m*
CH3-N
s Dz
R2
0
m/e= MR2t 97
m*
, CH3-N@
R,
m/e=M~,+97
free acids. These derivatives were not extensively examined, however, as they are rarely found in the body because of their rapid metabolism. For the barbiturates examined, blood levels of 0.2 mg or higher were sufficient to give a complete identifiable mass spectrum. Lower levels could be identified by extracting larger volumes of blood. The postulated fragmentation patterns of the methylated barbituric acid derivatives are presented in Figure 3, and the major fragments for a given compound are listed in Table I according to the scheme presented in Figure 3. The fragmentation patterns are very similar to those previously obtained for unmethylated barbituric acid derivatives (8-10). They reflect an even higher stability of the ring system than shown by the unmethylated barbiturates. Only when R1or RP is aromatic does significant cleavage of the ring system occur.
n
CONCLUSION
m/e = M ~ , t 1 5 5
m / e 169
m / e 112
Figure 3. Fragmentation patterns of methylated barbituric acid derivatives gram for barbiturate standards. Figure 2 shows a chromatogram from a seconal overdose. N-Methyl barbiturates could not be distinguished from their unmethylated homologs by this procedure. This was not considered a serious deficiency, since they are rapidly metabolized to their N-demethylated derivatives in the body and would be found as such. Preliminary studies with the thiobarbituric acid derivatives indicated that their methylated derivatives are more stable in gas chromatography than the
The above study shows that the GC-MS can be used to provide rapid identification of barbiturates in overdose cases. However, the methodology presented here requires a skilled operator to interpret the spectra obtained. Computerization of the mass spectrometer output and the use of computer search programs would alleviate the need for skilled interpretation. Work in this direction is in progress. RECEIVED for review May 22, 1972. Accepted October 16, 1972. (8) A. Costopanagiotis and H. Budzikiewicz, Moiiatsh. Cliem., 96, 1800 (1965). (9) H. Grutzmacher and W. Arnold, Tetrukedro/7 Lett., 13, 1365 (1966). (10) R. T. Coutts and R. A. Locock, J . Plzarm. Sci., 57,2096 (1968).
Effect of Oxygen on Response of the Electron-Ca pture Detector Francis W. Karasek and David M. Kane Department of Chemistry, Unicersity of Waterloo, Waterloo, Ontario, Canada SINCEITS CONCEPTION by Lovelock ( I ) , the electron-capture (EC) detector for gas chromatography has occupied an important position in analytical use because of its unique sensitivity and selectivity, particularly for compounds of biomedical and environmental significance. The importance of the detector has led to many studies of its characteristics, both to improve performance ( 2 ) and to understand its basic mechanism (3). These early studies and their conclusions were necessarily made from indirect experimental data. The recent development of the new technique of plasma Chromatography for (1) J. E. Lovelock and S. R. Lipsky, J . Amer. Chem. Soc., 82, 431 ( 1960). (2) P. G. Simmonds, D. C. Fenimore, B. C . Pettitt, J. E. Lovelock, and A. Zlatkis, ANAL,CHEM., 39, 1428 (1967). (3) W. E. Wentworth and E. Chen, J . Gas Chromatogr., 5 , 170 (1 967).
