Development of Isocratic RP-HPLC Method for …downloads.hindawi.com/journals/ijac/2018/4798530.pdfHPLC Method. e initial isocratic RP-HPLC method for separation of mixed standard,

Research ArticleDevelopment of Isocratic RP-HPLC Method for Separation andQuantification of L-Citrulline and L-Arginine in Watermelons

Rasdin Ridwan 1 Hairil Rashmizal Abdul Razak2

Mohd Ilham Adenan34 andWanMazlina Md Saad 1

1Centre of Medical Laboratory Technology Faculty of Health Sciences Universiti Teknologi MARA Puncak Alam Campus42300 Bandar Puncak Alam Selangor Malaysia2Centre of Medical Imaging Faculty of Health Sciences Universiti Teknologi MARA Puncak Alam Campus42300 Bandar Puncak Alam Selangor Malaysia3Faculty of Applied Sciences Universiti Teknologi MARA 40450 Shah Alam Selangor Malaysia4Atta-ur-Rahman Institute forNatural ProductDiscovery Level 9 BangunanFF3Universiti TeknologiMARA PuncakAlamCampus42300 Bandar Puncak Alam Selangor Malaysia

Correspondence should be addressed to Wan Mazlina Md Saad mazlinasaad14gmailcom

Received 30 December 2017 Revised 25 February 2018 Accepted 12 March 2018 Published 2 May 2018

Academic Editor Gunther K Bonn

Copyright copy 2018 Rasdin Ridwan et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich 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 consumedinMalaysia were investigated for their amino acid content L-citrulline and L-arginine by the isocratic RP-HPLCmethod Flesh andrind watermelons were juiced and freeze-dried samples were used for separation and quantification of L-citrulline and L-arginineThree different mobile phases 07 H

3P04 01 H

3P04 and 07 H

3P04 ACN (90 10) were tested on two different columns

using Zorbax Eclipse XDB-C18

and Gemini C18

with a flow rate of 05mLmin and a detection wavelength at 195 nm Efficientseparation with reproducible resolution of L-citrulline and L-arginine was achieved using 01 H

3P04on the Gemini C

18column

The method was validated and good linearity of L-citrulline and L-arginine was obtained with 1198772 = 09956 119910 = 01664119909 + 24142and 1198772 = 09912 119910 = 04100119909 + 34850 respectively L-citrulline content showed the highest concentration in red watermelonof flesh and rind juice extract (4381mgg and 4502mgg) whereas L-arginine concentration was lower than L-citrulline rangingfrom 339 to 1114mgg The isocratic RP-HPLC method with 01 H

3P04on the Gemini C

18column proved to be efficient for

separation and quantification of L-citrulline and L-arginine in watermelons

1 Introduction

Citrullus lanatus (Thunb) Matsum and Nakai commonlyknown 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 whichoriginated from theAfricanKalahariDesert [1]Watermelonshave high content of phytonutrients and are rich in dietaryantioxidants such as carotenoids (lycopene and 120573-carotene)polyphenolics ascorbic acid and significant amino acids [2]Watermelons are usually consumed by juicing the flesh bene-ficial in the prevention and improvement of health problemssuch as cardiovascular diseases erectile dysfunction hyper-tension and cancers [3] Figueroa et al [4] demonstrated

that watermelon juice supplementation improves aortichemodynamics by reducing the reflected wave amplitudein prehypertensive individuals A study by Poduri et al [5]reported that watermelon attenuated hypercholesterolemia-induced atherosclerosis in mice Commercial watermelonjuices provide enormous marketing potential and nutritiousdrinks for individuals to maintain a healthy lifestyle

Amino acids particularly L-citrulline and L-arginine areregarded as major types of phytonutrients present in water-melons whichmay contribute to their reputed and diversifiedhealth benefits [6] L-citrulline C

6H13N3O3(IUPAC name

2-amino-5-(carbamoylamino)pentanoic acid) (Figure 1) is anonessential amino acid firstly identified from watermelonCitrullus vulgaris Schrad [7 8] L-citrulline is a physiological

HindawiInternational Journal of Analytical ChemistryVolume 2018 Article ID 4798530 9 pageshttpsdoiorg10115520184798530

2 International Journal of Analytical Chemistry

O

NH

OH

O

（2

（2

Figure 1 Molecular structure of L-citrulline (1752 gmol)

NH

OH

O

（2

（

（

2

Figure 2 Molecular structure of L-arginine (1742 gmol)

endogenous amino acid to most living systems involved inprotein metabolism and removal of excess metabolic ammo-nia [9] It serves as a precursor for L-arginine and product ofnitric oxide (NO) cycle [10] L-arginine C

6H14N4O2(IUPAC

name (S)-2-amino-5-guanidinopentanoic acid) (Figure 2)is a semiessential and free form physiological amino acidthat functions as one of 20 building block proteins forbiological processes such as cell division ammonia removalwound healing and hormone release [7 8] Wu et al [11]demonstrated that supplementation of L-citrulline and L-arginine fromwatermelon juice improved serum levels of NOmetabolites and aortic endothelial-mediated vasodilation indiabetic rats

L-citrulline and L-arginine are present in all parts ofwatermelon fruits including flesh rind and seed [7] Astudy done by Rimando and Perkins-Veazie [12] reportedthat the rind of red watermelon and yellow watermeloncontains more L-citrulline at a concentration ranging from156 to 294mgg than flesh 79ndash285mgg Similar to theabove finding Jayaprakasha et al [13] reported that rinds ofCitrullus vulgaris varieties such as petite treat and jamboreewatermelon and also yellow crimson watermelon containedslightly higher L-citrulline ranging from 1395 to 2846mggthan flesh 1125ndash1673mgg These findings suggested thatwatermelon rind has an abundance of L-citrulline content incomparison to its content in flesh

Analyses of L-citrulline and L-arginine were routinelyconducted using capillary electrophoresis and quantificationby a spectrophotometric method however the method isless sensitive leading to discrepancies in the outcomes[6] L-citrulline and L-arginine are polar nonvolatile anddevoid of chromophores thus analysis by reverse-phase highperformance liquid chromatography (RP-HPLC) commonlyemployed a derivatization method using pre- or postcol-umn derivatization [14ndash17] Jayaprakasha et al [13] statedthat precolumn derivatization such as orthophthalaldehyde(OPA) naphthalene-23-dicarboxaldehyde or 4-dimethyl-aminoazobenzene-41015840-sulfonyl chloride (dabsyl chloride) wasable to provide accurate and stable chromatography baselinebut the reactions were unstable and affected by the sam-ple matrix [18] Postcolumn derivatization by ninhydrin istedious due to long analysis timeup to 72 hours and instability

of derivatization reagents that may cause poor compoundrecovery [19] Analysis of underivatized L-citrulline and L-arginine is warranted for rapid and effective quantification ofthese compounds Given that no amino acids content of L-citrulline and L-arginine in Malaysia watermelons has beenreported so far we have developed an isocratic RP-HPLCmethod for separation and quantification of L-citrulline andL-arginine in watermelons

