Fast Reversed-phase Liquid Chromatography Research Articles

Comprehensive Lipidomics Analysis of Bioactive Lipids in Complex Regulatory Networks

In the present work we describe the development of an analytical technique for simultaneous profiling of over 100 biochemically related lipid mediators in biological samples. A multistep procedure was implemented to extract eicosanoids and other bioactive lipids from the biological matrix, chromatographically separate them using fast reversed-phase liquid chromatography, tentatively identify new candidate eicosanoids through a matching process of retention times, isotope distribution patterns, and high-resolution orbitrap MS/MS fragmentation patterns, and subsequently quantify tentative candidates by means of analytical reference standards. Key new aspects of this profiling technique included the classification of bioactive lipids into 12 groups according to their calculated exact masses and the development of optimized liquid chromatographic conditions for these groups to achieve sufficient separation of the numerous isobaric and isomeric species, many of which exhibited virtually identical collision-induced dissociation behavior. Importantly, no analytical standards were required at this screening stage of the assay, and tentative identifications were achieved by matching results to selected reference species from each of the groups. The analytical figures of merit for the orbitrap assay such as linear dynamic range, limit of detection, limit of quantitation, and precision demonstrated that the performance of the assay was very similar to that of a quadrupole linear ion trap assay, which was used for validation purposes. The method allowed us to examine eicosanoid profiles within the signaling cascade in chronic lymphocytic leukemia (CLL) cells under basal conditions and following arachidonic acid stimulation. The preliminary screening based on high-resolution tandem mass spectrometry data along with isotope pattern and retention time matching revealed the presence of 15 bioactive lipids, belonging to a range of prostaglandin, leukotriene, and hydroxy and epoxy fatty acid lipid mediators produced by CLL cells.

On‐Column Hydrolysis Kinetics Determination of Boronic Pinacol Ester Intermediates for Use in Optimization of Fast HPLC Methods

Boronic acid and boronic ester intermediates are the basis for Suzuki coupling and Petasis reactions that are widely used in pharmaceutical synthetic schemes. The analysis of these compounds utilizing traditional reversed phase liquid chromatography (RPLC) is complicated by the potential of on‐column hydrolysis. In order to effectively develop and optimize RPLC methods for accurate analysis of these compounds, a better understanding of the potential on‐column hydrolysis needs to be achieved. Kinetic studies of this type of on‐column hydrolysis were performed utilizing a stop flow kinetic approach. The rate of on‐column hydrolysis was determined as a function of initial organic composition, mobile phase pH, and column temperature. In addition, the Arrhenius activation energy was calculated. A fast reversed phase liquid chromatography method was then developed and optimized to minimize on‐column hydrolysis effects based on the garnered information. The method was applied to successfully resolve ten different boronic acid and boronic pinacol ester functionalized compounds within five minutes. Thus, the wide range applicability of fast RPLC technology for accurate analysis of this specific class of compounds was demonstrated.

Application of Fast Reversed Phase Liquid Chromatography for Analysis of Pharmaceutical Related Boronic Acid and Boronic Pinacol Ester Functionalized Compounds

Boronic acid and boronic pinacol ester functionalized compounds are the basis for Suzuki coupling and Petasis reactions that are widely used in pharmaceutical synthetic schemes. The purity analysis of these compounds utilizing traditional reversed phase liquid chromatography methodology is complicated by the potential of on‐column hydrolysis. Therefore, a fast liquid chromatography method was developed to minimize on‐column hydrolysis effects. The method was applied as an in‐process control method for the separation of three process related impurities from a boronic pinacol ester functionalized intermediate with no appreciable on‐column hydrolysis observed. The method was then optimized to successfully resolve ten different boronic acid and boronic pinacol ester functionalized compounds within five minutes. Thus, the wide range applicability of fast liquid chromatographic technology to this specific class of compounds was demonstrated.

Ultrahigh-Throughput Proteomics Using Fast RPLC Separations with ESI-MS/MS

We describe approaches for proteomics analysis using electrospray ionization-tandem mass spectrometry coupled with fast reversed-phase liquid chromatography (RPLC) separations. The RPLC separations used 50-microm-i.d. fused-silica capillaries packed with submicrometer-sized C18-bonded porous silica particles and achieved peak capacities of 130-420 for analytes from proteome tryptic digests. When these separations were combined with linear ion trap tandem mass spectrometry measurements, approximately 1000 proteins could be identified in 50 min from approximately 4000 identified tryptic peptides; approximately 550 proteins in 20 min from approximately 1800 peptides; and approximately 250 proteins in 8 min from approximately 700 peptides for a S. oneidensis tryptic digest. The dynamic range for protein identification with the fast separations was determined to be approximately 3-4 orders of magnitude of relative protein abundance on the basis of known proteins in human blood plasma analyses. We found that 55% of the MS/MS spectra acquired during the entire analysis (and up to 100% of the MS/MS spectra acquired from the most data-rich zone) provided sufficient quality for identifying peptides. The results confirm that such analyses using very fast (minutes) RPLC separations based on columns packed with microsized porous particles are primarily limited by the MS/MS analysis speed.