Anal. Chem. 1988, 60, 787-792
to reproduce ECNI mass spectra on different instruments, an assessment must be made of the range of relative ion abundances that can be achieved, using lenses and ion source parameters, on different instruments. In addition, the presence of background impurities must be monitored and controlled. Registry No. 2, 50-32-8; 3,439-14-5; 4,38444-93-8; 5,3506527-1;6, 30746-58-8;7, 3268-87-9;8, 84-74-2;9, 1956-06-5;10, 87-86-5; 11, 5074-71-5; 12, 959-98-8; CHI, 74-82-8. LITERATURE CITED Dougherty, R. C. Anal. Chem. 1081, 53, 625-636A. Hunt, D. F.; Crow, F. W. Anal. Chem. 1978, 5 0 , 1781-1784. Oehme, M.; Stockl, D.; Knoppel, H. Anal. Chem. 1088, 58, 554-558. Stockl, D.; Budzlklewlcz, H. Org. Mass Spectrom. 1982, 17, 470-474. Chrlstophorou, L. G. EHP, Envlron. Health Perspect. 1080, 3 6 , 3-32. Chrlstophorou, L. 0.;McCorckle, D. L.; Christodoulldes, A. A. Electron Molecule Interactlons and Their Appllcations ; Chrlstophorou, L. G., Ed.; Academlc: Orlando, FL, 1984; pp 477-617. Slegel, M. W. Practlcal Spectroscopy Serles : Mass Spectrometty ; Merrit, C., McEwen, C. N. M., Eds.; Marcel Dekker: New York, 1979; Vol. 3, Part B, pp 297-306. Siegel, M. W. Int. J . Mass Spectrom. Ion Phys. 1983, 46,325-328. Sears, L. J.; Campbell, J. A.; Grlmsrud, E. P. Homed. Envlron. Mass Spectrom. 1087, 14,401-416. McEwen, C. N.; Rudat, M. A. J. Am. Chem. SOC. 1081, 103, 4343-4349. Jennlngs, K. R. Phllos. Trans. R . SOC. London, A 1070, 293, 125-133. McEwen, C. N. Mass Spectrom. Rev. 1086, 5 , 521-547.
787
Miwa, 8. J.; Garland, W. A.; Blumenthal, P. Anal. Chem. 1081, 53, 793-797. Crow, F. W.; Bjorseth, A.; Knapp, K. T.; Bennett, R. Anal. Chem. 1081, 53, 619-625. Greaves, J.; Bekesl, J. G.; Roboz, J. Homed. Mass Spectrom. 1082, 9 , 406-410. Stan, H. J.; Kellner, G. Blomed. Mass Spectrom. 1082, 9 , 483-492. Busch, K. L.; Norstrom, A.; Bursey. M. M.; Hass, J. R.; Nllsson, C. A. Homed. Mass Spectrom. 1970, 6 , 157-161. Stemmler, E. A. Ph.D. Thesis, Indiana University, Bloomlngton, IN, 1986. Low, G. K.-C.; Batley, G. E.; Lidgard, R. 0.; Duffield, A. M. Biomed. Environ. Mass Spectrom. 1988, 13,95-104. Gregor, I.K.; Gullhaus, M. Int. J. Mass Spectrom. Ion Processes 1084, 56, 167-176. Szulejko, J. E.; Howe, I.; Beynon, J. H.; Schlunegger, U. P. Org. Mass Spectrom. 1080, 75, 263-267. Laramee, J. A.; Arbogast, B. C.; Delnzer, M. L. Anal. Chem. 1088, 58, 2907-2912. Stemmler, E. A.; Hites, R. A. Anal. Chem. 1985, 57,684-692. Low, G. K.-C.; Duffield, A. M. Homed. Mass Spectrom. 1984, 1 1 , 223-229. Garland, W. A.; Mlwa, B. J. Biomed. Mass Spectrom. 1083, IO, 126-1 29. Eichelberger, J. W.; Harris, L. E.; Budde, W. L. Anal. Chem. 1075, 47, 995-1000. Stafford, G. C. EHP, Envlron. Health Perspect. 1980, 3 6 , 85-96. Mathews, D. E.; Hayes, J. M. Anal. Chem. 1076, 48, 1375-1382.
