Quantitative Determination of Iron-Siderophore Complexes in Peat by Isotope-Exchange Size-Exclusion UPLC-Electrospray Ionization High-Resolution Accurate Mass (HRAM) Mass Spectrometry

A method was developed for the quantification of iron-siderophorecomplexes by electrospray high-resolution accurate mass (HRAM) massspectrometry (MS) without the need for authentic standards. The bulkof iron-siderophore complexes was purified by solid-phase extraction(SPE) and concentrated by evaporation. The individual complexes wereidentified by fast size-exclusion chromatography (FastSEC)-OrbitrapMS(n) on the basis of the exact molecular mass (+/- 1 ppm)and MS2 or MS3 fragmentation. Their capabilityto readily exchange the natural Fe-56 for the added Fe-58 was demonstrated by SEC with ICP MS and ESI MS detection.The method was applied to the analysis of peat sampled in the easternpart of the French Pyrenean mountains. Nineteen siderophores belongingto four different classes were identified and quantified. The resultswere validated using ICP MS detection of iron by matching the sumof iron complexes determined by isotope exchange-ESI MS withineach peak observed by FastSEC-ICP MS.

Automated summary

A method for quantifying iron-siderophore complexes by electrospray high-resolution accurate mass (HRAM) mass spectrometry without the use of authentic standards was developed. The complexes were purified by solid-phase extraction (SPE) and identified by fast size-exclusion chromatography (FastSEC)-Orbitrap MS(n) based on molecular mass and fragmentation. The ability of the complexes to exchange Fe-56 and Fe-58 was demonstrated by SEC with ICP MS and ESI MS detection. The method was applied to analyze peat samples from the French Pyrenean mountains and successfully identified and quantified 19 siderophores belonging to different classes.