Research Article Development of Isocratic RP-HPLC Method for Separation and Quantification of L-Citrulline and L-Arginine in Watermelons Rasdin Ridwan , 1 Hairil Rashmizal Abdul Razak, 2 Mohd Ilham Adenan, 3,4 and Wan Mazlina Md Saad 1 1 Centre of Medical Laboratory Technology, Faculty of Health Sciences, Universiti Teknologi MARA, Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor, Malaysia 2 Centre of Medical Imaging, Faculty of Health Sciences, Universiti Teknologi MARA, Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor, Malaysia 3 Faculty of Applied Sciences, Universiti Teknologi MARA, 40450 Shah Alam, Selangor, Malaysia 4 Atta-ur-Rahman Institute for Natural Product Discovery, Level 9, Bangunan FF3, Universiti Teknologi MARA, Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor, Malaysia Correspondence should be addressed to Wan Mazlina Md Saad; mazlinasaad14@gmail.com Received 30 December 2017; Revised 25 February 2018; Accepted 12 March 2018; Published 2 May 2018 Academic Editor: G¨ unther K. Bonn Copyright © 2018 Rasdin Ridwan et al. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Watermelons (Citrullus lanatus) are known to have sufcient 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 quantifcation of L-citrulline and L-arginine. Tree diferent mobile phases, 0.7% H 3 P0 4 , 0.1% H 3 P0 4 , and 0.7% H 3 P0 4 : ACN (90 : 10), were tested on two diferent columns using Zorbax Eclipse XDB-C 18 and Gemini C 18 with a fow rate of 0.5 mL/min and a detection wavelength at 195 nm. Efcient separation with reproducible resolution of L-citrulline and L-arginine was achieved using 0.1% H 3 P0 4 on the Gemini C 18 column. Te method was validated and good linearity of L-citrulline and L-arginine was obtained with  2 = 0.9956,  = 0.1664 + 2.4142 and  2 = 0.9912,  = 0.4100 + 3.4850, respectively. L-citrulline content showed the highest concentration in red watermelon of fesh 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. Te isocratic RP-HPLC method with 0.1% H 3 P0 4 on the Gemini C 18 column proved to be efcient for separation and quantifcation of L-citrulline and L-arginine in watermelons. 1. Introduction Citrullus lanatus (Tunb.) Matsum. and Nakai, commonly known as watermelon, is a nonseasonal fruit which is cul- tivated abundantly in Malaysia and other tropical regions [1]. It belongs to the Cucurbitaceae plant family, which originated from the African Kalahari Desert [1]. Watermelons have high content of phytonutrients and are rich in dietary antioxidants such as carotenoids (lycopene and -carotene), polyphenolics, ascorbic acid, and signifcant amino acids [2]. Watermelons are usually consumed by juicing the fesh, bene- fcial in the prevention and improvement of health problems, such as cardiovascular diseases, erectile dysfunction, hyper- tension, and cancers [3]. Figueroa et al. [4] demonstrated that watermelon juice supplementation improves aortic hemodynamics by reducing the refected wave amplitude in prehypertensive individuals. A study by Poduri et al. [5] reported that watermelon attenuated hypercholesterolemia- induced atherosclerosis in mice. Commercial watermelon juices provide enormous marketing potential and nutritious drinks for individuals to maintain a healthy lifestyle. Amino acids, particularly L-citrulline and L-arginine, are regarded as major types of phytonutrients present in water- melons which may contribute to their reputed and diversifed health benefts [6]. L-citrulline, C 6 H 13 N 3 O 3 (IUPAC name: 2-amino-5-(carbamoylamino)pentanoic acid) (Figure 1), is a nonessential amino acid frstly identifed from watermelon, Citrullus vulgaris Schrad. [7, 8]. L-citrulline is a physiological Hindawi International Journal of Analytical Chemistry Volume 2018, Article ID 4798530, 9 pages https://doi.org/10.1155/2018/4798530