Science
The Separation of L Citrulline & L Arginine Using Isocratic HPLC Methodology Discussion

Question Description

I need help with a Chemistry question. All explanations and answers will be used to help me learn.

Rephrase/Summarize and discuss the attached document. Take out the parts that you feel are unnecessary for it to be summarized well (like the story).

Unformatted Attachment Preview

Hindawi International Journal of Analytical Chemistry Volume 2018, Article ID 4798530, 9 pages https://doi.org/10.1155/2018/4798530 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: Günther K. Bonn Copyright Β© 2018 Rasdin Ridwan et al. This 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 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% H3 P04 , 0.1% H3 P04 , and 0.7% H3 P04 : 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% H3 P04 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 = 0.1664π‘₯ + 2.4142 and 𝑅2 = 0.9912, 𝑦 = 0.4100π‘₯ + 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% H3 P04 on the Gemini C18 column proved to be efficient for separation and quantification of L-citrulline and L-arginine in watermelons. 1. Introduction Citrullus lanatus (Thunb.) Matsum. and Nakai, commonly known as watermelon, is a nonseasonal fruit which is cultivated 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 𝛽-carotene), polyphenolics, ascorbic acid, and significant amino acids [2]. Watermelons are usually consumed by juicing the flesh, beneficial in the prevention and improvement of health problems, such as cardiovascular diseases, erectile dysfunction, hypertension, and cancers [3]. Figueroa et al. [4] demonstrated that watermelon juice supplementation improves aortic hemodynamics by reducing the reflected wave amplitude in prehypertensive individuals. A study by Poduri et al. [5] reported that watermelon attenuated hypercholesterolemiainduced 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 watermelons which may contribute to their reputed and diversified health benefits [6]. L-citrulline, C6 H13 N3 O3 (IUPAC name: 2-amino-5-(carbamoylamino)pentanoic acid) (Figure 1), is a nonessential amino acid firstly identified from watermelon, Citrullus vulgaris Schrad. [7, 8]. L-citrulline is a physiological 2 International Journal of Analytical Chemistry O O N H (2 . OH .(2 Figure 1: Molecular structure of L-citrulline (175.2 g/mol). .( (2 . of derivatization reagents that may cause poor compound recovery [19]. Analysis of underivatized L-citrulline and Larginine is warranted for rapid and effective quantification of these compounds. Given that no amino acids content of Lcitrulline and L-arginine in Malaysia watermelons has been reported so far, we have developed an isocratic RP-HPLC method for separation and quantification of L-citrulline and L-arginine in watermelons. O OH N H .(2 Figure 2: Molecular structure of L-arginine (174.2 g/mol). endogenous amino acid to most living systems involved in protein metabolism and removal of excess metabolic ammonia [9]. It serves as a precursor for L-arginine and product of nitric oxide (NO) cycle [10]. L-arginine, C6 H14 N4 O2 (IUPAC name: (S)-2-amino-5-guanidinopentanoic acid) (Figure 2), is a semiessential and free form physiological amino acid that functions as one of 20 building block proteins for biological processes such as cell division, ammonia removal, wound healing, and hormone release [7, 8]. Wu et al. [11] demonstrated that supplementation of L-citrulline and Larginine from watermelon juice improved serum levels of NO metabolites and aortic endothelial-mediated vasodilation in diabetic rats. L-citrulline and L-arginine are present in all parts of watermelon fruits including flesh, rind, and seed [7]. A study done by Rimando and Perkins-Veazie [12] reported that the rind of red watermelon and yellow watermelon contains more L-citrulline at a concentration ranging from 15.6 to 29.4 mg/g than flesh, 7.9–28.5 mg/g. Similar to the above finding, Jayaprakasha et al. [13] reported that rinds of Citrullus vulgaris varieties such as petite treat and jamboree watermelon and also yellow crimson watermelon contained slightly higher L-citrulline ranging from 13.95 to 28.46 mg/g than flesh, 11.25–16.73 mg/g. These findings suggested that watermelon rind has an abundance of L-citrulline content in comparison to its content in flesh. Analyses of L-citrulline and L-arginine were routinely conducted using capillary electrophoresis and quantification