Beitri.ge zur Tabakforschung International ·Volume 12 ·No. 5 ·November 1984
Isolation and Identification of N' -Acylalkaloids of Cigarette Smoke *
by M. E. Snook, 0. T. Ch.,.tyk ood R.F. A,.,W,
Tobacco Safety Research Unit, Agricultural Research Seroice,
United States Department of Agricult.Nre, Athens, Georgia, U.S.A.
SUMMARY
Methods have been developed for the analyses of the minor high-boiling bases of smoke. Fused silica glass capillary columns coated with Superox-4 were prepared and used successfully for the GC analyses of the basic fraction of smoke, containing compounds covering a wide range of volatility and polarity (nicotine to norbarman). The Superox-4 capillary column also produced an excellent separation of the numerous Nacylalkaloids, which have been reported in tobacco and tobacco smoke. Several new N -acylalkaloids are reported. The described methodology should be a great asset in future isolation and identification studies of these and other, as yet to be characterized, minor bases of smoke and leaf.
ZUSAMMENFASSUNG
Zur Untersuchung der in germgen Mengen vorkommenden hochsiedenden Basen des Zigarettenrauches wurden Verfahren entwickelt. Mit Superox-4 beschichtete Quarzglaskapillarsiulen wurden erfolgreich zur gaschromatographischen Analyse der basischen Raucbfraktion eingesetzt, die Verbindungen einer weiten Spannbreite an Fhichtigkeit und Polaritit enthilt
* Prexntcd in pan a' me I 84th NaUoml Meeting of me American Chemical Sociny (Symposium on dlemistry of to~co and toba<:co smoke), Kansat City, Mo., 1982. Recc:ind: t9d. May 198}- aoo:pud: 22nd Mardll9U.
(Nicotin his Norharman). Auch konnte mit den Glaskapillarsiulen (Superox-4) die grolle Zahl der in Tabak. und Tabak.rauch bekannten N -Acylalkaloide sehr gut getrennt werden. Es wurden auch mehrere neue N-Acylalkaloide identifiziert. Die beschriebene Methodik kOnnte in der Zukunft bei der Isolierung und Identifizierung dieser und anderer noch zu bestimmender Basen in Blattabak und Rauch von grollem Nutzen sein.
RESUME
Des procedes ont CtC mis au point pour !'analyse des bases a point d'Cbullicion eleve qui sont presentes en faible quantitC dans la fumCe de cigarette. L'utilisation de colonnes capillaires de verre quartzeux revetues de Superox-4 permit d'analyser avec sucd:~s par chromatographie en phase gazeuse la fraction basique de la fumCe, laquelle contient des composes dont la volatilitC et la polaritC sont ues diverses (de la nicotine au norharman). Les colonnes capillaires (Superox-4) permirent Cgalement de sCparer dans d'excellentes conditions les nombreux N -acylalcaloides dont on connait !'existence dans le tabac et la fumee, ainsi que d'identifier de nouveaux composes de ce type. La mCthode employee pourrait s'averer trCs utile, a l'avenir, pour la separation et !'identification de bases dont la presence dans le tabac en feuilles et la fumee est connue ou non.
227
Table 1. Acylalkalolds of tobacco and tobacco smoke.
References R Molecular weight
leaf I smoke
Acylnornlcotines:
Formyl H- 176 (9, 23) (2)
Acetyl CH3- 190 (9) (2) .
Butanoyl CH3(CH2)2- 218 (22) (3).
Hexanoyl CH3(CH2)4- 246 (8) (3)
Heptanoyl CH3(CH2)5- 260
Branched heptanoyl (Ce) 260
Octanoyl CH3(CH2)8- 274 (8) (3)
Branched octanoyl (C7) 274
Octenoyl (C7:1) 272
6- + 7-Hydroxyoctanoyl (C70H)- 290 (21)
Carbomethoxy CH30- 206 (10)
Carboethoxy CH3CH20- 220 (22)
4-Dimethylaminobutanoyl (CH3)2N(CH2h- 261 (22)
Other acylalkaloids:
N' -Formylanatablne 188 (24) (2)
N' -Formylanabasine 190 (24, 25)
N' -Acetylanatablne 202 (2)
N' -Valerylanabasine 246 (3)
• identified In this work.
