Carotenoids are lipophilic natural pigments synthesized by all photosynthetic organisms and some non-photosynthetic prokaryotes and fungi. Typical carotenoids are C40 mushroom compounds and their derivatives consisting of eight isoprene units linked end to end. Some carotenoids have shorter (C30) or longer (C45 or C50) side carbon chains and are usually divided into two classes: xanthophylls and carotenes. In nature, more than 1100 carotenoids have been found, the common ones are lycopene, a-carotene, β-carotene, astaxanthin, lutein, zeaxanthin, cryptoxanthin and violet xanthin.
Carotenoids have a wide range of biological roles. In plants, carotenoids are integral to the correct composition of the plant photosynthetic system and are also the main pigments for coloring petals, fruits, and colored leaves. In human health, some carotenoids act as synthetic precursors of VA to improve vision, and some carotenoids have various effects such as scavenging free radicals, improving immunity, reducing the risk of cancer, and preventing cardiovascular disease. In addition, carotenoids are used as natural colorants in food and cosmetics and as additives in animal feed.
Animals (except for some species of aphids) cannot synthesize carotenoids and need to obtain them from food. The demand for carotenoid products is perhaps increasing and there is a need to fully exploit plant carotenoids. Reliable techniques for qualitative and quantitative analysis of carotenoids are needed to meet research and production needs.
Carotenoid Detection Method
- Spectrophotometric method
Spectrophotometric methods take advantage of the long conjugated double bond system (chromophores) possessed by carotenoid molecules to give them strong absorption peaks in the UV-visible region. Qualitative and quantitative analysis of carotenoids can be performed based on information such as the position of the highest peak, the shape of the absorption spectrum and the fine structure of the spectrum.
- High performance liquid chromatography (HPLC) and ultra-high performance liquid chromatography (UHPLC)
The HPLC method uses a single or a mixture of solvents of different polarities as the mobile phase, which is fed into the column using a high-pressure pump. Within the column, the different components are separated according to the equilibrium of the partition of the separated substance components between the stationary and mobile phases, and subsequently enter the detector for detection, thus enabling the analysis of the sample. The reversed-phase C18 column allows the separation and determination of carotenoids, but the C18 column has limited ability to separate the cis-trans isomers of carotenoids, while the C30 column provides better separation of non-polar carotenoids and geometric isomers that are difficult to separate on the C18 column.
UHPLC has higher signal-to-noise ratio, better separation, and greatly reduces the analysis time. Currently, the main UPLC columns used for the analysis of carotenoids include HSS C18 (for the separation of luteolin) and BEH C18 (for the separation of carotenoids).
- High performance liquid chromatography-mass spectrometry (HPLC-MS)
High performance liquid chromatography-mass spectrometry (HPLC-MS) combines the high separation performance of HPLC with the powerful structural identification capabilities of MS, and is widely used for qualitative, quantitative and structural identification of substances. For known substances, the molecular structure information (molecular mass and fracture pattern) obtained by mass spectrometry can be compared with published fragment ion abundance or mass spectrometry libraries. For unknown substances, online PDA or UV-Vis detectors and tandem mass spectrometry (MS/MS) are used to provide more valuable information for their identification.
Reference
- Arathi, B. P., Sowmya, P. R. R., Vijay, K., Baskaran, V., & Lakshminarayana, R. (2015). Metabolomics of carotenoids: The challenges and prospects–A review. Trends in Food Science & Technology, 45(1), 105-117.