 64 (1972). (4) M. Blurner, J. Saas, Science, 176,1120 (1972).
ACKNOWLEDGMENT The author thanks G. G. Tertipis for supplying the artificially weathered crude oil fractions.
RECEIVEDfor review April 19, 1976. Accepted June 21, 1976.
Gas Chromatography/Mass Spectrometric and Nuclear Magnetic Resonance Determination of Polynuclear Aromatic Hydrocarbons in Airborne Particulates M. L. Lee and M. Novotny" Department of Chemistry, Indiana University, Bloomington, Ind. 4740 1
K. D. Bartle Department of Physical Chemistry, University of Leeds, Leeds, England
A previously designed analytical scheme has been applied to the structural analysis of polynuclear aromatic hydrocarbons in air-pollutionparticulate matter. Well over 100 polycyclics, includingtrace alkylated compounds, were identifiedfrom their chromatographic behavior, mass spectra, and NMR spectra. High separating power of glass capillary columns provided resolution of isomeric compounds with different position of an alkyl group, the presence of which was also ascertained by Fourier-transform NMR.
A variety of studies have indicated that lung cancer deaths are more frequent in cities than in rural areas (1).When the available figures are standardized with respect to both smoking habits and age, the urban factor is found t o be responsible for approximately 25% of lung cancer deaths in cities. Although air pollution by chemical carcinogens may not be entirely responsible for this urban factor ( Z ) , it is to a great degree a contributory cause. Consequently, the interest in identification and routine measurements of a wide range of such carcinogens has steadily been increasing. Polynuclear aromatic hydrocarbons (PAH) are the largest known group of chemical carcinogens that are created during various combustion processes and found in airborne particulates. Many procedures, based on various chromatographic and spectroscopic principles, were developed for the analysis of PAH. Since the relevant methodology was reviewed by Sawicki (3) more than 10 years ago, a wider application of gas chromatography to this class of compounds and, in particular, the advent of combined gas chromatography-mass spectrometry can be considered as the most important developments. This is, perhaps, best exemplified by a recent work of Lao et al. ( 4 , 5 ) . Recent interest in the use of capillary columns stems from the complexity of PAH mixtures, the possible existence of numerous structural isomers, and the wide differences in tumor-promoting activity of various close isomers (6, 7). I t is clear that the resolving power of conventionally used packed columns is not sufficient for adequate separation of closely 1566
related PAH. Since mass spectra of such isomeric compounds show little difference, the prior chromatographic resolution is necessary for their identification. The use of high-resolution (capillary) columns was reported in the analysis of PAH in airborne particulates, automobile exhaust condensates, and engine oils by our group (8-10) and others (11, 12). Desirability of inert glass capillary columns in such analyses has been stressed (8,12). However, a complete analysis of PAH in various complex mixtures (including minor compounds) cannot rely on the use of combined gas chromatography-mass spectrometry alone. We have, therefore, developed an effective analytical scheme (8)that provides the means for selective enrichment of certain portions of a complex P A H profile and t h e positive identification of individual mixture components through the combination of several techniques. This methodology was recently used successfully in the analysis of over 150 PAH compounds in marijuana and tobacco smoke condensates (13). This communication describes application of the previously developed methodology ( 8 )to a detailed qualitative analysis of PAH in airborne particulates collected in typical urban and industrial areas. The particular emphasis is placed on separation and identification of hitherto unresolved toxicologically important isomers by combined capillary gas chromatography-mass spectrometry. The use of NMR spectroscopy for elucidation of the position of substitution for methylated PAH is another valuable contribution of this study. Some 120 P A H compounds from airborne particulates collected in Indiana cities are reported.
EXPERIMENTAL AND RESULTS Three hundred glass fiber filters (8 X 10 inches), through which Indianapolis and Gary city air has been drawn at the approximate rate of $3 m3 for 24 h each, were cut into pieces and successively extracted by batches in Soxhlet thimbles using methylene chloride as the solvent. The extraction procedure and sample workup through the solvent partition scheme, lipophilic-gel column chromatography and bulking of fractions were carried out as described in a previous publication (8).High-resolution gas chromatography using a glass capillary column coated with SE-52 methylphenylsilicone elastomer was
ANALYTICAL CHEMISTRY, VOL. 48, NO. 11, SEPTEMBER 1976
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Figure 1. Capillary-column gas chromatogram of the total polynuclear aromatic hydrocarbon fraction of air-particulate matter.