228
Figure 1. Scheme 1: Fractlonatlon of cigarette smoke condensate for bases (ether extraction).
SCHEME I
Cigarette smoke condensate (1 kg, 100%)
partition: j1 N aqueous NaOH - dlethyl ether
Dlethyl ether
1. 1 N HCI extraction 2. adjust extract to pH 12
(with cooling) 3. diethyt ether extraction of free bases
Free bases (56.3 g, 5.63%)
silicic I chromatography acid (SA) (1000 g SA)
I NaOH
F-A 81 benzene/ether (1:1)
F-B 61 benzene/ether (1:3)
F-C 61ether
F-0 6lacetone
F-E 31methanol
(6.49 g, 0.65%) (contalning 30% aza-arenes)
INTRODUCTION
(5.8 g, 0.56%) (11.4g, 1.1%)
Nicotine and related alkaloids are by far the major components in the basic fraction of both tobacco and tobacco smoke. In addition, cigarette smoke also contains large quantities of simple pyridines and pyrazines and their alkyl and vinyl derivatives (1). Among the minor bases, found in appreciable quantities in smoke, are N -acyl derivatives of nornicotine, anabasine and anatabine (2, 3) (Table 1) and the carboline bases, barman (1-methyl-9H-pyrido[3,4-b]indole) and norharman (9H-pyrido[3,4-b]indole) first reported by Poindexter and Carpenter (4, 5). For analyses of leaf and smoke compounds, we have developed fused silica capillary columns coated with the .. thermally stable polar phase Superox-4 (6). Use of these columns has, for the first time, allowed the efficient separation of not only the important bases barman and norharman, but also the resolution of several N -acylnornicotines and acylanatabines from the total free bases fraction of smoke. We will also detail our newly developed isolation procedures for the above compounds, which have resulted in the identification of several new N -acylnornicotines in cigarette smoke condensate (CSC).
* Reference to a company or product name does not imply approval or recommendation by the United States Department of Agriculture.
(16.9 g, 1.7%) (10.1 g, 1.0%)
gel chromatography
Blo:-Beads S-X12 (benzene) or Sephadex LH-20 (CHCI3)
Gel fractions
11.GC 2.GC-MS
Identifications
EXPERIMENTAL*
Preparation and Fractionation of Cigarette Smoke Condensate
All solvents were Burdick and Jackson "distilled-inglass" solvents. Cigarette smoke condensate was prepared under standard smoking conditions from commercially available cigarettes, as previously described (7).
Scheme I (see Figure 1)
Fractionation of 1 kg of cigarette smoke condensate to obtain the basic subfractions by this procedure has been described before (7). Fraction F-D contained the compounds of interest.
Scheme II (see Figure 2)
Twenty grams of cigarette smoke condensate were dissolved in 600 ml of 30 % methanol I chloroform (MeOH/CHCl3) and extracted with 150 ml of 2 N HCl followed by 4 X 100 ml 2 N HCl. Each aqueous HCl extract was cross extracted with 50 ml CHC13 and the CHC13 extracts were added to the original MeOH/
229
·Figure2. Scheme 11: Fractlonatlon of cigarette smoke condensate for bases (CHCI3 extraction).
