Carotenoid Quantification by HPLC

Carotenoid was extracted as described in our previous study (20 (link)). High-performance liquid chromatography (HPLC) was performed for carotenoid quantification in samples. Samples in a volume of 10 μL were loaded on an infinity Lab Proshell 120 EC-C18 column (4.6 × 150, ODS 4 μm). Two solvents A (96% methanol) and B (100% methyl-terc-butyl ether) were used as mobile phase in the following gradient to analyze carotenoid: min/solvent A%/solvent B% was (0/99/1; 8/60/40; 13/46/54; 15/0/100; 18/0/100; 21/99/1; 25/99/1) at a flow rate of 1 ml/min. Column thermostat temperature was set as 35°C and detection wavelength was set as 450 nm, using a diode-array detector (Agilent Technologies, Santa Clara, CA, United States). The following standards were used to identify the carotenoids in transformants: β-carotene, canthaxanthin, astaxanthin, echinenone (Sigma–Aldrich). The total carotenoids were quantified by a spectrophotometer at 450 nm and measured using an extinction coefficient of 2,500 (A1% = 2,500) as previously described (21 ).

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Naz T., Yang J., Nosheen S., Sun C., Nazir Y., Mohamed H., Fazili A.B., Ullah S., Li S., Yang W., Garre V, & Song Y. (2021). Genetic Modification of Mucor circinelloides for Canthaxanthin Production by Heterologous Expression of β-carotene Ketolase Gene. Frontiers in Nutrition, 8, 756218.

Publication 2021

diode-array detector β-carotene canthaxanthin astaxanthin echinenone

Astaxanthin Canthaxanthin Carotene Carotenoids Echinenone Ether Extinction High performance liquid chromatography Methanol Solvent Terc

Corresponding Organization :

Other organizations : Shandong University of Technology, National University of Malaysia, Al-Azhar University, University of Lahore, Consejo Superior de Investigaciones Científicas, Universidad de Murcia, Instituto de Química Física Blas Cabrera

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Variable analysis

independent variables

Carotenoid extraction method described in previous study (20)

dependent variables

Carotenoid quantification in samples
Total carotenoids measured by spectrophotometry at 450 nm

control variables

Sample volume (10 μL)
HPLC column (Infinity Lab Proshell 120 EC-C18, 4.6 × 150, ODS 4 μm)
Mobile phase gradient
Flow rate (1 mL/min)
Column thermostat temperature (35°C)
Detection wavelength (450 nm)

controls

Positive controls: β-carotene, canthaxanthin, astaxanthin, echinenone standards

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