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HPLC
The products were purified using the XDB-C18 separationcolumn (5 μm, 250 × 4.6 mm). Retinol and retinal were detected in a mobile phase consistingof methanol and acetonitrile in a ratio of 95:5 at the detection temperature of 25 ℃ with thedetection wavelength of 352 nm. The mobile phase consisted of methanol, acetonitrile, andisopropanol in a ratio of 3:5:2, and the detection temperature was 40 ℃. The detectionwavelength used for β-carotene was 450 nm.Squalene was detected at 210 nm usingmobile phase methanol at 40 ℃.
References：
Wang X, Xu X, Liu J, Liu Y, Li J, Du G, Lv X, Liu L. Metabolic Engineering of Saccharomyces cerevisiae for Efficient Retinol Synthesis. J Fungi (Basel). 2023 Apr 26;9(5):512. doi: 10.3390/jof9050512. PMID: 37233223; PMCID: PMC10219262.
HPLC systemequipped with a C18 column (5 μm × 4.6/250 mm)was used. Pigments were eluted at a flow rate of 0.7 mL/min with a 25-min gradient of ethyl acetate (0%–100%) inacetonitrile-water-triethylamine (9:1:0.01, vol/vol/vol) and detectedusing thediode array detector at 470 nm.
References：
Bi Y, Guo P, Liu L, Chen L, Zhang W. Elucidation of sterol biosynthesis pathway and its co-regulation with fatty acid biosynthesis in the oleaginous marine protist Schizochytrium sp. Front Bioeng Biotechnol. 2023 Apr 27;11:1188461. doi: 10.3389/fbioe.2023.1188461. PMID: 37180050; PMCID: PMC10174431.
UPLC
Chromatography was carried out with a Ultra-high performance liquid chromatography. Samples were measured by a C18 carotenoid column (100 mm × 2.1 i.d., 1.7 μm). The eluent was methyl tertiary butyl ether and methanol (V/V = 30:70) with the type of isocratic elution. And each eluent contained 0.01% BHT and 0.05% TEA (triethylamine) as modifiers in order to prevent the degradation of carotenoids on the column. The following rate was 0.5 mL/min, column temperature was 25 ℃, and the injection volume was 5 μL, analyzing with UV at 445 nm.
References：
Shen YH, Yang FY, Lu BG, Zhao WW, Jiang T, Feng L, Chen XJ, Ming R. Exploring the differential mechanisms of carotenoid biosynthesis in the yellow peel and red flesh of papaya. BMC Genomics. 2019 Jan 16;20(1):49. doi: 10.1186/s12864-018-5388-0. PMID: 30651061; PMCID: PMC6335806.
HPLC
HPLC analysis of chlorophyll and carotenoids were carried out using an Agilent 1260 instrument with a VWD detector . Samples (10 μL) were separated at 30℃ on a C18 column (150 mm × 3.9 mm i.d.; 4 μm particle size) using isopropanol and 80% acetonitrile–water at a flow rate of 0.5ml/min.
References：
Di H, Zhang Y, Ma J, Wei J, Wang Y, Li Z, Cui C, Fang P, Ma W, Li H, Sun B, Zhang F. Sucrose treatment delays senescence and maintains the postharvest quality of baby mustard (Brassica juncea var. gemmifera). Food Chem X. 2022 Mar 1;14:100272. doi: 10.1016/j.fochx.2022.100272. PMID: 35257095; PMCID: PMC8897633.
HPLC
HPLC analysis of carotenoids was carried out using an equipped with a variable wavelength detector (VWD). Samples (10 μL) were separated at 30℃ on a C18 column (150 × 3.9 mm id; 3 μm particle size) using isopropanol and 80% acetonitrile-water at a flow rate of 0.5 mL min?1 ; the absorbance was measured at 448 and 428 nm.
References：
Jian Y, Zhang C, Wang Y, Li Z, Chen J, Zhou W, Huang W, Jiang M, Zheng H, Li M, Miao H, Zhang F, Li H, Wang Q, Sun B. Characterization of the Role of the Neoxanthin Synthase Gene BoaNXS in Carotenoid Biosynthesis in Chinese Kale. Genes (Basel). 2021 Jul 24;12(8):1122. doi: 10.3390/genes12081122. PMID: 34440295; PMCID: PMC8393425.
