Targeted LC-MS/MS profiling highlights altered cerebrospinal fluid glutathione in cancer patients with CNS metastases

Brain metastasis is associated with substantial biological heterogeneity, yet cerebrospinal fluid (CSF) biomarkers that reflect redox-related metabolic alterations remain insufficiently characterized. Glutathione (GSH), a central regulator of oxidative stress and metabolic adaptation, may provide cerebrospinal fluid (CSF)-based metabolic information associated with central nervous system (CNS) metastatic disease. However, reliable quantification of GSH in CSF is analytically challenging because of its low abundance, strong polarity, and susceptibility to oxidation. We developed and validated a targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for CSF GSH quantification using dansyl chloride (Dns-Cl) derivatization. The method was optimized to improve analyte stability, chromatographic retention, ionization efficiency, and was validated for selectivity, linearity, sensitivity, precision, accuracy, recovery, matrix effect, and stability. The assay was then applied to CSF samples from patients with cancer brain metastasis to evaluate associations between CSF GSH levels and clinicomolecular features. The method showed satisfactory analytical performance, with good linearity over 10-500 ng/mL (r² = 0.9943), accuracy of 92.88%-104.85%, and intra- and inter-batch precision within 8.98%. In the clinical cohort, CSF GSH levels were significantly higher in patients with epidermal growth factor receptor (EGFR)-mutant tumors than in those with EGFR wild-type disease (p = 0.024). Higher CSF GSH levels were also observed in patients receiving furmonertinib than in those receiving other tyrosine kinase inhibitors (TKIs) (p = 0.0022), including within the EGFR-mutant subgroup (p = 0.0462). In multivariable regression adjusting for EGFR status, age, and sex, furmonertinib exposure remained independently associated with higher log-transformed CSF GSH levels (β = 0.1395, 95% CI 0.0158-0.2633). This study establishes a Dns-Cl derivatization-based LC-MS/MS platform for reliable quantification of GSH in CSF and provides an initial clinical characterization of CSF GSH metabolism in real-world patients with metastatic CNS disease. Stratified association analyses by molecular subtype and TKI treatment suggested distinct CSF GSH profiles in patients with brain metastasis.