High-performance liquid chromatography methods for carotenoid determination

Authors

DOI:

https://doi.org/10.14739/2409-2932.2026.2.355779

Keywords:

high-performance liquid chromatography, iquid chromatograph, carotenoids

Abstract

A relevant area of pharmaceutical research is the search for new compounds capable of inhibiting free radical oxidation at various stages of the pathological process. Taking into account the prospects for practical application, particular attention of researchers is focused on carotenoids as natural bioantioxidants characterized by high biological activity and minimal toxicity. The primary function of carotenoids is to protect cellular membranes and other structural components of the cell from the damaging effects of reactive oxygen species, which determines their important role in maintaining cellular homeostasis and preventing oxidative stress. In this context, an in-depth study of analytical quality control methods, particularly the determination of the purity of the most common groups of carotenoids, is of significant interest.

Aim of the study. The aim of this study was to generalize and systematize scientific data on the methods of analysis of biologically active carotenoids using high-performance liquid chromatography (HPLC), with the prospect of further application of the obtained results for the optimization of laboratory analytical methods.

Materials and methods. An information search was conducted using the following scientometric databases: Scopus, Web of Science, and PubMed. Analytical, descriptive, and generalization methods were applied to achieve the objectives of the study. The research materials included data from contemporary scientific literature sources addressing the determination of purity, identification, and structural analysis of biologically active carotenoids by HPLC.

Results. An analysis of available scientific literature sources was performed, focusing on approaches to the assessment of purity and structural characteristics of bioactive carotenoids using high-performance liquid chromatography. The main chromatographic parameters influencing the selectivity and sensitivity of the method, as well as sample preparation features, were considered.

Conclusions. Based on the analysis of current scientific literature, examples of qualitative and quantitative determination methods for two major groups of carotenoids –carotenes and xanthophylls – using HPLC were identified. A comparative analysis of these methods was carried out, taking into account chromatographic conditions, types of stationary and mobile phases, and detection approaches, which substantiates the selection of optimal analytical conditions for further scientific and practical research.

Author Biographies

I. F. Duiun, Zaporizhzhia State Medical and Pharmaceutical University

PhD, Senior Lecturer of the Department of Clinical Pharmacy, Pharmacotherapy, Pharmacognosy and Pharmaceutical Chemistry

I. M. Keitlin, Zaporizhzhia State Medical and Pharmaceutical University

PhD, Lecturer of the Department of Clinical Pharmacy, Pharmacotherapy, Pharmacognosy and Pharmaceutical Chemistry

