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.