SCHEME !I
Cigarette smoke condensate (20 g, 100 %)
1. dissolve In 30% MeOH I CHCI, 2. extract wHh 2 N HCI
AqueousHCI
Aqueous
1. adjusttopH 12 2. saturate with NaCI 3. extract wHh CHCh
CHCI,
Tevaporate
Total free bases (3.05g,15.25%)
silicic acid
CHCI,
chromatography
F-A 1 I benzene/ether
(0.63g, 3.15%)
F-B 0,51 ether
(0.21 g, 1.05%)
CHC13 solution. The combined aqueous HCl extracts were adjusted (with ice-cooling) to pH 12, with 15 N
aqueous NaOH, saturated with NaCl and extracted with CHC13 (3 X 100 ml). Evaporation gave 3.05 g (15.25 %) of a free bases fraction. The bases were redissolved in diethyl ether (ether or E) and deposited onto 20 g of silicic acid (SA), the ether was removed on a rotary evaporator and the SA-sample mixture was placed on top of a column of SA (100 g), packed in petroleum ether, and eluted with the following solvents: F-A, 11 benzene (B) I E (1 : 1); F-B, 0.51 E; F-C, 0.5 1 E; F-D, 1 1 acetone; F-E, 1 l MeOH. The fractions were evaporated on a rotary evaporator and the yields in grams and percentage composition of cigarette smoke condensate were: F-A, 0.63 g, 3.15 %; F-B+C, 0.21 g, 1.05 %; F-D, 1.34 g, 6.70 %; F-E, 1.06 g, 5.30 %.
Adsorption Gel Chromatography
The gel chromatographic system ~onsisted of four columns connected in series and packed with Bio-Beads ~-X12 as described previously (12). Fraction F-D from Scheme I (acetone eluant) was dissolved in 50 ml of
230
F-C 0,51 ether
F-D 11acetone
F-E 11methanoi
(1.34 g, 6.70%) (1.06g, 5.30%)
gel chromatography Bio-Beads S-X12 (benzene)
Gel fractions
1
1.GC 2. GC-MS
Identifications
benzene and aliquots were placed on the first gel column with a 1.0 ml loop injection valve. Six 1 ml aliquots of F-D were individually chromatographed through the gel columns. Benzene was pumped (Altex 110 pump) at a flow rate of 120 ml/ h and 8 ml fractions were collected. Eluted gel fractions (GF) from all the runs with the same number were combined. Column eluant was monitored at 280 nm ~ith a Chromatronix Model230 detector. In a similar manner, fraction F-E (MeOH eluant) from Scheme 11 was dissolved in 2 ml of benzene and 1 ml of the solution was chromatographed on the gel system.
Gas Chromatography (GC)
The glass capillary GC columns used were fused silica columns (10 m or 22 m X 0.3 mm ifiside diameter) statically coated with Superox8 -4 (2 mg I ml in methylene chloride), after pre-treatment and deactivation with Superox-4 (6). In addition, an SE-54 capillary column (30 m X 0.3 mm inside diameter) was used for the separation of N -acylnornicotines. GC analyses were performed on a Hewlett-Packard 5830 gas chromatograph. (The columns were operated at 8 p.s.i. and 34 cm I s he-
lium linear velQcity; split ratio of about lOO: 1; injector, 260 oc; FID, 310 "C; oven program, 120-250 "Cat 4"/min.)
Identification of Compounds
Standards: A convenient source of nornicotine, anabasine and anatabine was the end fraction of the slow fractional distillation of commercial technical grade nicotine. Treatment of this £faction with acid halides produced the desired N -acyl derivatives. The formyl derivatives were prepared by treating the alkaloids with formic acid (8) or N -formylimidazole (9). The carbomethoxy derivatives were prepared by the method of Hechtet al. (10) by reaction of the alkaloids with methylchloroformate.
Mass Spectrometry (MS)
GC-MS analyses were performed on a Hewlett-Packard 5985B GC/MS system. Separations were made on a 45 m X 0.3 mm inside diameter fused silica capillary column coated with Superox-4 (20 p."s.i. hdium column pressure; linear velocity: 30 cm Is at 100 "C; temperature program:-40 "C for 2 min, then 40 "C to 120 "Cat 20 "/min followed by 120 •c to 250 •c at 4 "/min). Injection of the sample was made in the splitless mode and the GC effluent was led into the mass spectrometer through an open-split interface, constructed in our laboratory ( 11 ).
