Development of Isocratic RP-HPLC Method for Separation and Quantification of L-Citrulline and L-Arginine in...

Watermelons (Citrullus lanatus) are known to have sufficient amino acid content. In this study, watermelons grown and consumed in Malaysia were investigated for their amino acid content, L-citrulline and L-arginine, by the isocratic RP-HPLC method. Flesh and rind watermelons were juiced, and freeze-dried samples were used for separation and quantification of L-citrulline and L-arginine. Three different mobile phases, 0.7% H₃P0₄, 0.1% H₃P0₄, and 0.7% H₃P0₄ : ACN (90 : 10), were tested on two different columns using Zorbax Eclipse XDB-C₁₈ and Gemini C₁₈ with a flow rate of 0.5 mL/min and a detection wavelength at 195 nm. Efficient separation with reproducible resolution of L-citrulline and L-arginine was achieved using 0.1% H₃P0₄ on the Gemini C₁₈ column. The method was validated and good linearity of L-citrulline and L-arginine was obtained with R² = 0.9956, y = 0.1664x + 2.4142 and R² = 0.9912, y = 0.4100x + 3.4850, respectively. L-citrulline content showed the highest concentration in red watermelon of flesh and rind juice extract (43.81 mg/g and 45.02 mg/g), whereas L-arginine concentration was lower than L-citrulline, ranging from 3.39 to 11.14 mg/g. The isocratic RP-HPLC method with 0.1% H₃P0₄ on the Gemini C₁₈ column proved to be efficient for separation and quantification of L-citrulline and L-arginine in watermelons.