2 Materials and Methods

21 Chemicals and Reagents L-citrulline (purity ge 99)and L-arginine (purity ge 98) standard were purchasedfrom Sigma-Aldrich (St Louis MO USA) Methanol andacetonitrile of HPLC grade were purchased from Merck(Germany) Phosphoric acid (purity ge 85) was purchasedfrom Sigma-Aldrich (St Louis MO USA) Deionized waterwas prepared using ultrapure water purifier system (ElgastatBucks UK)

22 Instrumentation The isocratic RP-HPLC method wascarried out using Thermo Scientific Dionex-UltiMate3000 HPLC system equipped with solvent reservoirs LPG-3400SD pump WPS-3000 autosampler injector TCC-3000column oven and DAD-3000 ultraviolet-visible (UV-Vis)diode array detectormodule operated at fourwavelengths peranalysis Chromeleon data software (Version 7) was used fordata analysis

23 Sample Preparation Citrullus lanatus (Thunb) Matsumamp Nakai of red watermelon and yellow crimson watermelonwas obtained fromSelangor Fruit Valley Selangor Seedswereremoved manually and the edible part was cut into cubesWatermelon flesh and rind were juiced and frozen at minus80∘Cfor at least 2 daysThe frozen juices were put in a freeze-drier(Labconco USA) for 4 days until completely driedThe driedjuice powders were kept at minus20∘C For the analysis sampleswere prepared in the form of juice extract and methanolextract Juice extract was prepared directly by dissolving thedried juice powder in dH

2O For methanol extract a known

quantity of dried juice powders was extracted with 30mLof MeOH and 1mL of 1 N HCl vortexed and sonicated for30 minutes The samples were macerated by cold macerationfor a period of 72 hours in an orbital shaker Methanolextracts were then filtered using Whatman filter paper Theresidues were reextracted twice using fresh solvent and thethree methanol extracts were pooledThe obtained methanolextracts were evaporated to dryness using a rotary vacuumevaporator at 60∘C and stored at 4∘C until analysis

24 Isocratic RP-HPLC Analysis

241 Standard Preparation Procedure A stock solution of L-citrulline and L-arginine was prepared individually in dH

2O

at 1mgmL and filtered through a 045 120583m syringe filter(Bioflow) Amixed standard solutionwas prepared bymixingan equal volume of each standard stock solution A seriesof working standard solutions was prepared by diluting thestock solution with dH

2O in the range of 01ndash1000 120583gmL

International Journal of Analytical Chemistry 3

Table 1 Selection of mobile phases for separation of mixedstandard L-citrulline and L-arginine by isocratic RP-HPLC

Mobile phase Ratio Solution mixture ()07 H

3P04

100 07 H3P04+ 993 dH

2O

01 H3P04

100 01 H3P04+ 999 dH

2O

07 H3P04 ACN 90 10 (07 H

3P04+ 993 dH

2O) +

100 ACN

242 Sample Preparation Procedure Juice extracts were pre-pared directly by dissolving the dried juices powder in dH

2O

at 5mgmL Crude methanol extracts were also dissolved indH2O at 5mgmL and vortexed for 15 minutes All extracts

were filtered through 045 120583m filters and injected to isocraticRP-HPLC

243 Chromatographic Analysis Preliminarily three differ-ent concentrations of ion-pair reagents phosphoric acid(H3P04) or addition of acetonitrile (ACN) as mobile phases

07 H3P04 01 H

3P04 and 07 H

3P04 ACN (90 10)

(Table 1) were tested for separation and determination of L-citrulline and L-arginine standard The column temperaturewas fixed at room temperature and UV-Vis detection wasperformed at 195 nm The RP-HPLC columns [ie ZorbaxEclipse XDB-C

18 250mm times 46mm 80 A 5120583m (Phe-

nomenex Torrance CA) and Gemini C18 250 times 46mm

110 A 3 120583m (Phenomenex Torrance CA)] were used Theanalysis proceeded for quantification of both compoundsL-citrulline and L-arginine in watermelons juice extractsand methanol extracts using the chosen column and mobilephase Gemini C

18eluted by 01 H

3P04with a flow rate

of 05mLmin at 195 nm Chromeleon software was used forquantification of L-citrulline and L-arginine The concentra-tion of L-citrulline and L-arginine content was quantifiedbased on the linear curve of standards The content ofcompounds was expressed as milligrams per gram (mgg) ofsample extracts

244 Method Validation The validation of the isocratic RP-HPLC method was performed for linearity of calibrationcurve limit of detection (LOD) limit of quantification(LOQ) accuracy and precision The linearity of the isocraticRP-HPLCmethod for quantification of compounds was con-structed using the concentration range of 01ndash1000120583gmL forL-citrulline and 01ndash500 120583gmL for L-arginineThe regressionequation was calculated in the form of 119910 = 119886119909 + 119887 where 119909is the concentration and 119910 is the peak area of compoundsLinearity was established by the coefficient of determination(1198772) LOD and LOQ were measured based on signal-to-noise ratio (SN) method LOD is the lowest concentration ofanalyte that can be detected with signal-to-noise ratio of 3 1and LOQ is the lowest concentration that can be quantifiedwith acceptable precision and accuracy with signal-to-noiseratio of 10 1 SN of 3 is considered acceptable for LOD whileLOQ is established at SN of 10 Precision of the method wasdetermined 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)

and interday (three different days) studies were performedat three different concentrations (Level 1 20120583gmL Level2 60 120583gmL Level 3 150120583gmL) The resulting peak areawas used to calculate SD and the relative standard deviation(RSD) Accuracy of themethod by recovery study was doneby adding a known amount of reference standard solution(three concentrations) to test samples The spiked extractsolutions were injected three times and the recovery wascalculated with the value of detected versus added amounts

3 Results and Discussion

31 Separation of L-Citrulline and L-Arginine by Isocratic RP-HPLC Method The initial isocratic RP-HPLC method forseparation of mixed standard L-citrulline and L-argininewas performed using selected mobile phases according toprevious literatures with slight modifications [8 13 20]Interaction between mobile phase and stationary phases inisocratic RP-HPLC is important for the determination ofsolutesrsquo retention time [21]