RESULTS AND DISCUSSION
GC Separarion of Smoke Alkaloids
The GC liquid phase, Superox-4, is a 4 X 106 molecular weight polyethylene glycol. It is similar in polarity to Carbowax 20M, but more thermally stable. Our recent preparation of fused silica glass capillary columns, coated with Suplirox-4 (6), has allowed successful GC analyses of many high-boiling polar compounds of cigarette smoke condensate. We have applied this capillary GC technology to the development of methods of analysis for high-boiling compounds of the basic fraction of cigarette smoke condensate. The Superox-4 columns were found to give excellent peak shapes for not only the important bases, barman and norharman, but also allowed for the efficient separation of a number of N -acylnornicotines, anabasines, and anatabines. The GC separation of a number of synthesized Nacylalkaloids on a Superox-4 column is shown in Figure 3. The chromatogram also includes some commonly found tobacco alkaloids: nicotine, nornicotine, myosmine, anabasine, nicotyrine, anatabine, 2,3'-dipyridyl, cotinine, and barman and norharman. As expected for the polar Superox-4 column, the N -acetylalkaloids eluted before the more polar formyl derivatives.
Isolation ofN -Acylalkaloids
We have developed procedures for the large scale (20-1000 g cigarette smoke condensate) fractionation of smoke bases. One of these procedures (Figure 1), based on our previously derived fractionation of cigarette smoke condensate for bioassays (7), involved partitioning of cigarette smoke condensate between aqueous NaOH and dietbyl ether to remove acidic materials. Extraction of the diethyl ether solution with aqueous HCl separated the bases, which were subsequently extracted and fractionated by silicic acid chromatography. In a second procedure (Scheme 11, Figure 2), cigarette smoke condensate was -dissolved in 30% CH,OH I CH Cl, and the bases were extracted into aqueous HCI. After adjustment of the acid extract to pH 12, the bases were extracted into CHCI3• Extraction of a smaller quantity of cigarette smoke condensate (Scheme 11) and without prior removal of an acid fraction allowed a more efficient extraction of basic material. Preliminary examination of the total free bases fraction from the two methods showed that ether extraction (Figure 1) failed to extract theN-acetyl- and formylalkaloids, but did extract the N -hexanoyl- and octanoylnornicotines. This was in agreement with Schumacher et al. (2) who found N-acetyl- and formylnornicotines and -anatabines in the water-soluble portion of smoke partitioned between water and ether. Also, Heckman and Best (3) found the N-butanoyl-, hexanoyl-, and octanoylnornicotines in an acid extract-of the ether soluble portion of smoke partitioned between water and ether. In comparison, the gas chromatogram of the total base fraction from Scheme 11 (Figure 4) showed the successful extraction of the N -acetyl- and formylalkaloids, as well as of the higher homologs. Both Schemes I and 11 employed silicic acid (SA) chromatography to separate and concentrate the Nacylalkaloids and norharman from the multitude of other bases in cigarette smoke condensate. The N -C,and C8-acylnornicotines as well as norharman eluted from the SA column with acetone. Gas chromatograms of the acetone eluant (fraction F-D) from Scheme I and Scheme 11 are given in Figures 5 and 6, respectively. The chromatograms also show the additional compounds identified in the fractions. N-Acetyl- and formylnornicotine required the mOre polar solvent, methanol, to be eluted from SA (Scheme Il). A gas chromatogram of the methanol fraction from Scheme 11 (Figure 7) also showed the presence of nicotine and cotinine in this fraction. Interestingly, N-acetyl- and formylanatabine·were concentrated mainly in the acetone fraction (Figure 6). Lipophilic adsorption gel chromatography proved to be an efficient method for obtaining relatively pure isolates of the norharmans and N -acylalkaloids suitable for identification of minor components. We have successfully utilized the Bio-Beads S-Xt2 gel to isolate and purify many different classes of compounds from tobacc~ and tobacco smoke, such as polynuclear aromatic hydrocarbons (12), aromatic ketones (13), aro-
231
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FigureS. Gas chromatogram of acetone fraction F-D from silicic acid chromatography of total bases
(Scheme I) on a Superox-4 column (1 0 m x 0.3 mm inside diameter, 130 ·c to 250 ·cat 4•/min).
