Biological, Medical, and Clinical Analysis

This is the first article in a four-part series exploring the quantitative assessment of drugs and their metabolites in biological fluids (such as blood, plasma, and urine) and tissue homogenates using liquid chromatography–mass spectrometry (LC–MS).

This article is the first of four on the bioanalysis of small-molecule drugs and metabolites by liquid chromatography–mass spectrometry (LC–MS). In this article, we provide an overview of the fundamentals, workflow, regulations, and modern trends on these quantitative assays.

A simple and rugged method to determine psychoactive drugs using automated SPE–LC–MS/MS is described.

The Column spoke to Martin Giera of Leiden University Medical Center about his innovative research using GC–MS in bioanalysis applications.

A look at the LC and LC–MS methods that are helping to overcome challenges in sample analysis, and how this can make adeno‑associated virus (AAV)–based gene therapy more accessible.

Biomarker studies using exhaled breath are rapidly emerging as a technique for early disease detection and precision medicine. By offering a completely non-invasive experience for patients as an alternative to painful biopsy procedures. A new approach has the potential to enhance patient compliance, while making clinical workflows simpler. Exhaled breath analysis, however, requires a highly sensitive analytical technique capable of accurately measuring the broad range of volatiles present in breath. In this article, we present a proof-of-concept study to demonstrate a reliable and sensitive method to detect analytes in breath samples. Using high‑resolution accurate mass (HRAM) mass spectrometry (MS), the method validates how low- and high-abundance biomarkers can be quantified from exhaled breath.

A novel modular bioreactor for dynamic in vitro studies has been set-up, connecting two-dimensional (2D) scaffolds and mimicking a multi-organ model, to study the absorption/metabolization of compounds. The effect of dietary methylglyoxal, a potentially exogenous and endogenous toxic compound, on a dynamic gastro-intestinal system has been evaluated. Bioreactors represent a powerful advance in comparison with conventional in vitro static assays and could be a potential alternative to animal testing in the future.

The Column spoke to Nelson Roberto Antoniosi Filho, a professor at the Chemistry Institute of the Federal University of Goiás (UFG), in Goiânia, Brazil, about his development of a gas chromatography–mass spectrometry (GC–MS) method for cancer diagnosis using cerumen.

Therapeutic drug monitoring (TDM) is an important method for determining both dose and optimal effective level of a drug in the blood, preventing side effects such as kidney damage. Using the antibiotic classes aminoglycoside and vancomycin as examples, an analysis method is presented that combines fully automated sample preparation with ultrahigh-pressure liquid chromatography–triple quadrupole-mass spectrometry (UHPLC–TQMS).

The Column spoke to Arcadius V. Krivoshein, Assistant Professor of Chemistry at the University of Houston–Clear Lake, USA, about his work developing EPP, an experimental anticonvulsant that can help to stop convulsions during epileptic seizures, and the role of chiral HPLC in this research.

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.

The discovery and development of biopharmaceuticals that target specific diseases can be transformative for people living with illness. However, bringing a new therapy to market is a prolonged and costly process mired in uncertainty. Ensuring safety, efficacy, and product quality is paramount. Biopharmaceuticals, by their nature, are highly complex. A myriad of heterogeneity can be intentionally functional, an unwanted consequence of manufacturing and storage, or generated by biological modification in vivo. Not all, but some post-translational modifications or biotransformations can impact development, manufacturing, safety, efficacy, and overall product quality. These critical quality attributes (CQAs) need to be identified, characterized, controlled, and monitored throughout the drug discovery and development cycle. Specialty measurement using mass spectrometry (MS) continues to play an ever‑increasing role across the continuum.

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.

The application of a novel approach that enables a selective proteolysis of the Fab region of monoclonal antibodies (mAbs) is described. The technique is called nano-surface and molecular-orientation limited (nSMOL) proteolysis. By restricting the proteolysis to the complementarity-determining region (CDR) of the mAb, there is considerable reduction in sample complexity and the time required for method set-up and optimization.

The chemical messages that animals use to communicate can trigger a range of responses in members of the same species. The Column spoke to Jorge Saiz from the Centre of Metabolomics and Bioanalysis (CEMBIO) at the University San Pablo CEU, Spain, about his research into the chemical secretions of lizards and the role of gas chromatography–tandem mass spectrometry (GC–MS/MS) in his work.

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.