G. V. Mazulin, Zaporizhzhia State Medical and Pharmaceutical University

PhD, Senior Lecturer of the Department of Pharmacognosy, Pharmacology and Botany

References

  1. Li Y, Zhao Y, Zhang H, Ding Z, Han J. The Application of Natural Carotenoids in Multiple Fields and Their Encapsulation Technology: A Review. Molecules. 2024;29(5):967. doi: https://doi.org/10.3390/molecules29050967
    | |
  2. Sun T, Rao S, Zhou X, Li L. Plant carotenoids: recent advances and future perspectives. Mol Hortic. 2022;2(1):3. doi: https://doi.org/10.1186/s43897-022-00023-2
    | |
  3. González-Peña MA, Ortega-Regules AE, Anaya de Parrodi C, Lozada-Ramírez JD. Chemistry, Occurrence, Properties, Applications, and Encapsulation of Carotenoids-A Review. Plants (Basel). 2023;12(2):313. doi: https://doi.org/10.3390/plants12020313
    | |
  4. Takaichi S. Distribution, biosynthesis, and function of carotenoids in oxygenic phototrophic algae. Mar Drugs. 2025;23(2):62. doi: https://doi.org/10.3390/md23020062
    | |
  5. Malyugina EA, Mazulin AV, Mazulin GV, Smoylovskaya GP, Logvin PA. [The study of the carotenoid content in the inflorescences of the spreading marigold]. Current issues in pharmacy and medicine: science and practice. 2013;(3):89-91. Ukrainian.
  6. Tufail T, Bader Ul Ain H, Noreen S, Ikram A, Arshad MT, Abdullahi MA. Nutritional Benefits of Lycopene and Beta-Carotene: A Comprehensive Overview. Food Sci Nutr. 2024;12(11):8715-41. doi: https://doi.org/10.1002/fsn3.4502
    | |
  7. Jurja S, Negreanu-Pirjol T, Vasile M, Hincu MM, Coviltir V, Negreanu-Pirjol BS. Xanthophyll pigments dietary supplements administration and retinal health in the context of increasing life expectancy trend. Front Nutr. 2023; 10:1226686. doi: https://doi.org/10.3389/fnut.2023.1226686
    | |
  8. Gholipour-Varnami K, Mohamadnia S, Tavakoli O, Faramarzi MA. A review on the biological activities of key carotenoids: Structures, sources, market, economical features, and stability. Food Biosci. 2025;68(106529):106529. doi: https://doi.org/10.1016/j.fbio.2025.106529
    |
  9. Varynskyi BO, Kaplaushenko AG. The development and validation of HPLC-DMD method for intermediate products impurities determination of morpholinium 2-((4-(2-methoxyphenyl)-5-(pyridine-4-yl)-4H-1,2,4-triazole-3-yl)thio)acetate in bulk drug. Zaporozhye Medical Journal. 2017;19(3):373-80. doi: https://doi.org/10.14739/2310-1210.2017.3.100947
  10. Singh A, Omer K. Recent advancement in therapeutic activity of carotenoids. In: Physiology. IntechOpen; 2024. Available from: https://doi.org/10.5772/intechopen.112580
  11. Amorim-Carrilho KT, Cepeda A, Fente C, Regal P. Review of methods for analysis of carotenoids. Trends Analyt Chem. 2014;56:49-73. doi: https://doi.org/10.1016/j.trac.2013.12.011
    |
  12. Vendruscolo RG, Fernandes AS, Fagundes MB, Zepka LQ, de Menezes CR, Jacob-Lopes E, et al. Development of a new method for simultaneous extraction of chlorophylls and carotenoids from microalgal biomass. J Appl Phycol. 2021;33(4):1987-97. doi: https://doi.org/10.1007/s10811-021-02470-8
    |
  13. Kurek MA, Aktaş H, Pokorski P, Pogorzelska-Nowicka E, Custodio-Mendoza JA. A comprehensive review of analytical approaches for carotenoids assessment in plant-based foods: Advances, applications, and future directions. Appl Sci (Basel). 2025;15(7):3506. doi: https://doi.org/10.3390/app15073506
    |
  14. Enggi CK, Mahardika F, Devara DM, Saputra MD, Wafiah N, Raihan M, et al. HPLC-UV method validation for quantification of β-carotene in the development of sustained release supplement formulation containing solid dispersion-floating gel in situ. J Pharm Biomed Anal. 2022; 221:115041. doi: https://doi.org/10.1016/j.jpba.2022.115041
    | |
  15. Meléndez-Martínez AJ, Mandić AI, Bantis F, Böhm V, Borge G, Brnčić M, et al. A comprehensive review on carotenoids in foods and feeds: status quo, applications, patents, and research needs. Crit Rev Food Sci Nutr. 2022;62(8):1999-2049. doi: https://doi.org/10.1080/10408398.2020.1867959
    | |
  16. Xu J, Lin J, Peng S, Zhao H, Wang Y, Rao L, et al. Development of an HPLC-PDA Method for the Determination of Capsanthin, Zeaxanthin, Lutein, β-Cryptoxanthin and β-Carotene Simultaneously in Chili Peppers and Products. Molecules. 2023;28(5):2362. doi: https://doi.org/10.3390/molecules28052362
