A Simple Method for Quantification of Five Urinary Porphyrins, Porphobilinogen and 5-Aminolevulinic Acid, Using Liquid Chromatography Tandem Mass Spectrometry

Abstract

Analysis of porphyrins and 5-aminolevulinic acid (ALA), porphobilinogen (PBG) in physiological liquids is required for diagnosis and follow-up of porphyrias. High performance liquid chromatography (HPLC) and liquid chromatography tandem mass spectrometry (LC-MS) methods with higher specificity and sensitivity have been developed. The major disadvantage of those methods is that they require longer extraction times due to their matrix effects. The present study suggests a simple, fast, sensitive, and specific assay for determination of Coproporphyrin, 5-carboxylporphyrin, 6-carboxylporphyrin, 7-carboxylporphyrin, Uroporphyrin I and ALA, PBG in urine sample by direct injection without sample pre-treatment using LC-MS. For the purposes of the present study LC-MS device was set to multiple reaction monitoring (MRM) and positive ion mode. Porphyrins and ALA, porphobilinogen were characterized by their MS/MS product ion, spectra. ALA, PBG and 5 porphyrins were detected simultaneously. Limit of detection for Coproporphyrin, 5-carboxylporphyrin, 6-carboxylporphyrin, 7-carboxylporphyrin, Uroporphyrin I were 2 nmol/L, where it was 5 lmol/L for ALA and 2 lmol/L for porphobilinogen. The present study suggests that the present method is very effective compared to many other available methods for it does not require pre-treatment, provides simultaneous results of ALA, PBG and 5 porphyrins quantitatively in a shorter span of time, and has suitable sensitivity and selectivity. LC-MS technique was used clinically for the determination of urine porphyrin levels.

A year-long study of the occurrence and risk of over 140 contaminants of emerging concern in wastewater influent, effluent and receiving waters in the Republic of Ireland

“…Many CECs survive the wastewater treatment process and, for combined systems in particular, sewer overflows of both treated and untreated wastewater have also been identified internationally as sources of aquatic CEC contamination, especially following heavy rainfall events (Munro et al, 2019). Receiving water CEC concentrations can range from ng•L −1 to μg•L −1 (Cantwell et al, 2018;Dogan et al, 2017;Letsinger et al, 2019), where CECs can have effects on living systems (Brumovský et al, 2017). In comparison to other EU countries, and despite marked rises in personal wealth and life expectancy, as well as indicators of higher medical interventions and reliance on pharmaceuticals (Lichtenberg, 2022), relatively little is known about the environmental occurrence and impact of CECs in the Irish aquatic environment.…”

Natural 5-Aminolevulinic Acid: Sources, Biosynthesis, Detection and Applications

“…Compared with a microbial fermentation broth with high 5-ALA content, samples with low 5-ALA concentrations such as blood, urine or tissue require more sensitive detection technology. The LC-MS/MS method was developed to simultaneously determine 5-ALA concentrations in fluids or tissues after solid-phase extraction, butanol derivatization, and quantification by selective reaction monitoring using 13 C 5 , 15 N-ALA and 2,4-13 C 2 -PBG internal standards (Zhang et al, 2011;Dogan et al, 2019). The assay was highly sensitive for 5-ALA (LLOQ = 0.05-0.005 µM), and required ~4 h from extraction to results.…”

Haem relieves hyperoxia-mediated inhibition of HMEC-1 cell proliferation, migration and angiogenesis by inhibiting BACH1 expression

“…As described in previous methods [15,16], HMEC-1 cells were seeded in 6-well plates (1 × 10 5 /well) for 12 h under normoxic conditions to allow adherence to the well bottom. The culture medium was changed, and the cells were incubated under normoxic or hyperoxic conditions and treated according to the designated group (0 μM haem, 20 μM haem or vehicle) for 48 h (the medium was changed every 24 h).…”