Identification of Conjugated Linoleic Acid (CLA) Isomers by Silver Ion-Liquid Chromatography/In-line Ozonolysis/Mass Spectrometry (Ag⁺‑LC/O₃‑MS)

A novel method for the identification of conjugated linoleic acid (CLA) isomers has been developed in which silver ion liquid chromatography is coupled to in-line ozonolysis/mass spectrometry (Ag⁺-LC/O₃-MS). The mobile phase containing CLA isomers eluting from the Ag⁺-LC column flows through a length of gas-permeable tubing within an ozone rich environment. Ozone penetrating the tubing wall reacts with the conjugated double bonds forming ozonolysis product aldehydes. These, and their corresponding methanol loss fragment ions formed within the atmospheric pressure photoionization (APPI) source, were detected by in-line MS and used for the direct assignment of double bond positions. Assignment of positional isomers is based entirely on the two pairs of diagnostic ions seen in the in-line O₃-MS mass spectra. In this way, de novo identification of CLA positional isomers, i.e. without requiring comparison to CLA standards, was achieved. The Ag⁺-LC/O₃-MS method was applied to the analysis of CLA isomers in a commercial CLA supplement, milk fat, and the lipid extract from a <i>Lactobacillus plantarum</i> TMW1460 culture. The results demonstrate how Ag⁺-LC/O₃-MS can be used for the direct and fast determination of CLA isomers at low concentrations and in complex lipid mixtures