by a spectrophotometric method; however, the method is less sensitive, leading to discrepancies in the outcomes [6]. L-citrulline and L-arginine are polar, nonvolatile, and devoid of chromophores; thus analysis by reverse-phase high performance liquid chromatography (RP-HPLC) commonly employed a derivatization method using pre- or postcolumn derivatization [14–17]. Jayaprakasha et al. [13] stated that precolumn derivatization such as orthophthalaldehyde (OPA), naphthalene-2,3-dicarboxaldehyde, or 4-dimethylaminoazobenzene-4σΈ€  -sulfonyl chloride (dabsyl chloride) was able to provide accurate and stable chromatography baseline, but the reactions were unstable and affected by the sample matrix [18]. Postcolumn derivatization by ninhydrin is tedious due to long analysis time up to 72 hours and instability 2. Materials and Methods 2.1. Chemicals and Reagents. L-citrulline (purity β‰₯ 99%) and L-arginine (purity β‰₯ 98%) standard were purchased from Sigma-Aldrich (St. Louis, MO, USA). Methanol and acetonitrile of HPLC grade were purchased from Merck (Germany). Phosphoric acid (purity β‰₯ 85%) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Deionized water was prepared using ultrapure water purifier system (Elgastat, Bucks, UK). 2.2. Instrumentation. The isocratic RP-HPLC method was carried out using Thermo Scientific Dionex-UltiMate 3000 HPLC system equipped with solvent reservoirs, LPG3400SD pump, WPS-3000 autosampler injector, TCC-3000 column oven, and DAD-3000 ultraviolet-visible (UV-Vis) diode array detector module operated at four wavelengths per analysis. Chromeleon data software (Version 7) was used for data analysis. 2.3. Sample Preparation. Citrullus lanatus (Thunb.) Matsum. & Nakai of red watermelon and yellow crimson watermelon was obtained from Selangor Fruit Valley, Selangor. Seeds were removed manually and the edible part was cut into cubes. Watermelon flesh and rind were juiced and frozen at βˆ’80∘ C for at least 2 days. The frozen juices were put in a freeze-drier (Labconco, USA) for 4 days until completely dried. The dried juice powders were kept at βˆ’20∘ C. For the analysis, samples were prepared in the form of juice extract and methanol extract. Juice extract was prepared directly by dissolving the dried juice powder in dH2 O. For methanol extract, a known quantity of dried juice powders was extracted with 30 mL of MeOH and 1 mL of 1 N HCl, vortexed, and sonicated for 30 minutes. The samples were macerated by cold maceration for a period of 72 hours in an orbital shaker. Methanol extracts were then filtered using Whatman filter paper. The residues were reextracted twice using fresh solvent and the three methanol extracts were pooled. The obtained methanol extracts were evaporated to dryness using a rotary vacuum evaporator at 60∘ C and stored at 4∘ C until analysis. 2.4. Isocratic RP-HPLC Analysis 2.4.1. Standard Preparation Procedure. A stock solution of Lcitrulline and L-arginine was prepared individually in dH2 O at 1 mg/mL and filtered through a 0.45 πœ‡m syringe filter (Bioflow). A mixed standard solution was prepared by mixing an equal volume of each standard stock solution. A series of working standard solutions was prepared by diluting the stock solution with dH2 O in the range of 0.1–1000 πœ‡g/mL. International Journal of Analytical Chemistry Table 1: Selection of mobile phases for separation of mixed standard, L-citrulline, and L-arginine by isocratic RP-HPLC. Mobile phase 0.7% H3 P04 0.1% H3 P04 0.7% H3 P04 : ACN Ratio Solution mixture (%) 100 100 0.7% H3 P04 + 99.3% dH2 O 0.1% H3 P04 + 99.9% dH2 O (0.7% H3 P04 + 99.3% dH2 O) + 100% ACN 90 : 10 2.4.2. Sample Preparation Procedure. Juice extracts were prepared directly by dissolving the dried juices powder in dH2 O at 5 mg/mL. Crude methanol extracts were also dissolved in dH2 O at 5 mg/mL and vortexed for 15 minutes. All extracts were filtered through 0.45 πœ‡m filters and injected to isocratic RP-HPLC. 2.4.3. Chromatographic Analysis. Preliminarily, three different concentrations of ion-pair reagents, phosphoric acid (H3 P04 ), or addition of acetonitrile (ACN) as mobile phases, 0.7% H3 P04 , 0.1% H3 P04 , and 0.7% H3 P04 : ACN (90 : 10) (Table 1), were tested for separation and determination of Lcitrulline and L-arginine standard. The column temperature was fixed at room temperature and UV-Vis detection was performed at 195 nm. The RP-HPLC columns [i.e., Zorbax Eclipse XDB-C18 , 250 mm Γ— 4.6 mm, 80 Å, 5 πœ‡m (Phenomenex, Torrance, CA), and Gemini C18 , 250 Γ— 4.6 mm, 110 Å, 3 πœ‡m (Phenomenex, Torrance, CA)] were used. The analysis proceeded for quantification of both compounds, L-citrulline and L-arginine in watermelons juice extracts and methanol extracts using the chosen column and mobile phase: Gemini C18 eluted by 0.1% H3 P04 with a flow rate of 0.5 mL/min at 195 nm. Chromeleon software was used for quantification of L-citrulline and L-arginine. The concentration of L-citrulline and L-arginine content was quantified based on the linear curve of standards. The content of compounds was expressed as milligrams per gram (mg/g) of sample extracts. 