Sample preparation for radio-profiling and metabolite identification

An equal volume of 2, 4, 8, 12, 24, and 96 h plasma sample was pooled by time-point across subject 03 ~ 06, to give six pooled plasma samples for metabolite profiling. An appropriate volume (3x, v/v) of acetonitrile was added to a portion of the pooled plasma sample, followed by vortexing ca. 2 min. After storage in a refrigerator for ca. 30 min, the mixture was centrifuged at ca. 10000 g at 4 °C for 10 min. The supernatant was then separated from the PES, which was suspended with water, followed by addition of methanol (2x, v/v) twice. The three extracts were combined and evaporated to dryness under a nitrogen stream by an N-Evap evaporator at room temperature. The residues were reconstituted with an appropriate volume of methanol:water (1:1, v/v) and centrifuged at ca. 10000 g at 4 ℃ for 10 min. The supernatant was subjected to HPLC radio-profiling.

Four pooled human urine samples (subject 03 ~ 06) were pooled from 0 to 432 h based on equal volume ratios from the individual patients across time intervals. In addition, three pooled urine samples (0–48, 48–120 and 360–432 h) (subjects 03–06) were prepared by pooling human urine samples across subjects and time points. Portion of the pooled urine sample was evaporated to a small volume under a nitrogen stream and extracted with methanol (3x, v/v) followed by centrifuged at ca. 10000 g at 4 ℃ for 10 min. The supernatant was reconstituted with an appropriate volume of methanol:water (1:1, v/v) and centrifuged at ca. 10000 g at 4 ℃ for 10 min. The supernatant was subjected to HPLC radio-profiling.

Four human fecal samples (0–432 h) were prepared by pooling feces from individual subjects (Subjects 03–06). In addition, three pooled human fecal samples (0–48, 48–120 and 360–432 h) were prepared by pooling feces samples across subjects and time points. The pooled fecal homogenate was extracted with methanol (3x, v/v) and the mixture was vortexed, and centrifuged at ca. 10000 g at 4 ℃ for 10 min. The supernatant was then separated from the PES, which was suspended with water, followed by addition of methanol twice. The three extracts were combined and evaporated to dryness under a nitrogen stream by an N-Evap evaporator at room temperature. The residues were reconstituted with an appropriate volume of methanol:water (1:1, v/v) and centrifuged at ca. 10000 g at 4 ℃ for 10 min prior to analysis.