LC–MS

The main objective of this review article is to provide a clear summary of the different methods that can be used to quantify endogenous small molecules. Because of the increased use of mass spectrometry (MS) in the field of bioanalysis, a special focus will be placed on quantification by liquid chromatography (LC)–MS. Practical recommendations to face this bioanalytical challenge, in particular in terms of method validation, will also be provided.

The main objective of this review article is to provide a clear summary of the different methods that can be used to quantify endogenous small molecules. Because of the increased use of mass spectrometry (MS) in the field of bioanalysis, a special focus will be placed on quantification by liquid chromatography (LC)–MS. Practical recommendations to face this bioanalytical challenge, in particular in terms of method validation, will also be provided.

This is the fourth in a series of articles exploring current topics in separation science that will be addressed at the HPLC 2019 conference in Milan, Italy, from 16–20 June.

Anneli Kruve previews the topic of nontargeted liquid chromatography–high-resolution Mass spectrometry for HPLC 2019.

Per- and polyfluoroalkyl substances (PFAS) are chemicals found in fire‑fighting foams and consumer products requiring water-resistant and stain-repellent properties. As a result of their unique chemical properties and long‑term widespread usage, these chemicals are an emerging human health concern. US Environmental Protection Agency (EPA) released analytical methods for PFAS measurement in 2009 and most recently in November of 2018. In this article, data generated using these methods with allowed analytical modifications is presented and demonstrates robustness and reproducibility while achieving low level detection limits in drinking water.

This article demonstrates how a user-friendly vacuum-jacketed column (VJC) has been designed without the need of a large internal diameter vacuum chamber and low- and high-vacuum pumps.

A fully automated method for the effective drug screening of large populations based on dried blood spot (DBS) technology is presented. DBSs were prepared, scanned, then spiked with deuterated standards, and directly extracted, before they were transferred online to an analytical liquid chromatography (LC) column and then to the electrospray ionization tandem mass spectrometry (ESI-MS/MS) system. The method was applied to DBS samples from two patients with back pain; codeine and oxycodone could be identified and quantified accurately below the level of misuse of 89.6 ng/mL and 39.6 ng/mL, respectively.

Liquid chromatography–mass spectrometry (LC–MS) platforms are continually developing to offer improved sensitivity to meet the analytical demands of today’s laboratories. However, choosing an inappropriate solvent can significantly undermine the quality of results, even when using the most advanced technology; a high-purity mobile phase with excellent batch-to-batch consistency is essential for reliable and reproducible results. This article discusses the importance of selecting the correct grade of solvent for LC–MS analyses and some of the challenges arising from an insufficiently pure mobile phase.

Researchers from the University of Gothenburg, in Gothenburg, Sweden, have investigated whether plasma amino acid levels differed among children with celiac disease using liquid chromatography–mass spectrometry (LC–MS) (1).

Food analysis is often handled less thoroughly than pharmaceutical analysis because of the smaller life-threatening risk expected from foodstuffs. However, food analysis is still a major focus for chromatographers from a scientific and an analytical point of view. Adoption of modern “in‑silico” techniques, such as chromatographic modelling, offer analysts new possibilities for method development.

Synthetic azo- and non-azo dyes were once commonly used as food colourings in many countries. Food safety regulators in Europe, the U.S, and other countries have now banned the use of these synthetic dyes in food because of their potential genotoxic and carcinogenic effects. In some countries, however, these dyes are still being used, especially in spices. There are currently no published legal limits for these illegal food dyes, but any detectable amount is deemed unacceptable. Thus, any analytical method used to test foods for these illegal dyes must be highly sensitive. Conventional methods are only able to provide limits of quantitation (LOQs) of 10–1000 ppb for these illegal food dyes. A reversed-phase ultrahigh-pressure liquid chromatography tandem mass spectrometry (UHPLC–MS/MS) method has been developed that reliably achieves LOQs that are three-to-four orders of magnitude lower than conventional methods while also providing improved accuracy and reproducibility.

Characterization of protein modifications is an essential aspect of biopharmaceutical development. Traditionally, the characterization process of chromatographic peaks involves manual, larger-scale fractionation to obtain a sufficient amount of material for further analytical studies. This article presents a fully automated process for online peak fractionation and reduction of therapeutic antibodies with subsequent quadrupole time-of-flight mass spectrometry (QTOF-MS) characterization. This innovative technique significantly accelerates MS peak characterization compared to traditional approaches and avoids the risk of unintended modifications of the variants as a result of the isolation process, for example, deamidation during storage of isoforms. This approach considerably reduces the required sample amount and can be used for the characterization of product-related impurities during early stage development.

Biotherapeutics must endure in-depth testing to validate their efficacy and safety before their release to the medical community. Characterization and quantitation of these large molecule medicines is traditionally performed with ligand binding assays or radiolabeling procedures. Issues with selectivity, accuracy, and unavailability of applicable assays for the characterization and quantitation of certain biotherapeutics means that liquid chromatography–mass spectrometry (LC–MS) is becoming an increasingly selected method for biotherapeutics testing. Typically used for small molecules, LC–MS can be adapted for larger molecule analysis with additional high throughput and multiplexing capabilities. New method development has turned LC–MS into a highly sensitive option for biotherapeutics validation.

