2.3. Measurement of cannabis consumption in plasma
To estimate cannabis consumption, THC and its metabolites, 11-hydroxy-THC (11-OHTHC) and 11-nor-9-carboxy-Δ-9-THC (THC-COOH) were quantified in plasma, as reported previously (Galindo et al., 2018). We followed the Waters Corporation protocol with some modifications (Zhang et al., 2016). Briefly, 1 ml of plasma was spiked with an internal standard d3-Δ-9-THC (10 μl of 1 μg/ml solution in MeOH), and the protein was precipitated with 2 ml of 0.1% formic acid in acetonitrile and centrifuged for 10 minutes at 3,500 rpm. The supernatant was diluted in 4 ml of MilliQ water and loaded to an Oasis Prime HLB 3 cm3, 60 mg column (Waters Co., USA). To wash the column, 2 ml of 25% methanol was added and it was eluted by adding 2 ml of 90:10 acetonitrile:methanol twice. A nitrogen stream was used at <39 °C and <15 psi pressure to dry the organic phase. Analytes were reconstituted in 50 μl of 90:10 ACN:MeOH and 50 μl of MilliQ water, and THC and its metabolites were quantified in an Agilent 1200 series HPLC system coupled to a 6410 Triple Quadrupole LC-MS (Agilent Technologies, USA) mass spectrometer with an electrospray interface. Nitrogen was employed as a drying and nebulizing gas.