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Flgure7. Gas chromatogram of methanol fraction F·E from silicic acid chromatography of total bases (Scheme 11) on a Superox-4 column (22 m x 0.3 mm inside diameter, 120 ·c to 250 ·c
at 4•/min).
i z
10
Methanol fraqtlon F- E (Scheme 11)
20
Time . (min)
! 8
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30
FigureS. UV trace (280 nm) for elution of fraction F-0 (Scheme I) for gel chromatography on Blo-Beads S-X12 (benzene).
Acetone fraction F- D (Scheme I)
Acyl.nomicotines
1- --1
1-Norharmans--t
~~~~~~~~~~~oo~~~~ro~~
Fraction number (8 ml fractions)
Flgure9. Gas chromatogram of selected gel fractions (Scheme 1, fraction F-0) containing harman and norharman on a Superox-4 column (10 m x 0.3 mm inside diameter, 130 ·c to 250 ·cat 4•/min).
Acetone fraction F- D (Scheme I) GF48-80
Harman
10 20
Time (min)
Norharman
30
matic nitriles (14}, phenolics (15, 16}, leaf lipids (17), and indoles and carbazoles (18). Aromatic compounds are retained by the gel to a greater extent than interfering aliphatic compounds and can be effectively concentrated and obtained in relatively pure form. The retention is based on a form of 1t- 1t electron interaction between solute and gel, as in the case of Bio-Beads (a styrene-divinylbenzene copolymer) in benzene. The adsorption effects are useful in separating different classes of compounds (i.e. aliphatic hydrocarbons, alcohols, aromatic compounds, phenols, etc.). However, the permeation properties of the gel operate within classes of aliphatic compounds and elutions are in order of decreasing molecular size (17). These properties of the Bio-Beads S-X12 gel were used successfully to isolate the norharmans and N-
Figure 10. Gas chromatogram of GF-39 from Blo-Beads S·X12 chromatography of fraction F-E (methanol, Scheme 11) on a SE-54 fused silica capillary column (30 m x 0.3 mm inside diameter, 100 ·c to 280 ·cat 4•/mln).
10
Methanol fraction F- E (Scheme 11) GF39
ll
20 30
Time (mln)
acylalkaloids from the more abundant nicotine and dipyridyl compounds. Fraction F-D (Figure 1) was chromatographed on .Bio-Beads S-X12 in benzene (Figure 8). Since the norharmans have greater 1t-electron densities than the other alkaloids, they were retained more by the gels. They eluted last from the gel systems in relatively pure form, as illustrated by the gas chromatogram of combined gel fractions 46-60 (Figure 9). The methyl derivative (harman) eluted before the parent (norharman) due to steric interruption of the 1t -1t interaction by the methyl group. Due to their greater molecular weight and overall molecular width, the N -C6- and C8-acylnornicotines eluted from Bio-Beads before nicotine. Their elution from the gels in gel fractions 34-38 indicated that these N -acylnornicotines have properties midway between aliphatic compounds (elution: GF 29-33) and aromatic nicotine and dipyridyl compounds (elution: GF 40+). This is due to the long alkyl chains in the molecules. In contrast, the short-chain N -Cc and C2-acylnornicotines were found to elute later (due to permeation effects) and eo-eluted with nicotine. This is shown in Figure 10, a chromatogram of GF-39 from the BioBeads separation of fraction F-D (methanol, Figure 2}, which contains both nicotine and the short-chain Nacylalkaloids. The chromatogram also shows that the N -formyl- and acetylnornicotines are cleanly separated on the SE-54 capillary column, in contrast to the Superox-4 column (Figure 7). * Gas chromatographic analyses of the individual gel
*However, for the unrefined basic fractions, the SE-54 columns caused tailing of separated peaks and were not used. '
237
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200
220
240
260
~~ ~'
= o
ao•o
18
9 1
00
, N
C
O! C
H2!
