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-

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at 4•/min).

i z

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Acyl.­nomicotines

1- --1

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~~~~~~~~~~~oo~~~~ro~~

Fraction number (8 ml fractions)

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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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I

I 11

I M

· 43

w

20

55

79

16

1 -

M+

-29

M

+-1

5

260

OJ Jl

oulh.l

ulmil!!

l ,.lll

llulle

!JIIIL

t 1 1!1

L h

.. I L

~~~~"

! 1

1!1

11,

ljll

• I'l

l I,

1"

I, ..1

~~~:,

1 ~1

2 ~5 I

i I

I I

I I

I I

I I

60

80

100

120

140

160

180

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)

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5. Poindexter, E. H., and R. F. Carpenter: The isola­tion of harmane and norharmane from tobacco and cigarette smoke; Phytochem. 1 (1962) 215-221.

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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.

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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.

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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