Chromatographic retention behaviour of monosubstituted benzene derivatives on porous graphitic carbon and octadecyl-bonded silica studied using molecular modelling and quantitative structure–retention relationships

Matteis, Cristina I. De; Simpson, David A.; Euerby, Melvin R.; Shaw, P. Nicholas

doi:10.1016/j.chroma.2011.12.090

Cited by 19 publications

(11 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Porous graphitic carbon (PGC) constitutes an interesting option, as it has been shown to separate highly polar or charged analytes (Pereira 2008;Michel and Buszewski 2009) and structurally closely related species, such as isomers (Gundersen 2001;De Matteis et al 2012). PGC consists of planar, two-dimensional sheets composed of hexagonally arranged carbon atoms where each sp 2 hybridized carbon is joined to three adjacent carbons, creating an extensive polyaromatic scaffold (West et al 2010).…”

Section: Introductionmentioning

confidence: 99%

Nano LC-MS using capillary columns enables accurate quantification of modified ribonucleosides at low femtomol levels

Sarin

Kienast

Leufken

et al. 2018

RNA

View full text Add to dashboard Cite

Post-transcriptional chemical modifications of (t)RNA molecules are crucial in fundamental biological processes, such as translation. Despite their biological importance and accumulating evidence linking them to various human diseases, technical challenges have limited their detection and accurate quantification. Here, we present a sensitive capillary nanoflow liquid chromatography mass spectrometry (nLC-MS) pipeline for quantitative high-resolution analysis of ribonucleoside modifications from complex biological samples. We evaluated two porous graphitic carbon (PGC) materials and one end-capped C18 reference material as stationary phases for reversed-phase separation. We found that these matrices have complementing retention and separation characteristics, including the capability to separate structural isomers. PGC and C18 matrices yielded excellent signal-to-noise ratios in nLC-MS while differing in the separation capability and sensitivity for various nucleosides. This emphasizes the need for tailored LC-MS setups for optimally detecting as many nucleoside modifications as possible. Detection ranges spanning up to six orders of magnitude enable the analysis of individual ribonucleosides down to femtomol concentrations. Furthermore, normalizing the obtained signal intensities to a stable isotope labeled spike-in enabled direct comparison of ribonucleoside levels between different samples. In conclusion, capillary columns coupled to nLC-MS constitute a powerful and sensitive tool for quantitative analysis of modified ribonucleosides in complex biological samples. This setup will be invaluable for further unraveling the intriguing and multifaceted biological roles of RNA modifications.

show abstract

Section: Introductionmentioning

confidence: 99%

Nano LC-MS using capillary columns enables accurate quantification of modified ribonucleosides at low femtomol levels

Sarin

Kienast

Leufken

et al. 2018

RNA

View full text Add to dashboard Cite

show abstract

“…Nonpolar analytes interact with the PGC surface via dispersive forces while polar ones are retained via charge induction on the polarizable PGC surface. The mobile phase is a major determinant of retention because it influences the ionization state of both analytes and the PGC surface . We hypothesized that failure to maintain the same PGC surface before and after running a gradient is a cause of the observed retention loss/variability on PGC.…”

mentioning

confidence: 99%

Understanding the Complexity of Porous Graphitic Carbon (PGC) Chromatography: Modulation of Mobile-Stationary Phase Interactions Overcomes Loss of Retention and Reduces Variability

2016

View full text Add to dashboard Cite

Porous graphitic carbon (PGC) is an important tool in a chromatographer’s armory that retains polar compounds with mass spectrometry (MS)-compatible solvents. However, its applicability is severely limited by an unpredictable loss of retention, which can be attributed to contamination. The solutions offered fail to restore the original retention and our observations of retention time shifts of gemcitabine/metabolites on PGC are not consistent with contamination. The mobile phase affects the ionization state of analytes and the polarizable PGC surface that influences the strength of dispersive forces governing retention on the stationary phase. We hypothesized that failure to maintain the same PGC surface before and after running a gradient is a cause of the observed retention loss/variability on PGC. Herein, we optimize the choice of mobile phase solvent in a gradient program with three parts: a preparatory phase, which allows binding of analytes to column; an elution phase, which gives the required separation/peak shape; and a maintenance phase, to preserve the required retention capacity. Via liquid chromatography/tandem mass spectrometry (LC-MS/MS) analysis of gemcitabine and its metabolites extracted from tumor tissue, we demonstrate reproducible chromatography on three PGC columns of different ages. This approach simplifies use of the PGC to the same level as that of a C-18 column, removes the need for column regeneration, and minimizes run times, thus allowing PGC columns to be used to their full potential.

show abstract

“…The functional groups of acidic glycans, phosphates and carboxyls, are readily deprotonated at pH 5.5 and bind more strongly to the instant dipoles of graphite (Figure S-4). ,, We could detach acidic glycans from PGC by the addition of low pH solvent, promoting the protonation of negatively charged species. Finally, different types of acidic glycans such as phosphorylated and sialylated glycans were also fractionated by simply changing the SPE solution.…”

Section: Resultsmentioning

confidence: 99%

Comprehensive Characterization of Biotherapeutics by Selective Capturing of Highly Acidic Glycans Using Stepwise PGC-SPE and LC/MS/MS

Seo

Park

et al. 2019

Anal. Chem.

View full text Add to dashboard Cite

The glycosylation of biologics is an important factor in pharmacological functions such as efficacy, safety, and biological activity and is easily affected by subtle changes in the cellular environment. Hence, comprehensive and in-depth glycomic characterization of biological products should be performed to ensure product quality and process consistency prior to regulatory approval, but it is still highly challenging due to glycan microheterogeneity produced by enzymatic machinery. In this study, we have developed a systematic methodology for the separation and characterization of various glycans of biotherapeutics using the combination of solid-phase extraction (SPE) and high resolution LC/MS. Neutral and multiple-acidic glycans were selectively fractionated by SPE with a porous graphitized carbon (PGC) cartridge according to their molecule size and polarity (acidity, pK a). Subsequent LC-MS and -MS/MS analyses enabled us to obtain glycan compositions, structures, and quantitative information. Indeed, we have successfully performed glycomic characterization of agalsidase-beta, a representative therapeutic enzyme containing both phosphorylated and sialylated glycans. In addition, a comparative analysis of functional glycans released from different batches of enzymes was performed to verify our method. These results suggest that stepwise PGC-SPE and LC/MS/MS pairwise assays can be used as an efficient tool to detect glycosylation changes of therapeutic glycoproteins including abundant acidic species in biologics or biosimilar development.

show abstract

Chromatographic retention behaviour of monosubstituted benzene derivatives on porous graphitic carbon and octadecyl-bonded silica studied using molecular modelling and quantitative structure–retention relationships

Cited by 19 publications

References 27 publications

Nano LC-MS using capillary columns enables accurate quantification of modified ribonucleosides at low femtomol levels

Nano LC-MS using capillary columns enables accurate quantification of modified ribonucleosides at low femtomol levels

Understanding the Complexity of Porous Graphitic Carbon (PGC) Chromatography: Modulation of Mobile-Stationary Phase Interactions Overcomes Loss of Retention and Reduces Variability

Comprehensive Characterization of Biotherapeutics by Selective Capturing of Highly Acidic Glycans Using Stepwise PGC-SPE and LC/MS/MS

Contact Info

Product

Resources

About