CH
2! C
H! C
H2
CH
3 I
Isol
ated
1: :
l ~~~
175
I •n
fele
o-C
r 80
I
CH
3
60
120
147
40
100
203
20
M ..
-1
5
M+
245
260
0 60
80
10
0 12
0 14
0 16
0 18
0 20
0 22
0 24
0 26
0
Figure 11. Gas chromatogram of GF-36 from Blo-Beads S-X12 chromatography of fraction F-D (acetone, Scheme I) on a Superox-4 column (10 m x 0.3 mm inside diameter, 130 ·c to 250 ·cat
4•tmin).
10 20
Time
Peaks c and d (C1 ,1 ) are described in the text.
fractions of the Bio-Beads separation of fraction F-D (acetone, Figure 1) (GF 34-38) showed that a relatively pure N -C6-C8-acylnornicotine isolate had been obtained. The gas chromatogram of GF-36 is shown in Figure 11. As expected, N -hexanoyl- and octanoylnornicotines were the major components present (the C8 isomer eluted from the gels slightly before the C6
isomer). In addition to the N -C6- and C8-acylnornicotines, several other isomers were characterized in this subfraction, including N -heptanoyl-, anteiso-heptanoyl-, iso-octanoyl- and four different isomers of octenoylnornicotines, which are being reported for the first time. MS data for these compounds are presented in Figures 12-14. Major MS fragmentation pathways, as reported by Bolt (8), are given in Figure 15. Comparative analyses of the .M+'-15, .M+'-29 and .M+'-43 ion intensities in the isolated N-n-C 7-, n-C8-, anteiso-C7-, and iso-C8-
acylnornicotines with the same ions in n-heptanoic acid and anteiso-heptanoic acid (19) and in previously identified n-, anteiso- and iso-hydrocarbons of tobacco (20) left no doubt as to the validity of the assigned structures. The detection of the N -octenoylnornicotines in
Q) c:
~ E 2 ~ uf
.~ '0 !! 't ~ fa 2:: .
, t
(mln)
cS ,-;-.
30
Acetone fraction F- D (Scheme I) GF36
40
smoke is very interesting, as Miyano et al. recently reported the presence of N-(6-hydroxyoctanoyl)- and N-(7-hydroxyoctanoyl)nornicotines (Table 1) in Japanese domestic tobacco (21 ). The N -octenoylnornicotines were found in the smoke of American commercial non-filter cigarettes. Although N -hydroxyoctanoylnornicotines have yet to be identified in American fluecured tobacco, they probably exist and are converted by the proposed pyrolytic dehydration pathway (Figure 16) to the octenoyl compounds. This scheme indicates three possible isomers would be formed; however, MSindicated four peaks in GF-36 (Figure 11) were Noctenoylnornicotines. Migration of the double bond, which is known to occur in pyrolysis reactions, could produce a fourth isomer ( N -( 4-octenoyl)nornicotine ). The presence of an ion of m/z 203 (Figure 15) in the spectra of all four peaks precludes migration of the double bond closer to the carbonyl than the y position, unless hydrogen transfer occurs. There is also the possibility of cis/trans isomers. Two peaks in GF-36 (Figure 11, marked c and d) were found to have greatly enhanced abundances of mlz 203 ions (Figure 14), relative to the usual base peak of mlz 189. It is postulat~d
239
N ~
0
100 80
60
40
20
0
10
0,
80-i
:J 20~
~n
r:~.§
'7~.
:-...N
C
O(C
H2)
eCH
3
60
80
N
I.
11 h1s
18
9 20
3
Fig
ure
13.
Mas
s sp
ect
ra o
f re
fere
nce
N -n
-oct
an
oyl
no
rnlc
otl
ne
an
d G
C-s
ep
ara
ted
(Is
olat
ed}
N -i
so
oct
an
oyl
no
rnlc
otl
ne
(*m
ass
num
bers
ind
icat
e en
hanc
ed i
on i
nten
sitie
s).