    | |
  17. European Pharmacopoeia Commission (EDQM). European Pharmacopoeia, monograph of Betacarotene. 11th ed. Strasbourg; 2024.
  18. Mazurets SI, Kovalov SV, Rudnyk AM. Fitokhimichne vyvchennia lipofilnoi fraktsii z lystia khmeliu zvychainoho. Zaporozhye Medical Journal. 2012;(3): 96-9. Ukrainian.
  19. Dincel D, Kepekci-Tekkeli SE, Önal C, Önal A, Sagirli O. Liquid chromatographic analysis of carotenoids in foods. J Chil Chem Soc. 2019;64(2):4492-5. Available from: https://jcchems.com/index.php/JCCHEMS/article/view/1220/323
    |
  20. Saini RK, Prasad P, Lokesh V, Shang X, Shin J, Keum YS, et al. Carotenoids: Dietary Sources, Extraction, Encapsulation, Bioavailability, and Health Benefits-A Review of Recent Advancements. Antioxidants (Basel). 2022;11(4):795. doi: https://doi.org/10.3390/antiox11040795
    | |
  21. Lyu X, Ying D, Zhang P, Fang Z. Effect of whole tomato powder or tomato peel powder incorporation on the color, nutritional, and textural properties of extruded high moisture meat analogues. Food Bioprocess Technol. 2024;17(1):231-44. doi: https://doi.org/10.1007/s11947-023-03133-x
    |
  22. Yu ZW, Wang J, Lin FH, He FM, Li HS, Wang SS, et al. A comprehensive study on solvent effect and establishment of n-hexane solvent system based normal-phase liquid chromatography × reversed-phase liquid chromatography for isolation of natural products. J Chromatogr A. 2024;1733:465278. doi: https://doi.org/10.1016/j.chroma.2024.465278
    | |
  23. Hsu BY, Lin CH, Kao TH. Development of a Rapid UPLC Method for Analysis of Carotenoids in Goji Berry Extract and Evaluation of Their Transformation Affected by Saponification. Molecules. 2024;29(23):5684. doi: https://doi.org/10.3390/molecules29235684
    | |
  24. Papapostolou H, Kachrimanidou V, Alexandri M, Plessas S, Papadaki A, Kopsahelis N. Natural Carotenoids: Recent Advances on Separation from Microbial Biomass and Methods of Analysis. Antioxidants (Basel). 2023;12(5):1030. doi: https://doi.org/10.3390/antiox12051030
    | |
  25. Dordai L. A fast HPLC method for the determination of beta-carotene and lycopene from Daucus carota. Nat Resour Sustain Dev. 2023;13(1):195-202. doi: https://doi.org/10.31924/nrsd.v13i1.128
  26. De Luca C, Buratti A, Krauke Y, Stephan S, Monks K, Brighenti V, et al. Investigating the effect of polarity of stationary and mobile phases on retention of cannabinoids in normal phase liquid chromatography. Anal Bioanal Chem. 2022;414(18):5385-95. doi: https://doi.org/10.1007/s00216-021-03862-y
    | |
  27. Jin H, Lao YM, Zhou J, Zhang HJ, Cai ZH. Simultaneous determination of 13 carotenoids by a simple C18 column-based ultra-high-pressure liquid chromatography method for carotenoid profiling in the astaxanthin-accumulating Haematococcus pluvialis. J Chromatogr A. 2017; 1488:93-103. doi: https://doi.org/10.1016/j.chroma.2017.01.088
    | |
  28. Lawler T, Liu Z, Nalbandyan M, Liu Y, Hammond B, Wallace RB, et al. Lutein and zeaxanthin supplement use is associated with increased macular pigment density over 15 years and greater contrast sensitivity in the Carotenoids in Age-Related Eye Disease Study of older-adult women. Investig Ophthalmol Vis Sci. 2021;62(8):2950. Available from: https://iovs.arvojournals.org/article.aspx?articleid=2775628
  29. Fraterrigo Garofalo S, Mallen V, Fino D. Extraction of carotenoids from tomato pomace using deep eutectic solvents composed of short and medium-chain fatty acids and menthol. Food Chem. 2025;484:144342. doi: https://doi.org/10.1016/j.foodchem.2025.144342
    | |
  30. Bas TG. Bioactivity and Bioavailability of Carotenoids Applied in Human Health: Technological Advances and Innovation. Int J Mol Sci. 2024;25(14):7603. doi: https://doi.org/10.3390/ijms25147603
    | |
  31. Schmidt M, Böhm V. Investigating alternative solvents regarding extractability of lipophilic food ingredients in spinach-tomato powder and algae materials. Food Anal Methods. 2025;18(8):1850-62. doi: https://doi.org/10.1007/s12161-025-02821-y
    |
  32. Jiang Y, Ye J, Hu Y, Zhang J, Li W, Zhou X, et al. Extraction and Synthesis of Typical Carotenoids: Lycopene, β-Carotene, and Astaxanthin. Molecules. 2024;29(19):4549. doi: https://doi.org/10.3390/molecules29194549