In this study separation for determination of mixedstandard L-citrulline and L-arginine was performed usinga hydrophilic anionic ion-pairing reagent with differentconcentrations of phosphoric acid (H

3P04) or addition of

acetonitrile (ACN) as mobile phases 07 H3P04 01

H3P04 and 07 H

3P04 ACN (90 10) The mobile phase at

the concentration of 07 H3P04 ACN (90 10) resulted in

L-citrulline and L-arginine were unretained and coeluted (119896value close to 0) as shown in Figure 3(a)Themixture of 07H3P04 ACN (90 10) is highly hydrophilic leading to rapid

elution of L-citrulline and L-arginine with poor separationPeaks of L-citrulline and L-arginine were slightly retainedand partially separated using 07 H

3P04(Figure 3(b))

However optimum resolution was not achieved by 07H3P04as 119896 value between L-citrulline and L-arginine is close

to 1 The mobile phase of 01 H3P04resulted in efficient

separation with reproducible peaks of L-citrulline and L-arginine although all chromatograms showed stable baseline(Figure 3(c)) This finding is in agreement with Fekete et al[22] who noted that 01 H

3P04acts as a good separation

agent by increasing the polarity and improving the reten-tion time of zwitterionic molecules including amino acidsDolan [23] supported the notion that 01 H

3P04adequately

provides reasonable buffering for amino acids separation byRP-HPLC This showed that a concentration less than 10H3P04as mobile phase provides efficient separation of amino

acids peptides or proteins as demonstrated by Shibue et al[24]Thus the mobile phase 01 H

3P04is proven to provide

efficient separation and the best resolution ofmixed standardL-citrulline and L-arginine

The study also evaluated separation of mixed standard intwo different columns Zorbax Eclipse XDB-C

18and Gemini

C18

using 01 H3P04 Zorbax Eclipse XDB-C

18did not

provide good separation and resolution of L-citrulline andL-arginine as shown in Figure 4(a) A study by Barberand Joseph [25] showed that polar compounds were lessseparated and not well resolved using Zorbax Eclipse XDB-C18

with a longer analysis time of 54 minutes Efficientseparation and resolution of L-citrulline and L-arginine from


1 2 3 4 5 6 7 8 9 100(min)

minus500

0

500

1000

1500

2000

2500

3000(m

AU)

(a)

minus500

0

500

1000

1500

2000

2500

3000

(mAU

)

1 2 3 4 5 6 7 8 9 100(min)

(b)

(1)

(2)

1 2 3 4 5 6 7 8 9 100(min)

minus500

0

500

1000

1500

2000

2500

3000

(mAU

)

(c)

Figure 3 Comparative chromatograms showing isocratic RP-HPLC separation of mixed standard L-citrulline and L-arginine in differentmobile phases (a) 07 H

3P04 ACN (90 10) L-citrulline and L-arginine were unretained and coeluted at 119896 value close to zero (b) 07

H3P04 L-citrulline and L-arginine were slightly retained and partially separated (c) 01 H

3P04 L-citrulline and L-arginine were efficiently

separated with reproducible peaks The peaks marked represent (1) L-arginine and (2) L-citrulline

1 2 3 4 5 6 7 8 9 100(min)

minus500

0

500

1000

1500

2000

2500

3000

(mAU

)

(a)

(1)

(2)

minus500

0

500

1000

1500

2000

2500

(mAU

)

1 2 3 4 5 6 7 8 9 100(min)

(b)

Figure 4 Comparative chromatograms showing isocratic RP-HPLC separation of mixed standard L-citrulline and L-arginine from 2different columns (a) Zorbax Eclipse XDB-C

18 5120583m and (b) Gemini C

18 3120583m efficient separation and the best resolution were achieved

by the Gemini C18column which showed that compounds are well separatedThe peaks marked represent (1) L-arginine and (2) L-citrulline

mixed standard were achieved using Gemini C18

as shownin Figure 4(b) L-citrulline and L-arginine are eluted at ashort retention time with L-arginine 4773min followed byL-citrulline at 5787min (Figure 5) Efficient separation of L-citrulline with a retention time of about 4min was achievedon the Gemini C

18column 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 asbase core and polymer media coated on top of the silicacore which exhibit silica-like mechanical properties of basematerial while similarly decreasing the number of residualsilanols [26] This result demonstrated that Gemini C

18is

the most suited column for efficient separation of mixedstandard L-citrulline and L-arginine


(1)

(a)

(b)

(c)

(2)

minus500

0

500

1000

1500

2000

2500

(mAU

)

1 2 3 4 5 6 7 8 9 100(min)

minus500

0

500

1000

1500

2000

2500

(mAU

)

1 2 3 4 5 6 7 8 9 100(min)

minus500

0

500

1000

1500

2000

2500

(mAU

)1 2 3 4 5 6 7 8 9 100

(min)

Figure 5 Comparative chromatograms showing isocratic RP-HPLC separation of individual andmixed standard L-citrulline and L-arginineusing Gemini C

18 (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 L-citrulline and L-arginine shown in Figure 5(b) demonstratedthat the reverse-phase (RP) mode provided efficientseparation and substantial retention achieved on both polarcompoundswithout the need for derivatization RPmode canefficiently be applied in this study although Brown et al [27]proposed the use of hydrophilic interaction liquid chroma-tography (HILIC) mode after cyano- and pentafluorophenyl-propyl stationary phases failed to retain target compoundsincluding L-citrulline and L-arginine HILIC mode isprimarily used when separation of very polar compoundsis needed or for incomplete chromatographic separation inRP mode [28] However HILIC mode required an expen-sive and robust system equipped with tandem mass spec-trometric (MS) detection for L-citrulline and L-arginineseparation in high biological matrix samples such as serumand plasma [16 27] Complete chromatographic separation

of L-citrulline and L-arginine by RP mode in this studyeliminates the use of HILIC mode

32 Validation of Isocratic RP-HPLC Method Method val-idation of isocratic RP-HPLC method was performed bydetermination of linearity LOD LOQ recovery and intradayand interday analysis The results of linearity LOD and LOQwere summarized in Table 2 Good linear regression equa-tions of L-citrulline and L-arginine standard are displayedbetween corresponding peak areas versus concentrations ofcompounds based on the correlation coefficients (1198772 =09956 119910 = 01664119909 + 24142 and 1198772 = 09912 119910 = 04100119909 +34850 resp) The LOD and LOQ for L-citrulline were042 120583gmL and 128 120583gmL while those for L-arginine were088 120583gmL and 266 120583gmL which demonstrated that iso-cratic RP-HPLCmethodwas efficiently sensitiveThemethodhad good accuracy that showed efficient recoveries for both


