Increasing the operating temperature of the liquid chromatography (LC) column has the same effect as reducing the diameter of the packing particles on minimizing the contribution of C-term in the van Deemter equation, flattening the curve of plate height vs. linear velocity in the high-speed region, thus allowing a fast LC analysis without the loss of plate count. While the use of smaller particles requires a higher pumping pressure, operating the column at higher temperature reduces the pressure due to lower liquid viscosity. At present, the adoption of high-temperature LC lags behind the ultra-high-pressure LC. Nevertheless, the availability of thermally stable columns has steadily improved and new innovations in this area have continued to emerge. This paper gives a brief review and updates on the recent advances in high-temperature liquid chromatography (HTLC). Recent efforts of hyphenating the capillary HTLC with mass spectrometry via a super-atmospheric pressure electrospray ionization is also reported.

Direct analysis in real time mass spectrometry (DART MS) is one of the first ambient ionization methods to be introduced and commercialized. Analysis by DART MS requires minimal sample preparation, produces nearly instantaneous results, and provides detection over a broad range of compounds. These advantageous features are particularly well-suited for the inherent complexity of natural product analysis. This review highlights recent applications of DART MS for species identification by chemotaxonomy, chemical profiling, genetic screening, and chemical spatial analysis from plants, insects, microbes, and metabolites from living systems.

Lipids, a class of biomolecules, play a significant role in the physiological system. In this study, gas-phase hydroxyl radicals (OH·) and atomic oxygens (O) were introduced into the collision cell of a triple quadruple mass spectrometer (TQ-MS) to determine the positions of the double bond in unsaturated phospholipids. A microwave-driven compact plasma generator was used as the OH·/O source. The reaction between OH·/O and the precursor ions passing through the collision cell generates product ions that correspond to the double bond positions in the fatty acyl chain. This double bond position specific fragmentation process initiated by the attachment of OH·/O to the double bond of a fatty acyl chain is a characteristic of oxygen attachment dissociation (OAD). A TQ-MS incorporating OAD, in combination with liquid chromatography, permitted a high throughput analysis of the double bond positions in complex biomolecules. It is important to know the precise position of double bonds in lipids, since these molecules can have widely different functionalities based on the position of the double bonds. The assignment of double bond positions in a mixture of eight standard samples of phosphatidylcholines (phospholipids with choline head groups) with multiple saturated fatty acyl chains attached was successfully demonstrated.

Hydrogen/deuterium exchange (HDX) coupled with pepsin digestion is useful for rapidly analyzing the kinetic properties of small amounts of protein. However, the analysis of HDX by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) is time-consuming due to a lack of dedicated software. Currently available software programs mainly calculate average mass shifts, even though the isotopic distribution width contains information regarding multiple protein conformations. Moreover, HDX reaction samples are typically composed of peptides that contain various numbers of deuterium atoms, which also hinders the rapid and comprehensive analysis of protein dynamics. We report here on the development of a software program “Scipas DX” that can be used to automatically analyze the hydrogen–deuterium isotopic distribution in peaks in HDX spectra and calculate the average number of atoms exchanged, the average deuteration ratio, the abundance ratio for exchanged atoms, and their fitted spectra with a high degree of accuracy within a few minutes. Analysis of the abundance ratio for exchanged atoms of a model protein, adenylate kinase 1, using Scipas DX indicate that the local structure at residues 83–106 and 107–117 are in a slow equilibrium, suggesting that these regions adopt multiple conformations that are involved in the stability and in switching between the active and inactive forms. Furthermore, precise HDX kinetics of the average deuteration ratio both confirmed the known induced conformations of two regions (residues 46–75 and 131–165) that are responsible for ligand binding and verified the novel structural dynamics of residues 107–117 and 166–196 following ligand binding to ligand-binding pockets 1 and 2, respectively. Collectively, these results highlight the usefulness and versatility of Scipas DX in MALDI-MS HDX-based analyses of protein dynamics.

We describe systematic troubleshooting of the carry-over of neuropeptide Y (NPY) in LC-MS analysis. The objective was to remove candidate parts of the LC-MS system that are responsible for carry over one-by-one. The findings indicate that the carry-over of NPY occurs on the column, particularly in the guard column and at the consumable seals of the sample-needle and high-pressure valves. The methodology demonstrates that it is possible to troubleshoot carry-over in an LC-MS system in a systematic and logical manner.