D4 alanine

All samples were processed with a standard operating procedure.150 µL aliquots of thawed plasma or urine were mixed with three internal standards (IS) [20 μL of d4-alanine (Sigma, USA, 10 mM), d5-phenylalanine (Sigma, USA, 10 mM), and d5-tryptophan (Sigma, USA, 10 mM)]. To precipitate protein from the plasma or urine samples, 400 µL of cold methanol was added, followed by freezing at - 20°C for 30 min. Then the supernatant was isolated by centrifugation at 12,000 rpm for 15 min at 4°C. The tissue sample was prepared by dissecting 30.00 ± 0.50 mg into new tubes. After adding three internal standards and 400 μL cold methanol, tissues were homogenized via TissueLyser II (QIAGEN, USA) and centrifuged (10,000g, 15 min, 4°C) to isolate the supernatant. The hair sample was washed with methanol and distilled water twice, then air-dried in a fume hood. 5.00mg ± 0.50 mg hair was prepared and added to three internal standards, and then incubated with 1 ml sodium hydroxide (1 M) at 54°C for 18 h. To precipitate the salt and protein, 1 ml of methanol was added to the hair extracts, followed by vortexing for 30 seconds, and centrifuged at 4000 g for 5 min. The supernatant was mixed with 50 µl of 4M NaOH. Then these mixtures underwent derivatization consecutively.