Table 2 Calibration data of L-citrulline and L-arginine standard reported from isocratic RP-HPLC method

Standard Concentrationrange (120583gmL) Regression equation

Correlationcoefficient

(1198772)

Limit ofdetection(120583gmL)

Limit ofquantification

(120583gmL)L-citrulline 001ndash1000 119910 = 01664119909 + 24142 09956 042 128L-arginine 001ndash500 119910 = 04100119909 + 34850 09912 088 266

Table 3 Recovery of L-citrulline and L-arginine standard reported from isocratic RP-HPLC method

Compounds Added concentration (120583gmL) Measured concentration (120583gmL) Recovery () RSD ()

L-citrulline100 10194 10194 17060 6124 10207 14630 3102 10338 100

L-arginine100 9987 9987 19660 5933 9888 17630 3102 10341 117

Table 4 Intraday and interday analysis of L-citrulline and L-arginine standard reported from isocratic RP-HPLC method

Compounds Concentration (120583gmL) Intraday (119899 = 3) () Interday (119899 = 3) ()Mean RSD Mean RSD

L-citrulline150 10125 123 10042 10960 10207 146 10344 10120 11644 203 11622 037

L-arginine150 10201 056 10383 20560 9626 104 9736 11320 9541 068 9594 033

compounds ranging from 9888 to 10341 (Table 3) TheRSD () for intraday and interday precision ranged from037 to 109 for L-citrulline and 033 to 205 for L-arginine in which both RSD le 2 (Table 4)These validationresults confirmed that the isocratic RP-HPLC method is pre-cise accurate and sensitive for simultaneous quantificationof L-citrulline and L-arginine

33 Quantification of L-Citrulline and L-Arginine Contents inTwo Different Watermelon Extracts Consumption of water-melon extracts rich in L-citrulline and L-arginine is proven tobe beneficial for diseases prevention Thus a rapid reliableand efficient isocratic RP-HPLC method is essential forsimultaneous quantification of these amino acids in juiceextracts andmethanol extractsThe chromatographic profilesof both extracts in red watermelon and yellow crimsonwatermelon are presented in Figure 6 Quantification of L-citrulline and L-arginine was performed using ChromeleonsoftwareThe content was calculated based on the calibrationcurve of L-citrulline and L-arginine standard achieved withgood correlation coefficients and linear regression equations1198772 = 09956 119910 = 01664119909 + 24142 and 1198772 = 09912119910 = 04100119909 + 34850 respectively The results are tabulatedin Table 5

Red watermelon juice extract showed slightly highyield of L-citrulline in rind 4502mgg compared to flesh

4381mgg Similar trends were shown in L-citrulline contentin rind and flesh of yellow crimson juice extract 1661mggand 1577mgg respectively This finding is in accordancewith the study by Jayaprakasha et al [13] which foundthat rinds from C vulgaris watermelon varieties of petitetreat and jamboree watermelon and yellow crimson water-melon contained significantly high L-citrulline 1395mgg2084mgg and 2846mgg respectively compared to flesh1125mgg 1673mgg and 1474mgg respectively usingRP-HPLC method L-arginine content in red watermelonjuice extract was higher in flesh 1110mgg compared torind 339mgg L-arginine content was approximately 3-fold lower than L-citrulline in red watermelon flesh juiceextract Consumption of watermelon flesh juice extract aidedin efficient conversion of significantly high L-citrulline apotent endogenous precursor to L-arginine in the kidneywhich resulted in increased plasma L-arginine concentrationFindings by Collins et al [29] proved that plasma L-arginineconcentration increased by 952 plusmn 35 120583M and 1080 plusmn 41 120583Mcompared to normal plasma baseline 864 plusmn 35 120583M after3 weeks of consumption of 780mL (sim1 g L-citrullineday)to 1560mL (sim2 g L-citrullineday) of watermelon juicesRecently a study by Bailey et al [30] supported the notionof the increased plasma L-arginine concentration by 116 plusmn9 120583M compared to placebo 67 plusmn 13 120583M after 2 weeks of300mLday watermelon juice consumption which contains


(1) (2)

minus50050100150200250300350400450

(mAU

)

1 2 3 4 5 6 7 8 9 100(min)

(a)

(2)(1)

minus500

0

500

1000

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2000

2500

(mAU

)

1 2 3 4 5 6 7 8 9 100(min)

(b)

(2)(1)

1 2 3 4 5 6 7 8 9 100(min)

minus50050100150200250300350400

(mAU

)

(c)

(1)(2)

1 2 3 4 5 6 7 8 9 100(min)

minus500

0

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2500

(mAU

)

(d)

(1)(2)

minus20020406080100120140160180

(mAU

)

1 2 3 4 5 6 7 8 9 100(min)

(e)

(1) (2)

minus500

0

500

1000

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2500

(mAU

)

1 2 3 4 5 6 7 8 9 100(min)

(f)

(1)

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1 2 3 4 5 6 7 8 9 100(min)

minus50050100150200250300350400

(mAU

)

(g)

(1)(2)

minus500

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)

1 2 3 4 5 6 7 8 9 100(min)

(h)

Figure 6 Comparative chromatographic profiles showing isocratic RP-HPLC separation of flesh and rind from juice extracts and methanolextracts of red watermelon and yellow crimson watermelon using Gemini C

18 Red watermelon (a) flesh juice extract (b) rind juice extract

(c) crude flesh extract and (d) crude rind extract Yellow crimson watermelon (e) flesh juice extract (f) rind juice extract (g) crude fleshextract and (h) crude rind extract The peaks marked represent (1) L-arginine and (2) L-citrulline


Table 5 L-citrulline and L-arginine contents (mgg) in juice extract and methanol extract of watermelon flesh and rind juices

Watermelon Compounds Juices Juice extract (mgg) Methanol extract (mgg)

RedL-citrulline Flesh 4381 1622

Rind 4502 2499

L-arginine Flesh 1110 642Rind 339 408

Yellow crimsonL-citrulline Flesh 1577 1391

Rind 1661 1603


sim34 g L-citrullineday L-citrulline content in crude fleshand rind extract of red watermelon and yellow crimsonwatermelon varied in the range of 1391ndash2499mgg whileL-arginine content was in the range of 408ndash841mgg L-citrulline and L-arginine content is much lower in methanolextracts compared to juice extracts Fish and Bruton [17]stated that methanol extracts may diminish the solubility ofamino acids thusmarked reduction in amino acids yieldThequantitative results confirmed that watermelon juice extractsmost effectively quantified higher yield of L-citrulline and L-arginine and this studyrsquos outcomes may possibly suggest thatjuice extraction method is best in optimizing amino acidsyield

