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Anal. Chem. 1982, 5 4 , 1802-1806
phenol incubation (quenched with an equal volume of cold methanol, centrifuged briefly, and injected into the chromatograph) is shown in Figure 6. Responses for hydroquinone and catechol were observed with relatively low gain detection. The chromatogram of a blank consisting of a control incubation with phenol added after quenching indicated that the dihydroxy peaks were not due to impurities in phenol (16). B ~ a u s metabolites e are present in the incubation at concentrations adequate for detection, uncertainties associated with solvent extraction are circumvented.
ACKNOWLEDGMENT The authors literature search assistance from J. R. Rice and S. M. Hommel. LITERATURE CITED (1) Snyder, R.; Longacre, S.L.; Wltmer, C. M.; Kocsls, J. J.; Andrews, L. S.; Lee, E. W. “Reviews in Blochemical Toxlcology”; Hodgson, E., Bend, J. R., Plhlpot, R. M., Eds., Elsevler/North Holland: New York, 1981; p 123. (2) Sammett, D.; Lee, E. W.; Kocsls. J. J.; Snyder, R. J. Toxlcol. Environ. Health 1961, 5 , 785. (3) Sloane, N. H. Biochim. Blophys. Acta 1965, 107, 599.
(4) Jerina, D.; Daly, J.; Wltkop, B.; Zaltzman-Nirenberg, P.; Udenfriend, S. Arch. Biochem. Biophys. 1968, 128, 176. (5) Tunek, A.; Platt, K. L.; Przybylski, M.; Oesch, F. Chem.-Biol. Interact. 1980, 33, 1. (6) Tunek, A.; Platt, K. L.; Bentley, P.; Oesch, F. Mol. Pharmacol. 1976, 14, 920. (7) Greenlee, W. F.; Chism, J. P.; Rlckert, D. E. Anal. Biochem. 1981, 112, 367. (8) Andrews, L. S.;Sasame, H. A.; Glllette, J. R. Life Sci. 1979, 25, 567. (9) Mlner, D. J.; Kissinger, P. T. Biochem. Pharmacol. 1979, 28, 3285. (10) Kissinger, P. T.; Bruntlett, C. s.; Shoup, R. E. Life sci. 1981,28,455. (11) Roston, D. A.; Klsslnger, P. T. “Liquid Chromatography/Electrochemlstry: Principles and Applications”; Klssinger, P. T., Ed.; BAS Press, in press. (12) Roston, D. A.; Klsslnger, P. T. Anal. Chem. 1981, 5 3 , 1965. (13) Lowry, 0. H.; Rosebrough, N. J.; Farr, A. L.; Randall, A. J. J. Blol. Chem. 1951, 193, 265. (14) Oettel, H. Naunyn-Schmiedbergs Arch. f x p fafhol. fharmakol. 1936, 183. 319. . .. (15) Nomiyama, K. Ind. Health 1965, 3 , 53. (16) Masoud, A. N.; Dubes, G. R. HRC C C , J . High Res. Chromafogr., Chromafogr. Commun. 1980, 3 , 133. (17) Roston, D. A.; Klssinger, P. T. Anal. Chem. 1982, 5 4 , 429. (18) MacCrehan, W. A.; Durst, R. A. Anal. Chem. 1961, 53, 1700. (19) Blank, L. J. Chromafogr. 1976, 117, 35.
~ . .
RECEIVED for review April 26, 1982. Accepted June 21, 1982.
