Quantifying Plant-Specialized Metabolites Using UHPLC

Scientists from the University of Turin in Turin, Italy developed a method to quantify plant-specialized metabolites for determining the quality of medicinal and aromatic plants. Their findings were published in the Journal of Chromatography A (1).

Green plants in botanical garden indoor. | Image Credit: © Татьяна Максимова - stock.adobe.com

There are many challenges that come with analyzing complex biological matrices, especially with extracts from plant matrices that may be used for pharmaceutical, medicinal, and food. Correctly identifying and quantifying compounds of interest is fundamentally important, representing an invaluable source of information regarding the genotype and phenotypic expression of the plant, geographical origin, potential biological activities, and quality assessment, among other factors. Selective extractions are carried out prior to analysis to simplify samples under investigation. Gas (GC) and liquid chromatography (LC) are the most common analytical techniques used for separating volatile and non-volatile compounds, respectively. That said, choosing the most suitable detector and quantification approach is still a critical issue.

The quantification of non-volatile specialized metabolites biosynthesized in medicinal and aromatic plants (MAPs) is usually completed using reversed-phase high-performance liquid chromatography (HPLC) with different detection systems alone or in series. Different detectors have advantages and disadvantages. For example, photodiode arrays (PDAs) are widely used for their versatility, high sensitivity, and wide range of linearity, but they only respond to compounds that absorb UV or visible light at a specific wavelength. Mass spectrometers (MS) do not require molecules of interest to have chromophores, or molecules in a material that absorb wavelengths of visible light (2). However, MS only respond to ionizable compounds and must be hyphenated to an LC system with an appropriate ionization interface.

In this study, the scientists aimed to compare the performance of different detectors (PDA and MS) used in HPLC and acquisition modes including selected ion monitoring (SIM) and selected reaction monitoring (SRM). From there, they analyzed how the techniques can assist with the quantification of specialized plant metabolites, such as phenolic and triterpenic compounds. The metabolites were quantified using this method with the reference standard and with a compound with similar UV and MS spectra (semi-quantification) to simulate common cases where reference standards are not accessible. Each approach was evaluated by the percentage relative error based on the actual concentration of the quantified compound. Precision was also tested in the presence of interferences and in two case studies: leaf extracts of Cynara cardunculus subsp. cardunculus L. and Punica granatum L.

Ultrahigh-performance liquid chromatography (UHPLC)–PDA-MS/MS is a popular analytical technique for identifying and quantifying various non-volatile molecules in plants. PDA and MS in SIM and SRM (both in positive and negative ionization mode) were tested, highlighting the advantages and disadvantages of each method. PDAs are highly versatile and sensitive, but they can only react to compounds that absorb UB or visible light at specific wavelengths. Meanwhile, MS do not require molecules of interest to have chromophores, though they must be hyphenated to an LC system to properly function.

The scientists do think it best to quantify each analyte of interest with appropriate reference standards, possibly through combining and comparing different acquisition methods to obtain a higher guarantee of correct results. If no commercial standard is available, potential errors that can occur during semi-quantification. Each compound used involves different chemical properties and will give different response factors with the considered acquisition modes. The best approach, according to the scientists, is to use PDA with a calibration curve of a surrogate standard with similar UV absorbance. With co-elutions, the MS response of the analyte of interest should be compared to the surrogate standard by applying the proposed correction method by calculating the relative CF.

References

(1) Marengo, A.; Menzio, G.; Cagliero, C.; Sgorbini, B.; Rubiolo, P. Quantification of Plant Specialized Metabolites for Quality Determination of Medicinal and Aromatic Plants: Findings in the Absence of a Reference Standard. J. Chromatogr. A 2025, 1745, 465696. DOI: 10.1016/j.chroma.2025.465696

(2) Chromophore. ScienceDirect 2009. https://www.sciencedirect.com/topics/chemistry/chromophore (accessed 2025-2-5)