Miniature Flow-Through Low-Temperature Plasma Ionization Source for Ambient Ionization of Gases and Aerosols.

The design and operation of an inexpensive, miniature low-temperature plasma ion source is detailed. The miniature low-temperature plasma ion source is operated in a "flow-through" configuration, wherein the gaseous or aerosolized analyte, caffeine or pyrolyzed ethyl cellulose, in a carrier gas is used as the plasma gas. In this flow-through configuration, the sensitivity for the caffeine standard and the pyrolysis products of ethyl cellulose is maintained or increased and the reproducibility of the ion source is increased. Changes in the relative intensity of ions from the aerosol produced by pyrolysis of ethyl cellulose are observed in the mass spectrum when the low-temperature plasma ion source is used in the flow-through configuration. Experiments suggest this change in relative intensity is likely due to differences in ionization efficiency rather than increased fragmentation of ethyl cellulose pyrolysis products during ionization. Flow-through low-temperature plasma ionization with the miniature ion source is shown to be a promising technique for the ionization of compounds in gases or aerosol particles.

Resolving powers of >7900 using linked scans: how well does resolving power describe the separation capability of differential ion mobility spectrometry.

Differential ion mobility spectrometry (DIMS) separations are described using similar terminology to liquid chromatography, capillary electrophoresis, and drift tube ion mobility spectrometry. The characterization and comparison of all these separations are typically explained in terms of resolving power, resolution, and/or peak capacity. A major difference between these separations is that DIMS separations are in space whereas the others are separations in time. However, whereas separations in time can, in theory, be extended infinitely, separations in space, such as DIMS separations, are constrained by the physical dimensions of the device. One method to increase resolving power of DIMS separations is to use helium in the DIMS carrier gas. However, ions have a greater mobility in helium which causes more ions to be neutralized due to collisions with the DIMS electrodes or electrode housing, i.e. the space constraints. This neutralization of ions can lead to the loss of an entire peak, or peaks, from a DIMS scan. To take advantage of the benefits of helium use while reducing ion losses, linked scans were developed. During a linked scan the amount of helium present in the DIMS carrier gas is decreased as the compensation field is increased. A comparison of linked scans to compensation field scans with constant helium is presented herein. Resolving powers >7900 are obtained with linked scans. However, this result highlights the limitation of using resolving power as a metric to describe DIMS separations.

Perspective on high-throughput bioanalysis to support in vitro assays in early drug discovery.

As the desire for a shortened design/make/test/learn cycle increases in early drug discovery, the pressure to rapidly deliver drug metabolism pharmacokinetic data continues to rise. From a bioanalytical standpoint, in vitro assays are challenging because they are amenable to automation and thus capable of generating a high number of samples for analysis. To keep up with analysis demands, automated method development workflows, rapid sample analysis approaches and efficient data analysis software must be utilized. This work provides an outline of how we implemented those three aspects to provide bioanalytical support for in vitro drug metabolism pharmacokinetic assays, which include developing hundreds of mass spectrometry methods and analyzing thousands of samples per week, while delivering a median bioanalytical turnaround time of 1-2 business days.

A Potent and Selective Kallikrein-5 Inhibitor Delivers High Pharmacological Activity in Skin from Patients with Netherton Syndrome.

Regulation of proteolytic activity in the skin plays a pivotal role in epidermal homeostasis. This is best exemplified in Netherton syndrome, a severe genetic skin condition caused by loss-of-function mutations in the gene serine protease inhibitor Kazal-type 5 encoding lympho-epithelial Kazal-type-related inhibitor, a serine protease inhibitor that regulates kallikrein (KLK)-related peptidase 5, 7, and 14 activities. KLK5 plays a central role in stratum corneum shedding and inflammatory cell signaling, activates KLK7 and KLK14, and is therefore an optimal therapeutic target. We aimed to identify a potent and selective small-molecule inhibitor of KLK5 amenable to epidermal delivery. GSK951 was identified using a structure-based design strategy and showed a half maximal inhibitory concentration of 250 pM for KLK5 and greater than 100-fold selectivity over KLK7 and KLK14. Cocrystal structure analysis identified the critical catalytic site interactions to a surrogate for KLK5. Topical application of GSK951-containing cream inhibited KLK5 activity in TgKLK5 mouse skin, reduced transepidermal water loss, and decreased proinflammatory cytokine expression. GSK951 achieved high concentrations in healthy human epidermis following topical application in a cream formulation. Finally, KLK5 protease activity was increased in stratum corneum of patients with Netherton syndrome and significantly inhibited by GSK951. These findings unveil a KLK5-specific small-molecule inhibitor with a high therapeutic potential for patients with Netherton syndrome.