Mesogenic Polysiloxane Stationary Phase for High-Resolution Gas Chromatography of Isomeric Polycyclic Aromatic Compounds Robert C. Kong and Milton L. Lee* Department of Chemistty, Brigham Young University, Provo, Utah 84602
Yoshinori Tominaga, Ram Pratap, Masatomo
Iwao,’
and Raymond N. Castle
Department of Chemlsfry, University of South Florlda, Tampa, Florlda 33620
The open tubular column gas chromatographlc properties of a new mesogenlc polyslloxane phase are descrlbed and compared wlth those of a poly(methylpheny1slloxane) (SE52). The former ISshown to yield hlgh column efflclency commensurate wlth Its gumllke character, yet concomitant hlgh selectlvlty for Isomers of polycycllc aromatlc hydrocarbons and sulfur heterocycles. The nematlc temperature range of the new phase, 70-300 ‘C, exceeds by several factors those of prevlously described llquld crystal stationary phases.
Polycyclic aromatic compounds (PAC) which comprise by far the largest class of chemical carcinogens known, are ubiquitous in air, water, sediment, food, tobacco smoke, and fossil fuels (1). Because of the attendant sample complexity, open tubular column gas chromatography has accordingly become an indispensable technique for analysis of these species. Slightly polar gum phases such as SE-52 or SE-54 have been shown to offer high column efficiency, some selectivity, and high thermal stability for PAC (1,2);however, many compounds are not well resolved even at high column Present address: Department of Chemistry, Faculty of Liberal Arts, Nagasaki University, 1-14 Bunkyo-Machi, Nagasaki 852, Japan.
efficiency with these phases, e.g., benz[a]anthracene/triphenylene/chrysene, the benzofluoranthenes, and many sulfur heterocyclic compounds (1-3). Moreover, only minor gains in selectivity can be achieved with more polar phases ( 4 , 5 ) . In contrast, mesogenic (nematic liquid-crystalline) phases which provide separations on the basis of molecular geometry have been shown to be highly selective for polycyclic aromatic hydrocarbons (PAH) and polycyclic aromatic sulfur heterocycles (PASH) which coelute on other phases (6-9). However, the mesogenic phases reported to date are accompanied by high volatility and poor column efficiency, the latter a result of slow kinetics of mass transfer. Laub et al. (l(F.12)overcame these limitations by blending, e.g., N,N‘-bis(p-butoxybenzylidene)a,a’-bi-p-toluidine(BBBT) with SE-52 which provided open tubular columns of concomitant high efficiency and high and adjustable selectivity. There is nonetheless some inconvenience in the fabrication of blended phase systems; and more importantly, dispersion of compounds such as BBBT in SE-52 does not lower the solid nematic transition temperature (186 “C) of the former. Thus, these mixed phases are not useful for analysis of solutes of high vapor pressure. Poly(mesogen/methyl)siloxanes (PMMS) offer the means of overcoming these difficulties while, at the same time, preserving the above noted advantages of blended phases. Ideally, such a phase would exhibit selectivity on the basis of solute geometry. In addition, the phase must provide acceptable chromatographic efficiency which, in terms of open
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4 5
4
it3
10,11
'7
120
TIME min IO 2.0 150 200 TEMPERATURE( C)
3,O 250
I
i i 13
TIME.min
49
30
250
TEMPERATUREI'C)
Figure 2.
Chromatogram of the sulfur heterocycle fraction of a coal
liquid on columns containing (a) SE-52 and (b) PMMS. Temperature was programmed from 120 'C to 260 'C at 4 'C min-I. Peak assignments are given in Table I.
Chromatogramis of methyidibenzothiophenes and four-ring PASH: (1) 1-methyidibenzothiophene,(2) 2-niethyldibenzothiophene, (3)3-methyidibenzothiophene, (4) 4-methyidibenzothiophene,(5)benzo [bInaphtho[2,I-dlthiophene, (6)benzo [ b ]napMho[ 1,2-d]thiophene, (7) phenanthro[g,lO-blthiophene, (8)phenanthro[4,3-b]thiophene,(9) anthra[ 1,2-b]thiophene,(10) benzo[b]naphtho[2,3-d]thiophene, (1 1) phenanthro[ 1,2-b]thiophisne,(12) phenanthro[3,4-b]thiophene,(13) anthra[2,l-b]thiophene, (14) phenanthrol2,l-b ]thiophene, (15) phenanthro [3,241]thiophjene, (16) phenanthro [2,3-b] thlophene; (a) C to 260 SE-52, (b) PMMS. Temperature was programmed from 120 ' 'C at 4 O C min-I. Figure 1.
