Method-Dependent Variation in Orange Peel Phytochemicals: Insights from LC-MS and Antioxidant Analysis

Aims: To evaluate method-dependent selectivity in the extraction of carotenoids and phenolics from orange (Citrus sinensis) peel using ultrasound-assisted (UAE), microwave-assisted (MAE), and conventional solvent (SAE) extraction, and to characterize the resulting extracts by LC-MS profiling and in vitro antioxidant assays. Study Design: Comparative experimental study using three extraction methods with multi-parameter bioactive profiling. Place and Duration of Study: Departments of Chemistry and Food Science and Technology, IBB University (IBBUL), Lapai, Niger State, Nigeria, and some analytical work conducted in the National Institute for Pharmaceutical Research and Development (NIPRD). Methodology: Orange peel was freeze-dried and extracted by UAE, MAE, and SAE using acetone. Total carotenoid content (TCC) was determined spectrophotometrically at 470 nm using the lutein-specific absorption coefficient (E0 = 2000). Individual carotenoid species were identified by UPLC-MS/MS (Acquity H-CLASS/Xevo TQD, APCI positive mode, Sunfire C18 column). Total phenolic content (TPC) and total flavonoid content (TFC) were quantified by Folin-Ciocalteu and aluminium chloride colorimetric methods, respectively. Antioxidant activity was assessed by DPPH radical scavenging (IC50) and ferric reducing antioxidant capacity (FRAC). Pearson correlation analysis was performed to integrate findings across parameters. Results: UAE yielded the highest TCC (131.33 +/- 3.51 microg/g), while SAE yielded the highest TPC (165.94 +/- 3.21 mg GAE/g) and the most potent DPPH activity (IC50 = 26.3 microg/mL). TCC and TPC were strongly negatively correlated (r = -0.929, P < .001), confirming inverse method-dependent selectivity. LC-MS identified 11 compounds, including beta-carotene, lutein, beta-cryptoxanthin, zeaxanthin, and zeinoxanthin; UAE yielded the highest proportions of all major carotenoid species. TPC was the dominant predictor of antioxidant performance (TPC vs FRAC: r = +0.981; TPC vs DPPH IC50: r = -0.963; both P < .001). Conclusion: UAE is the preferred method for carotenoid-targeted extractions, while SAE and MAE are more suitable where phenolic antioxidant capacity is the primary objective. MAE offers a sustainable intermediate option. The study provides a data-driven framework for compound-class-targeted extraction method selection in orange peel valorisation.