New trends in fast and high-resolution liquid chromatography: a critical comparison of existing approaches

Recent developments in chromatographic supports and instrumentation for liquid chromatography (LC) are enabling rapid and highly efficient separations. Various analytical strategies have been proposed, for example the use of silica-based monolithic supports, elevated mobile phase temperatures, and columns packed with sub-3 μm superficially porous particles (fused core) or with sub-2 μm porous particles for use in ultra-high-pressure LC (UHPLC). The purpose of this review is to describe and compare these approaches in terms of throughput and resolving power, using kinetic data gathered for compounds with molecular weights ranging between 200 and 1300 g mol⁻¹ in isocratic and gradient modes. This study demonstrates that the best analytical strategy should be selected on the basis of the analytical problem (e.g., isocratic vs. gradient, throughput vs. efficiency) and the properties of the analyte. UHPLC and fused-core technologies are quite promising for small-molecular-weight compounds, but increasing the mobile phase temperature is useful for larger molecules, for example peptides.     

Top-Down Proteomics and Direct Surface Sampling of Neonatal Dried Blood Spots: Diagnosis of Unknown Hemoglobin Variants

We have previously shown that liquid microjunction surface sampling of dried blood spots coupled with high resolution top-down mass spectrometry may be used for screening of common hemoglobin variants HbS, HbC, and HbD. In order to test the robustness of the approach, we have applied the approach to unknown hemoglobin variants. Six neonatal dried blood spot samples that had been identified as variants, but which could not be diagnosed by current screening methods, were analyzed by direct surface sampling top-down mass spectrometry. Both collision-induced dissociation and electron transfer dissociation mass spectrometry were employed. Four of the samples were identified as ??-chain variants: two were heterozygous Hb D-Iran, one was heterozygous Hb Headington, and one was heterozygous Hb J-Baltimore. The fifth sample was identified as the ??-chain variant heterozygous Hb Phnom Penh. Analysis of the sixth sample suggested that it did not in fact contain a variant. Adoption of the approach in the clinic would require speed in both data collection and interpretation. To address that issue, we have compared manual data analysis with freely available data analysis software (ProsightPTM). The results demonstrate the power of top-down proteomics for hemoglobin variant analysis in newborn samples.     

The reversible and stereoselective N- to C-bonded rearrangement of tris(2-pyridylmethyl)-1,4,7-triazacyclononanecobalt(III)

The hexaaminecobalt(III) complex [Co(tmptacn)]3+ (tmptacn = 1,4,7-tris(2'-pyridylmethyl)-1,4,7-triazacyclononane) undergoes a novel base-catalyzed N- to C-bonded rearrangement in which a tacn nitrogen is displaced by the alpha-carbon which deprotonates and binds to the metal ion as a carbanion. The X-ray structure establishes the configuration for the regio- and stereoselectively (100%) formed product. The reaction involves both ring expansions and ring contraction. The carbanion is part of a strained four-membered ring. The kinetics are reported for the N- to C-rearrangement, shown to be retentive for the optically resolved (+)-[Co(tmptacn)]3+ reactant, and also the kinetics for a competitive and somewhat faster base-catalyzed racemization reaction of this complex. The reaction is completely but very slowly reversed in acid, also with retention, and in D2O/D+ there is 1:1 D-incorporation into the two sets of inequivalent tacn carbons. Extensive 1D and 2D NMR studies establish mechanistic details, and alternative mechanisms are proposed for the forward and reverse reactions. In neutral solution, there is a competitive oxidation reaction for the reverse C- to N-bonded process, involving the regio- and stereoselective (100%) incorporation of an alpha-OH substituent into the tacn ring.     

para-Nitroaniline is a Promising Matrix for MALDI-MS Imaging on Intermediate Pressure MS Systems

para-Nitroaniline (PNA) is presented as a promising matrix for matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) on an intermediate-pressure ion source (~1 Torr) QqTOF instrument using an Nd:YVO₄ laser operated at 5 kHz. An imaging study was carried out to determine the utility of PNA at this pressure by analyzing 14 tissue sections. We demonstrate acquisition of high-quality imaging data over a 6-h period in the ion source. In this study, comparisons were made between PNA and α-cyano-4-hydroxycinnamic acid (CHCA) in positive ion mode to demonstrate the utility of PNA in these circumstances. PNA performed as well as or better than CHCA in terms of lipid ion intensities, resulting in lower levels of ion fragmentation and in lower incidences of analyte migration at the edges of the tissue sections when using airspray matrix deposition.      

Identification and Quantitative Determination of Dipropylene Glycol in Terpene Mixtures Using 13C NMR Spectroscopy

A method that allows direct identification and quantitative determination of dipropylene glycol (DPG) using ¹³C NMR spectroscopy was developed. The quantitative procedure was checked and validated with commercially available DPG, controlled with two DPG-added essential oils, and then applied to commercial “Extraits de parfum” (perfume extracts).     

Oxygen-induced changes in longitudinal relaxation times in skeletal muscle

The objective was to measure the effect of 100% oxygen inhalation on T1 relaxation times in skeletal muscle. Healthy volunteers were scanned using three different MRI protocols while breathing medical air and 100% oxygen. Measurements of T1 were made from regions of interest (ROIs) within various skeletal muscle groups. Dynamic data of subjects breathing a sequence of air-oxygen-air allowed the calculation of characteristic wash-in and -out times for dissolved oxygen in muscle. Contrary to previous findings, a statistically significant decrease in T1 in skeletal muscle was observed due to oxygen inhalation. We report approximate baseline characteristic values for the response of skeletal muscle to oxygen inhalation. This measurement may provide new biomarkers for evaluation of oxygen delivery and consumption in normal and diseased skeletal muscle.