2.4.4. Method Validation. The validation of the isocratic RPHPLC method was performed for linearity of calibration curve, limit of detection (LOD), limit of quantification (LOQ), accuracy, and precision. The linearity of the isocratic RP-HPLC method for quantification of compounds was constructed using the concentration range of 0.1–1000 πœ‡g/mL for L-citrulline and 0.1–500 πœ‡g/mL for L-arginine. The regression equation was calculated in the form of 𝑦 = π‘Žπ‘₯ + 𝑏, where π‘₯ is the concentration and 𝑦 is the peak area of compounds. Linearity was established by the coefficient of determination (𝑅2 ). LOD and LOQ were measured based on signal-tonoise ratio (S/N) method. LOD is the lowest concentration of analyte that can be detected with signal-to-noise ratio of 3 : 1 and LOQ is the lowest concentration that can be quantified with acceptable precision and accuracy with signal-to-noise ratio of 10 : 1. S/N of 3 is considered acceptable for LOD, while LOQ is established at S/N of 10. Precision of the method was determined as percentage relative standard deviation (%RSD) of peak area of intraday and interday analysis data. Intraday (three times in a day operation under the same conditions) 3 and interday (three different days) studies were performed at three different concentrations (Level 1: 20 πœ‡g/mL; Level 2: 60 πœ‡g/mL; Level 3: 150 πœ‡g/mL). The resulting peak area was used to calculate SD and the relative standard deviation (%RSD). Accuracy of the method by recovery study was done by adding a known amount of reference standard solution (three concentrations) to test samples. The spiked extract solutions were injected three times, and the recovery was calculated with the value of detected versus added amounts. 3. Results and Discussion 3.1. Separation of L-Citrulline and L-Arginine by Isocratic RPHPLC Method. The initial isocratic RP-HPLC method for separation of mixed standard, L-citrulline, and L-arginine was performed using selected mobile phases according to previous literatures with slight modifications [8, 13, 20]. Interaction between mobile phase and stationary phases in isocratic RP-HPLC is important for the determination of solutes’ retention time [21]. In this study, separation for determination of mixed standard, L-citrulline, and L-arginine was performed using a hydrophilic anionic ion-pairing reagent with different concentrations of phosphoric acid (H3 P04 ) or addition of acetonitrile (ACN) as mobile phases: 0.7% H3 P04 , 0.1% H3 P04 , and 0.7% H3 P04 : ACN (90 : 10). The mobile phase at the concentration of 0.7% H3 P04 : ACN (90 : 10) resulted in L-citrulline and L-arginine were unretained and coeluted (π‘˜ value close to 0) as shown in Figure 3(a). The mixture of 0.7% H3 P04 : ACN (90 : 10) is highly hydrophilic, leading to rapid elution of L-citrulline and L-arginine with poor separation. Peaks of L-citrulline and L-arginine were slightly retained and partially separated using 0.7% H3 P04 (Figure 3(b)). However, optimum resolution was not achieved by 0.7% H3 P04 as π‘˜ value between L-citrulline and L-arginine is close to 1. The mobile phase of 0.1% H3 P04 resulted in efficient separation with reproducible peaks of L-citrulline and Larginine although all chromatograms showed stable baseline (Figure 3(c)). This finding is in agreement with Fekete et al. [22] who noted that 0.1% H3 P04 acts as a good separation agent by increasing the polarity and improving the retention time of zwitterionic molecules including amino acids. Dolan [23] supported the notion that 0.1% H3 P04 adequately provides reasonable buffering for amino acids separation by RP-HPLC. This showed that a concentration less than 1.0% H3 P04 as mobile phase provides efficient separation of amino acids, peptides, or proteins as demonstrated by Shibue et al. [24]. Thus, the