Article: Revolutionising Biomarker Discovery: The Role of High-Performance Liquid Chromatography

Sly Saint · Jan 4, 2025

Introduction
Biomarkers can be found in body fluids (e.g., blood and urine) or tissues, and their detection is crucial for disease diagnosis and monitoring treatment. They can be proteins representing cellular and enzymatic changes or metabolites highlighting a specific physiological state.

There is a strong need for methods that can assist with the discovery of new biomarkers. Thanks to its sensitivity, versatility, and ability to analyze a wide range of samples from complex matrices, high-performance liquid chromatography (HPLC) is among the techniques that play a pivotal role in biomarker discovery.

The Basics of HPLC in Biomarker Discovery
HPLC separates individual compounds from mixtures based on how they interact with a stationary phase and a mobile phase under high pressure (100-500 bar). The technique is widely used in analytical chemistry and finds application various fields, ranging from forensics, to pharmaceutical development and clinical research.

HPLC is applicable to complex samples such as plasma, urine, and other biological fluids. The great resolution and reproducibility of HPLC allows for the separation, identification, and quantification of biomolecules such as proteins, lipids, hormones, and metabolites, and it is therefore a powerful tool for the discovery and monitoring of biomarkers.1

Advancements in HPLC Technology
Over the last few decades, HPLC technology witnessed significant advancements that led to enhanced sensitivity, resolution, and speed. A remarkable innovation was the development of ultra high-performance liquid chromatography (UHPLC).

Compared to standard HPLC, UHPLC uses smaller particle sizes and higher pressures. One of its major strengths is the use of sub-2-micron particles in stationary phases, which leads to a drastic increase of the chromatographic efficiency, faster separations, and increased peak resolution.2
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Renal stress biomarkers such as 8(F2a)-isoprostane and 4-hydroxy-2-nonenal (produced by increased lipid peroxidation during oxidative stress) can be detected in serum and urine samples by HPLC. Their increased concentrations are indicative of chronic renal disorders.4

A selective and sensitive method based on HPLC with diode-array detection (HPLC-DAD) was used to measure malondialdehyde (MDA) in goat plasma. MDA is as an oxidative stress biomarker widely used to predict the pattern of numerous diseases, including diabetes, hypertension, and atherosclerosis.

High-performance liquid chromatography with fluorescence detection (HPLC-FLD) finds application in the quantification of various types of steroids, and it is used to investigate their role in diseases and clinical conditions.5

For instance, HPLC-FLD allowed the determination of cortisol (a biomarker of chronic stress), cortisone, and their metabolites in plasma and urine. Cortisol levels were also measured in human hair samples with a 1 pg/mg detection limit.

A metabolomic study used UHPLC analysis to identify biomarkers in patients with unstable angina (UA), a coronary disease with high mortality and morbidity. Urine samples from 28 patients with UA were analyzed and compared with 28 healthy controls.

The study identified 16 biomarkers, including D-glucuronic acid, creatinine, succinic acid, and N-acetylneuraminic acid, and concluded it was possible to distinguish patients with UA from healthy ones.6
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Challenges and Limitations
Despite the numerous advantages of the technique, the complexity of biological samples and the presence of compounds that can interfere with the detection of target molecules pose a big challenge to biomarker discovery using HPLC.

Achieving improved specificity and reproducibility is crucial, and researchers are continuously working towards developing novel sample preparation techniques and optimized methods. In clinical settings, a major barrier to the adoption of this technology is also the need for suitably trained users, together with the cost of the instrumentation.4,5,6
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Revolutionising Biomarker Discovery: The Role of High-Performance Liquid Chromatography

Creekside · Jan 4, 2025

I do see potential problems, such as small cohorts "proving" a biomarker, but results in many false negatives in a larger population, and once "proven", it won't be challenged. Also, some diseases may not have a biomarker in the serum or CSF, and if a biomarker identifying technique is claimed to find any biomarker, the lack of one might get the disease labeled psychosomatic.

This could be a very useful tool, but the limitations do have to be taken into account.

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Article: Revolutionising Biomarker Discovery: The Role of High-Performance Liquid Chromatography

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Article: Revolutionising Biomarker Discovery: The Role of High-Performance Liquid Chromatography

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Creekside Senior Member (Voting Rights)

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