Vitamin A (retinylpalmitate) and E (α-tocopherol) are essential fat-soluble micronutrients that play critical roles in maintaining human health and their accurate quantification in food matrices such as eggs is vital for nutritional assessment, quality control and regulatory compliance. A simple, precise and robust reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated for the simultaneous estimation of Vitamins A and E in egg yolk. Chromatographic separation was achieved on an RP-C18 column (250 × 4.6 mm, 10 µm) using methanol and acetonitrile (30:70, v/v) as the mobile phase under isocratic conditions at a flow rate of 1.0 mL/min. Detection was performed at 264 nm for Vitamin E and 325–395 nm for Vitamin A. Sample preparation involved saponification with ethanolic potassium hydroxide, followed by liquid–liquid extraction and reconstitution in hexane. The method provided baseline resolution of Vitamin E (retention time ≈ 7 min) and Vitamin A (retention time ≈ 9 min) without interference from matrix components, confirming specificity. Linearity was observed across the ranges of 0.1–100 ppm for Vitamin E (r² = 0.999) and 0.1–1.3 ppm for Vitamin A (r² = 0.997). Precision studies showed excellent repeatability with %RSD values below 1%, while accuracy was confirmed with mean recoveries of 99.8% for Vitamin E and 100.1% for Vitamin A. Sensitivity was established by low LOD and LOQ values and robustness testing confirmed method reliability under minor variations in chromatographic conditions. This validated RP-HPLC method is rapid, accurate and reproducible, making it highly suitable for routine food quality control, nutritional evaluation and regulatory applications.
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