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Background: Dried blood spot (DBS) have been applied in many application as alternate analytical solution for limitations and challenges in conventional methods, and validated methods demand by DBS technique are still required in nicotine (Nic) research area.  Aim: The development and validation of a new bioanalytical method for simultaneous determination of Nic and cotinine (Cot) in human blood using DBS and LC-Orbitrap MS technique.  Methods: The DBS was punched at 6.35 mm diameter and extracted by 10% w/v of trichloroacetic acid solution, containing Nic-d3 as an internal standard (IS). The extracted samples were then injected into a Kinetex-C18 column and eluted by mobile phase of methanol:water:formic acid (10:90:0.001, v/v/v). Chromatographic effect for DBS was investigated by decentralized disk punching (peripheral area). The developed method was validated according to European and American guidelines for bioanalytical method validation.  Results: Nic, Cot and IS were detected accurately by Orbitrap-MS using heated-ESI source at positive ions m/z of 163.1235, 177.1028 and 166.1423, respectively. The established calibration ranges for Nic and Cot were linear between 5-250 ng/mL and 10-500 ng/mL, respectively. Within- and between-run measurements accuracy for Nic and Cot were all higher than 80 % for LLOQ and 85 % for QC levels, and the measurements precision for Nic and Cot were all within 15 %.  Conclusion: The developed method for Nic and Cot determination in human blood was successfully validated using DBS LC-Orbitrap MS technique. Nic and Cot investigation was not affected by DBS’s chromatographic influence.

Determination of Nicotine and Cotinine in Human Blood by Dried Blood Spot-LC-Orbitrap MS Technique