A new liquid chromatography/electrospray ionization mass spectrometry method for the analysis of underivatized aliphatic long‐chain polyamines: application to diatom‐rich sediments

Abstract

Natural polyamines are found in all three domains of life and long-chain polyamines (LCPAs) play a special role in silicifying organisms such as diatoms and sponges where they are actively involved in the complex formation and nanopatterning of siliceous structures. With chain lengths extending up to 20 N-methylated propylamine repeat units, diatom LCPAs constitute the longest natural polyamines. Mixtures of natural LCPAs are typically purified in bulk using ion-exchange, size-exclusion and dialysis and then analyzed either by direct infusion mass spectrometry or by MALDI-TOF. Here, we describe a novel ion-pairing liquid chromatographic method that allows baseline separation, detection and structural elucidation of underivatized aliphatic methylated and non-methylated LCPAs with a wide range of chain lengths. Complete separation of synthetic mixtures of LCPA species differing by either a propylamine or an N-methylpropylamine unit is achievable using this method and chromatographic separation of natural, diatom frustule bound LCPAs extracted from sediment core samples is greatly improved. Using electrospray ionization mass spectrometry (ESI-MS), we detected singly [M+H](+) and multiply [M+nH](n+) charged protonated ions. The abundance of multiply charged LCPA species increased linearly as a function of LCPA chain length (N) and multiprotonated ions [M+nH](n+) were more abundant for longer chain polyamines. The abundance of multiply charged LCPAs along with the concomitant disappearance of the singly charged protonated molecular ion significantly increases the complexity of the MS spectra, justifying the need for good chromatographic separation of complex LCPA mixtures. This analytical procedure will likely constitute a powerful tool for the characterization, quantification, as well as the purification of individual LCPAs in natural and synthetic samples for studies of silica precipitation as well as nitrogen and carbon isotopic analysis used in paleoceanographic studies.

Diatom microfossils from cretaceous and eocene sediments contain native silica precipitating long‐chain polyamines

“…A,B), and the relative abundance of the doubly protonated ions increased as a function of chain length. Such spectral features have previously been observed in the LC/ESI(+)‐MS spectra of synthetic aliphatic long‐chain polyamines and of natural LCPAs extracted from diatom frustules (Bridoux et al ., ).…”

“…Within each mass series, neighboring ions differ by 71 daltons. The presence of two terminally located secondary amine functionalities is confirmed by the presence of the fragments † and †† that correspond to an internal proton transfer reaction, creating a positively charged N‐terminated fragment (Bridoux et al ., ). (C) The suggested polyamine structure fits the fragmentation patterns observed in B).…”

“…Fragmentation of m/z 543 and m/z 742 resulted in two series of product ions, shifted by 14 mass units (Figs B and C). Within each mass series, neighboring ions differ by 71 daltons; a fragmentation pattern diagnostic of aliphatic long‐chain polyamines with various N‐methylated polypropyleneimine repeat chains, as confirmed by analysis of authentic methylated polypropyleneimine synthetic standards (Bridoux et al ., ). Finally, similar fragmentation studies were conducted on the doubly charged [M+2H] 2+ species (e.g., m/z 372; Fig.…”

“…Finally, similar fragmentation studies were conducted on the doubly charged [M+2H] 2+ species (e.g., m/z 372; Fig. A), which produced the expected fragments as reported previously (Bridoux et al ., ). Other, minor LCPAs also eluted in the HF‐extract, but the details of their structures could not be unambiguously identified.…”

“…Sediment samples were chemically cleaned of exogenous organic material prior to HF digestion (Ingalls et al, 2004;Bridoux et al, 2011). In brief, sediments were sequentially extracted three times each in 6 mL MeOH, then 6 mL DCM:MeOH, and finally in 6 mL DCM, in Teflon centrifuge tubes.…”