Smokeless

Journal of Chromatography, 619 (1993) 49--61 Biomedical Applications
Elsevier Science Publishers B.V., Amsterdam CHROMBIO. 7001

Gas chromatographic-mass spectrometric method for determination of anabasine, anatabine and other tobacco alkaloids in urine of smokers and smokeless tobacco users
P e y t o n J a c o b III*, Lisa Yu, G a n g Liang, A l e x a n d e r T. Shulgin and Neal L. BenowitzDivision of Clinical Pharmacology and Departments of Medicine and Psychiatry, University of California, San Francisco, San Francisco General Hospital Medical Center, Building 100, Room 235, 1001 Potrero Avenue, San Francisco, CA 94110 (USA)
(First received February 3rd, 1993; revised manuscript received May 19th, 1993)

ABSTRACT A selected ion monitoring method for determination of thetobacco alkaloids anabasine, anatabine, nornicotine, metanicotine, dihydrometanicotine, and 2,Y-bipyridyl in urine of smokers and smokeless tobacco users is described. The method involves conversion of the secondary amine alkaloids to tertiary amine derivatives by reductive alkylation using an aldehyde and sodium borohydride, and chromatography on a 5% phenylmethylsilicone capillary column. Thesederivatives have good chromatographic properties, allowing determination of concentrations as low as 1 ng/ml. The alkaloid 2,3'-bipyridyl is unaffected by the derivatization procedure and may be determined simultaneously with the other alkaloids. The structural analogues 2-(3-pyridyl)hexahydroazepine, 5-methyldihydrometanicotine, and 6-methyl-2,Y-bipyridyl were synthesized for use as internalstandards. Using the method, concentrations and 24 h excretion of anabasine, anatabine, and nornicotine in urine of twenty-two smokers, eight chewing tobacco users, and six oral snuff users were determined and compared with concentrations and excretion of nicotine and its metabolite cotinine. Excretion of nicotine and cotinine was similar in all tobacco users, but excretion of anabasine, anatabine, andnornicotine was substantially greater in urine of smokeless tobacco users, presumably due to absence of pyrolysis of these alkaloids in smokeless tobacco products.

INTRODUCTION

Nicotiana glauca, anabasine is the principal alka-

Tobacco and tobacco smoke contain a number of structurally related alkaloids (Fig. 1). In commercial tobaccos, the major alkaloid is nicotine, accounting forabout 95% of the total alkaloid content. Nornicotine and anatabine are the two most abundant minor alkaloids, present in roughly equal amounts, each accounting for about 2-3% [1]. Anabasine is generally present in concentrations of about 0.3% of the total alkaloids. Anabasine is present in numerous Nicotiana as well as other families [1]. In some, notably

* Corresponding author.0378-4347/93/$06.00 ©

loid. Tobacco and tobacco smoke contain smaller amounts of 2,Y-bipyridyl, metanicotine (Nmethyl-4-(3-pyridinyl)-3-buten-1-amine), dihydrometanicotine (3-[4-(methylamino)-1-butenyl]pyridine), and trace amounts of numerous other nitrogen-containing substances [2]. During the past several years, considerable information on the amount of nicotine absorbed by tobacco users has been obtained[3-5]. In contrast, nothing is known about the amounts of the other nicotine-related alkaloids absorbed during tobacco use. Nornicotine concentrations in urine of cigarette smokers has been reported [6,7] but, since it is also a nicotine metabolite, nornicotine in urine results both from direct absorption from

1993 Elsevier Science Publishers B.V. All rights reserved

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P. Jacob et al. /J. Chromatogr. 619 (1993) 4 9 4 1

LJ

0.3

~
COTININE

O
3

NICOTINE

NORNICOTINE

ANABASINE

ANATABINE

2,3'-BIPYRIDYL

METANICOTINE

DIHYDROMETANICOTINE

MYOSMINE

Fig. 1. Structures of tobacco alkaloids.

CO2C'•SiiH50• (CH3)3
0.3
L N~,~II
CH3

1. Nail 2. H H20, A +, 3. NaOH

~NaBH4

=
2-(3-PYRIDYL)HEXAHYDROAZEPINE

H2, Pd(C)
P

LN~

0.3...