Integrating mass spectrometry and hyperspectral imaging for protoporphyrin IX detection in malignant glioma tissue

Maximal safe tumor resection is crucial for the treatment of high-grade gliomas (HGG). 5-aminolevulinic acid (5-ALA)-mediated fluorescence-guided surgery enhances tumor visualization by inducing protoporphyrin IX (PpIX) accumulation. However, current fluorescence-based observation devices lack the sensitivity for detecting tumor cells in low-density infiltrative zones. Hyperspectral imaging (HI) offers a potential solution. In this study, HI-derived PpIX measurements were compared to those obtained from reversed-phase liquid chromatography coupled to mass spectrometry (LC–MS), a method that delivers accurate concentrations. Additionally, we investigated coproporphyrins (Cp) I and III, since they potentially interfere with PpIX determination. Pig brain was used as a surrogate for protocol development and acquisition of comparative HI and LC–MS reference data, which were subsequently used to evaluate the results obtained from 27 biopsies from nine patients undergoing 5-ALA-mediated tumor resection. During sample preparation for LC–MS, 80% PpIX and 45% combined Cp I & III were recovered from brain tissue. For LC–MS quantification of PpIX, accuracy ranged from 98 to 137%, and coefficient of variation was 5–14%, indicating sufficient precision. For HI, the values were 77–121% and 11–31%, respectively. Notably, HI significantly overestimated PpIX concentrations compared to those determined by LC–MS. This study highlights LC–MS as a reliable method for porphyrin quantification and suggests that HI workflows need further optimization for accurate tumor delineation in HGG.