Capillary Electrophoresis Optimization for Metabolite Separation in Hypogymnia physodes Using DoE: Validation Across Lichen Species
Abstract
1. Introduction
2. Results and Discussion
2.1. Phytochemical Characteristics of H. physodes
2.2. Optimization of H. physodes Metabolites CE Separation
2.3. Validation of Method
2.4. Application of the Method for Analyzing Various Lichen Species
3. Materials and Methods
3.1. Chemicals and Reagends
3.2. Lichen Material and Extraction Protocol
3.3. Equipment and CE Separation Conditions
3.4. Metabolite Identification and Validation of the Method
3.5. Optimization of Separation Efficiency Using the D-Optimal Design
3.6. Classification Analysis
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
- Ranković, B.; Kosanić, M. Chapter 12-Biotechnological Substances in Lichens. In Natural Bioactive Compounds; Sinha, R.P., Häder, D.-P., Eds.; Academic Press: Cambridge, MA, USA, 2021; pp. 249–265. ISBN 978-0-12-820655-3. [Google Scholar]
- Ranković, B. Lichen Secondary Metabolites: Bioactive Properties and Pharmaceutical Potential; Ranković, B., Ed.; Springer International Publishing: New York, NY, USA, 2015; ISBN 978-3-319-35516-0. [Google Scholar]
- Le Pogam, P.; Herbette, G.; Boustie, J. Analysis of Lichen Metabolites, a Variety of Approaches. In Recent Advances in Lichenology; Upreti, D.K., Divakar, P.K., Shukla, V., Bajpai, R., Eds.; Springer India: New Delhi, India, 2015; pp. 229–261. [Google Scholar]
- Baczewska, I.; Hawrylak-Nowak, B.; Ozimek, E.; Sęczyk, Ł.; Dresler, S. Enhanced Accumulation of Biologically Active Compounds in Lichens with Potential Functional Food Applications. Food Chem. 2024, 458, 140286. [Google Scholar] [CrossRef] [PubMed]
- Sveshnikova, N.; Yuan, T.; Warren, J.M.; Piercey-Normore, M.D. Development and Validation of a Reliable LC–MS/MS Method for Quantitative Analysis of Usnic Acid in Cladonia uncialis. BMC Res. Notes 2019, 12, 550. [Google Scholar] [CrossRef] [PubMed]
- Przybylska, A.; Gackowski, M.; Koba, M. Application of Capillary Electrophoresis to the Analysis of Bioactive Compounds in Herbal Raw Materials. Molecules 2021, 26, 2135. [Google Scholar] [CrossRef] [PubMed]
- Falk, A.; Green, T.K.; Barboza, P. Quantitative Determination of Secondary Metabolites in Cladina stellaris and Other Lichens by Micellar Electrokinetic Chromatography. J. Chromatogr. A 2008, 1182, 141–144. [Google Scholar] [CrossRef]
- Kreft, S.; Strukelj, B. Reversed-Polarity Capillary Zone Electrophoretic Analysis of Usnic Acid. Electrophoresis 2001, 22, 2755–2757. [Google Scholar] [CrossRef]
- Agilent ChemStation. Understanding Your ChemStation for CE System, 7/8th ed.; Agilent Technologies Inc.: Waldbronn, Germany, 2008. [Google Scholar]
- Jacyna, J.; Kordalewska, M.; Markuszewski, M.J. Design of Experiments in Metabolomics-Related Studies: An Overview. J. Pharm. Biomed. Anal. 2019, 164, 598–606. [Google Scholar] [CrossRef]
- Latkowska, E.; Bober, B.; Chrapusta, E.; Adamski, M.; Kaminski, A.; Bialczyk, J. Secondary Metabolites of the Lichen Hypogymnia physodes (L.) Nyl. and Their Presence in Spruce (Picea abies (L.) H. Karst.) Bark. Phytochemistry 2015, 118, 116–123. [Google Scholar] [CrossRef]
