An LC/MS Method Providing Improved Sensitivity: Electrospray Ionization Inlet

Abstract

Electrospray ionization inlet (ESII) combines positive aspects of electrospray ionization (ESI) and solvent-assisted ionization (SAI). Similar to SAI, the analyte solution is directly introduced into a heated inlet tube linking atmospheric pressure and the initial vacuum stage of the mass spectrometer. However, unlike SAI, in ESII a voltage is applied to the solution through a metal union linking two sections of fused silica tubing through which solution flows into the inlet. Here, we demonstrate liquid chromatography (LC) ESII/MS on two different mass spectrometers using a mixture of drugs, a peptide standard mixture, and protein digests. This LC-ESII/MS approach has little dead volume and thus provides excellent chromatographic resolution at mobile phase flow rates from 1 to 55 μL min. Significant improvement in ion abundance and less chemical background ions were observed relative to ESI for all drugs and peptides tested at flow rates from 15 to 55 μL min. At a low inlet tube temperature, ESII has an ionization selectivity similar to that of ESI but, at higher inlet temperatures, appears to have the attributes of both ESI and SAI.

Novel ionization processes for use in mass spectrometry: ‘Squeezing’ nonvolatile analyte ions from crystals and droplets

“…Using a temperature range from 70 to 400 °C on an Orbitrap Q‐Exactive Focus mass spectrometer produces for 3‐NBN matrix exceptionally high analyte ion abundance without the need for high voltages or a laser , while other matrices, especially typical MALDI matrices, produce less abundant analyte ions, multiply and singly charged, and only at high temperatures. The temperature can be applied directly or indirectly to the tube, typically used as an ESI inlet . Charge separation occurs after the matrix is introduced to sub‐atmospheric pressure.…”

“…A ‘sizzling’, ‘popping’, or ‘clicking’ sound can be heard when solution is being introduced into a hot inlet (Movie S2, supporting information). In introducing solution into a heated inlet using fused‐silica tubing as in SAI, a voltage ( v SAI) can be applied to the solution to generate a mass spectrum that combines ESI and SAI with higher sensitivity than either . Freezing water or methanol solutions is an alternative means of creating charge separation and observation of analyte ions.…”

“…A number of different mass spectrometers, in terms of analyzer types and vendors, have been used with the novel ionization processes demonstrating the importance of pressure, temperature, and collisions in optimizing ion formation . An example is shown in Figure for bovine serum albumin (BSA), 66 kDa, using mass spectrometers to ionize this 66 kDa protein intact and multiply charged using the new ionization processes.…”

“…Multiply charged protein ions are readily sequenced by an ETD approach providing a means of gas‐phase peptide/protein sequencing directly from surfaces . Liquid chromatography (LC) can be hyphenated with solvent‐based inlet ionization, as is the case with ESI, and fractionation is an obvious choice with vacuum ionization, as is the case with MALDI. Interfacing with IMS adds a useful separation of gas‐phase ions from mixtures and, when combined with LC approaches, permits powerful deconvolution of sample complexity, sometimes even of isomers .…”

“…MS TM , an NSF STTR Phase I and II supported university spin‐off company, has developed a manual multifunctional ionization platform (Figure ; Movie S3, supporting information) with modules for MAI, SAI, voltage SAI, ESI, and more recently an atmospheric pressure solids probe (ASAP) . In reproducibility studies, the platform delivered a standard deviation below 5% using MAI, faring as well as the commercial ESI source and better than the vacuum MALDI source on the same instrument . The designed sources and methods work well with various solvents present including dimethyl sulfoxide (DMSO), important for the pharmaceutical industry .…”