RECEIVED for review August 10,1987. Accepted December 10,1987. We thank the U.S. Department of Energy (Grant No. 80EV-10449),the U.S. Environmental Protection Agency (Grant No. R808865) (R.A.H. and E.A.S.), and NIEHS (ES00040) (M.L.D. and B.A.) for support.
Electron Capture Negative Ion Mass Spectra of Halogenated Diphenylethane Derivatives E. A. Stemmler and Ronald A. Hites* School of Public and Environmental Affairs and Department of Chemistry, Indiana University, Bloomington, Indiana 47405
The methane, electron capture, negative Ion mass spectra of 17 halogenated diphenyiethane, -ethene, and -ethanol derlvatlves (such as DDT, DDE, and dicofol) are reported. These three groups of compounds show spectral differences which reflect the nature of the base molecular structure. The diphenylethenes, where the ethene group provides conjugation between the aromatic rings, give intense molecular Ions and [M Clr Ions. Chlorine substitution on the aromatic ring Is necessary for molecular Ion stability. The dlphenyiethanes give complex spectra and weak molecular ions as a result of thelr ailphatlc character. The halogenated diphenylethanols also show significant fragmentation. Molecular ions and characterlstk bns that correspond to the dlhalobenzophenone anion are observed. The spectra of these compounds are sensitive to Isomeric substitution patterns; for example, para,pararvs ortho,para’ Isomeric pairs can be differentiated.
-
pherically transported DDT continues to be received in the U.S. and Canada from those countries that have not banned it (2). Mass spectrometry has been an important technique for that analysis and identification of DDT and related compounds. Their electron impact mass spectra have been reviewed by Hutzinger and Safe (3);in general, the molecular ions are weak, and no isomer specificity is observed (the para,para’ isomers give the same spectra as the ortho,para’ isomers). Furthermore, electron impact mass spectrometry offers no selectivity; that is, the sample matrix (for example, fish fat) ionizes just as well as the DDT-related analytes. For this reason, Dougherty et al. ( 4 , 5 )proposed the use of electron capture, negative ion (ECNI) mass spectrometry for the analysis of these compounds. With this technique, trace quantities of electrophilic compounds (such as DDT) can be detected in non-electron-capturing matrices. Dougherty et al. ( 5 )found that the ECNI spectra of DDT and related compounds were dominated by chlorine adduct ions ([M Cll-) and that para,para’ and ortho,para’ isomers could not be distinguished from one another. Because these older data were obtained with sample sizes of 10-100 wg, we suspected that data obtained from more realistic sample sizes (0.1-10ng) would be quite different (6). We were right. This
+
Although the use of l,l-bis(4-chlorophenyl)-2,2,2-trichloroethane (DDT) (5,see Figure 1)has been banned in the United States since 1970,it continues to be a problem. Its degradation products are abundant in fish ( I ) , and atmos-
0003-2700/68/0360-0787$01.50/00 1988 American Chemical Society
788
15, 1988
ANALYTICAL CHEMISTRY, VOL. 60, NO. 8, APRIL 3ighenyl Ethenes
Table I. Relative Sensitivity Determined under E1 and ECNI Conditions ions monitored h/z)
(1) P , P !2)
3
compd
(4)
(3)
P
D:phenyl
20
Ethanes C*CII
CCC]
CCI,
cb!Qcb/m Di,DC, \
CI
(5)
P P’
C’
( 7 ) P.P’
(5) S P
(9)
CHC,
I Z
CH,O
CF3
OCHl
JyfDX
ClHl
HICl
(13)
(le) x
= Cl (13) X = F
(11)
Ethanols a n d Benzhydrols o*
Og ,OCIHI
cl+{+cl
,
+cl$.i on
x x
,OCH:CH,;)
C
cx1
(14) (?5)
..*;*.,
,a
O i
= c: = 7
OH
(16)
!17)
an
Others
f:R)
x
(:9)
x
~
=
c:
r n, ,n
jyQ, O
I
(20)
7 10 17
ECNI
E1
490 130
250 318
3 6
281
139 246 235 235 221 341
0.02
70
248 345 428
(8) 0 . P ’
CCl,
Dipteny:
1 5
ECNI/EI
$11 0 I’
ZC-2 (231
/ !