- Baczewska, I.; Strzemski, M.; Feldo, M.; Hanaka, A.; Dresler, S. Green Extraction of Depsidones and Depsides from Hypogymnia physodes (L.) Nyl. Using Natural Deep Eutectic Solvents. Int. J. Mol. Sci. 2024, 25, 5500. [Google Scholar] [CrossRef]
- Latkowska, E.; Białczyk, J.; Węgrzyn, M.; Erychleb, U. Host Species Affects the Phenolic Compounds Content in Hypogymnia physodes (L.) Nyl. Thalli. Allelopath. J. 2019, 47, 221–232. [Google Scholar] [CrossRef]
- Olivier-Jimenez, D.; Chollet-Krugler, M.; Rondeau, D.; Beniddir, M.A.; Ferron, S.; Delhaye, T.; Allard, P.-M.; Wolfender, J.-L.; Sipman, H.J.M.; Lücking, R.; et al. A Database of High-Resolution MS/MS Spectra for Lichen Metabolites. Sci. Data 2019, 6, 294. [Google Scholar] [CrossRef]
- Çiçek, S.S.; Mangoni, A.; Hanschen, F.S.; Agerbirk, N.; Zidorn, C. Essentials in the Acquisition, Interpretation, and Reporting of Plant Metabolite Profiles. Phytochemistry 2024, 220, 114004. [Google Scholar] [CrossRef]
- Hancu, G.; Orlandini, S.; Papp, L.A.; Modroiu, A.; Gotti, R.; Furlanetto, S. Application of Experimental Design Methodologies in the Enantioseparation of Pharmaceuticals by Capillary Electrophoresis: A Review. Molecules 2021, 26, 4681. [Google Scholar] [CrossRef] [PubMed]
- Lauer, H.H.; Rozing, G.P. High Performance Capillary Electrophoresis: A Primer, 2nd ed.; Agilent Technologies: Waldbronn, Germany, 2009. [Google Scholar]
- Dresler, S.; Strzemski, M.; Baczewska, I.; Koselski, M.; Hassanpouraghdam, M.B.; Szczepanek, D.; Sowa, I.; Wójciak, M.; Hanaka, A. Extraction of Isoflavones, Alpha-Hydroxy Acids, and Allantoin from Soybean Leaves—Optimization by a Mixture Design of the Experimental Method. Molecules 2023, 28, 3963. [Google Scholar] [CrossRef] [PubMed]
- Chen, J.; He, L.; Abo, M.; Zhang, J.; Sato, K.; Okubo, A. Influence of Borate Complexation on the Electrophoretic Behavior of 2-AA Derivatized Saccharides in Capillary Electrophoresis. Carbohydr. Res. 2009, 344, 1141–1145. [Google Scholar] [CrossRef] [PubMed]
- Hancu, G.; Simon, B.; Rusu, A.; Mircia, E.; Gyéresi, A. Principles of Micellar Electrokinetic Capillary Chromatography Applied in Pharmaceutical Analysis. Adv. Pharml. Bull. 2013, 3, 1–8. [Google Scholar] [CrossRef]
- Hu, S.; Sun, T.; Li, R.; Zhang, D.; Zhang, Y.; Yang, Z.; Feng, G.; Guo, X. Comparison of the Performance of Different Bile Salts in Enantioselective Separation of Palonosetron Stereoisomers by Micellar Electrokinetic Chromatography. Molecules 2022, 27, 5233. [Google Scholar] [CrossRef]
- Yu, R.B.; Quirino, J.P. Bile Salts in Chiral Micellar Electrokinetic Chromatography: 2000–2020. Molecules 2021, 26, 5531. [Google Scholar] [CrossRef]
- Peres, R.G.; Micke, G.A.; Tavares, M.F.M.; Rodriguez-Amaya, D.B. Multivariant Optimization, Validation, and Application of Capillary Electrophoresis for Simultaneous Determination of Polyphenols and Phenolic Acids in Brazilian Wines. J. Sep. Sci. 2009, 32, 3822–3828. [Google Scholar] [CrossRef]
- Nowak, P.M.; Woźniakiewicz, M.; Gładysz, M.; Janus, M.; Kościelniak, P. Improving Repeatability of Capillary Electrophoresis—A Critical Comparison of Ten Different Capillary Inner Surfaces and Three Criteria of Peak Identification. Anal. Bioanal. Chem. 2017, 409, 4383–4393. [Google Scholar] [CrossRef]
