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

Abstract

Rapid, high-throughput, and quantitative evaluations of biological metabolites in complex milieu are increasingly required for biochemical, toxicological, pharmacological, and environmental analyses. They are also essential for the development, testing, and improvement of new commercial chemical products. We demonstrate the application of ultra-high performance liquid chromatography-mass spectrometry (uHPLC-MS), employing an electrospray ionization source and a high accuracy quadrupole time-of-flight mass analyzer, for the identification and quantification of a series of porphyrin derivatives in liver: a matrix of particular relevance in toxicological or pharmacological testing. Exact mass is used to identify and quantify the metabolites. Chromatography enhances sensitivity and alleviates potential saturation issues by fanning out the contents of a complex sample before their injection into the spectrometer, but is not strictly necessary for the analysis. Extraction and sample treatment procedures are evaluated and matrix effects discussed. Using this method, the known mechanism of action of a well-characterized porphyrinogenic agent was verified in liver extracts from treated rats. The method was also validated for use with bacterial cells. This exact-mass method uses workhorse instruments available in many laboratories, providing a highly flexible alternative to existing HPLC- and MS/MS-based approaches for the simultaneous analysis of multiple compounds in biological media.

Protoporphyrin IX Analysis from Blood and Serum in the Context of Neurosurgery of Glioblastoma

“…MS detection has a number of advantages over FLD [26,27,39]. Both high mass accuracy and the possibility of breaking the analyte molecules in the gas phase and measuring their fragments (MS/MS) contribute to unequivocal species identification, because complex samples can often not be fully chromatographically resolved.…”

“…Both high mass accuracy and the possibility of breaking the analyte molecules in the gas phase and measuring their fragments (MS/MS) contribute to unequivocal species identification, because complex samples can often not be fully chromatographically resolved. HPLC-MS was applied for the determination of porphyrin profiles in biological matrices [11,[39][40][41][42][43] using predominantly electrospray ionization (ESI) interfaces. Additionally, there were efforts to explore atmospheric pressure chemical ionization [42] for the purpose.…”

“…Regarding porphyrin detection in more complex matrices, Sullivan and coworkers [39] quantified twelve porphyrins including PPIX in liver extracts based on their mass, because most porphyrins co-eluted. MS/MS was not carried out.…”

“…MS/MS was not carried out. The method suffered from problems with peak-splitting and matrix suppression [39]. Furthermore, highly acidic extraction was performed so that this approach was not suitable for the individual determination of metal-free PPIX and ZnPPIX.…”

Extraction and preconcentration of hemin from human blood serum and breast cancer supernatant

“…So, HPLC has been applied for the determination of hemin in different biological samples using radioactivity emission if hemin has been labeled with 59 Fe , spectrophotometry with the absorbance being recorded at 400 nm or after its conversion to pyridine hemochromogen . In addition, UHPLC–MS has also been used for hemin determination . In another report, hemin was extracted using cloud point extraction technique and then quantified with fluorescence spectroscopy .…”