tubular column GC, prescribes almost exclusively a silicone gum. The first such phase reported (13) was a poly(mesogen/methyl)siloxane of nematic to isotropic temperature transition of only 97 "C. While the criterion of high column efficiency was adequately met by the material, the useful nematic range was obviously unacceptably low. Apfel et al. (14) therefore undertook the synthesis of mesogen polymers of considerably higher nematic/isotropic transition temperature, the chromatographic properties of the most promising of which are detailed here. Since the chromatographic separation of PAC is currently of widespread and intense interest, various mixtures of these were employed as solutes for purposes of illustration of the efficiency and selectivity of the PMMS phase. For comparison, chromatograms of the same mixtures are also shown with SE-52, a popular phase for samples of this type. In addition, the utility of tandem connection of lengths of pure phase columns as deduced from window diagrams (15) is described.
EXPERIMENTAL SECTION The PMMS stationary phase was provided by Richard J. Laub (Ohio State University); its synthesis will be presented in detail elsewhere (14). The capillary columns fabricated from untreated fused silica (Hewlett-Packard,Avondale, PA), were 19 m in length by 0.32 mm i.d. and were coated statically with SE-52 (Alltech
Associates, Deerfield, IL) or with PMMS. The coating solutions employed were 0.3% (3 mg ~ m -in~ n-pentane ) (SE-52) or in 30% n-pentane/70% dichloromethane (PMMS); all columns were conditioned overnight at 280 "C under helium flow, whereas the carrier routinely employed for generation of the chromatograms was hydrogen at an average linear velocity of 104 cm s-'. The solute standards were either obtained commercially or were synthesized (16, 17), while the PAH and PASH fractions of coal-derived samples were those employed in previous work (2) or obtained by using the fractionation scheme of Willey et al. (18). Solutions were injected in dichloromethanesolvent,and compound identifications were assigned from known relative retentions or from GC/MS data. The gas chromatograph was a HewlettPackard Model 5880 equipped with a flame ionization detector and operated in the split-injection (501) mode, while the GC/MS system was a Hewlett-Packard 5982A. Window-diagram data reduction was carried out in the usual way in terms of solute relative retentions and capacity factors (19).
RESULTS AND DISCUSSION Figure 1 contrasts the separation of four methyldibenzothiophene isomers as well as 12 four-ring PASH isomers with SE-52 (Figure l a ) and with PMMS (Figure lb). The selectivity of the latter phase clearly is different from that offered by the former where, in addition, only two groups of solutes (peak no. 7,8, and 12 and no. 13 and 16) have not been fully resolved. The PMMS phase, further, offers separation of the four-ring PASH isomers which is superior to that achieved with the mixed phase containing 20% BBBT in SE-52 (3). Figure 2 illustrates the separation of the PASH fraction of a coal liquid with SE-52 (Figure 2a) and with PMMS (Figure 2b). Again, the elution order of identified solutes (see Table I for peak assignments) is considerably different from one phase to another. Note that the methyldibenzothiophene isomers and the four-ring PASH isomers can be unambiguously identified.