Conditions: 11 m X 0.26 mm, i.d., glass capillary coated with SE-52 methyiphenylsllicone stationary phase: sample introduced through the precolumn technique (Ref. 14).Key: see Table II Table I. Methyl-Region Proton NMR Data for Final Air-Pollution Fractions (after High-F’ressure Liquid Chromatography) Proton N M R chemical shifts, p p m Literature reference
Fraction
Compound
f
1-Methylphenanthrene 2-Methy lphenanthrene 3-Methylphenanthrene 9-Methylphenanthrene
2.77 2.56 2.64 2.73
1-Methylan thracene 2-Methylanthracene
2.77 2.56
1-Methylfluoranthene 2-Methylfluoranthene 3-Methylfluoranthene 7-Methylfluoranthene 8-Methylfluoranthene
2.76 2.54 2.64 2.76 2.47
e
d
Observed
1-Methylpyrene 2-Methylpyrene 4-Methylpyrene
2.96bl C 2.80b.C 2.89 (1.1Hz doub1et)btc
3.00 2.80 2.91
1-Methylchrysene 2-Methylchrysene
2.730 2.540 2.590 2.85 (1Hz doublety
2.68 2.61 2.61
3-Methy lchrysene
6-Methylchrysene
b
a K. D. Bartle and J. A. S. Smith, Spectrochim. A c t a , Part A, 23, 1689 (1967). , b I. C. Lewis, J. Phys. Chem., 70, 1667 (1966). CA. Cornu, J. Ulrich, and K. Persand, Chim. Anal., 47, 357 (1965). d P . Durand, J. Parello, and N. P. Buu-Hoi, Bull. SOC.Chim. Fr., 2438 (1963). e E . Clar, A. Mullen, and U. Sanigok, Tetrahedron, 25, 5639 (1969). fFrom interpolation o f ’ graph of methyl shift against corresponding aromatic proton shift in parent hydrocarbon, cf., K. D. Bartle and D. W. Jones, A d v . Org. Chem., 8 , 317 ( 1 9 7 2 ) . g D . Cagniant, Bull. SOC.Chim. Fr., 2325 (1966).
used throughout the experiments to monitor the extent of fractionation, recording of sample “cuts” after liquid chromatography, and in the final analysis by combined GC-MS. A modified Model 1400 Varian gas chromatograph was used for the capillary column work. The total chromatographic profile of a small sample aliquot prior to LC fractionation is shown in Figure 1. Enrichment of the selected portions of this profile were further obtained through Sephadex LH-20 chromatography as described
previously (8).The fractions were collected and bulked according to the scheme determined by the elution of standard PAH (Figure 2). The comparison of fraction weights resulting from this chromatographic step can be seen in Figure 3. Only fractions 111through VI1 were of interest in this work; the lower aromatics are toxicologically less interesting, and the analytical methods for PAH higher than coronene have yet to be developed. High-resolution GLC monitoring of fractions I11 through VI1 sug-
ANALYTICAL CHEMISTRY, VOL. 48, NO. 11, SEPTEMBER 1976
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FRACTION NUMBER
rnl ELUTED
Flgure 2. Separation of standard polynuclear aromatic hydrocarbons on a Sephadex LH-20 column
Conditions: 115 cm X 1.5 cm, i.d., column; mobile phase: isopropanol, flow rate 6 ml/h. Fractions were collected at 1-h intervalswith a fraction collector (Buchler Instruments, Fort Lee, N.J.) and their uv absorption was measured
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Figure 3. Histogram showing the weight distribution of fractions obtained by Sephadex LH-20 gel chromatography of the air-pollution nitro-
methane extract
Figure 4. High-resolution liquid chromatograms of by Sephadex LH-20 fractionation.