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ANALYTICAL CHEMISTRY, VOL. 45, NO. 3, MARCH 1973
the first time permits one to obtain direct experimental data to observe the charged species being formed in the EC detector and their response and mobility under changing parameters. The plasma chromatograph, operating at atmospheric pressure, involves generation of charged particles in a carrier gas by a radioactive 63Ni source and an ion-molecule reactor whose products are subsequently separated in a coupled iondrift spectrometer. Both positive and negative species can be observed. The instrumentation is shown in Figure 1. Ions formed by the 63Ni source in the flowing carrier gas are moved by a n electrical field through the ion-molecule reactor section toward the drift spectrometer. A pulse of these charged species is injected into the drift spectrometer, where separation of ionic species occurs because of their different mobilities as they move through an inert gas at 760 Torr to reach the detector in a series of ion peaks recorded as a plasmagram. A variable delay gating technique on the scan grid permits recording of
GAS EXIT
Figure 1. Schematic diagram of the plasma chromatograph
P C TUBE HOUSING HEATED CARRIER GAS 10-500 mllMin
I
HEATED DRIFT GAS 100 3000
-
ml/Min
I1I
POSITIVE IONS ~H20l"U+
Figure 2. Plasmagrams of the charged species present in the plasma chromatograph with N Pcarrier. The change in electron current and formation of oxygen ions is seen upon injection of an air sample
ELECTRONS
e'
h I
c
I
0
the millisecond plasmagram scan in 1- t o 10-minute time spans. Details of the instrumentation has been described previously (4-6). The ability to create conditions found in the EC detector and observe the charged particles present and their behavior under differing conditions provides a unique opportunity to study many fundamental aspects of this detector. The presence of small amounts of air in the carrier gas used for the EC detector gas chromatograph has a serious deleterious effect on sensitivity and linearity of response in either the dc or pulsed mode of operation (7, 8). Data from the plasma chromatograph indicate that the reason for this effect is that electrons react with the oxygen in the air t o form ion-molecule complexes of the form (H~0),02-. This removes a significant portion of the electrons and prevents them from performing their function of reacting with the trace constituents in the gas chromatographic peaks. These constituents must then undergo the competing reactions of electron-capture and ionmolecule reaction with the (HzO),O,- species. (4) F. W. Karasek, Res./Decelop., 21 (3), 34 (1970). (5) F. W. Karasek, ANAL.CHEM., 43,1982 (1971). (6) F. W. Karasek and 0. S. Tatone, ibid., 44,1758 (1972). (7) J. E. Lovelock, ibid., 35,474 (1963). (8) G. G. Guilbault and C. Herrin, Anal. Chim. Acta, 36, 252 (1966).
I
'L
DRIFT TIME - GILLISECONDS
Table I. Experimental Conditions of Plasma Chromatograph for Data Obtained 125 "C, 190 "C Instrument temperature: 115 cm3/minute Reactant gas flow: 400 cm3/minute Drift gas flow: 6.0 cm Ion-molecule reaction space: 6.0 cm Ion-drift space: 250 V/cm Electric field: 0.2 millisecond Injection pulse: 0.2 millisecond Gating pulse: 2 minutes Recorded scan: Nitrogen-Linde, high purity Gases : grade (99.996 %) Air-Linde, very dry, purified grade
EXPERIMENTAL
The procedures and BETA-VI plasma chromatog:aph used in this work have been described previously (6). The experimental parameters used t o obtain plasmagrams are shown in Table I. Gases used in the plasma chromatograph were passed through a metal trap of 2.25-liter capacity packed with Linde Molecular Sieve 13X. This removes impurities and gives a very low water concentration, estimated t o be around 10 ppm. The air sample was syringe-injected directly from the cylinder and the chlorobenzene was Baker reagent grade. ANALYTICAL CHEMISTRY, VOL. 45, NO. 3, MARCH 1973
577
H
Figure 3. Composite plasmagrams obtained at 125 "C with a Nz carrier and an air carrier gas. While the negative species differ, the positive ions are the same in each case
E
n
-
I
lb
DRIFT TIM€-MILLISLCONDS
1as'c
NEGATIVE RfACTANT IONS
lnzOl,O~-
Figure 4. Plasmagrams obtained with an air carrier before and after injection of 10-9-gram sample of chlorobenzene
EUCTRON 18ACKGROUND
0
10 DRIFT TIME -MILLISECONDS
RESULTS AND DISCUSSION
The plasmagram of Figure 2 shows the charged species present when nitrogen is used as the carrier gas to simulate operating conditions in a n EC detector. The negative particles are thermal electrons. These electrons appear as a continuum across the plasmagram scan because the grids d o not close perfectly for such small, high-speed particles. If the grids could exclude the electrons, the plasmagram would show only a single peak with a drift time of 0.01 millisecond, the magnitude of this peak being about 15 times greater than that of the continuum. This event is too fast for the response of the recording system. The initial electron peak shown in Figure 2 appears because of the gating technique employed; during that initial interval the injection and scan gates are open simultaneously and the full electron current reaches the detector. The positive particles are predominantly of the form (HPO),H+. When a 10-milliliter sample of cylinder air (Linde, very dry, purified grade) is injected into the carrier gas of the plasma chromatograph, the charged species present become those shown in Figure 2 . One can note the initial disappearance of the electrons and the appearance of the (H20),02- ions, where n varies from 0 to 3 and is a function of water concentration and temperature. The identity of 578
ANALYTICAL CHEMISTRY, VOL. 45, NO. 3, MARCH 1973
these species formed with air has been established previously by use of a mass spectrometer directly coupled to the plasma chromatograph (9). The drift time scan was recorded over a 2-minute scan time, so that the rise of the curve toward the original electron level represents depletion of the air sample in the plasma chromatograph by the moving nitrogen carrier gas. By changing to a n air carrier gas, the effect of greater amounts of oxygen can be observed. This results in the appearance of the charged species shown in Figure 3. A greater portion of the electrons, about 90%, are converted to the (Hz0),02- particles. No significant change is observed in the positive reactant ions. We would expect to see the initial electron peak, but it is not observed in the 20-millisecond plasmagrams ; because of the relative gate widths, the peak is too narrow for the recording system to follow. The high sensitivity of detection of trace constituents in a sample by the plasma chromatograph depends upon reaction between reactant ions and the trace compounds. Depending upon the amount of air present in the carrier of a n EC detec(9) M. J. Cohen and F. W. Karasek, J . Chromatogr. Sci., 8, 330 (1970).
