2.2 | Hair collection and preparation
Hair samples were collected immediately after delivery 0.5 cm proximal from the scalp with processing as per a previously published protocol.16 All hair samples were stored at -20°C until processing for analysis. Hair samples (3.5mg ± 0.5 mg) were randomized, 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. Extracts were then neutralized by adding 67 μL sulphuric acid (3 M). To precipitate the salt and protein, 1 ml of methanol was then added, followed by vortexing for 30 seconds 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 30 uL of all hair extracts together and following the identical preparation steps to the samples.