ANALYTICAL CHEMISTRY, VOL. 54, NO. 11, SEPTEMBER 1982
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Table I. Peak Assignments for the Sulfur Heterocycle Fraction of a Coal Liquid (see Figure 2 ) no.
solute
2 3 4
5 6
7 8 9 10 11
12 13 14
15 16 17
120
1
T I M E , min
10 150
I
I20
solute naphthalene 2-methylnaphthalene 1-methylnaphthalene acenaphthylene acenaphthene dibenzofuran flu ore ne dibenzothiophene phenanthrene anthracene C -phenanthrenes fluoranthene acephenanthrene pyrene benzo [ a] fluorene benzo[ blfluorene benzo [ c ]phenanthrene nap h t h ace ne benz [alanthracene triphenylene chryse ne benzo [ b 3 fluoranthene benzo[h Ifluoranthene benzou] fluoranthene benzo[ alfluoranthene benzo[ e ] pyrene benzo[ alpyrene perylene indeno[ 1,2,3-cd]pyrene dibenz [a,h]anthracene benzo[ghi]perylene coronene
1 2
3 4 5
6 7 8 9 10 11
12 13 14 15
16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
30
200
250
TEMPERATURE[ C ,
17 20
14
I
no.
dibenzothiophene phenanthrene 4-methyldibenzothiophene 2-methyldibenzothiophene 3-methyldibenzothiophene 1-methyldibenzothiophene C,-dibenzothiophanes flu or anthe ne C,-dibenzothiophenes phenanthro[ 4,5-bcd]thiophene pyrene C -phen anthro [ 4,5- bcd ]thiophenes benz o[b ]naph tho[ 2 , l -d]thiophene benzo[ b]naphtho[ 1,2-d]thiophene benzo[ b]naphtho[ 2,3-d]thiophene C l-benzonaphthothiophenes C,-benzonaphthothiophenes
1
I
Table 11. Peak Assignments for PAC in a Standard Mixture (see Figure 3 ) and in a Coal Tar Sample (see Figure 4 )
150
20 200
I1
2225 26
TIME,min
40
69
250
TEMPERATURE(C1
Flgure 3. Chromatograms of standard PAH on columns containing (a) SE-52 and (b) PMMS. Temperature was programmed from 120 OC to 270 OC at 4 OC min-I. Peak assignments are given in Table 11.
Even more dramatic shifts in retentions are found for PAH, as shown in Figure 3 (see Table I1 for peak assignments): only benzo[a]fluoranthene (peak no. 25) remains unresolved from benzo[b]fluoranthene (peak no. 22) with the PMMS phase (Figure 3b). Much improved resolution was seen for isomeric groups: over three base-line-width separation between phenanthrene (peak no. 9) and anthracene (peak no. 10) and among chrysene (peak no. 21), triphenylene (peak no. 20), and benz[a]anthracene (peak no. 19); two to four base-line-width resolution among perylene (peak no. 28), benzo[a]pyrene (peak no. 27), and benzo[e]pyrene (peak no. 26); base line resolution of benzoplfluoranthene (peak no. 24) from benzo[k]fluoranthene (peak no. 23) and benzo[b]fluoranthene (peak no. 22), which coelute on SE-52. The enhanced resolution for PAH is much better than that obtained on packed columns coated
18
'b)
I21
TIME,min.
3,O
250 T E M P E R A T U R E I'Ct
,.
--L
Figure 4. Chromatograms of coal tar PAC on columns containing (a) SE-52 and (b) PMMS. Temperature was programmed from 120 OC to 270 'C at 4 OC min-'. Peak assignments are given in Table 11.
with other pure liquid crystals reported by Janini et al. (6, 7). Hence, this phase is particularly useful for the analysis
ANALYTICAL CHEMISTRY, VOL. 54, NO. 11, SEPTEMBER 1982
1 1
,
2 3 TIME min
-
' 4 4~
6 12 TIME m i n
2
'
13 14 TlME,mIli
6
1805
1.8
1'6
Flgure 5. Chromatograms, of the SIXmethylchrysene isomers on columns containing (a) SE-52 and (b) PMMS at 230 "C isothermal: (1) I-methylchrysene, (2) 2-methylchrysene, (3) :l-methylchrysene, (4) 4-methylchrysene, (5) 5-meithylchrysene, (6) 6-methylchrysene.