- Dresler, S.; Kubrak, T.; Rutkowska, E.; Gagoś, M.; Bogucka-Kocka, A.; Świeboda, R.; Wójcik, M. Comparison of Analytical Methods in Chemometric Fingerprinting of Metallicolous and Non-Metallicolous Populations of Echium vulgare L. Phytochem. Anal. 2016, 27, 239–248. [Google Scholar] [CrossRef]
- Krait, S.; Heuermann, M.; Scriba, G.K.E. Development of a Capillary Electrophoresis Method for the Determination of the Chiral Purity of Dextromethorphan by a Dual Selector System Using Quality by Design Methodology. J. Sep. Sci. 2018, 41, 1405–1413. [Google Scholar] [CrossRef] [PubMed]
- Singh, G. Linking Lichen Metabolites to Genes: Emerging Concepts and Lessons from Molecular Biology and Metagenomics. J. Fungi 2023, 9, 160. [Google Scholar] [CrossRef] [PubMed]
- Oran, S.; Sahin, S.; Sahinturk, P.; Ozturk, S.; Demir, C. Antioxidant and Antimicrobial Potential, and HPLC Analysis of Stictic and Usnic Acids of Three Usnea Species from Uludag Mountain (Bursa, Turkey). Iran. J. Pharm. Res. 2016, 15, 527–535. [Google Scholar] [PubMed]
- Carrasco, F.; Hernández, W.; Castro, N.; Guerrero, M.; Tamariz-Angeles, C.; Olivera-Gonzales, P.; Echevarría-Rodríguez, D.; Raposo, C.; Silva, L.A.; Rodilla, J.M. Identification and Determination of Usnic Acid and Fatty Acid from Various Lichen Species in Arequipa, Peru, as Well as Antibacterial and Antioxidant Capacity. Heliyon 2024, 10, e39703. [Google Scholar] [CrossRef]
- Thepnuan, P.; Sriviboon, C.; Rukachaisirikul, T.; Boonpragob, K. HPLC Analysis of Secondary Metabolites in the Lichen Parmotrema tinctorum from Different Substrates. In Proceedings of the Pure and Applied Chemistry International Conference 2013 (PACCON 2013), Chon Buri, Thailand, 23–25 January 2013. [Google Scholar]
- Hempel, G. Strategies to Improve the Sensitivity in Capillary Electrophoresis for the Analysis of Drugs in Biological Fluids. Electrophoresis 2000, 21, 691–698. [Google Scholar] [CrossRef]
- Kováčik, J.; Klejdus, B.; Štork, F.; Malčovská, S. Sensitivity of Xanthoria parietina to UV-A: Role of Metabolic Modulators. J. Photochem. Photobiol. B Biol. 2011, 103, 243–250. [Google Scholar] [CrossRef]
- Gackowski, M.; Przybylska, A.; Kruszewski, S.; Koba, M.; Mądra-Gackowska, K.; Bogacz, A. Recent Applications of Capillary Electrophoresis in the Determination of Active Compounds in Medicinal Plants and Pharmaceutical Formulations. Molecules 2021, 26, 4141. [Google Scholar] [CrossRef]
- Hanaka, A.; Dresler, S.; Mułenko, W.; Wójciak, M.; Sowa, I.; Sawic, M.; Stanisławek, K.; Strzemski, M. Phenolic-Based Discrimination between Non-Symptomatic and Symptomatic Leaves of Aesculus Hippocastanum Infested by Cameraria ohridella and Erysiphe flexuosa. Int. J. Mol. Sci. 2023, 24, 14071. [Google Scholar] [CrossRef]
- Zidorn, C. Plant Chemophenetics—A New Term for Plant Chemosystematics/Plant Chemotaxonomy in the Macro-Molecular Era. Phytochemistry 2019, 163, 147–148. [Google Scholar] [CrossRef]
Response | p-Value | R2 | Adj. R2 | Pred. R2 | Adeq Prec. | |
---|---|---|---|---|---|---|
Rs1 | Model | <0.0001 | 0.902 | 0.874 | 0.834 | 19.603 |
Lack of fit | 0.3151 | |||||
Rs2 | Model | 0.0008 | 0.516 | 0.423 | 0.296 | 8.834 |
Lack of fit | 0.7400 | |||||
Rs3 | Model | <0.0001 | 0.673 | 0.588 | 0.465 | 10.375 |