DRIFT IIMIS NEGAIIVE
POSlllVE
4.18 4.60 5.16 5.51
4.69 5.32 5.96 10.01 Ihokeround)
NEGATIVE IONS
Figure 5. Composite plasmagrams obtained with a NBcarrier and an air carrier at 190 "C. This temperature reduces electron conversion to negative ions
1I
3 6 1 . I
AIR CARRIER
MlFl llME -MILLISECONDS
NEGATIVE REACTANT IONS
Figure 6. Composite plasmagrams of negative ions obtained with an air carrier gas at different temperatures under higher resolution conditions of 0.1-millisecond gate widths
11
-
~
=i s
DRIFl llME
tor, there will be a mixture of electrons and the oxygen ions. The trace constituent in a GC peak will undergo reaction with both the electrons and the (HzO),Oz- species depending upon their relative numbers and reaction rates. Since the EC detector produces a GC peak by measuring a decrease in the standing electron current, this dual reactivity leads to anomalous sensitivity and linearity effects when oxygen is present, This is clearly illustrated in Figure 4, where an air carrier gas is used. Along with a low level of electron current, one can observe essentially the same pattern of negative reactant ions as appears in Figure 3. Upon injection of a trace sample of gram of chlorobenzene, formation of the C1- is seen from reactivity with both the electrons and the main peak of the negative reactant ions. The peaks indicated as product ions are most probably due to an ion-molecule reaction of the chlorobenzene with reactant ions. Temperature has an effect on the relative distribution of the (H,O),Oz- species and also on the relative amounts of the electrons and the total of these ions. This is shown by comparison of the data in Figure 5 with those in Figure 3. Plasmagrams for Figure 5 were taken under the same instrumental conditions as Figure 3 except that a temperature of 190 "C was used instead of 125 "C. At the higher temperature, only
- MILLISECONDS
about 20 % of the electrons are converted to the (H20),02ions. These negative ions also show a different relative composition. The identity of equivalent ions at the two temperatures can be established by correspondence of their calculated reduced mobility. Changing to higher resolution conditions by reducing gate widths to 0.1 millisecond gives the data shown in Figure 6 and Table 11, revealing essentially the same reduced mobility for equivalent ions. The presence of both reactive ions and electrons would lead to a different sensitivity and linearity in an EC detector at these two temperatures. The dependence of sensitivity on temperature that has been observed for the EC detector (3) could result, in part, from a similar situation where traces of electron-capturing compounds are present, whether from air, column bleed, or detector contamination. These data apply most directly to the dc mode of operating the EC detector using a nitrogen carrier gas. The nitrogen carrier used was a highly purified grade and was brought to a uniform water concentration, estimated to be 10 ppm or less, by passing through a large molecular sieve dryer. This still provides sufficient water to produce the positive and negative water clustered species observed. The addition of water to the carrier gas does not change the electron level appreciably ANALYTICAL CHEMISTRY, VOL. 45, NO. 3, MARCH 1973
579
Table 11. Drift Times and Calculated Reduced Mobilities (KO) for Negative Ions at Two Temperatures Drift time, Temperature, "C milliseconds cm2/Vsec 125 4.90 3.33 5.43 3.01 5.87 2.78 6.18 2.64 190 4.18 3.39 4.66 3.02 5.16 2.75 5.51 2.57
QK
-_.-.-.I 273
where 7 = drift time, I = length of cell 7 E T760 (6 cm), E = field gradient, T = absolute temperature, and p = pressure. 0
-
