Desmosine is a crosslinking pyridinium amino acid of elastin, which is a useful biomarker for the diagnosis of chronic obstructive pulmonary disease (COPD) by LC–MS/MS analysis. We previously reported a synthesis of desmosine-d4, which is useful as an internal standard for quantitative LC–MS/MS analysis of desmosines, by deuterogenation of an alkyne group; however, the isotopic purity of the desmosine-d4 was only ca. 50%. The present report describes a new synthesis of desmosine-d4 that improves the isotopic purity to ca. 90% by exchanging the protons of the amino groups to deuterium using deuterogenation.