(21)
-
pva
d
k
v
(221
(24:
Flgure 1. Structures of the diphenylethane derivatives: p ,p’DDE (1). o ,p’-DDE (2),p ,p’-DDD olefin (3), l11dichloro-2,2’di-4-tolylethylene (4), p,p’-DDT (5), O,p’-DDT (8), p,p’-DDD (7), o,p’-DDD (E), 1,lbis(4-chlorophenyl)-l,2,2,2-tetrachloroethane (9), methoxychlor (lo), perthane (ll),2,2-bis(4-chlorophenyl)-l,l,l-trifluoroethane (12),2,2b1s(4-fluorophenyl)-l11,l-trlfluoroethane (13), dicofol (14), 4,4-dichlorcl.cu-(trlfluorome~yl)benzhydrol (15),chlorobenrilate (lS),bromopropylate (17), 4,4‘dichlorobenzyhydrol (1E), 4,4’difluorobenzhydrol (19), and 4,4’dichlorobenzophenone (20), biphenyl (21), 1,l-diphenylethene (22), benzophenone (23), dlphenylfulvalene (24). paper reports on the ECNI mass spectra of the compounds shown in Figure 1, most of which are degradation products of DDT or are structurally related to DDT. These compounds have been divided into three groups: (a) diphenylethene derivatives [including compounds such as p,p‘- and o,p’-DDE ( 1 , 2 ) ] , (b) diphenylethane derivatives [including compounds such as p,p’- and o,p’-DDT (5, 6) and DDD (7, S)], and (c) diphenylethanol derivatives [including compounds such as dicofol and chlorbenzilate (14, 16)]. EXPERIMENTAL SECTION These studies were carried out on a Hewlett-Packard 5985B gas chromatographic mass spectrometer system. Samples were introduced through a 30 m X 0.25 mm, DB-5, fused silica column (J&W Scientific,Rancho Cordova, CA) with helium as the carrier gas. Methane reagent gas (99.99%, Air Products, Allentown, PA) was introduced through a modified transfer line (7) after passing through a trap of activated charcoal and molecular sieve. The ion source pressure, typically held at 0.5 Torr, was monitored by a Baratron capacitance manometer (MKS Instruments, Burlington, MA). The ion source temperature was held at 100 OC, and it was monitored by a thermocouple located in the ion source body. Electron energy was typically 230 eV; electron emission current was kept at 300 pA. The mass axis was calibrated with the m/z 633 and 452 ions from perfluorotributylamine and with the m / r 193 ion from pentafluorobenzonitrile (PCR Research Chemicals, Inc., Gainesville, FL). A 100 pg standard of decafluorotriphenylphosphine (PCR) was used to monitor instrument performance.
