|Byrdwell, W Craig|
|HARI, KOTAPTI - University Of Maryland|
|JAKUBEC, PAVEL - Charles University, Czech Republic|
|NOVAKOVA, LUCIE - Charles University, Czech Republic|
Submitted to: Journal of Chromatography
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/5/2022
Publication Date: 11/22/2022
Citation: Byrdwell, W.C., Hari, K.K., Goldschmidt, R.J., Jakubec, P., Novakova, L. 2022. Three-dimensional liquid chromatography with parallel second dimensions and quadruple parallel mass spectrometry for infant formula. Journal of Chromatography. 1661:462682. https://doi.org/10.1016/j.chroma.2021.462682.
Interpretive Summary: Complex samples require more separating power to isolate the components than normal separation techniques, like liquid chromatography, allow. Therefore, we had to develop new separation techniques that provide multiple dimensions of separation, in parallel. We used a three-dimensional separation, in which fats (triacylglycerols) are partially separated in the first dimension, further separated in a second dimension, and even further separated in a second second dimension. We combined three dimensions of separation with four detectors that separate compounds by mass (mass spectrometers). Then, we applied computer-aided analysis of lipids (called 'lipidomics') to identify and give peak areas for all identified compounds. Then, we used spreadsheets to calculate the percentage composition of fats in a model adult/infant formula. The techniques demonstrated here serve as a model for analysis of cow milk, beans, and other samples.
Technical Abstract: Three dimensions of chromatographic separation, using split-flow two-dimensional liquid chromatography (SF-2D-LC) with two parallel second dimensions, combined with quadruple parallel mass spectrometry (LC3MS4) is demonstrated for analysis of NIST SRM 1849a adult/infant formula. The first dimension, 1D, 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, 2D(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 second dimension, 2D(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 2Ds. The separation space in the 2D(2) was optimized using multi-cycle (aka, “constructive wraparound”) elution, which employed flow rate programming. In the 1D, 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 2D(2). These experiments can be represented: LC1MS2 × (LC1MS1 + LC1MS1) = LC3MS4, or three-dimensional liquid chromatography with quadruple parallel mass spectrometry.