Various forms of chromatography help scientists separate a sample’s components. High-performance liquid chromatography (HPLC), for example, can be used in drug discovery and development. The main objective is finding new techniques and tools to improve and accelerate the process.
“The key challenges in any chromatographic separation are basic, but fundamental,” says Kim Haynes, principal product marketing manager at Waters Corporation’s Chemistry Technology Center. “You need to be able to separate and measure the analytes of interest in a mixture, with a method that is accurate and reproducible.”
Those objectives might sound simple enough, but that’s not always the case. “With any chromatographic separation, the scientist’s goal is to create that separation using mobile phases and stationary phases,” Haynes explains. “However, there can be unintended interactions as well that turn these simple goals into difficult ones.”
Challenge-solving surfaces
The best separations arise from specific, controlled interactions. Consequently, non-specific binding and adsorption create challenges. “Both of these result in unintentional interactions of your sample with a component in the chromatographic flow path causing analyte loss, reproducibility problems, and, ultimately, wasted time and frustration,” Haynes says.
The analyte losses depend on the sample and the retention system being used. “Typically, scientists will try to passivate columns and surfaces with either acid or sacrificial sample, use complex mobile phases and/or additives, alternatives to stainless steel, or simply suffer with a method that is not as robust as they would like,” Haynes explains. “We suffered with these challenges ourselves and were well aware of the impacts on our results.”
To address those challenges, Haynes and her colleagues developed MaxPeak High Performance Surfaces (HPS). In 2019, Waters released the first product, QuanRecovery Vials and Plates, that included HPS to reduce non-specific binding due to hydrophobic interactions. “For example, many scientists automatically choose to use polypropylene with peptides, as they know peptides will bind to glass due to ionic interactions,” Haynes says. “However, hydrophobic peptides can be lost to polypropylene, as it is a hydrophobic surface and attracts the hydrophobic peptide.” With the QuanRecovery products, MaxPeak HPS reduces the hydrophobicity of the polypropylene to reduce the binding of hydrophobic peptides and proteins. “This yields better recovery, sensitivity, and reproducibility for these samples,” Haynes notes.
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To reduce non-specific adsorption, scientists at Waters looked closely at chromatographic separations and the fact that some analytes can adsorb to metal surfaces. For example, compounds with phosphorylation, acidic, or other functionalities might bind to stainless steel. “This non-specific adsorption results in lengthy passivation and conditioning steps, poor sensitivity, and peak shape, and reproducibility problems,” Haynes explains. In those cases, she says, scientists might “resort to complex mobile phases or additives that can cause ion suppression or solubility issues.”
To help scientists handle non-specific adsorption more effectively, Waters created another MaxPeak High Performance Surface and deployed it in the new PREMIER columns. These are “designed to reduce analyte/surface interactions by creating an inert surface barrier that prevents analyte losses,” Haynes says. “Based on an evolution of our hybrid organic/inorganic technology, PREMIER columns reduce the losses due to metal interactions without impacting the chromatographic performance of other analytes in the separation.”
Dealing with drugs
The science of drug discovery and development requires lots of separations. The need for separations really emerges when working with natural products. Natural-product samples include especially difficult materials to study, because they often consist of complex matrices and collections of compounds, which must be studied individually for clinical potential.
Some of the most interesting new drug discoveries come from nature. In Texas, a natural home to many venous snakes, a team of scientists used a variety of techniques, including HPLC, to study venoms. The researchers noted the value of snake venom for drug discovery and development, but pointed out that “a major challenge to drug discovery using snake venoms is isolating and analyzing the bioactive proteins and peptides in these complex mixtures.” The team added that advances in separation techniques, including multidimensional HPLC have “been critical to obtain an accurate picture of the startling complexity of venoms.”
Image: From natural products, such as snake venom, to analytes associated with the tricarboxylic acid cycle, scientists use separation techniques to isolate samples for analysis. Image courtesy of University of Central Florida
Scientists in another home to venomous snakes, Australia, also applied HPLC in search of compounds with drug-like capabilities. Studying the red-bellied black snake, the researchers looked for compounds with immunosuppressive traits. These scientists reported, “Deconvolution of the venom using reverse-phase HPLC identified four fractions responsible for the observed immunosuppressive activity.” They added that the data from this snake’s venom might produce “potential drug leads for T cell-associated conditions such as graft versus host disease, rheumatoid arthritis, and inflammatory bowel disease.”
Of course, drug discovery and development involve more than venomous snakes. At Waters, scientists used the PREMIER columns with MaxPeak High Performance Surfaces to test the analytes associated with the tricarboxylic acid cycle. “These compounds are especially challenging as they bind to metal surfaces, and the separation requires complex mobile phases while having limited sensitivity and reproducibility,” Haynes notes. “Our scientists were able to use the ACQUITY PREMIER CSH Phenyl-Hexyl column, utilizing MaxPeak HPS, to do this separation with a simple mobile phase of water, acetonitrile, and 0.1% formic acid.” She adds that this combination of technologies “improved the recovery, sensitivity, peak shape and reproducibility over what could be achieved with a standard column.”
In the world of drug discovery and development, from natural products to almost any other source of starting materials, scientists rely heavily on separation. The better the separation, the more accurately scientists can investigate the components of any potential drug material. In some cases, the method of separation will allow improved analysis. In other cases, advances in materials or technology will increase the ability to pick out specific elements of a sample. All of these improvements will contribute to advances in drug discovery and development. So, separation science contributes fundamentally to the health of people around the world, as well as their quality of life.