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

Abstract

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.

Fruit Morphology, Citrulline, and Arginine Levels in Diverse Watermelon (Citrullus lanatus) Germplasm Collections

“…Alcohols are commonly used for the extraction of citrulline and arginine instead of water. Ridwan et al (2018) [ 28 ] and this study ensure the use of water with two different extraction and measurement methods. Other authors either simply thawed, diluted, centrifuged, or used the supernatant for analysis [ 11 , 23 ].…”

“…The separation and analysis of citrulline and arginine in mixed standard and watermelon extracts were performed using selected mobile phases and a chromatographic column according to earlier reports, but with slight modifications [ 8 , 27 , 28 ]. Citrulline and arginine in watermelon flesh samples were efficiently separated in the Gemini C 18 column using 0.1% phosphoric acid in an aqueous solution as a mobile phase and isocratic elution mode.…”

“…Citrulline and arginine in watermelon flesh samples were efficiently separated in the Gemini C 18 column using 0.1% phosphoric acid in an aqueous solution as a mobile phase and isocratic elution mode. The Gemini column, a first-generation stationary phase obtained from Phenomenex, is end-capped with porous silica used as a base core, and a layer of embedded silica is coated on the top of the silica core [ 28 , 29 ]. In this column and the specified mobile phase condition, arginine and citrulline were eluted at 7.5 and 9.3 min, respectively ( Figure 2 ).…”

SEPARATION OF L-ARGININE AND L-CITRULLINE IN RED AND YELLOW CRIMSON WATERMELON (Citrullus lanatus) JUICES EXTRACT USING HPLC GRADIENT MODE

“…This present finding is in agreement with Agrafiotou et al [22] that showed high sensitivity detection of underivatised amino acids mixture at wavelength 200 nm compared to 190 nm and 210 nm. The lower wavelengths ranging from 195 nm to 210 nm were chosen in this study since most amino acids with appropriate functional group of carboxylic group (-COOH) including L-arginine and L-citrulline can only be detected at low UV wavelengths [5,22]. Hesse and Weller [20] supported the notion that lower UV range provides high sensitivity, but interference of solvents, buffers, and other additives is often prohibitive.…”

“…Yellow crimson, a yellow fleshed watermelon contains major carotenoid known as neoxanthin that plays specific role in protection against photooxidative stress [3]. Recent report from previous studies have shown that both red and yellow crimson watermelon juices contain high concentration of amino acids especially L-arginine and Lcitrulline [4,5]. These amino acids are crucial in nitric oxide production for improving cardiovascular health, immune boosting, and sexual health benefits [6].…”