The samples of 1-3,5-7,10,11,14,16,17, and 20 were obtained from the EPA Health Effects Research Laboratory, Office of Research and Development, Research Triangle Park, NC. The samples of 4, 9, 12, 13, and 15 were obtained from Alfred Bader Chemicals, Milwaukee, WI. The sample of 8 was obtained from Ultra Scientific, Hope, RI. All other chemicals (18, 19, 21-24) were obtained from Aldrich, Milwaukee, WI. All compounds were dissolved in isooctane (Burdick and Jackson Laboratories, Muskegon, MI) or toluene (MCB, Cincinnati, OH) at concentrations of 10-20 ng/pL, and 1-pL injections were used. Relative sensitivity determinations were made by using selected ion monitoring, where the base peak in the E1 or methane ECNI mass spectrum of each compound was used for quantitation. Low mass ions, such as C1- were not chosen. A 10 ng/pL standard was used for the E1 and the ECNI determinations of methoxychlor, and a 0.1 ng/pL standard was used for the other ECNI determinations. The spectra reported here were affected by sample concentration. When a few nanograms was injected, the spectra showed M- and fragment ions attributable to electron capture reactions. When larger amounts of sample were introduced, an [M + C1]ion was observed. The relative intensity of this ion increased with sample concentration. For example, the [M + Cl]-/M- ratio for o,p’-DDD (8)increased from 0.1 when 10 ng was injected to 0.5 when 300 ng was injected. This effect was previously observed for a group of hexachlorocyclopentadiene derivatives (6). The [M + C1]- ion comes from an intermolecular chlorine transfer between M- and a neutral sample molecule and is enhanced when concentrated samples are used. This reaction accounts for the discrepancy between the spectra reported here and the spectra of DDT and 11 related compounds that were reported by Dougherty et al. (5). RESULTS AND DISCUSSION The compounds shown in Figure 1have been divided into three groups. The methane ECNI mass spectra of these groups give characteristic fragmentation patterns. Features of these spectra will be discussed below in addition to unique ions observed in the spectra of the ortho,para’ versus the para,para’ isomers. Diphenylethene Derivatives. The diphenylethenes (1-4) gave simple spectra. With the ion source at 100 OC, the molecular ion was the most intense peak, and fragment ions were due to [M - C1]- and C1- (see Figure 2). The diphenylethene derivative substituted with methylphenyl groups (4) gave a spectrum dominated by Cl- (see Figure 2C). This demonstrates the importance of phenyl chlorines in providing molecular ion stabilization. The spectra of p,p’-DDE (1) and o,p’-DDE (2) showed major differences which will be discussed below. The chlorinated diphenylethenes respond well under methane ECNI conditions. ECNI sensitivity was estimated by comparison of electron impact (EI) and ECNI responses. Table I shows the ECNI to E1 response ratio for a number of representative compounds. 4,4’-Dichlorobenzophenone (20), a compound which gave M- and little additional fragmentation, has been included for comparison. Table I indicates that compounds such as p,p’-DDE (1) and 4,4’-dichlorobenzophenone (20) can be quantified with good sensitivity using
ANALYTICAL CHEMISTRY, VOL. 60, NO. 8, APRIL 15, 1988
789
M'
A
_.
CI
CI
gyn
CI
248 CHCI2
I
I
I
I
1
(M-Cl2)i
I/
/QfcncH3 (4)
( M - C1)241
f 1
I ' I ' I ' , ' I ' I ' I ' 1 ' 1 ' 1 ' / ' I ' I ' I ' , ' / ' 1 ' 1 ' 1 ' / ' , ~ , ' I ' 1 ' / ~ , ~ , ' , ' , ' I ~ , ~
I n u r n . . - - - . o o o 0 -0 w 0 0"
0 *- 0
.
"
g
~
g
~
~
~
M/z
M/z
Flgure 2. Methane ECNI mass spectra of (A) p,p'-DDE (i), (6) p,p'DDD defln (a), (C) l.ldichloro-2,2di4toIylethane(4): ion source temperature, 100 OC.
Flgure 3. Methane ECNI mass spectra of (A) p,p'-DDT (5), (B) p,p'DDD (7), and (C) l,l-b,ls(4chlorophenyl~l,2,2,2-tetrachlotoethane
ECNI maw spectrometry. This response enhancement results from the carbon-carbon and carbon-oxygen double bonds which provide conjugation between the two aromatic rings and, thus, enhance molecular ion stability. Selective sensitivity enhancement is also observed in other diphenyl systems. For example, when a solution, containing equal amounts of compounds 21-24, is analyzed under ECNI conditions, only benzophenone (23) and diphenylfulvalene (24) respond, giving M- ions. Diphenylethane Derivatives. The diphenylethane derivatives, such as p,p'-DDT (5) and p,p'-DDD (6), fragment in a way that reflects their aliphatic character. In contrast to the diphenylethenes, these compounds give weak or no molecular ions, even with the ion source at 100 "C. Losses of C1, C12,HC1, and HC12are seen, in addition to Cl-, Cl,, and HC12- ions (see Figure 3). The initial loss of HC1 from p,p'-DDT (5) and from p,p'DDD (6) or C12 from l,l-bis(4-chlorophenyl)-1,2,2,2-tetrachloroethane (9) gives the corresponding diphenylethene anion. For example, Scheme I shows that the loss of HC1 from p,p'-DDT (5) and the loss of C12 from l,l-bis(4-chlorophenyl)-l,2,2,2-tetrachloroethane(9) give an ion that corresponds to the molecular ion of p,p'-DDE (1) at m/z 316. As noted above, p,p'-DDE gives an [M - C1]- ion at m/z 281. This ion is also seen in the spectra of p,p'-DDT (5) and l,l-bis(4chlorophenyl)-1,2,2,2-tetrachloroethane(9) (see parts A and C of Figure 3). The abundance of the mlz 281 ion increases and that of m / z 316 decreases as a function of ion source pressure for p,p'-DDE (l), p,p'-DDT (5), and l,l-bis(4chlorophenyl)-1,2,2,2-tetrachloroethane (9). This behavior is shown in Figure 4 where the mlz 281 to mlz 316 ratio for p,p'-DDE and p,p'-DDT is plotted vs ion source pressure.
