Analysis of Nicotine, 3-Hydroxycotinine, Cotinine, and Caffeine in Urine of Passive Smokers by HPLC-Tandem Mass Spectrometry

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Abstract
Background: A method is described for the simultaneous analysis of nicotine and two of its major metabolites, cotinine and 3-hydroxycotinine, as well as for caffeine from urine samples. The method was developed to assess exposure of restaurant and hotel workers to environmental tobacco smoke. Methods: The method includes sample pretreatment and reversed-phase HPLC separation with tandem mass spectrometric identification and quantification using electrospray ionization on a quadrupole ion trap mass analyzer. Sample pretreatment followed standard protocols, including addition of base before liquid-liquid partitioning against dichloromethane on a solid matrix, evaporation of the organic solvent using gaseous nitrogen, and transferring to HPLC vials using HPLC buffer. HPLC separation was run on-line with the electrospray ionization-tandem mass spectrometric detection. Results: The detection limits of the procedure were in the 1 μg/L range, except for nicotine (10 μg/L of urine). Still lower detection limits can be achieved with larger sample volumes. Recoveries of the sample treatment varied from 99% (cotinine) to 78% (3-hydroxycotinine). Conclusions: The method described is straightforward and not labor-intensive and, therefore, permits a high throughput of samples with excellent prospects for automation. The applicability of the method was demonstrated in a small-scale study on restaurant employees.

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