Acetyl-DL-leucine (2-acetamido-4-methylpentanoic acid) is a small organic molecule derived from the branched-chain amino acid leucine, with N-acetylation of the amino group. It contains both an acetamide moiety and a terminal carboxylic acid, giving it amphiprotic character and enabling diverse acid–base and hydrogen-bonding interactions in solution. With a molecular formula of C8H15NO3 and a molecular weight of 173.21, it is well suited for use as a defined low-molecular-weight standard in analytical workflows. The presence of a chiral center and availability in the DL-racemic form make it useful for studies involving stereochemical resolution and chiral method development.

The compound’s combination of an amide and a carboxylic acid group provides distinct chromatographic and spectroscopic signatures valuable in method optimization for LC, LC–MS, and related techniques. Its moderate polarity, reflected in the polar surface area, allows controlled retention under both reversed-phase and hydrophilic interaction conditions, aiding in the tuning of separation parameters. As an N-acylated amino acid analogue, acetyl-DL-leucine is a convenient model substrate for examining peptide bond mimicry, acyl-transfer chemistry, and amide stability. These features make it a versatile tool compound for physico-chemical characterization, calibration, and mechanistic studies in amino acid and peptide-related research.