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A straightforward, high-throughput method was developed and fully validated for the simultaneous determination of the specific tobacco biomarkers nicotine and its main metabolite cotinine in a wide dynamic range and supporting the most common human biological matrices (urine, oral fluid and hair). Sample preparation was performed inside the very HPLC injection vials by pipetting 0.5 mL of the liquid samples, deuterated internal standards in alkaline solution and dichloromethane as extraction solvent. Solid samples (i.e. around 10 mg hair) were first submitted to alkaline digestion in the HPLC vials and processed accordingly. The organic phase (reached through the upper aqueous layer) was directly injected without further treatment. Instrumental analysis was performed using hydrophilic interaction (HILIC) ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Total chromatographic time was 2 min. The method covers a wide dynamic range making it fit-for-purpose for the analysis of samples covering entire populations, irrespective of the level of exposure or tobacco use. Calibration curves (r (2) > 0.995) covered the range 1-2000 ng/mL (or 0.05-100 ng/mg hair) for nicotine and 0.1-2000 ng/mL (or 0.005-100 ng/mg hair) for cotinine. Within-run and between-run precision and accuracy were typically below 10 %, and always below 20 % at the lower limit of quantification. The method was successfully applied to the analysis of samples from different projects involving multiple matrices.