This article presents two case studies regarding the characterization of protein-DNA complexes using two complementary multi-angle light scattering (MALS) techniques, namely size-exclusion chromatography (SEC–MALS) to determine absolute molar mass of each component, and composition-gradient MALS (CG–MALS) to quantify stoichiometry and affinity at binding sites in solution.
Post-translational modifications are potential critical quality attributes (pCQAs) routinely assessed in biotherapeutic development. Glycosylation is one of the most important attributes to assess because it affects protein function as well as antigen receptor binding. N-glycosylation of asparagine residues is the most common pCQA assessed during monoclonal antibody (mAb) therapeutic development. There are a few protocols to assess and quantitate N-glycans, but the most common approach is through an enzymatic release and labelling procedure, followed by separation and detection. This article demonstrates the method development considerations for sample preparation and chromatographic analysis of N-glycans of therapeutic mAbs.
The benefits of a robustness assessment for the analysis of a NIST mAb using a wide pore C4 LC column are described.
Forensic scientists often encounter highly complex analytical problems related to crime scenes that would benefit from the capabilities of GC×GC–MS. However, this technique has not been fully explored to help benefit forensic laboratories.
John McLean previews his plenary lecture at HPLC 2023, where he will describe emerging analytical strategies using liquid chromatography–ion mobility–mass spectrometry (LC–IM–MS) for untargeted molecular phenomics in systems, synthetic, and chemical biology.
A method is illustrated for the simultaneous analysis of ethyl glucuronide and ethyl sulfate in human urine samples.
William W. Carson, Ming Zhou, and Tom Kearney
The versatile size-exclusion ultrahigh-performance liquid chromatography (SE-UHPLC) platform method described here provides superior separation for bispecific monoclonal antibody formats compared to a previous method.
Application of multivariate statistical analysis to a combined data set demonstrated that SIFT-MS discriminates premium quality beef from eight sensory defects, and, therefore, could be applied as an instrumental grading tool, obviating sensory panel grading.
Application of multivariate statistical analysis to a combined data set demonstrated that SIFT-MS discriminates premium quality beef from eight sensory defects, and, therefore, could be applied as an instrumental grading tool, obviating sensory panel grading.
Application of multivariate statistical analysis to a combined data set demonstrated that SIFT-MS discriminates premium quality beef from eight sensory defects, and, therefore, could be applied as an instrumental grading tool, obviating sensory panel grading.
Using highly specific immunoaffinity solid-phase extraction to selectively extract and enrich aflatoxins prior to HPLC-FLD analysis, without derivatization, low LOQ values-far below the European regulations-can be achieved even in highly complex and fatty matrices.
This study assesses whether two commonly used plastic packaging materials are safe for use using liquid chromatography (LC) with high-resolution acccurate mass spectrometry (HRAMS) and data‑independent acquisition (DIA).
This study assesses whether two commonly used plastic packaging materials are safe for use. Their potential migrants are analyzed using data-independent acquisition and liquid chromatography coupled with high-resolution, accurate-mass mass spectrometry.
This study assesses whether two commonly used plastic packaging materials are safe for use using liquid chromatography (LC) with high-resolution acccurate mass spectrometry (HRAMS) and data‑independent acquisition (DIA).
Multi-detection GPC/SEC is described and new data showing how the technique is being used to determine the impact of sterilization on medical grade poly(L-lactide) is presented.
A fast, selective, and reproducible high performance liquid chromatography (HPLC) method was developed and validated for the analyses of third-generation cephalosporin antibiotics, namely, ceftriaxone, cefixime, and cefdinir in human plasma. The analysis was carried out on a 150 mm Ã- 4.6 mm, 5.0-µm C18 column. The mobile phase used was 80:20 (v/v) 50 mmM phosphate buffer (pH 5.0)–methanol at a flow rate of 1.0 mL/min with 230-nm UV detection.
The capability to separate and analyze a wide range of proteins in complex systems remains a prime requirement in the biochemical sciences. Intact protein separations are especially difficult as these large molecules can present different conformations, association states and amphoteric features with chromatographic surfaces. Combining high performance liquid chromatography (HPLC) and ultrahigh pressure liquid chromatography (UHPLC) with mass spectrometry (MS) has proven to be an effective approach for solving difficult problems involving protein analyses. Considerable effort has been made to develop columns for separating proteins with high efficiency for reversed-phase, ion-exchange, size-exclusion chromatography, hydrophilic interaction liquid chromatography (HILIC), and hydrophobic interaction chromatography (HIC). Even so, many situations still exist where insufficient resolution is available for accurate protein analysis even when high-resolution MS is available. This presentation provides a brief overview of new approaches being investigated in the author's laboratories for obtaining superior protein separations. This includes new approaches for obtaining better protein separations with columns of highly-efficient superficially porous silica particles and techniques using MS-friendly mobile phases with effective methods for changing protein selectivity (band spacings) by column type and organic mobile phase modifiers.
Forensic scientists often encounter highly complex analytical problems related to crime scenes that would benefit from the capabilities of GC×GC–MS. However, this technique has not been fully explored to help benefit forensic laboratories.
GC–MS is considered the gold standard in forensic trace evidence analysis because of its ability to chromatographically separate and analyze components in mixtures. Although GC×GC–MS has been used extensively in the oil and petroleum and flavour and fragrance industries, it has not been fully explored in the forensic sector. However, forensic scientists often encounter highly complex samples that would benefit from the capabilities of GC×GC–MS, such as, sexual lubricants, automobile paints, and tyres. GC×GC–MS analysis can allow for the deconvolution of coeluted components while providing increased sensitivity of minor components to help benefit any forensic laboratory.
Recent advances in sampling techniques in the pharmaceutical industry sparked significant interest in applying improvements to extraction methods for greater analyte detection and quantitation.
This application note outlines the performance benefits achieved with UCT’s LipiFiltr® cleanup cartridge for the analysis of pesticides in oil-based cannabis products using LC–MS/MS analysis.
This application note outlines the performance benefits achieved with UCT’s LipiFiltr® cleanup cartridge for the analysis of pesticides in oil-based cannabis products using LC–MS/MS analysis.
This application note outlines the performance benefits achieved with UCT’s LipiFiltr® cleanup cartridge for the analysis of pesticides in oil-based cannabis products using LC–MS/MS analysis.
Screening with multiple orthogonal HPLC methods provides a comprehensive alternative to single method drug impurity profiling with their complementary selectivity. One key challenge of the approach is to track the peaks across the orthogonal chromatograms and identify all unique impurities.
Biotherapeutic peptides and proteins are often PEGylated (covalently bonded to polyethylene glycol polymers) to improve bioavailability, reduce immunogenicity, and extend circulating half-life (1). Achieving the desired properties for each application depends on optimizing the number and site of polymers attached, chain length, and the degree of chain branching.