mobile phase 0.1% H3 P04 is proven to provide efficient separation and the best resolution of mixed standard, L-citrulline, and L-arginine. The study also evaluated separation of mixed standard in two different columns, Zorbax Eclipse XDB-C18 and Gemini C18 using 0.1% H3 P04 . Zorbax Eclipse XDB-C18 did not provide good separation and resolution of L-citrulline and L-arginine as shown in Figure 4(a). A study by Barber and Joseph [25] showed that polar compounds were less separated and not well resolved using Zorbax Eclipse XDBC18 with a longer analysis time of 54 minutes. Efficient separation and resolution of L-citrulline and L-arginine from International Journal of Analytical Chemistry 3000 3000 2500 2500 2000 2000 1500 1500 (mAU) (mAU) 4 1000 1000 500 500 0 0 βˆ’500 βˆ’500 0 1 2 3 4 5 (min) 6 7 8 9 10 0 1 2 3 (a) 4 5 (min) 6 7 8 9 10 (b) 3000 (1) 2500 (mAU) 2000 (2) 1500 1000 500 0 βˆ’500 0 1 2 3 4 5 (min) 6 7 8 9 10 (c) Figure 3: Comparative chromatograms showing isocratic RP-HPLC separation of mixed standard, L-citrulline, and L-arginine in different mobile phases: (a) 0.7% H3 P04 : ACN (90 : 10); L-citrulline and L-arginine were unretained and coeluted at π‘˜ value close to zero; (b) 0.7% H3 P04 ; L-citrulline and L-arginine were slightly retained and partially separated; (c) 0.1% H3 P04 ; L-citrulline and L-arginine were efficiently separated with reproducible peaks. The peaks marked represent (1) L-arginine and (2) L-citrulline. 3000 2500 2500 2000 1500 1500 (mAU) (mAU) 2000 1000 500 (2) 1000 500 0 0 βˆ’500 (1) 0 1 2 3 4 5 (min) 6 7 8 9 10 (a) βˆ’500 0 1 2 3 4 5 (min) 6 7 8 9 10 (b) Figure 4: Comparative chromatograms showing isocratic RP-HPLC separation of mixed standard, L-citrulline, and L-arginine from 2 different columns: (a) Zorbax Eclipse XDB-C18 , 5 πœ‡m, and (b) Gemini C18 , 3 πœ‡m; efficient separation and the best resolution were achieved by the Gemini C18 column which showed that compounds are well separated. The peaks marked represent (1) L-arginine and (2) L-citrulline. mixed standard were achieved using Gemini C18 as shown in Figure 4(b). L-citrulline and L-arginine are eluted at a short retention time with L-arginine, 4.773 min, followed by L-citrulline at 5.787 min (Figure 5). Efficient separation of Lcitrulline with a retention time of about 4 min was achieved on the Gemini C18 column due to the high degree similarity of column with polar compounds [13]. Gemini C18 is a new generation hybrid column end-capped with porous silica as base core and polymer media coated on top of the silica core which exhibit silica-like mechanical properties of base material while similarly decreasing the number of residual silanols [26]. This result demonstrated that Gemini C18 is the most suited column for efficient separation of mixed standard, L-citrulline and L-arginine. International Journal of Analytical Chemistry (a) 5 2500 2000 (mAU) 1500 1000 500 0 βˆ’500 (b) 0 1 2 3 4 2500 5 (min) 6 7 8 9 10 (1) 2000 (mAU) 1500 (2) 1000 500 0 βˆ’500 (c) 0 1 2 3 4 5 (min) 6 7 8 9 10 0 1 2 3 4 5 (min) 6 7 8 9 10 2500 2000 (mAU) 1500 1000 500 0 βˆ’500 Figure 5: Comparative chromatograms showing isocratic RP-HPLC separation of individual and mixed standard, L-citrulline, and L-arginine using Gemini C18 : (a) L-arginine, (b) mixed standard, and (c) L-citrulline. The peaks marked represent (1) L-arginine and (2) L-citrulline. The result from chromatography separation of Lcitrulline and L-arginine shown in Figure 5(b) demonstrated that the reverse-phase (RP) mode provided efficient separation and substantial retention achieved on both polar compounds without the need for derivatization. RP mode can efficiently be applied in this study although Brown et al. [27] proposed the use of hydrophilic interaction liquid chromatography (HILIC) mode after cyano- and pentafluorophenylpropyl stationary phases failed to retain target compounds including L-citrulline and L-arginine. HILIC mode is primarily used when separation of very polar compounds is needed or for incomplete chromatographic separation in RP mode [28]. However, HILIC mode required an expensive and robust system equipped with tandem mass spectrometric (MS) detection for L-citrulline and L-arginine separation in h ...
Purchase answer to see full attachment
Student has agreed that all tutoring, explanations, and answers provided by the tutor will be used to help in the learning process and in accordance with Studypool's honor code & terms of service.