4 Conclusion

The isocratic RP-HPLC method has been successfully devel-oped for separation and quantification of L-citrulline andL-arginine content in both watermelon extracts of fleshand rind using the selected mobile phase (01 H

3P04) in

Gemini C18 The established isocratic RP-HPLC method

provides evidence that L-citrulline and L-arginine are bestretained using Gemini C

18column The validated method

is robust sensitive accurate and precise with good linearity(1198772 ge 099) low values of LOD and LOQ recoverieswithin 9888ndash10341 and RSD precision less than 2Juice extract effectively yielded higher L-citrulline and L-arginine content by juice extraction method thus it ispotentially used for quantitative amino acids analysis Thepresent study procedure may provide a basis for separationand quantification of L-citrulline and L-arginine in localwatermelonsThe high content of L-citrulline and L-argininesuggested watermelons as a good source of nutraceutical andhealth benefits ingredients However further researches arenecessary to explore biological activities such as aphrodisiacproperties of these active constituents in watermelons tosupport their potential in human diet and prevention ofhealth related diseases

Conflicts of Interest

The authors declare that there are no conflicts of interest thatwould prejudice the impartiality of this scientific work

Acknowledgments

Theauthors gratefully acknowledge (1) theMinistry ofHigherEducation Malaysia through Fundamental Research GrantScheme [FRGS Grant no FRGS12016WAB01UITM023]and Institute of ResearchManagement amp Innovation (IRMI)Universiti Teknologi MARA (UiTM) for funding the study(2) Dr Maizatul Hasyima Omar from Phytochemistry UnitInstitute for Medical Research Kuala Lumpur for provid-ing analytical training and (3) Atta-ur-Rahman Institute(AuRIns)UiTMPuncakAlamCentre ofMedical LaboratoryTechnology and Centre of Postgraduate Study Faculty ofHealth Sciences UiTM Selangor Puncak Alam Campus forproviding facilities throughout this study

References

[1] M K A Mohammad M I Mohamed A M Zakaria HR Abdul Razak and W M M Saad ldquoWatermelon (Citrulluslanatus (Thunb)Matsum andNakai) juicemodulates oxidativedamage induced by low dose X-ray in micerdquo BioMed ResearchInternational vol 2014 Article ID 512834 6 pages 2014

[2] G K Jayaprakasha and B S Patil ldquoA metabolomics approachto identify and quantify the phytochemicals in watermelons byquantitative 1HNMRrdquo Talanta vol 153 pp 268ndash277 2016

[3] A Naz M S Butt M T Sultan M M N Qayyum andR S Niaz ldquoWatermelon lycopene and allied health claimsrdquoExperimental and Clinical Sciences Journal vol 13 pp 650ndash6662014

[4] A Figueroa M A Sanchez-Gonzalez P M Perkins-Veazieand B H Arjmandi ldquoEffects of watermelon supplementationon aortic blood pressure and wave reflection in individualswith prehypertension A pilot studyrdquo American Journal ofHypertension vol 24 no 1 pp 40ndash44 2011

[5] A Poduri D L Rateri S K Saha S Saha and A Daugh-erty ldquoCitrullus lanatus rsquosentinelrsquo (watermelon) extract reducesatherosclerosis in LDL receptor-deficient micerdquo The Journal ofNutritional Biochemistry vol 24 no 5 pp 882ndash886 2013

[6] W Liu S Zhao Z Cheng X Wan Z Yan and S R KingldquoLycopene and citrulline contents in watermelon (Citrulluslanatus) fruit with different ploidy and changes during fruitdevelopmentrdquo Acta Horticulturae vol 871 pp 543ndash550 2010

[7] A R Davis C L Webber III W W Fish T C Wehner SKing and P Perkins-Veazie ldquoL-citrulline levels in watermeloncultigens tested in two environmentsrdquo HortScience vol 46 no12 pp 1572ndash1575 2011


[8] A Shafaei A F Aisha M J Siddiqui and Z Ismail ldquoAnalysisof L-citrulline and L-arginine in Ficus deltoidea leaf extractsby reverse phase high performance liquid chromatographyrdquoPharmacognosy Research vol 7 no 1 pp 32ndash37 2015

[9] E Curis I Nicolis CMoinard et al ldquoAlmost all about citrullinein mammalsrdquo Amino Acids vol 29 no 3 pp 177ndash205 2005

[10] M J Romero D H Platt R B Caldwell and R W CaldwellldquoTherapeutic use of citrulline in cardiovascular diseaserdquo Car-diovascular Drug Reviews vol 24 no 3-4 pp 275ndash290 2006

[11] G Wu J K Collins P Perkins-Veazie et al ldquoDietary supple-mentation with watermelon pomace juice enhances arginineavailability and ameliorates the metabolic syndrome in zuckerdiabetic fatty ratsrdquo Journal of Nutrition vol 137 no 12 pp 2680ndash2685 2007

[12] A M Rimando and P M Perkins-Veazie ldquoDetermination ofcitrulline in watermelon rindrdquo Journal of Chromatography Avol 1078 no 1-2 pp 196ndash200 2005

[13] G K Jayaprakasha K N Chidambara Murthy and B S PatilldquoRapid HPLC-UV method for quantification of L-citrulline inwatermelon and its potential role on smooth muscle relaxationmarkersrdquo Food Chemistry vol 127 no 1 pp 240ndash248 2011

[14] GWuandC JMeininger ldquoAnalysis of Citrulline Arginine andMethylarginines Using High-Performance Liquid Chromatog-raphyrdquoMethods in Enzymology vol 440 pp 177ndash189 2008

[15] L Wang R Xu B Hu et al ldquoAnalysis of free amino acids inChinese teas and flower of tea plant by high performance liquidchromatography combined with solid-phase extractionrdquo FoodChemistry vol 123 no 4 pp 1259ndash1266 2010

[16] S Shin S-M Fung S Mohan and H-L Fung ldquoSimultaneousbioanalysis of L-arginine L-citrulline and dimethylargininesby LC-MSMSrdquo Journal of Chromatography B vol 879 no 7-8pp 467ndash474 2011