gested the necessity for further fractionation prior to spectral investigations. Consequently, these fractions were further resolved using high-pressure liquid chromatography with a polar chemically bonded stationary phase (oxypropionitrile/PorasilC, 37-75 p, from Waters Associates, Framingham, Mass.) and n-hexane as a mobile phase. A Model 4100 Varian liquid chromatograph with a conventional uv detector was used. Liquid chromatograms of fractions I11 through VI1 are shown in Figure 4, B through F, respectively. The shaded portions of the chromatograms, which are designated by lower case letters, a through f , represent fractions collected at the column outlet for final identification purposes. This purification step provided further enrichment desirable for GC-MS and proton NMR studies. Figure 4(A) represents high-pressure liquid chromatography of a mixture of selected PAH standard compounds. The final fractions that usually contained a parent PAH plus its alkylated homologues were analyzed by Fourier-transform proton NMR spectroscopy using a Bruker HX-90 spectrometer. The number of scans-varied between 4 000 and 22 000 for pulse widths of 4 ps. Chemical shifts were measured relative to internal tetramethylsilane. After an appropriate dilution with methylene chloride and transfer 1568
0
ANALYTICAL
fractions obtained
Chromatograms 8,C, D, E, and F, represent 111, IV, V, VI, and VII. Chromatogram A represents chromatography of standard compounds. Conditions: 4.25 m X 2.0 mm, i.d., column packed with oxypropionitrile/Porasii C. Mobile phase: nhexane, flow rate 2 ml/min. Key: (1) Benzene; (2) Biphenyl; (3)Fluorene; (4) Anthracene; (5)Benzo [a]fluorene;(6),Triphenylene;(7) Benzo[a]pyrene;(8) Perylene; (9)Dibenz [a,c]anthracene
of this solution onto a concentration precolumn ( 2 4 ) ,the individual fractions were analyzed by combined capillary GC-MS. Chromatograms of these fractions obtained by the total-ion-current monitor of the Hewlett-Packard Model 5980A dodecapole mass spectrometer, are shown in Figures 5 through 10. Peak designation corresponds to that used for monitoring the total PAH profile (Figure 1). Electron-impact ionization spectra were obtained with an electron energy of 70 eV. Chromatographic peaks were scanned at the rate of 100 amu/s. The results of proton NMR spectroscopic investigations are shown in Table I, and the PAH identified by combined GC-MS are listed in Table 11.
CHEMISTRY, VOL. 48, NO. 11, SEPTEMBER 1976
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Figure 5. Enriched fraction f of Figure 4 recorded by the total-ion-current monitor of the mass spectrometer.
Figure 7. Enriched fraction d of Figure 4 recorded by the total-ion-current monitor of the mass spectrometer Conditions: same as Figure 1. Key: see Table iI
Conditions: same as Figure 1. Key: see Table iI
hm
Flgure 6. Enriched fraction e of Figure 4 recorded by the total-ioncurrent monitor of the mass spectrometer
Figure 8. Enriched fraction c of Figure 4 recorded by the total-ion current monitor of the mass spectrometer
Conditions: same as Figure 1. Key: see Table I1
Conditions: same as Figure 1. Key: see Table II
DISCUSSION T h e total GC profile of the airborne PAH fraction, shown in Figure 1,demonstrates the most superior resolution of such mixture components achieved t o date. It indicates that short, but efficient, glass capillary columns can be used to resolve numerous PAH isomeric compounds. Of particular significance are t h e chromatographic results obtained with compounds of relatively high molecular weight (i.e., dibenzan-
thracenes, dibenzopyrenes, and coronene) at only moderately high column temperatures. The use of LC fractionation procedures to selectively enrich trace PAH for identification purposes appears t o have even more utility in the analysis of airborne particulates than in the previously reported studies of the composition of tobacco and marijuana smoke (13) because of the relatively low content of alkylated PAH as compared with t h e parent compounds.
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Table 11. List of Polynuclear Aromatic Hydrocarbons Identified by Gas Chromatography/Mass Spectrometry Peak No.
1
2 3
4 5
6
M o l wt
184 178 178 186 186 192, 198 198 198 192 192 190 192 192 220 212 204 20 6 206 206 206 206 206 202 202 206 206 208 218 202 21 8 218 218 218 220
Compound
Dibenzothiophene Phenanthrene Anthracene Unknown Unknown Unknown, methyldibenzothiophene
Peak No.
