Investigation of liquid chromatography-mass spectrometry analysis of a peptide aldehyde SJA6017 with identifying its hemiacetal, gem-diol, and enol ether

Abstract

The quantitative analysis of SJA6017, a peptide aldehyde inhibitor of calpain (Calpain Inhibitor VI), has encountered challenges in preclinical drug studies. The complex reverse-phase HPLC chromatographic behavior exhibits two peaks, each containing multiple species. An liquid chromatography-mass spectrometry (LC-MS/MS) study proposed an explanation for this phenomenon, caused by the amide aldehyde structure of SJA6017. Four chemical species corresponding to the two HPLC peaks have been identified as SJA6017 and its methyl hemiacetal, methyl enol ether, and gem-diol. In many instances of preclinical studies, methanol is favored as a substitute for DMSO. The hemiacetal is formed when the amide-activated peptide aldehyde reacts with methanol, which can then be further dehydrated in the mass spectrometer ion source under high temperature to form the methyl enol ether. The hemiacetal and gem-diol can also be decomposed to SJA6017 in the ion source. Additionally, the amide-activated peptide aldehyde can easily hydrate to the gem-diol of SJA6017 during sample incubation or sample preparation. The hemiacetal and gem-diol of SJA6017 are stable enough to have different retention times in the liquid chromatography, which explains why SJA6017 appears as two peaks, each containing multiple species. An LC-MS/MS tandem quadrupole mass spectrometer quantitative analysis method is proposed, enabling the analysis of these types of samples. This work serves as both an illustrative example and a cautionary note for mass analysis, sample incubations, and sample preparations involving compounds of peptide aldehyde, including similar aldehyde-containing metabolites, especially when methanol is present. This study provides the information needed to understand peptide aldehyde behavior at various steps of preclinical in vitro studies in the presence of methanol. It has assisted in the development of the SJA6017 bioanalysis method and will also aid in the development of bioanalysis methods for similar peptide aldehydes.

Keywords: LC–MS/MS; enol ether; gem‐diol; hemiacetal; peptide aldehyde.