of complex mixtures, as demonstrated in Figure 4, which shows chromatograms of the PAH fraction of coal tar (see Table I1 for peak assignments). With only SEI-52, triphenylene, chrysene, benzo[ blfluormthene, benzo[k]fluoranthene, and benzoljlfluoranthene cannot be unambiguously identified. Improved selectivity of PMMS over $33-52 was also obtained for the six methylchrysene isomers, as shown in Figure 5. On SE-52,4-, 5-, and 6-methylchrysene coelute, whereas 3- and 4-methylchrysene are not fully separated on PMMS. Therefore, a blend of the two phases would appear to be superior to either pure material. A window diagram (cf. ref 15) of relative retention against phase composition indicated that a 5050 blend of the two phases would provide base line (66) separation of all eiolutes provided that the resultant at k'> 10). A 4a column system yielded 1100000 plates (Nreq separation required, on tlhe other hand, only 45000 plates, that is close to the efficiency expected on coupling together the two pure phase columns with allowance made for connection dead volume. Figure 6a shows the chromatogram obtained from the capillaries tandem connected (20) in the order SE52:PMMS, where the resultant separation is almost exactly that predicted, i.e., 4a resolution. Reversal of the order of the columns, Figure 6b, yields only a slight decrease in the extent of separation relative to, that in Figure 6a but does result in a significant increase in absolute retention. The effect is expected since the pressure drops across column segments are both finite and nonequivalent (21, 22). The favorable chromatographic behavior of PMMS as demonstrated above is attributed to the multiple properties of the PMMS phase derived from its chemical structure, Le., polysiloxane backbone and mesogenic side chains. The polysiloxane backbone imparts the desired gumlike character to the PMMS phase, which facilitates the homogeneous coating of the phase onto the untreated fused silica surface, and, therefore, yields high efficiency. For example, PMMS yields 2200 plates/m (triphenylene solute; 220 "C) which compares favorably with about 3000 platen/m for pure SE-52, 1710 plates/m for 20% BBBT/SE-52, and 858 plates/m for 50% BBBT/SE-52 coated on untreated fused silica tubes (31, compared to 880 plates/ m for pure BBBT coated on treated glass capillaries (11). 011 the other hand, the high Selectivity of PMMS is accounted for by the mesogenic side chains. The attachment of a mesogenic moiety to the polysiloxane backbone does not destroy its chromatographic properties. Furthermore, the nematic range of the PMMS phase extends from 70 "C to 300 "C (differential scanning calorimetry), a wide nematic range usually not seen for liquid crystals, although
I
2
'
6
'
10
TIME
14
'
i'8
22
m!n
Flgure 6. Chromatograms of the six methylchrysene isomers on tandem-connected columns containing SE-52 and PMMS in the order of (a) SE-52:PMMS and (b) PMMS:SE-52 at 230 "C isothermal. Peak assignments are the same as in Figure 5.
a noticeable drop in column efficiency was found below ca. 110 "C. The PMMS phase also shows better thermal stability than that of liquid crystal/SE-52 mixed phases, which is attributed to its lower volatility and gumlike character. The bleeding of PMMS from untreated fused silica capillaries began around 240 "C as compared to below 230 "C for mixed phases of liquid crystals and SE-52 ( 3 ) . Furthermore, the bleeding was only minimal at higher temperature (e.g., 270 "C) similar to SE-52, when compared to the mixed phases. This observed high thermal stability of PMMS permits its use at higher column temperatures for longer periods of time and for low volatile solutes such as indeno[1,2,3-cd]pyrene,dibenz[a,h]anthracene, and benzo[ghi]perylene. Although solutes generally encounter longer retention on PMMS compared to SE-52, shorter column lengths can be used to reduce the elution temperature and analysis time. Therefore, the PMMS phase can be used for the analysis of PAC containing from two to at least five rings and is particularly useful as a complementary stationary phase to SE-52 or similar phases for the separation of isomers. In addition, the coupling of lengths of pure phase columns for optimizing selectivity as opposed to blending different phases in a single column appears to be an alternative dependent solely on practical convenience.
