Does SPE Improve LC–MS/MS Plant Hormone Analysis?

When scientists measure plant hormones using liquid chromatography–tandem mass spectrometry (LC–MS/MS), they often clean up the raw plant extracts first using solid-phase extraction (SPE). The idea is that removing unwanted material from the sample will reduce interference from everything else in the extract, which can throw off the measurements. But whether this cleanup step truly helps is rarely checked. Researchers at Colorado State University in Fort Collins put it to the test. They prepared plant extracts using three different methods: two that included a cleanup step, each using a different type of filtering material, and one that skipped the cleanup altogether. They then added known amounts of specially labeled plant hormones to the finished extracts. This allowed them to measure how much the rest of the extract interfered with the readings, how much of each hormone made it through the preparation process, and how well each method performed overall. A paper based on their research was published in the Journal of Mass Spectrometry.¹

Why Is It So Hard to Measure Plant Hormones Accurately?

Plant hormones are small, chemically varied molecules that act as messengers inside plants. They control how a plant grows and functions by attaching to specific receptors, a bit like keys fitting into matching locks.^2-4To accurately track how much of each hormone is present in different parts of a plant and how those levels change over time, scientists most often use ultrahigh-performance liquid chromatography-tandem mass spectrometry(UHPLC–MS/MS). This technique is popular because it can detect tiny amounts of specific hormones with great precision and can handle large numbers of samples quickly.²However, UHPLC–MS/MS can be thrown off by other substances in the sample, a problem known as the matrix effect. This can make results less accurate in two ways. First, the hormones being measured may not be fully separated from everything else in the sample during preparation or analysis. Second, these other substances can interfere with how the instrument detects the hormones, making their signals appear either stronger or weaker than they really are.^5,6

The cleanup step can remove many of the substances that cause this interference. However, it can also cause some of the hormones themselves to be lost along the way, which affects how much of each hormone makes it through the process. This, together with any remaining interference, determines how well the process works overall.^3,4

“Despite the widespread adoption of SPE as a purification step in phytohormone extraction protocols,” write the authors of the paper,¹ “few studies have directly and systematically compared SPE-based and non-SPE-based methods with respect to matrix effect, recovery efficiency, and processes efficiency within the same experimental system. This makes it difficult for researchers to determine whether the added cost, complexity, and reduced throughput associated with SPE will translate into improved quantification for their tissue and phytohormones of interest.”

Does Cleaning Up Plant Extracts Improve Plant Hormone Measurements?

The team tested six very different types of plant hormones in extracts made from both tomato fruit and tomato leaves. They found that skipping the cleanup step didn't lead to any significant interference in the measurements. The cleanup methods also rarely did any better than the no-cleanup method when it came to how much of each hormone made it through the process, how well the process worked overall, or how consistent the results were.¹

There was one notable exception. When tomato fruit extracts were cleaned up using one of the filtering materials (known as HLB), the results for three specific hormones were especially consistent. These included a hormone that controls plant growth and two others that help regulate cell division.¹

The authors of the paper believe that their findings matter for research that looks at many plant hormones at once to understand how they work together. As scientists increasingly try to measure lots of different hormones in a single test, they need preparation methods that work well for a wide variety of chemicals, rather than being tailored to just a few. The results, in the opinion of the researchers, suggest that simple, fast methods that skip the cleanup step, such as one known as the Trapp extraction, offer a reliable starting point for this kind of work. Cleanup methods, on the other hand, may work better for some hormones than others, which can make it harder to compare results across different hormones and different parts of the plant.¹

The researchers note that preparation methods will keep improving as they're tested on a wider range of plants and plant tissues, and as more labeled hormone standards become available. This, in turn, should help scientists measure many different plant hormones at once and better understand how these hormones interact.¹

References

1. Juarez Guzman, C. A.; Yao, L.; Broeckling, C. D. et al. Comparison of Extraction Methods for the Quantification of Phytohormones From Tomato Fruits and Leaves by LC-MS/MS. J Mass Spectrom. 2026, Oct;61(10):e70117. DOI: 10.1002/jms.70117
2. Li, J.; Li, C.; Smith, S. M. Hormone Metabolism and Signaling in Plants; Elsevier, 2017,.
3. Gasperini D.; Howe, G. A. Phytohormones in a Universe of Regulatory Metabolites: Lessons From Jasmonate. Plant Physiol. 2024, 195 (1), 135–154. DOI: 10.1093/plphys/kiae045
4. Takeuchi, J.; Fukui, K.; Seto, Y. et al. Ligand-receptor Interactions in Plant Hormone Signaling. Plant J. 2021, 105 (2), 290-306. DOI: 10.1111/tpj.15115
5. Chen, Y.; Wang, Y.; Liang, X. et al. Mass Spectrometric Exploration of Phytohormone Profiles and Signaling Networks. Trends Plant Sci. 2023,28 (4), 399-414. DOI: 10.1016/j.tplants.2022.12.006
6. Trufelli, H.; Palma, P.; Famiglini, G. et al. An Overview of Matrix Effects in Liquid Chromatography-Mass Spectrometry. Mass Spectrom Rev. 2011, 30 (3), 491-509. DOI: 10.1002/mas.20298