PhD Scientific Days 2025

Poster Session III. - H: Pharmaceutical Sciences and Health Technologies

Quantification of Endogenous Protoporphyrin IX in Rat Plasma using Liquid Chromatography with Fluorescence Detection

Name of the presenter

Széles Aliz

Institute/workplace of the presenter

Doctoral College Semmelweis University; HUN-REN Research Centre for Natural Sciences; Toxi-Coop Toxicological Research Centre

Authors

Aliz Széles¹, Katalin Monostory², Tibor Renkecz³

1: Doctoral College Semmelweis University; HUN-REN Research Centre for Natural Sciences; Toxi-Coop Toxicological Research Centre
2: HUN-REN Research Centre for Natural Sciences, Magyar tudósok körútja 2., H-1117 Budapest, Hungary
3: Toxi-Coop Toxicological Research Centre, Magyar jakobinusok tere 4., H-1122 Budapest, Hungary

Text of the abstract

Protoporphyrin IX (PPIX) is a central intermediate in the heme biosynthetic pathway with critical roles in both physiological functions and pathological conditions, including porphyrias and cancer. Its photoreactive properties make it a key compound in photodynamic therapy (PDT). Accurate quantification of PPIX in biological matrices is essential for understanding its metabolic profile and therapeutic potential.
We aimed to develop a sensitive liquid chromatography method with fluorescence detection (LC-FLD) for the determination of endogenous PPIX in rat plasma.
A reverse-phase LC-FLD method was developed using mesoporphyrin IX dihydrochloride as the internal standard, with a calibration range from 10 to 700 ng/mL. Method validation was performed in line with the ICH M10 bioanalytical guideline.
Unlike xenobiotics, for which well-established workflows exist, the development and validation of a bioanalytical method for an endogenous compound poses additional challenges. Since the analyte is naturally present in the biological matrix, a true blank sample—free of the target compound—cannot be obtained. Therefore, alternative strategies recommended by the ICH M10 guideline had to be employed, such as the use of a surrogate matrix or a surrogate analyte approach.
Mean recoveries were 93.1%, 104%, 102%, and 101% at high, medium, low, and endogenous concentration levels, respectively. Plasma samples were stable at - 75 °C ± 10 °C for at least 14 days, and under three freeze-thaw cycles, as well as for at least 24 hours on the benchtop at room temperature.
Chromatograms demonstrated clear separation of PPIX and the internal standard. Accuracy and precision remained within ±15% across all tested concentrations. No significant matrix effects or carryover were detected. The method is suitable for the reliable quantification of endogenous PPIX in rat plasma.
The developed LC-FLD method enables sensitive and selective quantification of PPIX in rat plasma. This technique can support both pharmacokinetic studies and research into porphyrin metabolism or PDT efficacy, offering valuable insights into the therapeutic use and biological significance of PPIX.
This work was supported by the 2024-2.1.2-EKÖP-KDP-2024-00002 University Research Scholarship Programme of the Ministry for Culture and Innovation from the source of the National Research, Development and Innovation Fund.