Analysis of Lutein Content in Microencapsulated Marigold Flower Extract ( Tagetes erecta L.) Using UHPLC-Q-Orbitrap-HRMS and Its Cytotoxicity in ARPE-19 Cells

**Background:** Lutein, a carotenoid abundant in marigold flowers (Tagetes erecta L.), is a potent antioxidant that accumulates in the macular region of the eye and protects against blue light damage. However, lutein is susceptible to degradation by oxygen, heat, and light. Conventional extraction uses toxic organic solvents, and lutein stability in finished products is a challenge. This study aimed to develop a green extraction method using virgin coconut oil, evaluate the cytotoxicity of the extract in ARPE-19 cells, optimize microencapsulation to protect lutein, and validate a UHPLC-Q-Orbitrap-HRMS method for lutein quantification.

**Methods:** Dried marigold petals were extracted with virgin coconut oil (1:5 w/w) using either ultrasonication (COU, 20 min) or microwave-assisted extraction (COM, 1 min at 900 W). The oil extracts were saponified to obtain lutein-rich fractions. Lutein content was analyzed by UHPLC-Q-Orbitrap-HRMS with ESI positive mode and SRM transition m/z 568.4275→145.0927. Method validation followed ICH guidelines. Cytotoxicity was assessed in ARPE-19 cells via MTT assay after 24 h treatment with lutein-rich fractions at concentrations equivalent to 1, 2, 4, 8, and 16 µM lutein. Microencapsulation was performed by spray drying (inlet 170°C) using maltodextrin and gum acacia as wall materials at four oil-to-wall ratios (1:2, 1:3, 1:4, 1:5) with 30% total solids. Powder characterization included moisture content, yield, encapsulation efficiency (%ME), and lutein content.

**Key Results:** Extraction yields were 72.10% (COU) and 74.23% (COM). The validated UHPLC method showed linearity (R²=0.9984) from 75–500 ng/mL, LOD 4.50 ng/mL, LOQ 13.64 ng/mL, recovery 90.53–125.20%, and intra-day/inter-day RSD <7% and <5%, respectively. COM yielded significantly higher lutein content (27.22 ± 1.17 ng/µg marigold powder) than COU (23.11 ± 0.49 ng/µg). Cytotoxicity assays showed no significant reduction in ARPE-19 cell viability at any tested concentration (p<0.05). Microencapsulation yields ranged from 62.01% to 70.20%, moisture content 3.83–4.88%. %ME increased with wall-to-oil ratio: F1 (1:2) 35.33%, F2 (1:3) 52.16%, F3 (1:4) 72.29%, F4 (1:5) 82.60%. Lutein content in microcapsules was highest in F4 (6.18 ± 0.17 µg/g powder) and lowest in F3 (4.69 ± 0.25 µg/g). SEM showed dents without cracks on particle surfaces.

**Clinical Implications:** The study demonstrates that microwave-assisted extraction with virgin coconut oil is a viable green method for obtaining lutein-rich marigold extract. The validated LC-MS/MS method provides a reliable tool for quality control of lutein in raw materials and finished products. The absence of cytotoxicity in ARPE-19 cells at up to 16 µM supports the in vitro safety of the extract for potential use in eye health supplements. Microencapsulation with a 1:5 oil-to-wall ratio effectively protects lutein, achieving high encapsulation efficiency and lutein retention. These findings lay groundwork for developing stable, value-added functional ingredients from marigold and coconut oil, though further in vivo toxicity and efficacy studies are needed.