Comprehensive Dual Liquid Chromatography with Quadruple Mass Spectrometry (LC1MS2 × LC1MS2 = LC2MS4) for Analysis of...

Online two-dimensional (2D) comprehensive liquid chromatography (LC × LC) has become increasingly popular. Most LC × LC separations employ one or more detectors at the outlet of the second dimension, ²D, with very short runs to avoid undersampling. We used six detectors, including dual parallel mass spectrometry (LC1MS2), for detection of the first dimension, ¹D. We made an argentation (silver-ion) UHPLC column from a strong cation exchange column for ²D, coupled with UV and LC1MS2 detection. LC1MS2 in ¹D combined with LC1MS2 in ²D, plus five other detectors, constituted LC2MS4 in a comprehensive LC1MS2 × LC1MS2 2D-LC separation. Electrospray ionization (ESI) high resolution accurate mass (HRAM) mass spectrometry (MS) and atmospheric pressure chemical ionization (APCI) MS were used in parallel for ¹D detection, while atmospheric pressure photoionization (APPI) MS and ESI-MS were used for detection of ²D. The LC1MS2 used for ¹D allowed quantification of triacylglycerol (TAG) molecular species of Parinari curatellifolia and other seed oils, while the ²D allowed isomers of TAG containing 18:3 fatty acyl chains as well as TAG regioisomers to be separated and identified. The LC1MS2 in ¹D allowed identification of oxo-TAG species by HRAM MS and quantification of 806.3 ± 1.3 and 1101 ± 22 μg/g of α- and γ- tocopherols, respectively, in P. curatellifolia by APCI-MS. It is now feasible to use silver-ion UHPLC as the ²D separation in LC × LC and to use multiple mass spectrometers across both dimensions to perform conventional quantitative analysis and to take advantage of the newest LC × LC separation technology to identify isomers that are otherwise difficult to separate.