147
106
189
175
203
217
100
120
140
160
180
200
220
231*
25
9*
189
CO
CH
2 C
H2
CH
2 C
H2
CH
CH
3 17
5 I.
I I
CH
3 14
7
120
106
.. 11
I I
11
203
70
I 13
0 I
!t&
; I
7R
92
0 ~ t'
''·"'
'i
eel 11
'1"'
... , ..
0 I 'I
"" .•.
, '"
"''I
I , "'
I' ..
I"
'"
I ·"
'"I
'I".
I ·'
"' I
. 'I'
I 11
I
I I
60
80
100
120
140
160
180
200
220
231
A.f+
-43
I.
I
240
Rtm
lnln
ce
N'-n
-oc:
tano
ylno
rnlc
otln
e
245
Isol
ated
hl
o-C
.
260
A.f+
274
A.f+
A.f+
-15
A.f+
-29
"""'
274
245
I I
240
260
Figure 15. Principal mass fragmentation pathways for N' -acylnornlcotlnes according to Bolt (20).
m/z203
~D ~~.§ '~~
N 0=0 I
R A= ~H15 : M+= 274 A = ~H13 : M+ = 272 A = CeH13 : M+ = 260 A = C5H11 : M+ = 246
+
.. mlz 190
·+ N c;-o
CH3
~ ~ m/z1" ~Ng ~~,
mlz 175
mlz 147
Figure 16. Postulated pyrolytlc syntheses of N'-octenoylnornlcotlnes.
this is due to cleavage of a stable allylic radical ( ·CH2CH = CHCH2CH3) from the side chain of the molecular ion (Figure 15) competing favorably with the usual ~-cleavage and y-hydrogen transfer (McLafferty rearrangement (8)). We could find no evidence for the presence of N -carbomethoxy-, carboethoxy-, butanoyl-, nonanoyl- or decanoylnornicotines in the gel isolate of Scheme I. The butanoyl isomer was probably lost in the NaOH extraction of Scheme I as were the formyl and acetyl isomers. The C9 and C 10 isomers apparently are not found in smoke.
REFERENCES
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2. Schumacher, J. N., C. R. Green, F. W. Best and M. P. Newell: Smoke composition, An extensive investigation of the water-soluble portion of cigarette smoke; J. Agric. Food Chem. 25 (1977) 310-320.
3. Heckman, R. A., and F. W. Best: An .investigation of the lipophilic bases of cigarette smoke condensate; Tob. Sci. 25 (1981) 33-39.
(N' -(7-0ctenoyl) nomicotine)
(N' -(6-0ctenoyl) nomicotine)
(N' -(5-0ctenoyl) nomicotine)
4. Poindexter, E. H., and R. F. Carpenter: Isolation of harmane and norharmane from cigarette smoke; Chem. Ind. (London) 1962, 176.
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241
N ~
N
too,
oY
.
N
CO
(CH
2)2C
stis
8
0'
60
40
1
20
0 aO
N
Flg
ure
14
. M
ass
spe
ctra
of
GC
-se
pa
rate
d (
Iso
late
d)
N-o
cte
no
yln
orn
lco
tln
es.
Upp
er s
pect
rum
ref
ers
to
peak
s a
and
b of
Fig
ure
11. L
ower
spe
ctru
m (p
ostu
late
d N
'-(5-
octe
noyl
)nor
nico
tine
isom
ers)
refe
rs
to p
eaks
c a
nd d
.(*
mas
s nu
mbe
r in
dica
tes
enha
nced
ion
inte
nsity
.)
189
175
147
QR
19
6 12
0
100
120
140
160
180
200
220
100
lt75
I
189
203*
CO
lCH
2 C
H2i
CH
2CH
= C
HC
H2C
H3
147
80
10
6
60
120.
