Proposed guidelines for sample preparation and ESI-MS analysis of humic substances to avoid self-esterification

Standard humic substances (HS) were stored in ¹³C-labelled methanol, ethanol, 2-propanol and d₃-methanol to investigate whether they underwent self-esterification with the solvents or not. Data from stable carbon isotope analysis, ¹³C NMR spectroscopy and electrospray ionisation mass spectrometry (ESI-MS) showed that significant self-esterification of carboxyl groups began immediately after dissolution. The major implication is that data collected in previous studies using these solvents in conjunction with HS are likely to be compromised. Self-esterification was minimised by varying parameters such as % water, temperature, concentration, storage time and buffer. Solvent reactivity was found to be: ethanol > 2-propanol > methanol. The study proposes preliminary guidelines for the preparation and storage of HS and their analysis by ESI-MS. The primary recommendation is to avoid the use of these solvents and use alternatives such as acetonitrile and water.