Scheme I
(9): ion source temperature, 100 OC.
CI
CI, I ,CI C
CI
H
p.p'-DDT +e
(5)
-HC1
-2c1
m/z
316
- c1
m/z
281
Because these ions behave in a similar fashion, as a function of ion source pressure, a diphenylethene intermediate is likely in the fragmentation of diphenylethanes. An intense ion at mlz 248 is seen in the spectrum of p,p'-DDD (7) resulting from loss of C12 (see Figure 3B). This ion is also seen in the spectrum of p,p'-DDT (5) (see Figure 3A) where it results from the loss of three chlorines and the addition of a hydrogen. The mlz 248 ion from 7 may result
790
1988
ANALYTICAL CHEMISTRY, VOL. 60, NO. 8, APRIL 15, I
(M-Cl2)-
A 0 - 1
/'
< o s -
E
/
c'
CI
,'
;
246
CI
CI
MI
& (2 l) mCl
p-DDE/'
3161
o,p'-DDE
l
(M-,C1) 281
E -
I,,, , , , , , , , , , , , , ,
," 0 2
I
I
I
1
,
,
, ,, , , ,
,
,
1
,
,
,I,.
, , . , , I ! ' . , , , , , I! , , ,_, ,
/'
,
I!, ,
i
I
, . , ,_ I
(M-H-Cl3)
B 01
02
i
05
0 4
2 4 6 /'
O C
I o n S o u r c e P r e s s u r e (torr)
Figure 4. Plot of the [mlz 281]/[mlz 3161 ratio vs ion source pressure for p ,p'-DDE (1) and p ,p'-DDT (5): ion source temperature, 100 "C.
+e
- CI2 I
1
1.2-phenyl shift
M/Z
I
CI
Flgure 5. Methane ECNI mass spectra of (A) o,p'-DDE (2), (B) o,p'-DDT (6), and (C) o,p'-DDD (8): ion source temperature, 100 "C.
