Gas chromatographic—mass spectrometric method for determination of anabasine, anatabine and other tobacco alkaloids in urine of smokers and smokeless tobacco users

Abstract

A selected ion monitoring method for determination of the tobacco alkaloids anabasine, anatabine, nornicotine, metanicotine, dihydrometanicotine, and 2,3′-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. These derivatives 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-2,3′-bipyridyl were synthesized for use as internal standards. 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, and nornicotine was substantially greater in urine of smokeless tobacco users, presumably due to absence of pyrolysis of these alkaloids in smokeless tobacco products.