EXTH-48. PROTOPORPHYRIN IX LEVELS IN PATIENTS’ PLASMA, BLOOD, AND BRAIN TUMOR BY A VALIDATED LC-MS/MS METHOD

Abstract BACKGROUND Protoporphyrin IX (PPIX) is an intermediate molecule in the heme biosynthesis pathway. Cancer cells, having a higher metabolic rate than normal cells, accumulate PPIX when an exogenous dose of its precursor, 5-aminolevulinic acid (5-ALA), is administered. 5-ALA is currently approved by the U.S. FDA for the visualization of high-grade gliomas (HGG) and fluorescence guided surgery. 5-ALA and PPIX are also central to experimental treatments of photodynamic and sonodynamic therapy for HGG patients. Accurate quantitation of PPIX in brain tumor, plasma, and blood is necessary to evaluate its pharmacokinetics and hence optimize its therapeutic benefit in the context of experimental therapies and tumor visualization. METHODS Plasma was used as a matrix for PPIX quantitation in plasma and brain homogenate. Brain homogenates (1:7) were prepared in 5 mM ammonium formate containing 0.2% formic acid. A separate plasma curve was used for blood quantitation. Before protein precipitation, blood and corresponding calibrators were diluted five times with water. PPIX was extracted from human plasma, blood, and brain homogenate samples using methanol containing PPIX-d6 (internal standard). Sample preparation was performed under low light conditions (< 5 lux). Chromatographic separation of the analyte was achieved isocratically on Phenomenex Kinetex™ EVO C18 column, with a total run time of 3.5 min. Analyte detection was performed on Sciex QTRAP 6500+ MS/MS detector. RESULTS The method was validated for 1-2000 nmol/L range. Using plasma as a surrogate matrix, the maximum coefficient of variation for intra- and inter-day precision and accuracy were within acceptable ranges for all matrices. CONCLUSIONS A new rapid and sensitive LC-MS/MS method was successfully validated for the PPIX quantitation in human plasma, blood, and brain. It is currently used in a sonodynamic therapy clinical trial (NCT04559685) to evaluate PPIX pharmacokinetic profile in plasma and blood, and to determine levels in brain tumors of HGG patients.