biostudies-literatureUnknown15(1)Knight WProtoporphyrin IX (PPIX) is a fluorescent metabolite in the heme biosynthesis pathway, and cancer cells accumulate it when 5-aminolevulinic acid (5-ALA), a precursor, is administered. In the U.S., 5-ALA is approved for visualizing high-grade gliomas (HGG) during fluorescence-guided surgery. PPIX is also central to experimental photodynamic and sonodynamic therapies for HGG. Additionally, PPIX measurement is critical for diagnosing and monitoring porphyrias. Despite the need for a sensitive and rapid bioanalytical method for accurate quantification of PPIX in biospecimens, no reliable validation of an LC-MS/MS method is available due to challenges related to its chemical instability, poor solubility, and tendency to aggregate. This work is the first to present a fully validated, sensitive, and rapid LC-MS/MS method for determining PPIX levels in human plasma, blood, and brain tumors. The method overcomes stability concerns, achieving a 3.5-min total run-time with a concentration range of 1-2000 nmol/L for plasma and tumors, and 10-2000 nmol/L for blood. Application of the method in a clinical trial, which assesses sonodynamic therapy for HGG patients, shows significant PPIX production, peaking in plasma and blood six hours post-5-ALA administration. In recurrent HGG patients, PPIX levels were notably higher in gadolinium-enhancing tumor regions compared to non-enhancing areas, indicating preferential accumulation in tumors.Scientific reports21521https://www.ebi.ac.uk/biostudies/studies/S-EPMC12217841biostudies-literatureProtoporphyrin IX plasma and blood pharmacokinetics and brain tumor distribution determined by a validated LC-MS/MS method.PMC12217841Sanai NKnight WMargaryan TShaik KTovmasyan AfalseProtoporphyrin IX plasma and blood pharmacokinetics and brain tumor distribution determined by a validated LC-MS/MS method.Protoporphyrin IX (PPIX) is a fluorescent metabolite in the heme biosynthesis pathway, and cancer cells accumulate it when 5-aminolevulinic acid (5-ALA), a precursor, is administered. In the U.S., 5-ALA is approved for visualizing high-grade gliomas (HGG) during fluorescence-guided surgery. PPIX is also central to experimental photodynamic and sonodynamic therapies for HGG. Additionally, PPIX measurement is critical for diagnosing and monitoring porphyrias. Despite the need for a sensitive and rapid bioanalytical method for accurate quantification of PPIX in biospecimens, no reliable validation of an LC-MS/MS method is available due to challenges related to its chemical instability, poor solubility, and tendency to aggregate. This work is the first to present a fully validated, sensitive, and rapid LC-MS/MS method for determining PPIX levels in human plasma, blood, and brain tumors. The method overcomes stability concerns, achieving a 3.5-min total run-time with a concentration range of 1-2000 nmol/L for plasma and tumors, and 10-2000 nmol/L for blood. Application of the method in a clinical trial, which assesses sonodynamic therapy for HGG patients, shows significant PPIX production, peaking in plasma and blood six hours post-5-ALA administration. In recurrent HGG patients, PPIX levels were notably higher in gadolinium-enhancing tumor regions compared to non-enhancing areas, indicating preferential accumulation in tumors.2025-01-01T00:00:00Z2025 Jul2026-05-26T11:04:10.874Z2026-05-24T03:07:14.339ZS-EPMC122178414059597610.1038/s41598-025-05780-w