40-1
55
70
20
0 60
80
10
0 12
0
175
130
140
160
180
189
M-6
9
203
200
217 22
0
231
231
Isol
ated
N
'-oc
teno
ylno
mlc
otln
e
243
257
240
260
~
272
Isol
ated
N
'-(5
-oct
enoy
l)no
mlc
otln
e
243
240
257 26
0
M+
272
11. Arrendale, R. F., R. F. Severson, W. J. Chamber· lain, M. E. Snook and 0. T. Chortyk: Analysis of complex mixtures of tobacco and tobacco smoke by capillary GC/MS; Abstr. Pap. 34th Southeast Regional Meeting, American Chemical Society, Birmingham, Ala., in conjunction with the 6th An· nual University of Alabama Symposium on devel· opments in mass spectroscopy, November 1982.
12. Snook, M. E., R. F. Severson, H. C. Higman, R. F. Arrendale and 0. T. Chortyk: Polynuclear aro· matic hydrocarbons of tobacco smoke: Isolation and identification; Beitr. Tabak.forsch. 8 -(1976) 250-272.
13. Chamberlain, W. J., M. E. Snook, J. L Baker and 0. T. Chortyk: Gel permeation chromatography of oxygenated components of cigarette smoke con· densate; Anal. Chim. Acta 111 {1979) 235·241.
14. Chamberlain, W, J., M. E. Snook, 0. T. Chortyk and J. L Baker: Characterization of an aryl nitrile fraction of cigarette smoke condensate; Tob. Sd. 25 (1981) 15-17.
15. Snook, M. E., P. J. Fortson and 0. T. Chonyk: Application of gel chromatography to characterize more completely the phenols of cigarette smoke; Tob. Sci. 24 (1980) 30·36.
16. Snook, M. E., P. J. Fortson and 0. T. Chortyk: Isolation and identification of phenolic acids from tobacco leaf; Beitr. Tabak.forsch. lot. 11 (1981) 19· 26.
17. Snook, M. E., R. F. Severson, R. F. Arrendale, P. J. Fonson and 0. T. Chonyk: Behavior of hydro· lyzed tobacco leaf lipids during gel chromatogra· phy; Tob. Sci. 23 (1979) 38·42.
18. Snook, M. E., R. F. Arrendale, H. C. Higman and 0. T. Chortyk: Isolation of indoles and carbazoles from cigarette smoke condensate; Anal. Chem. 50 (1978) 80-82.
19. Stenhagen, E., S. Abrahamsson and F. W. McLaf· ferty (eds.): Registry of mass spectral data, Vol. 1, John Wiley and Sons, Inc., New York, 1974.
20. Chonyk, 0. T., R. F. Severson and H. C. Higman: Chromatographic determination of hydrocarbon waxes in tobacco leaf and smoke; Beitr. Tabak· forsch. 8 (1975) 204-210.
21. Miyano, M., N. Yasumatsu, H. Matsushita, and K. Nishida: 1'·(6·Hydroxyoctanoyl)nornicotine and 1'-(7·hydroxyoctanoyl)nornicotine, two new alka· loids from Japanese domestic tobacco; Agric. Bioi. Chem. 45 (1982) 1029·1032.
22. Matsushita, H., Y. Tsujino, D. Yoshida, A. Saito, T. Kisaki, K. Kato and M. Noguchi: New minor aJ. kaloids in flue·cured tobacco leaf; Agric. Bioi. Chem. 43 (1979) 193-194.
23. Piade, J. J., and D. Hoffmann: Chemical studies on tobacco smoke, LXVII. Quantitative determination of alkaloids in tobacco by liquid chromatography; J. Liquid Chromatogr. 3 (1980) 1505-1515.
24. Miyano, M., H. Matsushita, N. Yasumatsu and K. Nishida: New minor alkaloids itt Burley tobacco; Agric. Bioi. Chem. 43 (1979) 1607-1608.
25. Matsushima, S., T. Ohsumi and S. Sugawara: Composition of trace alkaloids in tobacco leaf lamina; Agric. Bioi. Chem. 47 (1983) 507·510.
Acknowledgements
The authors wish to thank Patricia F. Mason for her fine technical assistance.
Authors' address:
Tobacco Safety Research Unit, Agricultural Research SeNJice, U.S. Department of Agriculture, P. 0. Box 5677, Athens, Georgia, 30613, U.S.A.
243
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