[17] W W Fish and B D Bruton ldquoQuantification of L-citrullineand other physiologic amino acids in watermelon and variouscucurbitsrdquo Cucurbitaceae vol 2010 pp 152ndash154 2010

[18] PMarkowski I Baranowska and J Baranowski ldquoSimultaneousdetermination of L-arginine and 12 molecules participating inits metabolic cycle by gradient RP-HPLC method Applicationto human urine samplesrdquo Analytica Chimica Acta vol 605 no2 pp 205ndash217 2007

[19] D J Dietzen A L Weindel M O Carayannopoulos et alldquoRapid comprehensive amino acid analysis by liquid chro-matographytandemmass spectrometry Comparison to cationexchange with post-column ninhydrin detectionrdquo Rapid Com-munications inMass Spectrometry vol 22 no 22 pp 3481ndash34882008

[20] M Sadji P M Perkins-Veazie N F Ndiaye et al ldquoEnhancedL-citrulline in parboiled paddy rice with watermelon (Citrulluslanatus) juice for preventing sarcopenia A preliminary studyrdquoAfrican Journal of Food Science vol 9 no 10 pp 508ndash513 2015

[21] MW DongModern HPLC for Practicing Scientists JohnWileyand Sons 2006

[22] S Fekete J-L Veuthey and D Guillarme ldquoNew trends inreversed-phase liquid chromatographic separations of thera-peutic peptides and proteins Theory and applicationsrdquo Journalof Pharmaceutical and Biomedical Analysis vol 69 pp 9ndash272012

[23] J Dolan A Guide to HPLC and LC-MS Buffer Selection ACE2009

[24] M Shibue C T Mant and R S Hodges ldquoEffect of anionicion-pairing reagent hydrophobicity on selectivity of peptide

separations by reversed-phase liquid chromatographyrdquo Journalof Chromatography A vol 1080 no 1 pp 68ndash75 2005

[25] W E Barber andM Joseph Eclipse XDB-CN Provides ExcellentSelectivity and Resolution for Urea Pesticides 2004 httpswwwagilentcomcslibraryapplications5989-0930EN lowpdf

[26] E M Borges ldquoSilica hybrid silica hydride silica and non-silica stationary phases for liquid chromatographyrdquo Journal ofChromatographic Science (JCS) vol 53 no 4 pp 580ndash597 2015

[27] C M Brown J O Becker P M Wise and A N HoofnagleldquoSimultaneous determination of 6 L-arginine metabolites inhuman and mouse plasma by using hydrophilic-interactionchromatography and electrospray tandem mass spectrometryrdquoClinical Chemistry vol 57 no 5 pp 701ndash709 2011

[28] B Buszewski and S Noga ldquoHydrophilic interaction liquidchromatography (HILIC)-a powerful separation techniquerdquoAnalytical and Bioanalytical Chemistry vol 402 no 1 pp 231ndash247 2012

[29] J K Collins G Wu P Perkins-Veazie et al ldquoWatermelon con-sumption increases plasma arginine concentrations in adultsrdquoNutrition Journal vol 23 no 3 pp 261ndash266 2007

[30] S J Bailey J R Blackwell E Williams et al ldquoTwo weeksof watermelon juice supplementation improves nitric oxidebioavailability but not endurance exercise performance inhumansrdquo Nitric Oxide Biology and Chemistry vol 59 pp 10ndash20 2016

TribologyAdvances in

Hindawiwwwhindawicom Volume 2018


International Journal ofInternational Journal ofPhotoenergy


Journal of

Chemistry


Advances inPhysical Chemistry

Hindawiwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2018

Bioinorganic Chemistry and ApplicationsHindawiwwwhindawicom Volume 2018

SpectroscopyInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Medicinal ChemistryInternational Journal of


NanotechnologyHindawiwwwhindawicom Volume 2018

Journal of

Applied ChemistryJournal of



Biochemistry Research International


Enzyme Research


Journal of

SpectroscopyAnalytical ChemistryInternational Journal of


MaterialsJournal of



BioMed Research International Electrochemistry

International Journal of


Na

nom

ate

ria

ls


Journal ofNanomaterials

Submit your manuscripts atwwwhindawicom


O

NH

OH

O

（2

（2

Figure 1 Molecular structure of L-citrulline (1752 gmol)

NH

OH

O

（2

（

（

2

Figure 2 Molecular structure of L-arginine (1742 gmol)

endogenous amino acid to most living systems involved inprotein metabolism and removal of excess metabolic ammo-nia [9] It serves as a precursor for L-arginine and product ofnitric oxide (NO) cycle [10] L-arginine C

6H14N4O2(IUPAC

name (S)-2-amino-5-guanidinopentanoic acid) (Figure 2)is a semiessential and free form physiological amino acidthat functions as one of 20 building block proteins forbiological processes such as cell division ammonia removalwound healing and hormone release [7 8] Wu et al [11]demonstrated that supplementation of L-citrulline and L-arginine fromwatermelon juice improved serum levels of NOmetabolites and aortic endothelial-mediated vasodilation indiabetic rats

L-citrulline and L-arginine are present in all parts ofwatermelon fruits including flesh rind and seed [7] Astudy done by Rimando and Perkins-Veazie [12] reportedthat the rind of red watermelon and yellow watermeloncontains more L-citrulline at a concentration ranging from156 to 294mgg than flesh 79ndash285mgg Similar to theabove finding Jayaprakasha et al [13] reported that rinds ofCitrullus vulgaris varieties such as petite treat and jamboreewatermelon and also yellow crimson watermelon containedslightly higher L-citrulline ranging from 1395 to 2846mggthan flesh 1125ndash1673mgg These findings suggested thatwatermelon rind has an abundance of L-citrulline content incomparison to its content in flesh

Analyses of L-citrulline and L-arginine were routinelyconducted using capillary electrophoresis and quantificationby a spectrophotometric method however the method isless sensitive leading to discrepancies in the outcomes[6] L-citrulline and L-arginine are polar nonvolatile anddevoid of chromophores thus analysis by reverse-phase highperformance liquid chromatography (RP-HPLC) commonlyemployed a derivatization method using pre- or postcol-umn derivatization [14ndash17] Jayaprakasha et al [13] statedthat precolumn derivatization such as orthophthalaldehyde(OPA) naphthalene-23-dicarboxaldehyde or 4-dimethyl-aminoazobenzene-41015840-sulfonyl chloride (dabsyl chloride) wasable to provide accurate and stable chromatography baselinebut the reactions were unstable and affected by the sam-ple matrix [18] Postcolumn derivatization by ninhydrin istedious due to long analysis timeup to 72 hours and instability

of derivatization reagents that may cause poor compoundrecovery [19] Analysis of underivatized L-citrulline and L-arginine is warranted for rapid and effective quantification ofthese compounds Given that no amino acids content of L-citrulline and L-arginine in Malaysia watermelons has beenreported so far we have developed an isocratic RP-HPLCmethod for separation and quantification of L-citrulline andL-arginine in watermelons

