Liquid chromatography is a well-established separation technique used across a broad range of applications to characterise the various components of a mixture. It may be used, for example, in research to study biological responses, in medicine to identify potential biomarkers of disease or treatment response, in environmental analysis to monitor for the presence of pollutants, or in pharmaceutical development and production to identify active drug components or screen for impurities.
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The ever-increasing demands on liquid chromatography has fuelled ongoing improvements and enhancements of the technique to provide better and faster separations. With the need to achieve complete separation of more and more complex compounds with greater precision, ultra-high performance liquid chromatography (UHPLC) has become standard practice across numerous disciplines.
The importance of this technique is highlighted by the fact that UHPLC will be the focus of the 30th James L Waters Symposium at Pittcon 2019.
Conventional liquid chromatography was a breakthrough that enabled a mixture to be separated into different fractions. These were collected by allowing the diluted sample to pass through a packed column under the force of gravity. It was used mainly as purification technique since the process cannot be readily reproduced.
The process was enhanced by using an automated high-pressure (around 6,000 psi) pump in high performance liquid chromatography (HPLC). As technologies advanced, it subsequently became possible to achieve even more complex separations by elevating the pressure that could be applied to above 15,000 psi (UHPLC). This was achieved in part by reducing the column particle size to only 3 microns. In addition, the flow cell in UHPLC is typically smaller too (<1μL compared with 10 μL in HPLC), which reduces dispersion to maintain narrow precise peaks.
Consequently, UHPLC provides high performance, high speed, and high-resolution separation of complex mixtures.
UV detection is typically used to track target components of the eluent since it is highly precise (RSD <0.2%), allowing sensitive detection of trace impurities down to 0.05%. However, to achieve accurate quantisation with UV detection, it is important to physically separate all the impurities. Gradient methods are invariably used to increase the separation power for mixtures including components of diverse polarities, but complete separation requires optimizing each operating parameter, such as type of column, mobile phase, for the particular mixture and this is accomplished through a challenging and time-consuming method development process.
In his talk at Pittcon 2019 entitled "UHPLC in Method Development of Stability-Indicating Methods" Michael Dong describes how to quickly tailor UHPLC methodology to meet specific separation requirements.
Recently, multidimensional UHPLC separations have been used to enable the complete separation of highly complex samples without the need for complicated method development. Multidimensional separations massively increase peak capacity and enable results to be obtained more quickly.
In addition to increasing performance, multidimensional separations have several other benefits over single-dimension UHPLC. These include separation of impurities that co-elute with the target compound, identification of impurities with no UV chromophores, and use of mass spectroscopy, which is not compatible with the phosphate buffers used to improve chromatographic performance.
Either the entire sample or just a fraction of interest can be re-analysed in the second dimension (Zhang et al, 2013a and 2013b). A number of different techniques may be used in series to obtain a comprehensive profile of the sample, for example, non-UV absorbing chromophores can be detected using a charged aerosol detector in the second dimension and characterized using mass spectroscopy in the third dimension (Li et al, 2014).
At Pittcon 2019, Kelly Zhang will explore the latest developments in this area in her talk entitled. "Recent Advances in Multidimensional Liquid Chromatography for Pharmaceutical Analysis".
UHPLC in the pharmaceutical industry
With advances in the mass production of UHPLC instrumentation, the use of UHPLC in the pharmaceutical industry has grown quickly in recent years. It is no longer only a specialised research tool but it also in integral part of routine analysis. Indeed, the technique has been validated according to ICH Q2 guidelines as providing the required specificity, sensitivity, linearity, accuracy and precision for quality and process control in the pharmaceutical industry (Yang et al 2017; Wang et al 2019).
To ensure the efficacy and safety of their products, pharmaceutical companies must be confident that every batch contains the correct amount of active pharmaceutical ingredient (API) and any impurities do not exceed the specified limit (eg, <0.5%). As it cannot be known which impurities may be present, such purity evaluation relies heavily on the separation technologies involved. For this reason, regulations now stipulate that the HPLC method must be able to completely separate the main component, the API, and every impurity. Achieving this can necessitate lengthy process development, and so the speedy delivery of well characterised quality drugs to patients is a major challenge in pharmaceutical analysis.
The challenge is being further compounded by the development of more and more complex drug modalities. For example, drugs may be chiral, recombinant proteins or protein-drug constructs, all of which are challenging to separate.
Multi-dimensional UHPLC has facilitated the quality control of such specialised drugs. It has successfully separated antibody-drug conjugate from unconjugated or degraded products (Li et al 2015). Similarly, it differentiates between maleimide functionalized polyethylene glycol (PEG) with from numerous subtly different hydrolysis products (Wang et al 2019).
The importance of UHPLC in the pharmaceutical industry will be a hot topic at Pittcon 2019. Coverage will include the presentation "Characterization and Quality Control of Recombinant Protein Therapeutics Using UHPLC" by Jennifer Rea in which she will explore the value of UHPLC in the characterisation of protein-based therapeutics, and Michael Dong's talk "Implementing Ultra-High-Pressure LC (UHPLC) in Pharmaceutical Analysis". Michael Dong is also one of the tutors for the Pittcon 2019 short course entitled "Modern HPLC/UHPLC for Practicing Scientists 1: Fundamentals and Pharmaceutical Applications".
Find out more at Pittcon 2019
The research and technologies highlighted here will be covered in more detail in the symposia, oral presentations and short courses at Pittcon 2019. Check out the Pittcon 2019 guide to learn more about these talks, symposia and courses and their presenters.
Numerous market-leading producers of UHPLC equipment, including Agilent, Shimadzu and Waters Corporation, will also be present at Pittcon 2019 to discuss the latest additions to their capabilities and additional analytical requirements.
- Li Y, et al. Characterization and Stability Study of Polysorbate 20 in Therapeutic Monoclonal Antibody Formulation by Multidimensional Ultra High-performance Liquid Chromatography−Charged Aerosol Detection−Mass Spectrometry. Analytical Chemistry 2014;86:5150−5157.
- Li Y, et al. A size exclusion-reversed phase two dimensional-liquid chromatography methodology for stability and small molecule related species in antibody drug conjugates. Journal of Chromatography A 2015;1393:81–88.
- Wang J, et al. Multi-arm PEG-maleimide conjugation intermediate characterization and hydrolysis study by a selective HPLC method. Journal of Pharmaceutical and Biomedical Analysis 2019;164:452–459.
- Yang SH, et al. Validation of a two-dimensional liquid chromatography method for quality control testing of pharmaceutical materials. Journal of Chromatography A 2017;1492:87‑97.
- Zhang K, et al. Two-dimensional HPLC in Pharmaceutical analyses. American Pharmaceutical Review 2013a. Available at https://www.americanpharmaceuticalreview.com/Featured-Articles/151949-Two-Dimensional-HPLC-in-Pharmaceutical-Analysis/
- Zhang K, et al. Analysis of pharmaceutical impurities using multi-heartcutting 2D LC coupled with UV-charged aerosol MS detection. Journal of Separation Science 2013b;36:2986–2992.
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