Polyphenols are a well-known group of antioxidants widely diffused as secondary metabolites in plants, vegetables, and fruit. The Column spoke to Nicola Marchetti from the Department of Chemistry and Pharmaceutical Sciences at the University of Ferrara in Ferrara, Italy, about his research into the characterization of polyphenols in red chicory using high performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS).

Tips for effective use of chromatography and mass spectrometry for the analysis of antibody–drug conjugates, glycoengineered proteins, and biosimilars.

In metabolomics, LC–MS is a popular technique because of its ability to separate a wide range of metabolites, but the presence of many highly polar analytes in these samples poses a challenge. Benzoyl chloride derivatization can be a practical solution, improving both sensitivity and selectivity.

Wastewater analysis has become an established approach for retrieving additional epidemiological information about the use of illicit drugs, alcohol, and tobacco at the population level. Here, we present an overview of the recent analytical frameworks and workflows for target and suspect analyses using low- and high-resolution mass spectrometry and discuss the latest advances in wastewater-based epidemiology (WBE).

Leading separation scientists share their perspectives on current challenges in separation science and where the field is heading.

Practical examples of how to correct for matrix effects in food testing to obtain reliable quantitative data using LC–MS and GC–MS

We present an empirical assessment of summation integration involving 490 low-pressure GC–MS/MS analyses of 70 pesticides in 10 common fruits and vegetables over the course of 10 days.

Lipidomics, the analysis of lipids by mass spectrometric methods, revolutionized lipid science (1). It provides detailed quantitative information on hundreds of lipid species and opens new possibilities to gain an insight into lipid biology. This helps not only to explain the vital role of lipid species as membrane building blocks, but also to unravel their bioactive functions. Thus, lipid species can act as signaling molecules and modulate membrane properties, which are essential for organelle and membrane protein function. Moreover, the first examples demonstrated their potential as novel biomarkers to monitor human health.

Upon testing with 919 samples, this method produced excellent results.

A cross-disciplinary team of researchers in Tasmania from the fields of separation science, proteomics and metabolomics, immunology and zoology are on a mission to save the Tasmanian devil from extinction using metabolic fingerprinting of serum to identify biomarkers for Devil Facial Tumour Disease (DFTD). The Column spoke to Naama Karu, Rodrigo Hamede Ross, and Richard Wilson to find out more.

Parallel artificial liquid membrane extraction (PALME) is a miniaturized version of liquid–liquid extraction (LLE) and is based on two 96-well plates in a sandwich-like configuration. With a very simple workflow, 96 samples can be processed simultaneously in PALME, providing analyte enrichment, highly efficient sample cleanup, and direct compatibility with liquid chromatography–mass spectrometry (LC–MS). The consumption of hazardous organic solvents is also almost eliminated using PALME as the sample preparation technique. This article summarizes current experiences with PALME, based on work both in a university laboratory and in an analytical services contract laboratory.

Using a liquid chromatography–mass spectrometry (LC–MS) method in conjunction with two complementary types of chromatographic retention modes - reversed phase and aqueous normal phase - various compounds present in mesquite flour extracts were identified. Because of the diverse types of chemical constituents found in such natural product extracts, a single chromatographic mode may not be sufficient for a comprehensive characterization. However, the combination of reversed-phase and aqueous normal phase LC can encompass a wide range of analyte polarity. This characterization of the composition of mesquite flour could be used in future studies to elucidate the beneficial health effects of its consumption.

Part IV of this series takes a closer look at clustering. Clustering can be very useful at observing your data when the sample dimensionality is large. This is a barbarian term meaning that diversity among your samples may be wide. In that case, the space reduction provided by principal component analysis (PCA) is not always convincing, because the simplification provided by a single two-dimensional plot erases too much information. Clustering allows you to preserve more information.

Tips and tricks to help you recognize and solve the most common SPE problems.

How to optimize the key variables in LC–MS analysis

Here we propose an exemplary workflow for the analysis of phenolic extracts (i.e. wine) enabling confident differential analysis using high performance liquid chromatography in combination with low-field drift tube ion mobility quadrupole time-of-flight mass spectrometry (HPLC×IMS-QTOFMS). In this workflow, single-field collisional cross section values from low-field drift-tube IMS using nitrogen as drift gas (DTCCSN2) are readily extracted in addition to a retention time and a high resolution mass spectrum for each compound. “Alternating frames” experiments utilizing post-drift tube fragmentation also allow drift time-aligned MS/MS spectra to be obtained. Molecular feature extraction was highly repeatable with average precision values of 0.28% for retention time, 0.18% for drift time, and 1.5 ppm m/z determined for 233 molecular features found in all six technical replicates. The improved selectivity of this strategy increases confidence in intersample molecular feature alignment (i.e. compound identity confirmation), including the resolution of co-eluting isomeric compounds.

The power of nontargeted metabolite profiling is illustrated in a study focused on the determination of molecular markers in malting barley that are predictive of desirable malting quality for brewing applications. The metabolite extraction, detection, and analysis methods are high throughput and reproducible, and therefore, this approach represents a practical addition to the plant breeder’s molecular toolbox.