Validated LC-MS/MS quantification of kynurenine pathway metabolites and pathway-balance ratios in brain tissue and microglial culture supernatant: application to neonatal stroke

Kynurenine pathway (KP) metabolite imbalance is implicated in perinatal brain injury, and KP metabolites are measured in human plasma and cerebrospinal fluid as markers of neuroinflammation. Methods validated in neonatal brain tissue or glial culture supernatant to the same standard are scarce, limiting comparisons across preclinical and clinical measurements. We optimised LC-MS/MS detection of tryptophan (TRYP), kynurenine (KYN), kynurenic acid (KYNA), 3-hydroxykynurenine (3-HK), 3-hydroxyanthranilic acid (3-HAA), quinolinic acid (QA), anthranilic acid and picolinic acid after protein precipitation and Enhanced Matrix Removal-Lipid clean-up, validated it against Clinical and Laboratory Standards Institute (CLSI) acceptance criteria in brain tissue homogenate and cell culture supernatant, and applied it to a rat model of neonatal stroke and immune-activated BV2 microglia, reporting KYN/TRYP, KYNA/KYN, 3-HK/3-HAA and QA/KYNA ratios. The lower limit of quantification was 6.53-34.17 nM; imprecision was 0.57-11.47%, accuracy was 84.9-108.3%, recovery was 83.2-112.3%, and matrix effect was 87.8-108.8%, with linear calibration (R2 > 0.99) over 6.5-2000 nM. KYN/TRYP and QA/KYNA ratios were higher in the stroke hemisphere than in control brain at 1, 7 and 14 days (p<0.001), and KYNA/KYN was lower; lipopolysaccharide- and cytokine-stimulated BV2 microglia showed the same change in all four ratios (p<0.01). The assay quantifies eight KP metabolites and four pathway-balance ratios in brain tissue and microglial supernatant within clinical acceptance limits, allowing direct comparison with KP ratios reported in human plasma and cerebrospinal fluid. Changes in these ratios after neonatal stroke identify KYN/TRYP and QA/KYNA as candidate targets for neonatal biofluid studies.