Biological, Medical, and Clinical Analysis

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.

A multiresidue method has been developed and validated for the analysis of methylxanthines (caffeine and its metabolites) and cotinine in human plasma.

5-Fluorouracil (5-FU) is a low-molecular-weight anticancer drug in clinical use for several solid tumors in humans. Currently, the most widely used methodology for 5-FU quantitation is liquid chromatography–tandem mass spectrometry (LC–MS-MS) with either liquid–liquid extraction (LLE), protein precipitation, or a combination of both as sample cleanup procedures.

This column installment traces the early development of SEC from its inception to its acceptance as a modern analytical technique.

Joe Pesek answers questions about IEX, HILIC, polar-embedded, and polar-endcapped columns, and aqueous normal phase chromatography.

How to use these tools to develop an efficient and robust process

Detailed examples of light-scattering techniques are presented as well as how their performance compares to MS and other methods.

Sample clean-up using solid-phase extraction (SPE) and similar methods can sometimes be both time-consuming and expensive. The purification of complex plant extracts requires special care in particular. A robust and sensitive online SPE sample preparation method is described for the determination of steviol glycosides.

This work demonstrates the potential of the simultaneous on-line hyphenation of asymmetric flow field flow fractionation (AF4) to inductively coupled plasma-mass spectrometry (ICP-MS) and ultra violet-visible/multi angle light scattering (UV-vis/MALS) detection as a promising and simple tool to obtain information on the multi-element speciation and absolute molecular mass of human plasma proteins such as albumin and transferrin: the two main proteins that have been proposed to bind and transport metal ions in this biological fluid.

Smaller particles, increasingly clever ways to overcome mass transfer limitations, further reductions in plate height, shorter separation times, higher pressure, and enhanced peak capacity dominate thinking in the high performance liquid chromatography (HPLC) community today. For healthcare applications, however, we need to think differently about how to improve analytical separations.

The Column spoke to Martin Giera of Leiden University Medical Centre, Leiden, The Netherlands, about developments in biomarker discovery.

Surfactants present a real challenge to the analytical chemist as they are a very complex class of chemical compounds.