Cotinine and trans 3'-hydroxycotinine in dried blood spots as biomarkers of tobacco exposure and nicotine metabolism

Abstract

Tobacco use is the major preventable cause of premature death in the United States. Second-hand smoke (SHS) exposure also contributes to a number of premature deaths as well as other negative health outcomes. An accurate assessment of tobacco smoke exposure is critical to understanding these disease processes. The plasma concentration of cotinine, the primary metabolite of nicotine, is widely accepted as a quantitative measure of tobacco and SHS exposure. However, it is not always feasible to collect plasma. Dried blood spots (DBS), which are collected routinely from newborns and often from young children for lead screening, provide an alternative sampling method. We have developed a quantitative high throughput liquid chromatography tandem mass spectrometry method for the analysis of cotinine in DBS. The limit of quantitation was 0.3 ng/g (~ 0.2 ng/ml plasma). Cotinine levels in DBS from 83 smokers and 99 non-smokers exposed to SHS were determined. Plasma cotinine concentrations in these subjects ranged from <0.02 to 443 ng/ml. Cotinine was detected in DBS from 157 subjects, and the correlation between cotinine in plasma and DBS was excellent, 0.992 (P<0.001). We also determined the ratio of trans 3'-hydroxycotinine to cotinine, a measure of nicotine metabolism, in DBS from smokers. This ratio in DBS was well correlated with the ratio in plasma, 0.94 (P<0.001). In a small study, we confirmed the feasibility of using extant DBS collected for lead screening to assess SHS exposure in children.

Figure 1

LC/MS/MS analysis of DBS from (A) a smoker, (B) a SHS exposed individuals, and (C) a non-smoker (plasma cotinine <0.02ng/ml). The SRM data from time 0 to the vertical dotted line is for d₀ and d₃-3'-HCOT and past the line is for d₀ and d₃- cotinine. The retention times varied based on the age/condition of the column, cotinine and 3'-HCOT elute 0.03min prior to the respective deuterated standard. In panel C the peak at 7.16, overlaps with, but does not co-elute with cotinine.

Figure 2

Correlation between DBS cotinine and plasma cotinine (♦ smokers, ◊ self reported non-smokers exposed to SHS). The insert is for plasma cotinine values <10ng/ml.

Figure 3

Correlation between replicate analyses of punches from two DBS from the same subjects

Figure 4

Correlation between DBS 3'-HCOT/Cotinine ratio and plasma 3'-HCOT/Cotinine ratio in subjects with cotinine values ≥ 10ng/ml.