A rapid and green HPLC method for squalene determination in amaranth oil using a polar-embedded stationary phase and ethanol mobile phase

A reversed-phase high-performance liquid chromatographic (RP-HPLC) method for the quantitative determination of squalene in amaranth oil was developed and validated. As a preliminary step, the chromatographic separation of amaranth oil components was evaluated on eight columns with different bonded stationary phases, including C8, phenyl, pentafluorophenyl (PFP), and polar-embedded phases. Among the tested columns, a Supelcosil LC-ABZ (150 × 4.6 mm, 5 μm) column with an embedded polar group provided the most favorable selectivity and shorter analysis time compared with conventional C18 columns. The mobile phase composition was optimized by varying the ethanol content, and a final mobile phase containing 91% ethanol provided efficient separation of squalene and retention time about 5 min. Method selectivity was confirmed by separation of squalene from α-tocopherol acetate (Rs = 5.7) and satisfactory resolution from oil triglycerides (Rs = 9.3). Validation studies demonstrated satisfactory repeatability and intermediate precision, with relative standard deviations not exceeding 1.0%. Solution stability was confirmed for up to 20 h, with changes in peak area below 0.1%. Corn oil was used as a validation matrix due to its similar fatty acid composition to amaranth oil, ensuring relevant assessment of matrix effects. The developed RP-HPLC procedure is suitable for accurate, precise, and reliable determination of squalene in amaranth oil, offering a fast and environmentally friendly analytical approach.