Three-dimensional liquid chromatography with parallel second dimensions and quadruple parallel mass spectrometry for adult/infant formula analysis

Three dimensions of chromatographic separation, using split-flow two-dimensional liquid chromatography (SF-2D-LC) with two parallel second dimensions, LC x 2LC, combined with quadruple parallel mass spectrometry (LC3MS4) is demonstrated for analysis of NIST SRM 1849a adult/infant formula. The first dimension, D-1, was a conventional non-aqueous reversed-phase (NARP) HPLC separation using two C18 columns in series, followed by detection using an ultraviolet (UV) detector, a fluorescence detector (FLD), with flow then split to a corona charged aerosol detector (CAD), and then dual parallel mass spectrometry (MS), conducted in atmospheric pressure photoionization (APPI) and electrospray ionization (ESI) modes. The first second dimension, D-2(1), UHPLC was conducted on a 50.0 mm C30 column using a NARP-UHPLC parallel gradient for separation of short-chain triacylglycerols (TAGs) from long-chain TAGs, with detection by UV and ESI-MS. The second dimension, D-2(2), UHPLC was conducted using a 100.0 mm C30 column with a NARP-UHPLC parallel gradient for improved separation of TAG isomers, with detection by UV, an evaporative light scattering detector, and high-resolution, accurate-mass (HRAM) ESI-MS. Transferred eluent dilution was used to refocus peaks and keep them sharp during elution in both (2)Ds. The separation space in the D-2(2) was optimized using multi-cycle (aka, "constructive wraparound") elution, which employed flow rate programming. In the D-1, calibration lines for quantification of fat-soluble vitamins were constructed. A lipidomics approach to TAG identification and quantification by HRAM-ESI-MS was applied to the D-2(2). These experiments can be represented: LCIMS2 x (LC1MS1 LC1MS1) = LC3MS4, or three-dimensional liquid chromatography with quadruple parallel mass spectrometry. Published by Elsevier B.V.