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

The importance of glycosylated structures in modern biology and medicine has been beyond dispute for many years, but there are still gaps in biochemical understanding. The current realization that virtually all major human diseases have been associated with glycosylation changes demands in-depth structural studies of these highly complex glycobiomolecules. Glycoscience with its many directions and a broad scope in both prokaryotic and eukaryotic systems is currently securing its place at the centre stage of modern biological research.

In the biomedical research of molecular bases of both normal and pathological biological processes, it is currently necessary not only to detect, identify, and quantify individual compounds, but also to study their interactions with endo- and exogenous compounds. Obviously, for these purposes it is crucial to develop new advanced high‑performance analytical methods providing high sensitivity, high selectivity, and high throughput. These challenging requirements are well met by capillary electromigration (CE) methods. They have developed in the last three and half decades into high‑performance separation techniques suitable for the analysis of a wide spectrum of both low- and high‑molecular mass bioactive compounds.

The technical requirements for a successful LC–MS/MS method for the quantitation of biopharmaceuticals are presented and the advantages and disadvantages compared to ligand-binding assays are evaluated.

Sample preparation techniques in bioanalysis are multistep, time-consuming, and labour-intensive procedures that can take up 60–80% of the total analysis time. Sample preparation is often the limiting step of fast bioanalysis and the most error-prone part of the analytical method. There is currently a focus on improving the sample preparation process by shortening sample preparation time, cutting the cost of analysis, decreasing sample volume and solvent consumption, reducing the number of sample preparation steps, and adapting the whole process for automation. This article explores microextraction techniques, selective approaches, on-line sample preparation, and dried matrix spots that aim to provide solutions to sample preparation problems in bioanalysis.

The new generation of high-resolution mass spectrometry (HRMS) systems offers high sensitivity, dynamic range, resolution, accuracy, and scan-to-scan reproducibility, enabling high-throughput quantitative analyses in combination with information-rich qualitative data. The most recently released HRMS systems offer an alternative to triple quadrupole (TQ)-MS systems. This provides a huge opportunity to obtain quantitative and qualitative information from one analysis, but also requires a different mindset and expertise to make the right choices and compromises to get the most information from your sample.

Ian Wilson was a principal speaker at The Chromatographic Society’s Diamond Jubilee meeting on “Advances in Microcolumn and Related Separation Technologies”, which took place in London on 22 March this year. In this article Ian and co-author Rob Plumb elaborate on the theme of the meeting.

The last decade has seen a series of advances in the field of liquid chromatography that have resulted in improvements for many clinical diagnostic services. These innovations have included the expansion of superficially porous particle columns, new or improved stationary phase options, and “user-friendly” multiple-channel HPLC instrument options that allow sequential analysis-a boon for low and moderate throughput laboratories with limited hardware. As a result, diagnostic services are able to offer faster turn-around-times and measure analytes in patient types and disease states that were previously problematic. This article presents examples of the impact these innovations have had in a number of hospital settings.

The characterization of transcripts, proteins, peptides, and metabolites in cells is important to study disease mechanisms and develop novel therapeutics. Peter Nemes - from the George Washington University, Washington, USA - spoke to The Column about the important role of capillary electrophoresis–electrospray ionization (CE–ESI) and time-of-flight mass spectrometry (TOF-MS) in this area of research.

Hydrophilic interaction chromatography (HILIC) was introduced more than two decades ago and has garnered much attention. Characterized by a hydrophilic stationary phase used in combination with an aqueous organic mobile phase, numerous improvements have been achieved and HILIC is now considered as an attractive alternative to reversed-phase phase liquid chromatography (LC) for many applications. HILIC provides several advantages over reversed-phase LC for the analysis of polar compounds, including higher retention of polar metabolites, enhanced mass spectrometric sensitivity, moderate back-pressure - even at high flow rates, or when used with sub-2-µm particle size - and orthogonal selectivity. Several important technical developments have been proposed during the last decade that foster its use in metabolomics. This review presents an overview of the most recent technical improvements and applications of HILIC analysis in untargeted clinical metabolomics and discusses important practical considerations, including the selection of the optimal column chemistry, appropriate eluents, sample preparation, and data analysis.

Advances in Liquid Chromatography–Tandem Mass Spectrometry (LC–MS–MS)-Based Quantitation of Biopharmaceuticals in Biological Samples

Advances in Sample Preparation: Removing Phospholipids from Biological Samples

The biologically active form of vitamin D is an important analytical target in both research and clinical practice.