Final Answer

Hello😊 I've attached the final copy of your summary along with its outline down below. In case you need any changes made, don't hesitate to reach out to me for help, I'll be happy to revise it for you. Thank you for your patience and cooperation!Stay safe and take careπŸ˜‰ Bye for now!

Running head: RESEARCH SUMMARY

DEVELOPMENT OF ISOCRATIC RP-HPLC METHOD FOR SEPARATION AND
QUANTIFICATION OF L-CITRULLINE AND L-ARGININE IN WATERMELONS
SUMMARY
Name
Institution
Date

1

RESEARCH SUMMARY

2

Topic
The topic of the article deals with the separation of compounds present in
watermelons using isocratic HPLC methodology. Readers should read this article to
understand the amount of L-Citrulline and L-Arginine in watermelon.
Abstract
The purpose of this study was to investigate the content of amino acids in
watermelons. It was a lab-based experimental research study that used the Isocratic RPHPLC method to separate the materials of watermelon. ...

DrHill (7418)
Duke University

Anonymous
Thanks for the help.

Anonymous
Outstanding. Studypool always delivers quality work.

Anonymous
Tutor was very helpful and took the time to explain concepts to me. Very responsive, managed to get replies within the hour.

Studypool
4.7
Trustpilot
4.5
Sitejabber
4.4
Similar Questions
Related Tags