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Dynamic pH junction focusing and isoelectric focusing in capillary electrophoresis mass spectrometry Wang, Lingyu

Abstract

Capillary electrophoresis−mass spectrometry (CE−MS) is complementary to liquid chromatography−mass spectrometry (LC−MS) because of the orthogonal separation mechanism. An electrospray ionization (ESI) interface is the pivotal device in CE-MS and its emitter location relative to the MS inlet orifice has a significant influence on both ESI stability and detection sensitivity. A flow-through microvial interface provides a path for ion transmission between the CE and the MS systems. Based on the microvial interface with its small dilution volume and asymmetric emitter geometry, CE−MS concentration sensitivity can arrive at the nanomolar level with often-used mass spectrometers. To further improve the concentration sensitivity in CE–MS, dynamic pH junction focusing has been studied. By a focusing mechanism similar to isoelectric focusing (IEF), pH junction method can achieve a significant on-line preconcentration effect on weakly alkaline and zwitterionic analytes prior to the CE separation. Dynamic pH junction and its technical variant, dynamic pH barrage focusing have been investigated both theoretically and experimentally regarding peak symmetry, capturing efficiency, quantitative performance and compatibility to MS detection. Simulation-based optimization provided theoretical evidences that both configurations can capture targeted analyte with negligible or no loss during analyte focusing, giving adequate calibration curve linearity. Most theoretical predictions, alongside the capability of selective focusing in complex matrices, were verified by experimental evaluations with optical or MS detection. High resolution capillary isoelectric focusing mass spectrometry (cIEF−MS) enabled by the microvial interface is an attractive method for the charge heterogeneity characterization of biopolymers. Peptide and protein were used to demonstrate the feasibility of two online electrolyte configurations. pI resolution up to 0.02 pH in neutrally-coated capillaries demonstrated the high separation power. Isotopically resolved mass spectra collected by an Orbitrap MS instrument provided protein structural information. Analyses of intact monoclonal antibodies (mAb), including infliximab and adalimumab, an unidentified mAb and a reference mAb, showed the potential of cIEF–MS for charge variant separation and online identification in the biopharmaceutical industry. Automated cIEF–MS analysis shortened separation and identification process of mAb charge variants from days to approximately 2 h.

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