I1
Scheme I11 m/z
240
m/z
248
from loss of Clz to give the intermediate structure shown in Scheme 11. This ion may undergo a 1,2-phenyl or hydrogen shift to give either a dichlorostilbene or a bis(ch1oropheny1)ethene anion at m / z 248. The cause of this rearrangement would be formation of a conjugated system better able to stabilize the negative charge. 1,2-Phenyl shifts are known to occur in the rearrangement reactions of radical or anionic species (8), suggesting that formation of the stilbene structure may occur. Dichlorostilbenes are also reduction products from p,p'-DDT (9). The corresponding reaction could also occur to give the m / z 248 ion in the spectrum of p,p'-DDT (5) if a hydrogen-chlorine exchange occurred prior to ionization. Such exchange reactions have been observed in the spectra of other chlorinated compounds (6). In contrast to the behavior of p,p'-DDT (5) and p,p'-DDD (7),methoxychlor (10) and perthane (11) respond poorly under ECNI conditions. The spectra are dominated by C1-, and in the molecular ion region, weak [M - C1]- ions are observed. The ECNI/EI responses of 5, 7, and 10 are shown in Table I. Both p,p'-DDT and p,p'-DDD show a lower relative response than p,~'-DDE.This is due, in part, to the increased degree of fragmentation shown by the diphenylethanes. Methoxychlor (lo),with electron-donating substituents on the aromatic ring, gives the poorest response. The fluoro-substituted diphenylethanes (12 and 13) exhibit less fragmentation when compared with their chlorinated analogues. This reflects the increased strength of the carbon-fluorine bond. A molecular ion is observed for 2,2-bis-
&[b ffn. cI
-HCI
ClXC/CI
;1
CI
(yC1
cn c,
\
m/z
o p -DDT
-Clp
m/z
316
246
(6)
cI
H\
,CI
&.[& &YlCI
-HCI
c,
0
p
-DDD
-HC'
;1
\
m,z
282
(8)
(4-chlorophenyl)-l,l,l-trifluoroethane (12) with no other significant fragmentation when the ion source is held at 100 "C. At 250 "C, M- is still intense, and ions are observed at C1-, [M - Cll-, [M - FJ, and [M - H]-. l,l-Bis-(4-fluorophenyl)-2,2,2-trifluoroethane(13) gives an [M - HI-ion with the ion source a t 100 and 250 "C. Para,Para' and Ortho,Para' Isomers. The ortho,para' and para,para' isomers of DDE (1, 2), DDT (5, 6), and DDD (7,8) give easily distinguished spectra. While the para,para' isomers give the fragment ions described above, the ortho,para' isomers all show ions at m / z 246 (see Figure 5). This ion is very intense and is observed with the ion source at both 100 and 250 "C. The m / z 246 ion probably results from interaction of the chlorine on the ortho position with the sub-
ANALYTICAL CHEMISTRY, VOL. 60, NO. 8, APRIL 15, 1988
791
Scheme IV O-f-H
I
ci-f'
-H2
m/z m/z
(18) x=cI (19)
X=F
250
X=CI
218
X=f
CCI)
D i c o f ol
Scheme V -
O-!-H
HCCIS
B
Ctl)
Dicofol (14)
o'/'\ocH
1
250 , ( M - .H C O ~ C Z H ~ ) ~
OH
/
C l * { O C l
(CHI)
Bromopropylate
m/z
338
(17)
stituents on the 2 carbon of the ethene or ethane molecule. In the case of o,p'-DDE (Z),this interaction results in the loss of Clz (see Scheme 111). The diphenylethanes, o,p'-DDT (6) and o,p'-DDD (8), initially lose HC1, generating diphenylethenes, which lose Clz and HCI, respectively (see Scheme 111), to give mlz 246. Diphenylethanols and Benzhydrols. The spectra of four diphenylethanols (14-17) and two benzhydrols (18 and 19) were acquired. The spectra of the chloro- and fluoro-substituted benzyhydrols (18 and 19) show intense [M - HJ ions. This ion is the only peak observed in the spectrum of the fluoro-substituted benzhydrol (19). The chloro-substituted benzhydrol(18) also gives M-. This loss of H2 would generate a dihalobenzophenone anion (see Scheme IV). This provides conjugation between the two aromatic rings and enhances the negative ion stability. The diphenylethanols show similar fragmentation. Compounds substituted with two chlorines on the phenyl rings (14-16) give an ion at m / z 250, and bromopropylate (17) gives an ion at mlz 338 (see Figure 6). These ions correspond to the dihalobenzophenone anion and are formed by the losses shown in Scheme V. Another abundant fragment ion is seen in the spectra of the bis(chloropheny1)ethanols at mlz 262. One hypothesis for the formation of the mlz 262 ion in the spectrum of dicofol is shown in Scheme VI. Note that m / z 297 is present in the spectrum of dicofol (see Figure 6A). Unexpectedly, this ion is also seen in the spectra of several diphenylethenes and -ethanes, particularly when the ion source is at 250 "C. This ion and an ion at m / z 250 appear in the spectrum of p,p'-DDT obtained with hydroxide as a reactive ion (10);this suggests that the m / z 262 ion may be generated by reactions with trace amounts of hydroxide originating from water or air in the reagent gas. In the case of dicofol(l4) and its fluoro-substituted analogue (E), fragment ions appear at [M - OH - C1]- and [M - OH - F]-, respectively. These losses may result in formation of the corresponding diphenylethenes.
