Separation and fragmentation study of isocoproporphyrin derivatives by UHPLC-ESI-exact mass MS/MS and identification of a new isocoproporphyrin sulfonic acid metabolite

Abstract

Isocoproporphyrin and its derivatives are commonly used as biomarkers of porphyria cutanea tarda, heavy metal toxicity and hexachlorobenzene (HCB) intoxication in humans and animals. However, most are isobaric with other porphyrins and reference materials are unavailable commercially. The structural characterisation of these porphyrins is important but very little data is available. We report here the separation and characterisation of isocoproporphyrin, deethylisocoproporphyrin, hydroxyisocoproporphyrin and ketoisocoproporphyrin, isolated in the faeces of rats fed with a diet containing HCB, by ultra high performance liquid chromatography-exact mass tandem mass spectrometry (UHPLC-MS/MS). Furthermore, we report the identification and characterisation of a previously unreported porphyrin metabolite, isocoproporphyrin sulfonic acid isolated in the rat faeces. The measured mass-to-charge ratio (m/z) of the precursor ion was m/z 735.2338, corresponding to a molecular formula of C36H39N4O11S with an error of 0.3 ppm from the calculated m/z 735.2336. The MS/MS data was consistent with an isocoproporphyrin sulfonic acid structure, derived from dehydroisocoproporphyrinogen by sulfonation of the vinyl group. The metabolite was present in a greater abundance than other isocoproporphyrin derivatives and may be a more useful biomarker for HCB intoxication.

Mass-spectrometric profiling of porphyrins in complex biological samples with fundamental, toxicological, and pharmacological applications

“…These methods, moreover, may require significant column- and analyte-specific optimization in order to render them quantitative. Sophisticated MS/MS methods, on the other hand, have been developed for highly sensitive monitoring of specific porphyrias [4, 22, 22–24], but may not be generally accessible or flexible enough for toxicological, pharmacological, or basic research applications. High resolution (±0.005 mass units) electrospray ionization time of flight mass spectrometric methods allow for the definitive identification of compounds of interest, even against a complex background, via their exact masses.…”

“…These can result from inborn errors in metabolism; additionally, an extensive array of xenobiotic agents chemically induce porphyrias via the selective inhibition of enzymes in the pathway. Examples include heavy metals such as lead, arsenic, aluminum, iron, and mercury [3–8]; aromatic compounds including benzene, dioxins, pyridine, and polychlorinated biphenyls [4, 9–12]; and a variety of pharmaceuticals, agrochemicals, and complex natural products [13–16]. Additionally, many xenobiotics are known to be metabolized by mammalian cytochrome P450s [17], generating modified porphyrin products which themselves interfere with heme biosynthesis.…”

“…Many existing mass spectrometry-based studies on porphyrins have focused on identifying specific analytes or even particular isomers of analytes for diagnostic purposes, with an emphasis on tandem MS/MS [4, 22, 22–24]. Because they are highly refined to detect their target analytes, these methods can be exquisitely sensitive.…”

“…Because they are highly refined to detect their target analytes, these methods can be exquisitely sensitive. Accordingly, they have been applied most extensively to the clinical monitoring of porphyrias where the biomarkers of disease are very well known [2, 4, 25–29]. However, of particular importance to the toxicology, pharmacology, and research labs are highly flexible methods.…”