A validated method for urinary cotinine quantification used to classify active and environmental tobacco smoke exposure

The aims of this research were to develop and validate a simple, sensitive method to determine un-conjugated urinary cotinine (COT-U) levels and to investigate its ability to discriminate active and environmental tobacco smoke (ETS) exposure. For this assay, urine was diluted with water, cotinine-d3 was added as an internal standard, and the sample was separated by a C18 column and analysed by triple quadrupole mass spectrometry. The quantification limit was 0.1 μg/L, range of linearity was 0.1-4000 μg/L, intra- and inter-run precision were <10%, and accuracy was within 13% of the theoretical value. Investigation of the matrix effect showed that the internal standard controlled sources of bias. The assay was applied to 168 adults who classified themselves as non-smokers with (9.5%) or without (67.9%) ETS exposure, active smokers (20.2%), and those who did not report smoking information (2.4%). Median COT-U levels were 1.3, 0.6, 687, and 57 μg/L, respectively. Based on a critical evaluation of self-classification and COT-U levels, we proposed a 30- μg/L cut-off value to identify active smoking. The ability of COT-U levels to distinguish ETS exposure was evaluated among non-smokers, but a wide overlap between groups with and without ETS exposure prevented the identification of a reliable cut-off value. A 2-μg/L COT-U cut-off value correctly identified 95.4% of self-classified non-ETS exposure and 33.3% of self-classified ETS exposure. This method reliably measured a wide range of COT-U levels. The 30-μg/L cutoff value appropriately classified active tobacco smoke exposure, but the classification of ETS exposure needs further research.