CONCLUSIONS The ECNI mass spectra of chlorinated diphenylethene, -ethane, and -ethanol derivatives reflect the degree of unsaturation or the substituents present on the diphenylethane base structure. The conjugated diphenylethenes respond well under ECNI conditions and give simple spectra consisting of
M/Z
Flgure 6. Methane ECNI mass spectra of (A) dicofol (14), (B) chlorbenzilate (IS), and (C) bromopropylate (17): ion source temperature, 100 OC.
Scheme VI CCI)
Dicofol
CI
-HC12
I 1,Z-phenyl
1
1.2-0
m/z
I
shift
297
1
-c1
/O-
'C
/I
CI
m/z
262
DCD CI
m/z
262
M- and [M - C1]- ions. The diphenylethanes give weak molecular ions and [M - HClI-, [M - C1J and [M - HClJ ions. These complex spectra reflect the aliphatic nature of the diphenylethane structure. Spectra of chlorinated diphenylethenes and -ethanes, with different aromatic ring substituents, show that substitution of an electron-donating substituent on the aromatic ring results in spectra dominated by C1-.
Anal. Chem. 1988, 60,792-797
792
A characteristic ion at m l z 246 differentiates ortho,para’ and para,para’ isomers. The diphenylethanols give characteristic ions at mlz 250 and 338 for 4-chlorophenyl- and 4-bromophenyl-substituted compounds, respectively. Registry NO,1,72-55-9; 2,3424-82-6; 3, 1022-22-6;4,5432-01-9; 5 , 50-29-3; 6, 789-02-6; 7, 72-54-8; 8, 53-19-0; 9, 3563-45-9; 10, 72-43-5; 11,72-56-0; 12, 361-07-9; 13, 789-03-7; 14, 115-32-2; 15, 630-71-7; 16,510-15-6; 17, 18181-80-1; 18, 90-97-1; 19, 365-24-2; 20,90-98-2; 21,9262-4; 22, 530-48-3; 23,119-61-9; 24, 2175-90-8; methane, 74-82-8.
(3) Safe, S.;Hutzinger, 0. Mass Spectrometry of Pesticides and Pollutants; CRC Press: Cleveland, OH, 1973; pp 113-121. (4) Dougherty, R. C. Anal. Chem. 1981, 5 3 , 625A-636A. (5) Dougherty. R. C.; Roberts, J. D.; Biros, F. J. Anal. Chem. 1975, 47, 54-59. (6) Stemmier, E. A.; Hites, R. A. Anal. Chem. 1985, 5 7 , 684-692. (7) Jensen, T. E.; Kaminsky, R.; McVeety, B. D.; Wozniak, T. J.; Hites, R. A. Anal. Chem. 1982, 5 4 , 2388-2390. (8) Lowry, T. H.; Richardson, K. S. Mechanism and Theory in Organic Chemistry; Harper and Row: New York, 1981. (9) Forrest, J.; Oliver, S.; Waters, W. A. J. Chem. SOC. 1946, 333-339. (10) Stray, H.; Mano, S.; Mikaisen, A.; Oehme, M. HRC CC, J . High Resoiut. Chromafogr. Chromatogr. Common. 1984, 7 , 74-82.
LITERATURE CITED
RECEIVED for review May 20, 1987. Accepted December 17,
(1) Jaffe, R.; Stemmier, E. A.; Eitzer. B. D.; Hites, R. A. J. Great Lakes Res. 1985, 1 7 , 156-166. (2) Rapaport, R. A.; Urban, N. R.; Capel, p. D.; Baker, J. E.; Looney, 8. B.; Elsenreich, S. J.; Gorham, E. Chemosphere 1985, 14. 1167-1173.
1987. This work was supported by the V.S. Department of Energy (Grant No. 80EV-10449) and the U.S. Environmental Protection Agency (Grant No. R808865).