2 Materials and Methods

21 Chemicals and Reagents L-citrulline (purity ge 99)and L-arginine (purity ge 98) standard were purchasedfrom Sigma-Aldrich (St Louis MO USA) Methanol andacetonitrile of HPLC grade were purchased from Merck(Germany) Phosphoric acid (purity ge 85) was purchasedfrom Sigma-Aldrich (St Louis MO USA) Deionized waterwas prepared using ultrapure water purifier system (ElgastatBucks UK)

22 Instrumentation The isocratic RP-HPLC method wascarried out using Thermo Scientific Dionex-UltiMate3000 HPLC system equipped with solvent reservoirs LPG-3400SD pump WPS-3000 autosampler injector TCC-3000column oven and DAD-3000 ultraviolet-visible (UV-Vis)diode array detectormodule operated at fourwavelengths peranalysis Chromeleon data software (Version 7) was used fordata analysis

23 Sample Preparation Citrullus lanatus (Thunb) Matsumamp Nakai of red watermelon and yellow crimson watermelonwas obtained fromSelangor Fruit Valley Selangor Seedswereremoved manually and the edible part was cut into cubesWatermelon flesh and rind were juiced and frozen at minus80∘Cfor at least 2 daysThe frozen juices were put in a freeze-drier(Labconco USA) for 4 days until completely driedThe driedjuice powders were kept at minus20∘C For the analysis sampleswere prepared in the form of juice extract and methanolextract Juice extract was prepared directly by dissolving thedried juice powder in dH

2O For methanol extract a known

quantity of dried juice powders was extracted with 30mLof MeOH and 1mL of 1 N HCl vortexed and sonicated for30 minutes The samples were macerated by cold macerationfor a period of 72 hours in an orbital shaker Methanolextracts were then filtered using Whatman filter paper Theresidues were reextracted twice using fresh solvent and thethree methanol extracts were pooledThe obtained methanolextracts were evaporated to dryness using a rotary vacuumevaporator at 60∘C and stored at 4∘C until analysis

24 Isocratic RP-HPLC Analysis

241 Standard Preparation Procedure A stock solution of L-citrulline and L-arginine was prepared individually in dH

2O

at 1mgmL and filtered through a 045 120583m syringe filter(Bioflow) Amixed standard solutionwas prepared bymixingan equal volume of each standard stock solution A seriesof working standard solutions was prepared by diluting thestock solution with dH

2O in the range of 01ndash1000 120583gmL




3P04

100 07 H3P04+ 993 dH

2O

01 H3P04

100 01 H3P04+ 999 dH

2O

07 H3P04 ACN 90 10 (07 H

3P04+ 993 dH

2O) +

100 ACN


2O




07 H3P04 01 H

3P04 and 07 H

3P04 ACN (90 10)


18 250mm times 46mm 80 A 5120583m (Phe-



18eluted by 01 H










H3P04 and 07 H





3P04(Figure 3(b))






3P04adequately






18and Gemini

C18


18did not




1 2 3 4 5 6 7 8 9 100(min)

minus500

0

500

1000

1500

2000

2500

3000(m

AU)

(a)

minus500

0

500

1000

1500

2000

2500

3000

(mAU

)

1 2 3 4 5 6 7 8 9 100(min)

(b)

(1)

(2)

1 2 3 4 5 6 7 8 9 100(min)

minus500

0

500

1000

1500

2000

2500

3000

(mAU

)

(c)






1 2 3 4 5 6 7 8 9 100(min)

minus500

0

500

1000

1500

2000

2500

3000

(mAU

)

(a)

(1)

(2)

minus500

0

500

1000

1500

2000

2500

(mAU

)

1 2 3 4 5 6 7 8 9 100(min)

(b)









is a new


18is



(1)

(a)

(b)

(c)

(2)

minus500

0

500

1000

1500

2000

2500

(mAU

)

1 2 3 4 5 6 7 8 9 100(min)

minus500

0

500

1000

1500

2000

2500

(mAU

)

1 2 3 4 5 6 7 8 9 100(min)

minus500

0

500

1000

1500

2000

2500

(mAU

)1 2 3 4 5 6 7 8 9 100

(min)










(1198772)






L-citrulline100 10194 10194 17060 6124 10207 14630 3102 10338 100

L-arginine100 9987 9987 19660 5933 9888 17630 3102 10341 117



L-citrulline150 10125 123 10042 10960 10207 146 10344 10120 11644 203 11622 037

L-arginine150 10201 056 10383 20560 9626 104 9736 11320 9541 068 9594 033






(1) (2)

minus50050100150200250300350400450

(mAU

)

1 2 3 4 5 6 7 8 9 100(min)

(a)

(2)(1)

minus500

0

500

1000

1500

2000

2500

(mAU

)

1 2 3 4 5 6 7 8 9 100(min)

(b)

(2)(1)

1 2 3 4 5 6 7 8 9 100(min)

minus50050100150200250300350400

(mAU

)

(c)

(1)(2)

1 2 3 4 5 6 7 8 9 100(min)

minus500

0

500

1000

1500

2000

2500

(mAU

)

(d)

(1)(2)

minus20020406080100120140160180

(mAU

)

1 2 3 4 5 6 7 8 9 100(min)

(e)

(1) (2)

minus500

0

500

1000

1500

2000

2500

(mAU

)

1 2 3 4 5 6 7 8 9 100(min)

(f)

(1)

(2)

1 2 3 4 5 6 7 8 9 100(min)

minus50050100150200250300350400

(mAU

)

(g)

(1)(2)

minus500

0

500

1000

1500

2000

2500

(mAU

)

1 2 3 4 5 6 7 8 9 100(min)

(h)








Rind 4502 2499



Rind 1661 1603



4 Conclusion


3P04) in







Acknowledgments


References






































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls







3P04

100 07 H3P04+ 993 dH

2O

01 H3P04

100 01 H3P04+ 999 dH

2O

07 H3P04 ACN 90 10 (07 H

3P04+ 993 dH

2O) +

100 ACN


2O




07 H3P04 01 H

3P04 and 07 H

3P04 ACN (90 10)


