Combined analysis of r-1,t-2,3,c-4-tetrahydroxy-1,2,3,4-tetrahydrophenanthrene and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol in smokers' plasma

Abstract

Polycyclic aromatic hydrocarbons (PAH) and tobacco-specific nitrosamines, such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), are widely accepted to be two important types of lung carcinogens in cigarette smoke. In this study, we have developed a method to estimate individual uptake of these compounds by quantifying r-1,t-2,3,c-4-tetrahydroxy-1,2,3,4-tetrahydrophenanthrene (PheT) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in 1 mL of smokers' plasma. PheT and NNAL are biomarkers of PAH and NNK uptake, respectively. [D10]PheT and [pyridine-D4]NNAL were added to plasma as internal standards. The plasma was treated with beta-glucuronidase to release any conjugated PheT and NNAL. The analytes were enriched by solid-phase extraction on a mixed mode cation exchange cartridge and the PheT fraction was further purified by high-performance liquid chromatography. The appropriate fractions were analyzed by gas chromatography-negative ion chemical ionization-mass spectrometry for PheT and liquid chromatography-electrospray ionization-mass spectrometry for NNAL. The method was sensitive (limits of quantitation: PheT, 13 fmol/mL; NNAL, 3 fmol/mL), accurate, and precise. Levels of PheT and NNAL in plasma from 16 smokers averaged 95 +/- 71 and 36 +/- 21 fmol/mL, respectively, which are approximately 1% to 2% of the amounts found in urine. This method should be useful in molecular epidemiology studies of carcinogen uptake and lung cancer in smokers.