    | |
  33. Morón-Ortiz Á, Mapelli-Brahm P, Meléndez-Martínez AJ. Sustainable Green Extraction of Carotenoid Pigments: Innovative Technologies and Bio-Based Solvents. Antioxidants (Basel). 2024;13(2):239. doi: https://doi.org/10.3390/antiox13020239
    | |
  34. Gebregziabher BS, Zhang S, Qi J, Azam M, Ghosh S, Feng Y, et al. Simultaneous determination of carotenoids and chlorophylls by the HPLC-UV-VIS method in soybean seeds. Agronomy (Basel). 2021;11(4):758. doi: https://doi.org/10.3390/agronomy11040758
    |
  35. Hammond BR, Renzi-Hammond L. The influence of the macular carotenoids on women's eye and brain health. Nutr Neurosci. 2023;26(8):720-6. doi: https://doi.org/10.1080/1028415X.2022.2084125
    | |
  36. Rathi DN, Rashed AA, Noh M. Determination of retinol and carotenoids in selected Malaysian food products using high-performance liquid chromatography (HPLC). SN Appl Sci. 2022;4(4). doi: https://doi.org/10.1007/s42452-022-04955-8
    |
  37. Singh H, Singh G, Bhatia R. Development and validation of analytical method for simultaneous estimation of lutein, lycopene, and beta-carotene using reversed-phase high-performance liquid chromatography. Pharmaspire. 2020;12(2):55-60. Available from: https://www.isfcppharmaspire.com/uploads/228/13830_pdf.pdf
  38. Alnokkari AS. Optimizing analytical methods to determine lycopene levels in Syrian tomatoes from various regions. Agric Sci Dig - Res J. 2023;(Of). Available from: https://doi.org/10.18805/ag.df-550
    |
  39. Przybylska S, Tokarczyk G. Lycopene in the Prevention of Cardiovascular Diseases. Int J Mol Sci. 2022;23(4):1957. doi: https://doi.org/10.3390/ijms23041957
    | |
  40. Arballo J, Amengual J, Erdman JW Jr. Lycopene: A Critical Review of Digestion, Absorption, Metabolism, and Excretion. Antioxidants (Basel). 2021;10(3):342. doi: https://doi.org/10.3390/antiox10030342
    | |
  41. Bhavin SE, Anuradha G. Development and validation of an RP-HPLC method for estimating nutraceutical lutein. Int J Health Sci (IJHS). 2022;6(S1):9039-50. doi: https://doi.org/10.53730/ijhs.v6ns5.9039
  42. Sutharsan J, Adler L, Jones A, Arcot J. Quantification of macular carotenoids over a wide dynamic range in plant matrices and Caco-2 cells using a single transferable analytical method. Foods. 2026;15(6):981. doi: https://doi.org/10.3390/foods15060981
    | |
  43. Melfi MT, Nardiello D, Cicco N, Candido V, Centonze D. Simultaneous determination of water- and fat-soluble vitamins, lycopene and beta-carotene in tomato samples and pharmaceutical formulations: Double injection single run by reverse-phase liquid chromatography with UV detection. J Food Compost Anal. 2018;70:9-17. doi: https://doi.org/10.1016/j.jfca.2018.04.002
    |
  44. Craft NE, Springs DG, Chavan J. Lutein HPLC method comparison and validation. FASEB J. 2016;30(S1). doi: https://doi.org/10.1096/fasebj.30.1_supplement.689.5
  45. Olmedilla-Alonso B, Granado-Lorencio F, Castro-Feito J, Herrero-Barbudo C, Blanco-Navarro I, Estévez-Santiago R. Bioavailability of Lutein from Marigold Flowers (Free vs. Ester Forms): A Randomised Cross-Over Study to Assess Serum Response and Visual Contrast Threshold in Adults. Nutrients. 2024;16(10):1415. doi: https://doi.org/10.3390/nu16101415
    | |
  46. Wu B, Li X, Zheng X, Zhao J, Qiao L, Zheng J. A simple and robust HPLC method to analyze lutein in wheat. Methods X. 2022; 9:101926. doi: https://doi.org/10.1016/j.mex.2022.101926
    | |
  47. Anwar S, Nayak JJ, Alagoz Y, Wojtalewicz D, Cazzonelli CI. Purification and use of carotenoid standards to quantify cis-trans geometrical carotenoid isomers in plant tissues. Methods Enzymol. 2022;670:57-85. doi: https://doi.org/10.1016/bs.mie.2022.01.005
    | |

Additional Files

Published

2026-06-26

How to Cite

1.
Duiun IF, Keitlin IM, Mazulin GV. High-performance liquid chromatography methods for carotenoid determination. Current issues in pharmacy and medicine: science and practice [Internet]. 2026Jun.26 [cited 2026Jun.26];19(2):216-2. Available from: https://pharmed.zsmu.edu.ua/article/view/355779

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Section

Review