In Vitro Permeation Test (IVPT) for Pharmacokinetic Assessment of Topical Dermatological Formulations.

In vitro assessment of topical (dermal) pharmacokinetics is a critical aspect of the drug development process for semi-solid products (e.g., solutions, foams, sprays, creams, gels, lotions, ointments), allowing for informed selection of new chemical entities, optimization of prototype formulations during the nonclinical stage, and determination of bioequivalence of generics. It can also serve as a tool to further understand the impact of different excipients on drug delivery, product quality, and formulation microstructure when used in parallel with other techniques, such as analyses of rheology, viscosity, microscopic characteristics, release rate, particle size, and oil droplet size distribution. The in vitro permeation test (IVPT), also known as in vitro skin penetration/permeation test, typically uses ex vivo human skin in conjunction with diffusion cells, such as Franz (or vertical) or Bronaugh (or flow-through) diffusion cells, and is the technique of choice for dermal pharmacokinetics assessment. Successful execution of the IVPT also involves the development and use of fit-for-purpose bioanalytical methods and procedures. The protocols described herein provide detailed steps for execution of the IVPT utilizing flow-through diffusion cells and for key aspects of the development of a liquid chromatography-tandem mass spectrometry method intended for analysis of the generated samples (epidermis, dermis, and receptor solution). © 2020 Wiley Periodicals LLC. Basic Protocol 1: In vitro permeation test Support Protocol: Dermatoming of ex vivo human skin Basic Protocol 2: Bioanalytical methodology in the context of the in vitro permeation test.

Variables Affecting the Internal Energy of Peptide Ions During Separation by Differential Ion Mobility Spectrometry.

Differential ion mobility spectrometry (DIMS) devices separate ions on the basis of differences in ion mobility in low and high electric fields, and can be used as a stand-alone analytical method or as a separation step before further analysis. As with other ion mobility separation techniques, the ability of DIMS separations to retain the structural characteristics of analytes has been of concern. For DIMS separations, this potential loss of ion structure originates from the fact that the separations occur at atmospheric pressure and the ions, during their transit through the device, undergo repeated collisions with the DIMS carrier gas while being accelerated by the electric field. These collisions have the ability to increase the internal energy distribution of the ions, which can cause isomerization or fragmentation. The increase in internal energy of the ions is based on a number of variables, including the dispersion field and characteristics of the carrier gas such as temperature and composition. The effects of these parameters on the intra-DIMS fragmentation of multiply charged ions of the peptides bradykinin (RPPGFSPFR) and GLISH are discussed herein. Furthermore, similarities and differences in the internal energy deposition that occur during collisional activation in tandem mass spectrometry experiments are discussed, as the fragmentation pathways accessed by both are similar. Graphical Abstract ᅟ.

Improved Differential Ion Mobility Separations Using Linked Scans of Carrier Gas Composition and Compensation Field.

Differential ion mobility spectrometry (DIMS) separates ions based on differences in their mobilities in low and high electric fields. When coupled to mass spectrometric analyses, DIMS has the ability to improve signal-to-background by eliminating isobaric and isomeric compounds for analytes in complex mixtures. DIMS separation power, often measured by resolution and peak capacity, can be improved through increasing the fraction of helium in the nitrogen carrier gas. However, because the mobility of ions is higher in helium, a greater number of ions collide with the DIMS electrodes or housing, yielding losses in signal intensity. To take advantage of the benefits of helium addition on DIMS separations and reduce ion losses, linked scans were developed. In a linked scan the helium content of the carrier gas is reduced as the compensation field is increased. Linked scans were compared with conventional compensation field scans with constant helium content for the protein ubiquitin and a tryptic digest of bovine serum albumin (BSA). Linked scans yield better separation of ubiquitin charge states and enhanced peak capacities for the analysis of BSA compared with compensation field scans with constant helium carrier gas percentages. Linked scans also offer improved signal intensity retention in comparison to compensation field scans with constant helium percentages in the carrier gas.