Behavior of Liposomes in Flow Injection Systems Laurie Locascio-Brown,* Anne L. Plant, and Richard A. Durst Center for Analytical Chemistry, National Bureau of Standards, Gaithersburg, Maryland 20899
We have examlned the hydrodynamic behavlor and stablllty of llposomes In flow InJectIonanalysls, In order to evaluate thelr usefulness as analytlcal reagents In contlnuous flow systems. UpoaKHnes can be prepared wlth large numbers of water-soluble detectable or reactlve molecules, Le., fluorescent, electroactlve, or enrymatlc molecules, trapped In an aqueous center, and thus have the potential for providing tremendous M a l enhancement In many assay formats. We have found that liposomes contalnlng the fluorescent selfquenching dye, carboxyfluoresceln, are stable In the flow system and do not release thek trapped contents even at flow rates of 2 mL mln-‘. These structures are approxlmately 0.1 pm In diameter and showed flow profile behavior which Is quite dlfferent from solution (small molecule) behavlor in straight open tubing. Dmerences in concentration profiles for solution and liposome samples have been examined under condltlons of Induced radlai mlxlng In a knitted delay tube and a packed bead column. No aspects of ilposome behavlor have been observed that are not explalned by their small diffusJon coefflclent. Under condltlons of approprlate assay formats, Hpomnes wlll be hnpottant slgnai enhancement tools for flow Inlectlon analysls.
Flow injection analysis (FIA) has many advantages in analytical chemistry ( I ) . Besides providing on-line, automated reaction systems, FIA is a versatile technique that is being exploited for many diverse applications including biomedical applications such as soluble enzyme activity measurements (2), macromolecular affinity constant determinations (3),and immunoreactions (4). A review of current applications of FIA in clinical chemistry is provided by Linares et al. (5). One practical difficulty in clinical diagnostics employing FIA systems has been associated with the nonideal hydrodynamic behavior of red blood cells (6). The present work addresses the behavior of macromolecular assemblies and heterogeneous samples in FIA and hopefully will enhance the usefulness of FIA in applications involving biological samples. We are studying the use of phospholipid vesicles, or liposomes, in FIA. Liposomes are spherical structures that form
spontaneously when phospholipid molecules are dispersed in an aqueous solution. The bilayer membranes of liposomes are similar to cellular membranes and surround an entrapped aqueous volume. Liposomes are similar to red blood cells in this way, but they are at least an order of magnitude smaller. Liposomes can be prepared with as many as 1 X lo5watersoluble molecules trapped inside of their enclosed volume. These structures have great potential for application in drug delivery (7) and are also attractive for use in diagnostic and other assay systems where they provide signal enhancement in the form of the many molecules that can be released from them for detection. Other possible uses of liposomes in FIA systems include measurement of activity of membrane-associated proteins or quantification of phospholipid esterase activity. Memon and Worsfold (8)have used microemulsions of micelles (lipid structures that do not contain an aqueous center) for signal enhancement in fluorescence FIA. In addition, micelles have been used to quantify Tb(II1) in FIA (9), and bilayer vesicles have been used in a FIA method for cyanide analysis (IO). Our objective is to use liposomes containing a marker compound as analytical tools for signal enhancement in an immunochemical-based FIA system. The interaction between an antibody and antigen is converted to an optically detectable signal through an encapsulated marker which is released from the liposome cavity by lysis with detergent. A single binding event may, therefore, be amplified by a factor of lo5. Our goal is to study the feasibility of performing a competitive assay, although both competitive and noncompetitive assay formats can be used. Successful application of liposomes in competitive reactions requires that liposomes and solution species have identical and reproducible flow behavior so that liposomes and soluble analytes are uniformly mixed within the reacting volume. The criteria for development of a noncompetitive reaction scheme are less rigorous; however, the competitive reaction will not involve a prereactor incubation step thus resulting in a faster sample throughput time. In this work, we examine and compare the behavior of liposomes in various FIA system configurations so that appropriate liposome assay formats and system designs can be developed. We have examined the mixing behavior of liposomes and solution in the absence of chemical interactions on an unmodified,
This article not subject to U.S. Copyright. Published 1988 by the American Chemical Society