Lack of fit | 0.0520 | |||||
Rs4 | Model | <0.0001 | 0.922 | 0.892 | 0.846 | 19.957 |
Lack of fit | 0.041 | |||||
Rs5 | Model | <0.0001 | 0.843 | 0.787 | 0.684 | 16.981 |
Lack of fit | 0.438 | |||||
E | Model | <0.0001 | 0.731 | 0.664 | 0.562 | 13.371 |
Lack of fit | 0.733 |
Compound | Calibration Curve | Correlation Coefficient | LOD (µg/mL) | LOQ (µg/mL) |
---|---|---|---|---|
3-Hydroxyphysodic acid | y = 1.466 x + 0.03137 | 0.998 | 0.2408 | 1.530 |
Physodic acid | y = 2.001 x − 0.06959 | 0.998 | 0.1865 | 1.184 |
Physcion | y = 2.690 x − 0.00064 | 0.995 | 0.1384 | 0.879 |
Evernic acid | y = 2.146 x − 0.02295 | 0.997 | 0.1597 | 1.014 |
Atranorin | y = 2.015 x + 0.00373 | 0.999 | 0.1775 | 1.127 |
Usnic acid | y = 2.210 x − 0.0423 | 0.998 | 0.2019 | 1.282 |
Physodalic acid | y = 0.689x + 0.0254 | 0.998 | 0.4752 | 3.018 |
Salazinic acid | y = 1.251 x + 0.0034 | 0.994 | 0.2514 | 1.597 |
Protocetraric acid | y = 0.554 x + 0.0610 | 0.996 | 0.0807 | 0.513 |
Compound | MT (min) | MTSD | MTRSD(%) | CPASD | CPARSD(%) |
---|---|---|---|---|---|
3-Hydroxyphysodic acid | 7.50 | 0.056 | 0.747 | 0.012 | 7.179 |
Physodic acid | 8.17 | 0.074 | 0.902 | 0.019 | 9.730 |
Physcion | 8.24 | 0.045 | 0.549 | 0.010 | 4.547 |
Evernic acid | 8.68 | 0.132 | 1.523 | 0.003 | 1.882 |
Atranorin | 8.94 | 0.102 | 1.144 | 0.006 | 8.152 |
Usnic acid | 9.01 | 0.148 | 1.611 | 0.008 | 2.996 |
Physodalic acid | 9.34 | 0.075 | 0.806 | 0.010 | 7.237 |
Salazinic acid | 13.91 | 0.119 | 0.857 | 0.001 | 3.969 |
Protocetraric acid | 14.59 | 0.136 | 0.934 | 0.011 | 4.883 |
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. |
© 2025 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Dresler, S.; Hałka-Grysińska, A.; Baczewska, I.; Wójciak, H.; Hawrylak-Nowak, B.; Kováčik, J.; Mykhailenko, O.; Zidorn, C.; Sagan, J.; Hanaka, A. Capillary Electrophoresis Optimization for Metabolite Separation in Hypogymnia physodes Using DoE: Validation Across Lichen Species. Int. J. Mol. Sci. 2025, 26, 4828. https://doi.org/10.3390/ijms26104828
Dresler S, Hałka-Grysińska A, Baczewska I, Wójciak H, Hawrylak-Nowak B, Kováčik J, Mykhailenko O, Zidorn C, Sagan J, Hanaka A. Capillary Electrophoresis Optimization for Metabolite Separation in Hypogymnia physodes Using DoE: Validation Across Lichen Species. International Journal of Molecular Sciences. 2025; 26(10):4828. https://doi.org/10.3390/ijms26104828
Chicago/Turabian StyleDresler, Sławomir, Aneta Hałka-Grysińska, Izabela Baczewska, Hanna Wójciak, Barbara Hawrylak-Nowak, Jozef Kováčik, Olha Mykhailenko, Christian Zidorn, Joanna Sagan, and Agnieszka Hanaka. 2025. "Capillary Electrophoresis Optimization for Metabolite Separation in Hypogymnia physodes Using DoE: Validation Across Lichen Species" International Journal of Molecular Sciences 26, no. 10: 4828. https://doi.org/10.3390/ijms26104828
APA StyleDresler, S., Hałka-Grysińska, A., Baczewska, I., Wójciak, H., Hawrylak-Nowak, B., Kováčik, J., Mykhailenko, O., Zidorn, C., Sagan, J., & Hanaka, A. (2025). Capillary Electrophoresis Optimization for Metabolite Separation in Hypogymnia physodes Using DoE: Validation Across Lichen Species. International Journal of Molecular Sciences, 26(10), 4828. https://doi.org/10.3390/ijms26104828