Biomarkers are molecules that indicate normal or abnormal processes in relation to conditions or diseases and provide valuable information in medical research and diagnosis. Therefore, methods are greatly needed to identify new biomarkers and detect existing ones with high specificity and selectivity.
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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
Several instruments are now capable of operating at pressures as high as 1500 bar. In addition, improvements in column technology allow to use columns with an internal diameter of 2.1 mm versus the 4.6 mm typical of many HPLC columns, with reduced viscous heating when operating at ultra-high-pressure conditions.
Other advances include the development high throughput methods, particularly useful in clinical laboratories, with the use of autosamplers, reduced detector cell volumes and improvements in data collection and analysis. These aspects are extremely valuable when analyzing hundreds of samples.
Overall, HPLC advancements have a strong impact on biomarker discovery by allowing sample analysis with unprecedented efficiency and accuracy and with shorter analysis times without compromising resolution.
HPLC in Clinical Applications
Major clinical applications for HPLC include screening for metabolic disorders and toxicological investigations. It is a useful tool in the identification of biomarkers for cancer, stress-related disorders, and other diseases.
By identifying and quantifying these compounds, it is possible to have more information for the early diagnosis and treatment of diseases, contributing to the development of diagnostic tests and personalized medicine.
HPLC is capable of distinguishing between hemoglobin types and is one of the methods that can be used to separate and identify the HbA1c glycated hemoglobin, a biomarker for the diagnosis of diabetes.3
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
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
Future Directions and Potential
Integration with other technologies, such as mass spectrometry (MS) and bioinformatics, can further enhance the capabilities of HPLC. The identification of novel biomarkers will open avenues for targeted therapies and precision medicine.
Significant advancements can also be achieved by further reducing column dimension, reducing solvent consumption, and enhancing flow rate compatibility towards MS detectors. However, this requires a radical change in instrument design.
Further decreasing the column internal diameter will also allow to operate at higher pressure. Continuous efforts to enhance the sensitivity of MS detection are also being made, including the improvement of pretreatment procedures, ionization techniques, and mass analyzers.5,6
Conclusion
There is a variety of examples that showcase how HPLC can be a powerful technique with various applications. In particular, through continuous innovation and integration with complementary technologies, HPLC can provide a key contribution to biomarker discovery and drive advancements in medical research, supporting the early diagnosis and treatment of diseases.6
References
- Denoroy, L., Zimmer, L., Renaud, B. & Parrot, S. (2013). Ultra high performance liquid chromatography as a tool for the discovery and the analysis of biomarkers of diseases: A review. Journal of Chromatography B, 927, 37-53.https://doi.org/10.1016/j.jchromb.2012.12.005. Available: https://www.sciencedirect.com/science/article/pii/S1570023212007325
- De Vos, J., Stoll, D., Buckenmaier, S., Eeltink, S. & Grinias, J. P. (2021). Advances in ultra-high-pressure and multi-dimensional liquid chromatography instrumentation and workflows. Analytical Science Advances, 2, 171-192.https://doi.org/10.1002/ansa.202100007. Available: https://chemistry-europe.onlinelibrary.wiley.com/doi/abs/10.1002/ansa.202100007
- Pohanka, M. (2021). Glycated Hemoglobin and Methods for Its Point of Care Testing. Biosensors (Basel), 11.10.3390/bios11030070.
- Dhama, K., Latheef, S. K., Dadar, M., Samad, H. A., Munjal, A., Khandia, R., Karthik, K., Tiwari, R., Yatoo, M. I., Bhatt, P., Chakraborty, S., Singh, K. P., Iqbal, H. M. N., Chaicumpa, W. & Joshi, S. K. (2019). Biomarkers in Stress Related Diseases/Disorders: Diagnostic, Prognostic, and Therapeutic Values. Front Mol Biosci, 6, 91.10.3389/fmolb.2019.00091.
- Hameedat, F., Hawamdeh, S., Alnabulsi, S. & Zayed, A. (2022). High Performance Liquid Chromatography (HPLC) with Fluorescence Detection for Quantification of Steroids in Clinical, Pharmaceutical, and Environmental Samples: A Review. Molecules, 27.10.3390/molecules27061807.
- Liu, Y., Li, Y., Zhang, T., Zhao, H., Fan, S., Cai, X., Liu, Y., Li, Z., Gao, S., Li, Y. & Yu, C. (2020). Analysis of biomarkers and metabolic pathways in patients with unstable angina based on ultra‑high‑performance liquid chromatography‑quadrupole time‑of‑flight mass spectrometry. Mol Med Rep, 22, 3862-3872.10.3892/mmr.2020.11476.