2.6. Biological sample pretreatment

An aliquot of 2 ml plasma for positive ion detection was suspended in 8 ml methanol. Another aliquot of 2 ml plasma for negative ion detection was suspended in 200 µl 10% (v/v) hydrochloric acid and 8 ml methanol, and then mixed by vortex for 3 min to precipitate protein, followed by centrifugation at 10 000g for 10 min. The supernatants were evaporated to dryness under nitrogen gas at room temperature, and the residues were dissolved in 200 µl 70% (v/v) methanol. After centrifugation at 12 000g for 10 min, 10 µl of the supernatant was injected into the HPLC-MS/MS system for analysis.

Urine sample (3 ml) was dissolved in 12 ml methanol, and then mixed by vortex for 3 min to precipitate protein, followed by centrifugation at 10 000g for 10 min. The supernatant was evaporated to dryness under nitrogen gas at room temperature, and the residue was dissolved in 600 µl 70% methanol. After centrifugation at 12 000g for 10 min, 10 µl of the supernatant was injected into the HPLC-MS/MS system for analysis.

Bile sample (3 ml) was dissolved in 12 ml methanol, and then mixed by vortex for 3 min to precipitate protein, followed by centrifugation at 10 000g for 10 min. The supernatant was evaporated to dryness under nitrogen gas at room temperature, and the residue was dissolved in 1.5 ml 70% methanol. After centrifugation at 12 000g for 10 min, 10 µl of the supernatant was injected into the HPLC-MS/MS system for analysis.

Feces were dried at 37 °C and grinded into powder. Feces sample (1.5 g) was extracted with 30 ml 70% methanol in an ultrasonic bath for 30 min, followed by filtration. Filtrate (2 ml) was evaporated to dryness under nitrogen gas at room temperature, and the residue was dissolved in 400 µl 70% methanol. After centrifugation at 12 000g for 10 min, a 10-µl aliquot of the supernatant was injected into the HPLC-MS/MS system for analysis.