ACKNOWLEDGMENT The authors thank R. J. Laub for providing the mesogenic polysiloxane stationary phase. LITERATURE CITED (1) Lee, M. L.; Novotny, M. V.; Bartle, K. D. "Analytical Chemistry of Polycyclic Aromatic Compounds"; Academic Press: New York, 1981; Chapter 2. (2) Lee, M. L.; Wright, B. W. J . Chromatog. Sci. 1980, 18, 345-358.
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Anal. Chem. 1982, 54, 1806-1811 Kong, R. C.; Lee, M. L.; Tominaga, Y.; Pratap, R.; Iwao, M.; Castle, R. N. J. Chromatogr. Sci., in press. Lysyuk, L. S.; Koroi, A. N. Chromatographia 1977, IO, 712-719. Borwitzky, H.; Schomburg, G. J. Chromatogr. 1979, 170, 99-124. Janini, G.M.; Johnston, K.; Zieiinski, W. L., Jr. Anal. Chem. 1975, 47, 670-674. Janini, G. M.; Muschik, G. M.; Zieiinski, W. L., Jr. Anal. Chem. 1978, 48, 809-813. Zielinskl, W. L., Jr.; Scanian, R. A.; Miller, M. M. J. Chromatogr. 1981, 209, 87-95. Haky, J. E.; Muschik, G. M. J. Chromatogr. 1981, 214, 181-170. Laub, R. J.; Roberts, W. L. I n "Polynuclear Aromatic Hydrocarbons: Chemistry and Biological Effects"; Proceedings of the Fourth International Symposium on Polynuclear Aromatic Hydrocarbons; Bjorseth, A., Dennis, A. J., Eds.; Batteile Press: Columbus, OH, 1980; pp 25-58. Laub, R. J.; Roberts, W. L.; Smith, C. A. HRC CC, J. High Resolut. Chromatogr Chromatogr. Commun 1980, 3 , 355-356. Laub, R. J.; Roberts, W. L.; Smith, C. A. I n "Polynuclear Aromatic Hydrocarbons: Chemical Analysis and Biological Fate"; Proceedings of the Fifth International Symposium on Polynuclear Aromatic Hydrocarbons; Cooke, M. W., Dennis, A. J., Eds.; Batteiie Press: Columbus, OH, 1981; pp 287-295. Finkeimann, H.; Laub, R. J.; Roberts, W. L.; Smith, C. A. I n Polynuclear Aromatic Hydrocarbons: Chemical Analysis and Biological Fate"; Proceedings of the Sixth International Symposium on Polynuclear Aromatic Hydrocarbons; Cooke, M. W., Dennis, A. J., Eds.;
.
.
Batteiie Press: Columbus, OH, 1982, in press. (14) Apfei, M. A.; Flnkelmann, H.; Laub, R. J.; Luhmann, B.-H.; Price, A,; Roberts, W. L.; Smith, C. A., to be submitted for publication in Makromol. Chem ., Rapld Commun . (15) Laub, R. J. Am. Lab. (Fairfleld, Conn.) 1981, 13 (3), 47-56. (16) Iwao, M.; Lee, M. L.; Castle, R. N. J. Heterocycl. Chem. 1980, 17, 1259-1264. (17) Tominaga, Y.; Lee, M. L.; Castle, R. N. J. Heterocycl. Chern. 1981, 18,967-972. (18) Wiiiey, C.; Iwao, M.; Castle, R. N.; Lee, M. L. Anal. Chem. 1981, 53, 400-407. (19) Laub, R. J.; Purneil, J. H.; Summers, D. M.; Williams, P. S. J. Chromatow. 1978, 155. 1-8. (20) Lser, D. W.; Wright, 8. W.; Lee, M. L. HRC CC J. High Resoluf. Chromatogr. Chromatogr. Commun. 1981, 4, 406-407. (21) Vilialobos, R.; Brace, R. 0.; Johns, T. I n "Gas Chromatography"; Noebeis, H. J., Wail, R. F.,Brenner, N., Eds.; Academic Press: New York, 1961; pp 39-51. (22) Viilaiobos, R.; Turner, C. C. I n "Gas Chromatography"; Fowler, L., Ed.; Academic Press: New York, 1963; pp 105-118.
