Phenylalanine d5

Hair sample preparation was conducted according to the published protocol by Sulek et al with minor modifications (Sulek et al. 2014 (link)). All hair samples were randomised prior to sample preparation and weighed to 3.5 mg ± 0.5 mg. All weighed hair samples were washed with distilled water and methanol twice. Three internal standards, 20 μL of D4-alanine (Sigma, USA, 10 mM), D5-phenylalanine (Sigma, USA, 10 mM), and D2-tyrosine (Sigma, USA, 10 mM) were added to the hair samples and incubated with 1 ml potassium hydroxide (1 M) at 54 °C for 18 h. The hair extracts were then neutralized by adding 67 μL sulphuric acid (3 M). To precipitate the salt and protein, 1 ml of methanol was added to the hair extracts, followed by vortexing for 30 min, and centrifugation at 4000 g for 5 min. The 350 μL of supernatant was concentrated to dryness in a SpeedVac (Labconco, Kansas, USA) at 37 °C for 6 h and stored at − 20 °C prior to derivatization. Quality control (QC) samples were also prepared by combining a small portion of all hair extracts together and following the identical preparation steps to the samples.

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.

HPLC- or MS-grade solvents acetonitrile (ACN), methanol (MeOH), and chloroform (CHCl₃) were purchased from Merck (Darmstadt, Germany) or Sigma-Aldrich (Steinheim, Germany or St. Louis, MO). MS grade water or ultrapure water was purchased from Merck (Darmstadt, Germany) or using a Milli-Q purity system (Millipore, Billerica, MA).
MeOH-dissolved internal standard solution (ISS-1) containing L-methionine sulfone, D-camphor-10-sulfonic acid sodium salt, leucine-d3, phenylalanine-d5, tryptophane-d5, succinic acid-13C4, and cholic acid-d4 (all from Sigma-Aldrich, Steinheim, Germany) was used to normalize signal intensity of the CE-TOF/MS data. ACN-dissolved ISS-2 consisting of N,N-diethyl-2-phenylacetamide, trimesic acid, disodium 3-hydroxynaphthalene-2,7-disulfonate (all from Wako, Japan), and 3-aminopyrrolidine dihydrochloride (Aldrich, St. Louis, MO) was used to adjust the migration time of the CE-TOF/MS analysis. Internal standards of free fatty acid (FFA) 16:0-d4 (Cambridge Isotope Laboratories, Tewksbury, MA, USA), and FFA 22:0-d4 (ten Brink laboratories, Amsterdam, Netherlands) dissolved in CHCl₃/MeOH (2:1, v/v) were used to normalize signal intensity of the UHPLC-Q-TOF/MS-based FFA analysis.