LC-MS
The carotenoids contents were determined using an HPLC system equipped with a C30 column (150 ×4.6 mm, 3 μm) and a UV/VIS detector. The mobile phase consisted ofA (acetonitrile: methanol = 3:1) and B (methyl tert-butyl ether), and a gradient elution program (Lee et al., 2001) was used. The flow rate was 1.0 ml/min, and the column temperature was 40℃. The signals were detected at 450 nm. Identification of lutein and zeaxanthin was conducted on AB TripleTOF 5600 plus system equipped with an APCI source operated in the positive ionization mode. The column and temperature settings were the same as HPLC. The mobile phase was A (methanol) and B (acetonitrile: isopropanol = 1:1) with linear increase of B from 0 to 100% in 30 min followed by dropping to 0 in 10 min. The source voltage was +5.5 kV, and the source temperature was 500℃. The pressure of Gas 1 (Air) and Gas 2 (Air) was set to 50 psi. The pressure of Curtain Gas (N2) was set to 35 psi. The m/z scan range of production was set as 50–1500.
References：
Bian Q, Zhou P, Yao Z, Li M, Yu H, Ye L. Heterologous biosynthesis of lutein in S. cerevisiae enabled by temporospatial pathway control. Metab Eng. 2021 Sep;67:19-28. doi: 10.1016/j.ymben.2021.05.008. Epub 2021 May 30. PMID: 34077803.
HPLC
HPLC analysis of chlorophylls and carotenoids was carried out using an Agilent 1260 instrument with a variable wavelength detector (VWD) (Agilent Technologies, Inc., Palo Alto, USA).Samples (10 μL) were separated at 30 ℃ on a Waters C18 column (150 × 3.9 mm i.d.; 4 μm particle size) using isopropanol and 80% acetonitrile–water at a flow rate of 0.5 mL min?1. Absorbance was detected at 448 and 428 nm.
References：
Sun B, Di H, Zhang J, Xia P, Huang W, Jian Y, Zhang C, Zhang F. Effect of light on sensory quality, health-promoting phytochemicals and antioxidant capacity in post-harvest baby mustard. Food Chem. 2021 Mar 1;339:128057. doi: 10.1016/j.foodchem.2020.128057. Epub 2020 Sep 11. PMID: 32947106.
HPLC
The analyses of β-carotene were performed on an HPLC system equipped with a C18 column (4.6mm×250mm). The mobile phase is composed of acetonitrile, methanol, and isopropanol with a volume ratio of 5:3:2, followed by a flow rate of 1 mL/min at 40 ℃. The UV/VIS signals were detected at 450 nm, and the analysis time of each sample is 40 min.
References：
Dou W, Zhu Q, Zhang M, Jia Z, Guan W. Screening and evaluation of the strong endogenous promoters in Pichia pastoris. Microb Cell Fact. 2021 Aug 9;20(1):156. doi: 10.1186/s12934-021-01648-6. PMID: 34372831; PMCID: PMC8351359.
HPLC
Samples (20 μL of extract) were analyzed using a C30 column (4.6 × 250 mm, 5 μm) coupled with a diode array detector at 450nm,with HPLC-grade methyl tertiary butyl ether and methanol (V:V = 30:70) as the solvent. The flow rate was 1.0 mL/min, and the temperature was 25℃.
References：
Zhao B, Sun M, Li J, Su Z, Cai Z, Shen Z, Ma R, Yan J, Yu M. Carotenoid Profiling of Yellow-Flesh Peach Fruit. Foods. 2022 Jun 7;11(12):1669. doi: 10.3390/foods11121669. PMID: 35741867; PMCID: PMC9222759.
HPLC
The carotenoid content was chromatographically quantified using HPLC-PDA. Carotenoids were determined using a C30 column (4.6 × 250 mm i.d., 5 μm). As previously described, the mobile phases were MTBE as eluent A, methanol as eluent B, and H2O as eluent C. A gradient program was used, where the initial condition was 60% B/40% C; 0–5 min, 80% B/20% C; 5–10 min, 15%A/81% B/4% C; 10–60 min, 85%A/11% B/4% C; 60–71 min, 100% B; 71–72 min, then back to the initial condition for re-equilibration. The flow was 1.0 mL/min, and the injection volume was 10 μL. The wavelength and oven temperature were set to 450 nm, 470 nm, and 27℃, respectively.
References：
Wei J, Li Y, Ye Z, Li Y, Zhou Z. Citrus Carotenoid Extracts Exert Anticancer Effects through Anti-Proliferation, Oxidative Stress, and Mitochondrial-Dependent Apoptosis in MCF-7 Cells. Foods. 2023 Sep 18;12(18):3469. doi: 10.3390/foods12183469. PMID: 37761178; PMCID: PMC10529845.