RECEIVED for review April 21, 1982. Accepted June 21, 1982. This work was supported by the Department of Energy, Office of Health and Environmental Research, Contract No. DEAC02-79EV10237.
Fused Silica Capillary Gas Chromatography/Negative Chemical Ionization Mass Spectrometry for Determination of Catecholamines and Their 0-Methylated Metabolites Jeffrey T. Martin, Jack D. Barchas, and Kym F. Faull" Nancy Pritzker Laboratory of Behavioral Neurochemistry, Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, California 94305
Determination of the pentafluoropropionyl derlvatlve of normetanephrine by GC/MS In the negative chemical ionlratlon mode yields a 200-fold and 350-fold Increase In sensitivity compared with determinatlon done In the posltlve chemical lonlratlon and electron impact modes, respectlvely. I n light of this flndlng, we have prepared two classes of derlvatlves for the catecholamlnes and their 0-methyl metabolites suitable for GCIMS appllcatlons In the NCI mode. The two derlvatlratlon schemes are discussed and the NCI mass spectral characterlstlcs of the correspondlng derlvatlves are compared. I n addltlon, the pentafluoropropionyl derivatives of the catecholamlnes and their 0-methylated metabolltes were found to have vastly improved chromatographic characteristics when fused slllca capillary columns were used compared to conventlonal packed columns. This Improvement, which Is attrlbuted to the reduced chemlcal reactlvlty of the fused slllca caplllary columns, offers advantages for trace level analysls of these compounds. Finally, a practical appllcatlon of this work Is Illustrated In the form of a sensltlve assay for normetanephrlne In human cerebrosplnal fluld.
In recent years, a number of workers have attempted to correlate levels of biogenic amines in various body fluids with specific psychiatric and neurologic disease states. For some of the most intensely studied disorders, this goal has remained elusive. This is in part because of the minute amounts of these compounds found in biological fluids and because any differences in the concentrations of these compounds between
the disease state and the control population may be quite subtle. These factors underscore the need for dependable and sensitive analytical procedures. The development of gas chromatography/mass spectrometry (GC/MS) and subsequently of selective ion monitoring techniques has provided a combination of sensitivity and selectivity unequaled by other methods which have found application in this area. The recent refinements of fused silica capillary gas chromatography ( I , 2) and electron capture negative chemical ionization mass spectrometry (NCI) (3) have led to further improvements in the specificity and sensitivity attainable with GC/MS. In order to be applicable to the GC/MS method, a molecule must be sufficiently nonpolar, volatile, and thermally stable to traverse the chromatographic column. Furthermore, to attain the enhanced sensitivity available in the electron capture NCI mode, the molecule must have a positive electron affinity. Few biological molecules meet these requirements in and of themselves. Accordingly, chemical derivatization is necessary. Derivatizing reactions conferring both volatility and electron capturing ability have found wide application in the area of gas chromatography with electron capture detection. Thus, a great deal of literature is already available regarding the preparation of derivatives which are potentially suitable for NCI GC/MS work. Perfluoroacylating reagents have perhaps found the most widespread utility as derivatizing reagents for electron capture gas chromatographic applications. These derivatives owe much of their prominence in this area to the volatility and electron capturing properties conferred by the perfluorocarbon group and the presence of the carbonyl group which synergistically helps to delocalize the captured electron and stabilize
0003-2700/82/0354-1806$01.25/00 1982 American Chemical Society