Mol wt
62 63 64 65
228 2 34 2 34 244
6.6 67 68 69 70 71 72 73 74 75 76
228 228 258 24 2 240 242 248 258 248 248 242
Compound
Benzo[ c ]phenanthrene Naphthobenzothiophene Naphthobenzothiophene Ethylmethylpyrene or ethylmethylfluorantheneb Benz[a ]anthracene Chrysene Unknown Methylbenzo [clphenanthrene Methylbenzo [gh ilfluoranthene Methylbenzo [ c ]phenanthrene Methylnaphthobenzothiophene Unknown
Methyldibenzothiophene Methyldibenzothiophene 3-Methylphenanthrene 2-Methylphenanthrene 4H-cyclopenta[def]phenanthrene 9-Methy lphenanthrene 1-Methylphenanthrene Methylnaphthobenzothiophene Unknown Methylnaphthobenzothiophene Ethyldibenzothiophenea Methylchrysene or methylbenz [a]Methyl-4H-cyclopenta [def]phenanthrene anthracene Ethylphenanthrene or ethylanthracenea Methylchrysene or methylbenz [ a 177 242 Ethylphenanthrene or ethylanthracenea 18 anthracene Ethylphenanthrene or ethylanthracenea 19 Methylchrysene or methylbenz[a]78 242 Ethylphenanthrene or ethylanthracenea 20 anthracene Methylchrysene or methylbenz [a]Ethylphenanthrene or ethylanthracenea 21 79 242 Ethylphenanthrene or ethylanthracenea anthracene 22 Methylchrysene or methylbenz [ a ] Fluoranthene 80 242, 23 anthracene Benzacenaph thy lene 240 24 Binaphthyl Ethylphenanthrene or ethylanthracenea 254 81 25 Binaphthyl Ethylphenanthrene or ethylanthracenea 82 254 26 Benzo[def]dibenzothiophene 83 254 , Binaphthyl 27 84 Ethylchrysene or ethylbenz [a] anthracenea 256 Ethyl-4H-cyclopenta [def]phenanthrenea 28 85 256 Ethylchrysene or ethylbenz [a ]anthracenea Pyrene 29 86 256 Ethylchrysene or ethylbenz[ alanthracenea Ethyl-4H-cyclopenta[ de flphenanthrenea 30 87 Ethylchrysene or ethylbenz [alanthracenea 256 Ethyl-4H-cyciopenta [ def ] phenanthrenea 31 88 Ethylchrysene or ethylbenz [a] anthracenea 256 Ethyl-4H-cyclopenta[def] phenanthrenea 32 89 Ethylchrysene or ethylbenz[a]anthracenea 256 Ethyl-4H-cyclopenta[deflphenanthrenea 33 90 Ethylchrysene or ethylbenz[a]anthracenea 256 Ethylmethylphenanthrene or ethylmethyl34, 91 268 Methylbinaphthyl anthraceneb 35 92 Methylbinaphthyl 268 Methylfluoranthene 216 36 93 Methylbinaphthyl 268 Methylfluoranthene 37 216 94 Methylbinaphthyl 268 Methylfluoranthene 216 38 95 252 Benzo[j]fluoranthene Methylfluoranthene 216 .39 Benzo[ k Ifluoranthene 252 96 216 Methylfluoranthene 40 252 97 Benzofluoran thene Benzo [alfluorene 216 41 98 Benzo[ e Ipyrene 252 2-Methylpyrene and benzo[ b ]fluorene 216 42 252 99 Benzo [a] pyrene Ethylmethylphenanthrene or ethylmethyl220 43 100 Perylene 252 anthraceneb 101 Methylbenzopyrene or methylbenzo266 4-Methylpyrene 44 216 fluoranthene 1-Methylpyrene 216 45 102 266 Methylbenzopyrene or methylbenzoUnknown 230 46 fluoranthene Ethylmethyl-4H-cyclopenta[deflphen232 47 103 266 Methylbenzopyrene or methylbenzoanthreneb fluoranthene Ethylmethyl-4H-cyclopenta[def]phen'232 48 104 266 Methylbenzopyrene or methylbenzoanthreneb fluoranthene Ethylmethyl-4H-cyclopenta[deflphen232 49 105 266 Methylbenzopyrene or methylbenzoanthreneb fluoranthene Ethylmethyl-4H-cyclopenta[def]phen232 50 106 306 Quaterphenyl anthreneb 107 264 Unknown Ethylmethyl-4H-cy clopenta [de f 3 phen232 51 108 C 276 anthreneb 109 C 276 Ethylmethyl-4H-cyclopenta[deflphen232 52 110 C 276 anthreneb 111 Dibenzanthracene 278 Ethylmethyl-4H-cyclopenta[deflphen232 53 112 Dibenzanthracene 278 anthreneb 113 Benzo [ghilperylene 276 Ethylpyrene or ethylfluoranthenea 54 230 114 Anthanthrene 276 Ethylpyrene or ethylfluoranthenea 230 55 115 Methyldibenzanthracene 292 Ethylpyrene or ethylfluoranthenea 230 56 Dipheny lacenaphthalene 304 244 Ethylmethylfluoranthene or ethylmethyl- 116 57 117 302 Dibenzopyrene pyreneb 118 Dibenzopyrene 302 Ethylpyrene or ethylfluoranthenea 2 30 58 d 300 Ethylmethylfluoranthene or ethylmethyl- 119 59 244 Coronene 120 300 pyreneb Dibenzopyrene 121 302 Naphthobenzothiophene 234 60 122 Dibenzopyrene 302 Benzo[ghi jfluoranthene 226 61 benzo[b,mno]fluoranthene, dibenzo[e,rnno]fluoranthene, Could also be dimethyl-. b Could also be trimethyl- or dibenzo[f, m n o Ifluoranthene. Further possibilities are the propyl-. C Compounds with molecular weight 276 can be benzo derivatives of cyclopenta[cd]pyrene and cyclopentaany of the following: indeno[ 1,2,3-cd]pyrene, indeno[l,2,3cdlfluoranthene, cyclopenta[cd Iperylene, phenanthro[ 10,- [cdlfluoranthene. d Possibilities include the cyclopenta de1,2,3-cdef]fluorene, acenaphth[ 1,2-a]acenaphthylene, dirivatives of compounds with molecular weight 276. 7 8 9 10 11 12 13 14 15 16 17
