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Analysis of biomarkers in wastewater is increasingly seen as an important tool for valuation of health, nutrition and use of various substances by humans. Some biomarkers are used for population estimates, since the actual values of population size in cities can differ significantly from official counts, which inevitably leads to errors in assessing the impact of various factors to people calculated per capita. Cotinine and 5-hydroxyindole-3-acetic acid (5-HIAA) are the main metabolites of nicotine and serotonin, respectively, which are excreted by urine and can be used as biomarkers for population estimates. The determination of these metabolites is more preferred than the detection of nicotine and serotonin themselves in terms of correctness of the results. There are numerous methods for the determination of cotinine and 5-HIAA in urine by HPLC-MS/MS. However, there are only few works in the literature devoted to the determination of these compounds in wastewater with detection limits up to 1 μg/L. Considering such low and varied concentrations of cotinine and 5-HIAA in wastewater, it is vital to develop a reliable and highly sensitive procedure for the determination of these compounds. A technique of liquid-liquid extraction and subsequent quantitative determination of cotinine and 5-HIAA in wastewater by high-performance liquid chromatography in combination with tandem mass spectrometric detection was developed. Poroshell Hilic column was used as a stationary phase which has an alternative selectivity compared to traditional C18 columns. Developed procedure is characterized by low detection limits (0.1 μg/L for cotinine, 0.2 μg/L for 5-HIAA) and good selectivity. This technique provides reliable assessment of the concentration of cotinine and 5-HIAA in wastewater of Moscow region.