ИСТИНА

Smoking is one of the most serious problems nowadays and generally causes various diseases and premature death. Cigarette smoking turned into a direct or indirect cause of death of 3 million people in the world every year. In 2016, an estimated 31% of Russian adults were current cigarette smokers. Of these, 20% smoked everyday per 1 pack or more. Russia has one of the highest adult smoking rates in the European Region. After announcement of anti-smoking laws in Russia and restriction of smoking in public places, it becomes particularly important to determine the number of smokers in populated areas in real time. Cotinine is an alkaloid found in tobacco and is also the predominant metabolite of nicotine. Cotinine has an in vivo half-life of approximately 15-20 hours, and is typically detectable in urine for several days (up to one week) after the use of tobacco. Cotinine is widely used as a biomarker for exposure to tobacco smoke. Cotinine levels <5 ng/mL in urine are considered to be consistent with no active smoking. Values of 5-20 ng/mL are associated with passive smoking, 20-50 ng/mL – light smoking, and levels above 1000 ng/mL are seen in active smokers. This work presents selective determination of cotinine in urine and wastewater using HPLC coupled with spectrometric and mass-spectrometric detection. An analyzing sample was spiked with D3-cotinine as an internal standard. The mixture was subsequently vortex-mixed and centrifuged. For solid phase extraction the prepared cartridges were conditioned prior loading methanol and hydrochloric acid. An aliquot of previously prepared sample was loaded on the column. The columns were washed with methanol, acetone, acetonitrile and water in order to remove matrix compounds. Elution was performed with methanol, ammonia and water. The eluate was conditioned to pH 5 and injected to chromatograph. Various chromatographic columns were tested during this work. Column Synergy Hydro-RP (250 x 4.6 mm, 4μm) was chosen as the most effective column for determination of cotinine. Formic acid and acetonitrile were chosen as a mobile phase with the injection volume of 10μL. Appropriate validation was performed for the spectrometric and mass-spectrometric detection. The limits of detection for cotinine were found to be 1 μg/mL for spectrometric and 0.2 ng/mL for mass-spectrometric detection. As analyzing objects, urine of active smokers (more than 10 cigarettes a day) and wastewater after first filtering from different villages in Russia are selected. Cotinine free-urine from non-smokers and deionized water were used as a blank matrix to perform validation parameters.