1570
ANALYTICAL CHEMISTRY, VOL. 48, NO. 11, SEPTEMBER 1976
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Figure 10. Enriched fraction a of Figure 4 recorded by the total-ioncurrent monitor of the mass spectrometer Conditions: same as Figure 1. Key: see Table II
Figure 9. Enriched fraction b of Figure 4 recorded by the total-ioncurrent monitor of the mass spectrometer Conditions: same as Figure 1. Key: see Table II
Although the application of N M R spectroscopy in this work necessitated considerably larger quantities of the initial sample than would be needed for GC-MS work alone, it provided rather unique information on the presence of methylated PAH, and provided some verification of results obtained by GC-MS and from retention data of standard compounds. The limited sensitivity of this powerful identification method and unavailability of reference d a t a have prevented the analysis of P A H higher than the methylchrysenes. However, in addition to the compounds listed, the presence of benzo[k]pyrene, benzo [b] fluoranthene, benzo E]fluoranthene, benzo[k]fluoranthene, dibenz[a,c]anthracene, picene, dibenz[a,h]anthracene, benzo[ghi]perylene, a n d indeno[ 1,2,3cdlpyrene were suggested from proton NMR spectra of the aromatic region and uv spectra obtained on the higher molecular weight fractions. It is clear t h a t mass spectra alone are not sufficiently indicative of various PAH isomers. Although confirmation of the identities of many of the P A H listed in Table I1 was accomplished by comparison of GC retention times with those of acquired standards, the accumulation of many more standard compounds will be necessary to provide the positive identification of all compounds. Even though the positive identification of a number of isomers could not be obtained in this work because of the lack of standards, the value of capillary columns in resolving PAH with the same molecular weight, but different position of alkyl group(s) is clearly indicated. T h e importance of distinguishing such compounds from each other because of their different tumor-promoting properties (6, 7) has often been pointed out. Another important feature of high-resolution (capillary) GC lies in the future development of high-precision measurement techniques and standardized acquisition of retention data. As indicated by the GC-MS results of this work, only limited peak overlapping occurs within the studied PAH mixtures. Thus, retention characteristics can be looked upon much more favorably here than in packed-column work. T h e degree of confidence in a compound identity that would be satisfactory for routine air-pollution control work could, perhaps, be furnished by purely chromatographic means. Although the potential of GC-MS methods t h a t utilize re-
petitive scanning of packed-column chromatograms with further computer-aided data processing ( 1 5 ) in the analysis of complex mixtures is presently beyond dispute, the mass spectrometer has difficulties in distinguishing various isomers present in such mixtures without prior separation as distinct GC peaks. Composition of a P A H sample extracted from airborne particulates will obviously be dependent on the extraction and purification procedure involved. T h e present methodology has been oriented toward the analysis of ordinary (neutral) PAH. However, of particular interest is the presence of a number of sulfur-containing heterocyclics (see Table 11). I t is interesting to note the absence in our samples of polycyclics with partially saturated rings that were reported by Lao et al.
