Autism spectrum disorder (ASD) has been associated with disruptions in tryptophan (TRP) metabolism, affecting the production of key neuroactive metabolites. Investigating these metabolic pathways could yield valuable biomarkers for ASD severity and progression. We included 44 children with ASD and 44 healthy controls, members of the same family. ASD group average age was 10.7 years while control group was 9.4 years. Analysis of TRP metabolites in urine samples was performed using a ultra-high performance liquid chromatography (UHPLC) and tandem mass spectrometry (MS/MS) in selected reaction monitoring detection mode (SRM).

Tryptophan metabolite analysis was performed as per Pavlova et al., 2017. In brief, urine samples were removed from −80 °C and thawed on ice for 90 min. A 250 μL urine aliquot was transferred to a deep well plate and dried via vacuum evaporator (GeneVac at a maximum. temperature of 40 °C). 200 μL 80% isopropanol was added to the dried aliquot, sonicated for 30 s, and vortexed (500 rpm, room temperature, 10 min). The extract was centrifuged at 4000 rpm and 150 μL supernatant transferred to a new deep well plate. The remaining 50 μL sample was re-extracted following the same procedure, and the 200 μL supernatant was removed and combined with the prior 150 μL (i.e., total volume 350 μL). A 40 μL aliquot of the extract was transferred to a new 96- well plate, dried via centrifugal evaporator, and stored at −80 °C. Prior to the mass spectrometry analysis, the urine extract was reconstituted in 20 μL 5% isopropanol containing 4000 nM [13C6] indole-3-acetatic acid and 400 nM anthranilic acid [13C6]), vigorously vortexed, and centrifuged for 10 min at 4000 RPM. In addition, for the analysis of tryptophan and kynurenine, urine extracts were diluted 500×, and 40 μL diluted urine transferred to 96- well plate, dried via centrifugal evaporator, and stored at −80 °C. Prior to the mass spectrometry analysis, the diluted urine extract was reconstituted in 20 μL of 5% isopropanol containing 200 nM L-tryptophan [13C11][15N2]. Analysis was performed using a UHPLC system (1260 series Agilent, CA, USA ) coupled with a triple quadrupole mass spectrometer (AJS 6495A, Agilent, CA, USA). Samples were injected (2 µL) on the analytical column (C18 CSH; 1.7 µm, 2.1 mm i.d. × 100 mm; cat. #186005297; Waters, MA, USA). The column temperature was held at 40 °C. The mobile phase consisted of solution A (0.1% FA in water) and solution B (0.1% FA in 95% ACN). The flow rate was 300 µL/min. The gradient elution program: 0.0 min 5% B; 5 min 5% B; 10 min 95% B; 11.99 min 95% B; 12 min 5% B; 14 min 5% B. A standard-flow electrospray source operated in positive ion mode (capillary voltage 3.5 kV; gas flow rate 15 L/min at 160 °C; sheath gas flow 12 L/min at 250 °C; nozzle voltage 500 V). A total of 88 transitions were monitored via dynamic SRM mode, with a 2 min window scheduled around metabolite experimental RT.

The study population consisted of 88 children, 44 with ASD (36 boys and 8 girls) and 44 healthy children (23 boys and 21 girls), who were members of the same family. In the ASD group, the subjects’ average age was 10.7 years in the range of 4.9–17.0 years. The control group included 44 neurotypical children (siblings), without any acute or chronic illness, who were on average 9.4 years of age, in the range of 0.9–16.7 years. None of the children received any supplementary vitamin or magnesium intake. Children in the study group were diagnosed with ASD by either an expert pediatrician or a neuropsychiatrist in collaboration with a psychologist. The diagnosis was made using a multidisciplinary approach, which combined a clinical evaluation with a psychological assessment. Children were grouped according to the criteria detailed and summarized by DSM-5. Additional behavioral ratings were based on a standardized behavior classification for children with ASD, developed by the local educational authori-ty to provide additional school support and on Childhood Autism Rating Scale (C.A.R.S.). The CARS questionnaire was filled out by all parents together with a psy-chologist. 15 mL of morning urine samples were collected into sterile urine container and stored at −80 °C until analysis.