Phytochemical Profiles and Multifunctional Bioactivities of Chili Pepper Varieties Assessed by UHPLC-q-TOF-MS

Chili peppers (Capsicum spp.), especially C. annuum, C. baccatum, and C. chinense, are cultivated worldwide for their flavor, nutritional value, and bioactive compounds. These include capsaicinoids, phenolics, carotenoids, and vitamins, which have antioxidant, anti-inflammatory, analgesic, neuroprotective, and enzyme-inhibiting properties relevant to conditions such as Alzheimer’s disease, obesity, type II diabetes, and skin hyperpigmentation. Capsaicinoid levels vary by species, cultivar, and environmental factors, with C. chinense varieties ranking among the world’s spiciest.

A recent study examined 19 chili pepper varieties grown in Italy, applying ultrahigh-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-q-TOF-MS) for phytochemical profiling and in-house assays to assess inhibitory activity against acetylcholinesterase, butyrylcholinesterase, pancreatic lipase, α-glucosidase, and tyrosinase, alongside antioxidant capacity (DPPH) and total phenolic content (Folin–Ciocalteu). It is reported to be the first comprehensive evaluation of these enzyme targets in multiple Capsicum varieties combined with high-resolution mass spectrometry profiling, aiming to link phytochemical composition with functional properties and support diverse dietary inclusion. LCGC International spoke to Aristeidis S. Tsagkaris, associate professor of theDepartment of Food Analysis and Nutrition at the University of Chemistry and Technology Prague (Czech Republic), lead author of the paper that resulted from this research (1).

What advantages does ultrahigh-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry(UHPLC-qTOF-MS) offer over conventional HPLC when profiling complex plant extracts such as those from Capsicum spp.?
UHPLC-qTOF-MS offers superior resolution, speed, and sensitivity compared to conventional HPLC. In profiling complex plant matrices like Capsicum spp., UHPLC enables better separation of co-eluting phytochemicals due to smaller particle size columns and higher pressures. The qTOF-MS adds high mass accuracy and full-scan acquisition, allowing for retrospective data analysis. Its accurate mass measurements help in molecular formula prediction and identification of suspects. That is of outmost importance when dealing with complex, polyphenol- and capsaicinoid-rich samples.

In this study, a suspect screening workflow was applied. Could you explain what suspect screening entails and how it differs from targeted or untargeted approaches in chromatography?
Suspect screening involves searching MS data for known compounds, already reported in the literature, without using analytical standards. Unlike targeted analysis, where only pre-defined analytes are quantified, or untargeted profiling, which captures all detectable features, suspect screening uses a spectral database (molecular weight, formula, or MS/MS spectra) to tentatively identify known compounds. In our study, this method allowed for annotation of a wide range of phytochemicals while avoiding the need for analytical standards, improving throughput and discovery potential. Our primary goal was not quantification but characterizing the 19 different varieties of Capsicum spp.

How would you design a validated quantitative UHPLC method for capsaicinoid and phenolic compound analysis in chili pepper extracts? What parameters would you consider?
First, the sample preparation would need to be optimized. Spiking of target analytes in minimum two levels (one high concentration level and one low) would be performed, and the method trueness and repeatability would be assessed by preparing at least six different replicates. Other crucial performance characteristics such as linearity in matrix matched calibration curves, detectability (LODs/LOQs) and intermediate precision would be accessed as well. The use of internal standards would be also important to acquire accurate results. Optimizing also the chromatographic conditions can impact the performance, for example, column selection or mobile phase composition.

The article notes that chromatographic results were only qualitative. What steps are necessary to transition from qualitative to quantitative analysis in phytochemical profiling?
This overlaps a bit with the previous question. Briefly, analytical standards for the target compounds must be acquired. Calibration curves need to be established across a suitable concentration range, ideally in the sample matrix to correct for matrix effects. Internal standards enhance accuracy and reproducibility. Quantitative method validation—addressing precision, accuracy, recovery, LOD/LOQ, and robustness—is required. Additionally, sample preparation must be standardized.

Ethanol extracts showed more detected compounds and higher antioxidant activity than aqueous extracts. How could solvent polarity affect compound extraction and chromatography results?
This was something we expected. Ethanol is less polar than water achieving better recovery of non-polar bioactive compounds such flavonoids or capsaicinoids. More non-polar solvents may yield better separation of hydrophobic compounds, while aqueous extracts often show poor retention of polar analytes on reversed-phase columns. Matching solvent polarity with target analyte properties is key to efficient extraction and optimal chromatographic performance.

What role does preparative HPLC play in the isolation of bioactive compounds, and how does it differ from analytical HPLC in terms of purpose and setup?
Preparative HPLC is used for isolating and purifying larger quantities of bioactive compounds for structural elucidation or bioassays. It operates at higher flow rates and uses larger columns, focusing on yield and purity. Analytical HPLC, in contrast, is designed for high-resolution detection and quantitation at microgram levels, with optimized sensitivity and precision. Preparative systems tolerate higher sample loads and less resolution, while analytical setups prioritize separation efficiency. Both are complementary—analytical HPLC guides preparative isolation by identifying target retention times and profiles.

Reference

1. Tsagkaris, A. S.; Cafarella, C.; Rigano, F. et al. Bioactive Compounds in 19 Chili Pepper Varieties Cultivated in Italy: Suspect Screening and in vitro Enzyme Inhibitory Effect. Food Res. Intl. 2025, 219, 117051. DOI: 10.1016/j.foodres.2025.117051