(4,5). It is a well-known fact that pyrogenesis of PAH is strongly dependent on the combustion conditions. Thus, the content of PAH sampled in an industrial area is a composite of the emissions from various industries and transportation vehicles within that area. It is reasonable to expect PAH to differ quantitatively (and, perhaps, even qualitatively) among different cities and even locations within each city (9).Capillary columns and their superior resolving power provide a very attractive tool for routine screening for a number of PAH pollutants, analyzing the composition of automobile exhaust gases, studying the model combustion processes, etc.
ACKNOWLEDGMENT We thank Patrick McCoy, Department of Health, Gary, Ind., and W. Benson, Department of Public Works, Indianapolis, Ind., for providing samples of airborne particulates. We are also grateful to M. Cole for recording NMR spectra.
LITERATURE CITED (1) L. B. Lave and E. P. Seskin, Science, 169, 723 (1970). (2) J. R. Williams and C. G. Justus, J. Air Pollut. Control Assoc., 24, 1063 (1974). ( 3 ) E. Sawicki, Chemist. Analyst, 53, 24, 56, and 88 (1964). (4) R. C. Lao, R . S. Thomas, H. Oja, and L. Dubois, Anal. Chem., 45, 908
(1973). (5) R. C. Lao, R. S. Thomas, and J. L. Monkman, J. Chromatogr., 112, 681 (1975). (6) W. Carruthers, H.N. M. Stewart, and D. A. M. Watkins, Nature (London), 691 (1967).
ANALYTICAL CHEMISTRY, VOL. 48, NO. 11, SEPTEMBER 1976
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,
(7) D. Hoffman, W. E. Bondinell, and E. L. Wynder, Science, 183, 215 (1974). ( 8 ) M. Novotny, M. L. Lee, and K. D. Bartle, J. Chromatogr. Sci., 12, 606 (1974). (9) K. D. Bartle, M. L. Lee, and M. Novotny. lnt. J. Environ. Anal. Chem.. 3.349 (1974). (IO M. L. Lee, K. D. Bartle, and M. Novotny, Anal. Chem., 47, 540 (1975). (11) G. Grimmer and H. Bohke, Fresenius' 2.Ana/. Chem,, 261,310 (1972). (12) T. Doran and N. G. McTaggart, J. Chromatogr. Sci., 12, 715 (1974).
(13) M. L. Lee, M. Novotny and K. D.Bartle, Anal. Chem., 48, 405 (1976). (14) M. Novotny and R. Farlow, J. Chromatogr., 103, 1 (1975). (15) R. A. Hites and K. Biemann, Anal. Chem., 43, 681 (1971).
RECEIVEDfor review January 30,1976. Accepted June 3,1976. This work was supported by Grant No. M P S 75-04932 from t h e National Science Foundation.
Qualitative and Quantitative Analyses of Bansyl Derivatives of Dopamine and Some of Its Metabolites in Urine Samples by Electron Impact and Field Desorption Mass Spectrometry W. D. Lehmann," H. D. Beckey, and H.-R. Schulten Institute of Physical Chemistry, University of Bonn, 5300 BONN, Wegelerstr. 12, West Germany
The derivatives of dopamlne, 3-O-methyldopamlne, and 40-methyldopamine produced by reactlon wlth 5-di-n-butylamlnonaphthalene-1-sulfonylchloride (BANS-CI) were separated by thln-layer chromatographyand identified by electron impact and field desorption mass spectrometry. The base peaks In the electron impact spectra were formed by loss of C3H7 from the molecular ion. In contrast, the field desorption spectra showed only the molecular Ions. The BANS derlvatlves were isolated from urine samples by thln-layer chromatography. Two novel mass spectrometric methods were used for their quantltation: 1) A twln dlrect Introduction system for calibration in the electron impact mode (external standard) and 2) stable Isotope dilution in connection wlth electron Impact and field desorption mass spectrometry (Internal standard). The advantages and drawbacks of both technlques are descrlbed and thelr utlllty for physiological and pharmacokinetic studies is discussed.
