Compounds ingested by humans, be it as drugs or as part of the food chain, can cause severe toxicity when broken down in the human body. If active, a metabolite can interact with other targets, such as enzymes, proteins or different receptors and produce undesirable side effects. Identifying compounds’ metabolites is therefore essential throughout regulatory drug and agrochemical product development to ensure human safety.
“During the early drug discovery phase, the knowledge of the metabolic site of the new chemical entity is essential for selecting the compound with the most favorable pharmacokinetic properties,” explains Nuria Piñeiro Costas, Study Director of In Vitro DMPK at Charles River Laboratories. “In drug development, identifying circulating metabolites can help demonstrate the biotransformation pathways of drugs, and essentially, understand the effect of the drug itself.”
But it is not only human drugs that need to undergo metabolite profiling and identification in the course of safety assessments; the agrochemical and veterinary medicine sectors are also affected. For instance, an animal may be given a compound to cure a particular ailment. When the animal is eaten, the compound metabolites enter the human food chain.
The same goes for plant protection products: “On the agrochemical side, we could be using a fungicide or herbicide to help our crops grow,” says James O’Neill, Scientific Manager High-Resolution Mass Spectrometry at Charles River Laboratories. “But when we ingest these crops, secondary metabolism effects can occur. Animal and plant organisms will change the administered compounds and our liver may change them further. So it is critical to identify and understand those metabolites and their concentrations to ensure their ongoing safety assessment and continued availability on the market.”
Challenges of Metabolite Profiling and Identification
The complexity of metabolic pathways means that researchers are confronted with a number of challenges during metabolite profiling and structure elucidation. One problem is the unpredictability of biochemical reactions: “For agrochemicals, there are often unexpected or unusual biotransformations,” says Piñeiro Costas. “This can increase the difficulty of finding and understanding the reactions and the resulting metabolites.”
Another key problem is sensitivity. When researchers analyze samples, they encounter thousands of background compounds, which can be naturally occurring in animals or plants.
“It’s very much like finding a needle in a haystack sometimes,” O’Neill explains. “The concentration of background compounds is typically many times higher than that of the metabolites. So one of the major challenges is to be able to resolve the unwanted background of endogenous compounds from those compounds that are of interest.”
“Being able to differentiate between the compounds of interest within the complex matrix is essential,” Piñeiro Costas adds. “This makes sensitivity and resolution the two key challenges in metabolite profiling and identification.”
Gaining accuracy with high-resolution mass spectrometry
Using specific sample preparation techniques and refined liquid chromatography-mass spectrometry (LC-MS) systems as well as supporting software packages, researchers can address sensitivity and resolution issues.
High-resolution mass spectrometry (HR-MS) especially comes with a high degree of precision and great sensitivity. “HR-MS is what we call a positive confirmation technique that allows us to positively identify molecules,” says O’Neill. “Without HR-MS there would be very few other means to help identify molecules. Essentially, HR-MS delivers increased certainty around the nature and chemical identity of a compound and its metabolites.”
Using radiochemicals, researchers can also label drug or agrochemical compounds. These radiolabels enable the observation of metabolites within a sample and the detection of their concentration. With the help of high-resolution mass spectrometry, researchers can then gather information on the mass to charge ratio and isotope fine structure, which can provide clues around the structure and therefore likely the metabolism of the parent compound.
Other methods cannot keep up with HR-MS
Although other methods for metabolite profiling and identification exist, they do not offer both the precision and sensitivity of HR-MS. For example, while high-resolution mass spectrometry can measure the mass of molecules as far as six decimal places, low-resolution mass spectrometry measures only nominal mass with reliability.
“In certain situations, you may have hundreds or thousands of compounds with the same nominal mass, but by comparison, there are a lot fewer compounds with the same accurate mass measured to six decimal places,” O’Neill explains. “This is where the precision and accuracy of high-resolution really kicks in.”
Another technology used for metabolite identification is nuclear magnetic resonance (NMR). However, although NMR remains the gold standard for absolute structure determination, it lacks the sensitivity and tolerance for sample complexity, which are inherent in unpurified metabolism samples, says O’Neill.
“For a standard NMR – and we’re not talking about anything too fancy here – you ideally need analyte mass at the milligram level for sensible experiment timescales,” O’Neill explains. “But mass spectrometers nowadays can measure samples at the femtogram level, which is many times lower. The best NMRs in the country can work with just a few micrograms of analyte, however, the concentrations in metabolism studies can be much lower than that.”
The future of metabolite profiling and identification
As technologies evolve, the process of metabolite profiling and structure elucidation for drug, agrochemical and veterinary medicine product development will become more and more refined.
“Data processing techniques that support the screening of metabolites, such as software packages, will develop further to make the lives of chemists easier by improving effectively the metabolite identification processes,” says Piñeiro Costas. “There will probably also be a combination of technologies, like LC-MS with NMR, that will allow researchers to access structural information more easily without the need for large sample sizes.”
O’Neill agrees: “From my point of view there will always be advancements in the quality of mass spectrometry equipment. Sensitivity levels are getting better and better all the time, and the dynamic range of the instruments is continuously improving, allowing us to measure different metabolites at different concentrations, all in the same run.”
In the future, O’Neill also sees the regulatory framework shifting. Currently, there is comparatively less work done on the chirality of metabolites in the pharmaceutical industry, whereas it is already being conducted in the agrochemical sector. However, this is a necessary precaution as specific compounds can change toxicity from one enantiomeric form to another. Regulators will have more influence on this in the future, says O’Neill.
Images via Charles River Laboratories and Header image by Shutterstock.com
Author: Larissa Warneck, Science Journalist, Labiotech.eu