Blood was collected and immediately mixed with four volumes of methanol (with 1 μM nicotine D3 as an internal standard) to quench the enzyme. The samples were centrifuged at 10,000 rpm for 30 min, and the supernatant was transferred to clean tubes and evaporated in a rotatory evaporator Genevac. The residual was redissolved in 5% NH4OH in water and extracted by an Oasis HLB 96-well μElution Plate (Waters). The elution was evaporated in a rotatory evaporator Genevac and redissolved in Hepes buffer and 2% trifluoroacetic acid for liquid chromatography–mass spectrometry (LC-MS).

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