Although there are a great number of biologically active amines (biogenic amines), in recent years analytical interest has been focused on a relatively small number of these compounds, in particular, on those amines which act as inhibitors or transmitters of signals in the nervous system. These include small aliphatic amines (respectively, quaternary ammonium bases) such as acetylcholine, and phenolic and catecholic amines such as tyramine and dopamine. In general, two procedures are used for the identification and quantitation of aromatic amines in body fluids or tissue: 1)Extraction and separation of the amines by chromatography and formation of highly fluorescent derivatives which are quantitated by fluorometry (1, 2). 2) Relatively volatile derivatives of the amines are produced which are subsequently estimated by a coupled gas chromatograph-mass spectrometer unit (3, 4 ) . T h e sensitivity of both methods is sufficignt for determination of biogenic amines in naturally occurring concentrations (Le., in the range 1 to 100 ng/ml). Possible sources of error are: for method 1, the relatively unspecific identification by thin-layer chromatography (TLC) and fluorometry; for method 2 ions with m / e values considerably below the molecular weight must conventionally be used for the identification of these derivatives, since these compounds undergo strong fragmentation under electron impact. In quantitative determination, problems may arise from background and column bleeding. 1572
In this paper, a new method for qualitative and quantitative analysis of dopamine (DA), 3-0-methyldopamine (3-MDA), and 4-0-methyldopamine (4-MDA) is introduced. Defined, fluorescent derivatives of high molecular weight were generated by reaction with 5-di-n-butylaminonaphthalene-1-sulfonyl chloride (BANS-Cl) ( 5 , 6 ) .The BANS derivatives were isolated by T L C followed by identification and quantitation by two independent methods: electron impact (EI) and field desorption (FD) mass spectrometry (MS) (7).
EXPERIMENTAL Dopamine and 4-0-methyldopamine as hydrochlorides were purchased from Aldrich Chemical Co., Milwaukee; 3-0-methyldopamine hydrochloride was obtained from Regis Chemical Co., Ill. Dopamine-(a-d&d*) hydrochloride was obtained from Merck, Sharp, and Dohme, Canada. All compounds were of analytical grade. BANS-Cl was kindly supplied by N. Seiler, Max-Planck-Institut fur Hirnforschung, Frankfurt, W.-Germany. The reaction of the amines with BANS-CI was carried out according to a procedure described by Seiler et al. for 5-dimethylaminonaphthalene-1-sulfonyl chloride (DANS-Cl) (8). An acidified aqueous solution (sulfuric acid, pH 1) of the amine (about 10 pg/ml) was mixed with 6 ml of a solution of BANS-C1 in acetone (about 1mg/ml) and saturated with sodium carbonate. After 3 h at room temperature, the reaction mixture was decanted from the salt, which was washed once with 1ml of acetone. Saturating of the reaction mixture with potassium dihydrogen orthophosphate was followed by the evaporation of acetone under a stream of nitrogen. The residual aqueous phase was mixed with 3 ml of methanol and extracted with 2 ml of n-heptane or toluene. The extract was concentrated to dryness under a stream of nitrogen and the residue redissolved in 100 pl of ethyl acetate. Aliquots of this solution were submitted to TLC. Thin-layer chromatography was carried out on glass plates 20 X 20 cm coated with 300 Mm Silica gel G layers (Merck AG, Darmstadt) using ascending chromatography in a solvent vapor-saturated atmosphere. The thin-layer plates were developed using cyclohexane/ ethyl acetate (4:l) as eluent. Fluorescence excitation at 364 nm was used for visualization of the BANS derivatives. The fluorescent spots were scraped off and extracted with 500 p1 of ethyl acetate. After appropriate concentration under a stream of nitrogen, the extract was transferred into the microcrucible of the direct introduction system of the E1 mass spectrometer. Alternatively, for FD analysis, the extract was applied to a high temperature activated wire emitter by the syringe technique (7). The E1 mass spectra were recorded on a modified CH-4 mass spectrometer under standard conditions (70 eV electron energy, 20 FA emission, 150 "C ion source temperature). In order to obtain the complete E1 mass spectrum of the tris-BANS-DA derivative (mol wt 1104), the acceleration voltage was lowered to 2700 V. As shown in Figure 1, the ion source of the E1 mass spectrometer was equipped
ANALYTICAL CHEMISTRY, VOL. 48, NO. 11, SEPTEMBER 1976
