B-189 Comprehensive and Sensitive UHPLC-MS/MS Analysis for Urinary Porphyrins

Abstract Background Porphyrins are critical biomolecules involved in heme biosynthesis, with altered urinary excretion patterns indicating metabolic disorders like porphyria. These disorders arise from enzymatic deficiencies that disrupt heme synthesis, leading to porphyrin accumulation. Existing analytical techniques, including spectrofluorometry, HPLC, and mass spectrometry, often require extensive sample preparation, large volumes, or derivatization. We developed and validated a robust UHPLC-MS/MS method for selective porphyrin quantification in human urine, using a streamlined one-step extraction and reversed-phase liquid chromatography. This method enables precise differentiation of porphyrin profiles in healthy individuals and patients with liver disease, facilitating biochemical and clinical research. Methods Urine samples (75 µL) were acidified with 6 M formic acid, vortex-mixed, centrifuged, and directly injected into a Shimadzu LCMS-8045 system. Chromatographic separation was achieved using a Shim-pack GIST C18 column (4.6 × 75 mm, 3 µm) with a gradient mobile phase comprising acetonitrile, water, and 0.1% formic acid. Multiple reaction monitoring (MRM) transitions were optimized for eight porphyrins, ensuring high specificity and sensitivity. The method was validated for linearity, accuracy, and precision using calibrators and QC samples, ensuring bio-analytical standards. Results The optimized UHPLC-MS/MS method demonstrated excellent chromatographic resolution, with enhanced porphyrin ionization in positive electrospray mode. Method validation showed strong linearity (R² > 0.99) across clinically relevant concentrations. Intra- and inter-day precision were within 10%, and recovery exceeded 85%. Control sample analysis confirmed accuracy within specified QC ranges. Comparative analysis of urinary porphyrins from healthy individuals and liver disease patients revealed distinct metabolic patterns, highlighting the method’s clinical utility. This streamlined approach significantly improves throughput and minimizes sample preparation challenges. Conclusion A robust and validated UPLC-MS/MS method was developed and applied for the simultaneous quantification of porphyrins in human urine. The method demonstrated high accuracy, precision, and repeatability, with a simple and reproducible extraction process. By analyzing urine samples from both healthy individuals and liver disease patients, distinct metabolic patterns of porphyrins were identified, highlighting the method’s potential for clinical testing.