Blood samples were collected into a 10 mL EDTA tube and then centrifuged at 3,000 rpm for 8 min at 25°C to obtain plasma. Saliva samples (2–3 mL) were collected without any stimulation into a 50 mL tube by expectoration after gargling with water. Both plasma and saliva samples were stored at −80°C and thawed at room temperature before use. The plasma samples were diluted 1:100 with phosphate buffer saline before measurement because they had about 100-fold higher DTG concentration ranges than saliva samples and diluted samples permitted to minimize contamination and loss of sensitivity. Plasma and saliva samples (36 μL) were spiked with 4 μL of DTG-d5 (23.8 ng/mL) that correspond to 95.2 pg and each mixed 1:1 with acetonitrile to precipitate the proteins [9] and centrifuged at 12,000 rpm for 3 min at 25 °C to remove precipitated material. Then, 40 μL of supernatant were dried in a vacuum centrifuge and dissolved in 20 μL of 5 mM formic acid-25% (v/v) ethanol.

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