and only affects the relative concentration of ionic species slightly by shifting the equilibrium to favor the higher water clusters. When air is present in the carrier gas, and the negative species are a mixture of electrons and water-clustered negative ions (Figure 5 ) , the addition of water does result in a decrease of electrons with the formation of larger amounts of the negative ionic species. These observations tend to suggest that removal of water from the carrier gas of an EC detector is more important when air and other electron-capturing contaminants are present. While a more complete and definitive work is needed t o determine the exact effect these observations would have on the EC detector in its pulsed mode of operation with a n argon/ methane mixture, some data have been obtained. The addition of a 10-ml sample of argon/lOY, methane to the nitrogen carrier gas results in some formation of methane ion-molecules
of lower mobilities by reaction with the water-clustered protons which remain the dominant species. In the negative mode, the presence of the argon/methane mixture gives about the same results as does water; in the presence of air, the relative electron concentration decreases with formation of greater amounts of ion-molecules ; when only electrons are present as in pure nitrogen, the argon/methane has little effect o n the electron level. These effects are all a function of temperature. Work is currently under way to more completely define the pulsed mode of operation. CONCLUSIONS
These data indicate that the presence of small amounts of oxygen or similar electron-capture compounds in a carrier gas for an E C detector will considerably reduce sensitivity and linearity by formation of reactive negative ion-molecule complexes from the electrons. This effect is greater a t lower temperatures and is somewhat related to water concentration. These, and other similar studies, provide a means of understanding many aspects of the EC detector mechanism and characteristics. Recent work (6) has produced evidence for both the direct and intermediate ion types of dissociative capture with halogen compounds. Other work is in progress with the plasma chromatograph to identify the reactions occurring with electron capture and to elucidate the kinetics of these processes. RECEIVED for review August 31, 1972. Accepted November 3, 1972. The research for this paper was supported by the Defence Research Board of Canada, Grant Number 9530116, and the National Research Council of Canada, Grant Number A5433. Financial support to F. W. Karasek was provided by the National Science Foundation, Research Grant No. GP 31824.
Identification of Noncannabinoid Phenols in Marihuana Smoke Condensate Using Chemical Ionization Mass Spectrometry A. F. Fentiman, Jr., R. L. Foltz, and G . W. Kinzer Batteiie, Columbus Laboratories, Columbus, Ohio 43201
INCREASING USE OF MARIHUANA has generated interest in the constituents of the plant. The cannabinoids have been studied extensively because certain of these phenolic compounds are psychotomimetically active ( I ) . Since the principal way by which marihuana constituents are taken into the human body is by smoking, the composition of the smoke is of particular interest. The cannabinoids are efficiently volatilized and absorbed in the lungs during the smoking process (2), but little is known of the composition of the smoke, particularly in regard to other potentially physiologi-
cally active compounds. One class of compounds of concern is the noncannabinoid phenols. Among the various noncannabinoid phenols that have been identified in the marihuana plant are eugenol and guaiacol ( 3 ) as well as ferulic acid and p-hydroxycinnamic acid (melilotic acid) (4). However, the noncannabinoid phenolic constituents of marihuana smoke have not been investigated. We report here the identification of various noncannabinoid phenols in marihuana smoke condensate using gas chromatography/mass spectrometry (GC/MS) and the chemical ionization (CI) mass spectra thus generated.
(1) J. L. Neumever and R. A. Shagourv. .I. Pliarm. Sci.. 60, 1433 (1971). (2) E. B. Truitt, Jr., Plinrm. Ret., 23,273 (1971).
(3) Y . Obata and Y . Ishikawa, BUN. Agr. Cliem. SOC.Jup., 24, 670 (1960). (4) E. C . Bate-Smith, J . Lim. SOC.Lo/idon (Bota/zy),58, 95 (1962).
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