18 250mm times 46mm 80 A 5120583m (Phe-



18eluted by 01 H










H3P04 and 07 H





3P04(Figure 3(b))






3P04adequately






18and Gemini

C18


18did not




1 2 3 4 5 6 7 8 9 100(min)

minus500

0

500

1000

1500

2000

2500

3000(m

AU)

(a)

minus500

0

500

1000

1500

2000

2500

3000

(mAU

)

1 2 3 4 5 6 7 8 9 100(min)

(b)

(1)

(2)

1 2 3 4 5 6 7 8 9 100(min)

minus500

0

500

1000

1500

2000

2500

3000

(mAU

)

(c)






1 2 3 4 5 6 7 8 9 100(min)

minus500

0

500

1000

1500

2000

2500

3000

(mAU

)

(a)

(1)

(2)

minus500

0

500

1000

1500

2000

2500

(mAU

)

1 2 3 4 5 6 7 8 9 100(min)

(b)









is a new


18is



(1)

(a)

(b)

(c)

(2)

minus500

0

500

1000

1500

2000

2500

(mAU

)

1 2 3 4 5 6 7 8 9 100(min)

minus500

0

500

1000

1500

2000

2500

(mAU

)

1 2 3 4 5 6 7 8 9 100(min)

minus500

0

500

1000

1500

2000

2500

(mAU

)1 2 3 4 5 6 7 8 9 100

(min)










(1198772)






L-citrulline100 10194 10194 17060 6124 10207 14630 3102 10338 100

L-arginine100 9987 9987 19660 5933 9888 17630 3102 10341 117



L-citrulline150 10125 123 10042 10960 10207 146 10344 10120 11644 203 11622 037

L-arginine150 10201 056 10383 20560 9626 104 9736 11320 9541 068 9594 033






(1) (2)

minus50050100150200250300350400450

(mAU

)

1 2 3 4 5 6 7 8 9 100(min)

(a)

(2)(1)

minus500

0

500

1000

1500

2000

2500

(mAU

)

1 2 3 4 5 6 7 8 9 100(min)

(b)

(2)(1)

1 2 3 4 5 6 7 8 9 100(min)

minus50050100150200250300350400

(mAU

)

(c)

(1)(2)

1 2 3 4 5 6 7 8 9 100(min)

minus500

0

500

1000

1500

2000

2500

(mAU

)

(d)

(1)(2)

minus20020406080100120140160180

(mAU

)

1 2 3 4 5 6 7 8 9 100(min)

(e)

(1) (2)

minus500

0

500

1000

1500

2000

2500

(mAU

)

1 2 3 4 5 6 7 8 9 100(min)

(f)

(1)

(2)

1 2 3 4 5 6 7 8 9 100(min)

minus50050100150200250300350400

(mAU

)

(g)

(1)(2)

minus500

0

500

1000

1500

2000

2500

(mAU

)

1 2 3 4 5 6 7 8 9 100(min)

(h)








Rind 4502 2499



Rind 1661 1603



4 Conclusion


3P04) in







Acknowledgments


References






































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls





1 2 3 4 5 6 7 8 9 100(min)

minus500

0

500

1000

1500

2000

2500

3000(m

AU)

(a)

minus500

0

500

1000

1500

2000

2500

3000

(mAU

)

1 2 3 4 5 6 7 8 9 100(min)

(b)

(1)

(2)

1 2 3 4 5 6 7 8 9 100(min)

minus500

0

500

1000

1500

2000

2500

3000

(mAU

)

(c)






1 2 3 4 5 6 7 8 9 100(min)

minus500

0

500

1000

1500

2000

2500

3000

(mAU

)

(a)

(1)

(2)

minus500

0

500

1000

1500

2000

2500

(mAU

)

1 2 3 4 5 6 7 8 9 100(min)

(b)









is a new


18is



(1)

(a)

(b)

(c)

(2)

minus500

0

500

1000

1500

2000

2500

(mAU

)

1 2 3 4 5 6 7 8 9 100(min)

minus500

0

500

1000

1500

2000

2500

(mAU

)

1 2 3 4 5 6 7 8 9 100(min)

minus500

0

500

1000

1500

2000

2500

(mAU

)1 2 3 4 5 6 7 8 9 100

(min)










(1198772)






L-citrulline100 10194 10194 17060 6124 10207 14630 3102 10338 100

L-arginine100 9987 9987 19660 5933 9888 17630 3102 10341 117



L-citrulline150 10125 123 10042 10960 10207 146 10344 10120 11644 203 11622 037

L-arginine150 10201 056 10383 20560 9626 104 9736 11320 9541 068 9594 033






(1) (2)

minus50050100150200250300350400450

(mAU

)

1 2 3 4 5 6 7 8 9 100(min)

(a)

(2)(1)

minus500

0

500

1000

1500

2000

2500

(mAU

)

1 2 3 4 5 6 7 8 9 100(min)

(b)

(2)(1)

1 2 3 4 5 6 7 8 9 100(min)

minus50050100150200250300350400

(mAU

)

(c)

(1)(2)

1 2 3 4 5 6 7 8 9 100(min)

minus500

0

500

1000

1500

2000

2500

(mAU

)

(d)

(1)(2)

minus20020406080100120140160180

(mAU

)

1 2 3 4 5 6 7 8 9 100(min)

(e)

(1) (2)

minus500

0

500

1000

1500

2000

2500

(mAU

)

1 2 3 4 5 6 7 8 9 100(min)

(f)

(1)

(2)

1 2 3 4 5 6 7 8 9 100(min)

minus50050100150200250300350400

(mAU

)

(g)

(1)(2)

minus500

0

500

1000

1500

2000

2500

(mAU

)

1 2 3 4 5 6 7 8 9 100(min)

(h)








Rind 4502 2499



Rind 1661 1603



4 Conclusion


3P04) in







Acknowledgments


References






































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls





(1)

(a)

(b)

(c)

(2)

minus500

0

500

1000

1500

2000

2500

(mAU

)

1 2 3 4 5 6 7 8 9 100(min)

minus500

0

500

1000

1500

2000

2500

(mAU

)

1 2 3 4 5 6 7 8 9 100(min)

minus500

0

500

1000

1500

2000

2500

(mAU

)1 2 3 4 5 6 7 8 9 100

(min)










(1198772)






L-citrulline100 10194 10194 17060 6124 10207 14630 3102 10338 100

L-arginine100 9987 9987 19660 5933 9888 17630 3102 10341 117



L-citrulline150 10125 123 10042 10960 10207 146 10344 10120 11644 203 11622 037

L-arginine150 10201 056 10383 20560 9626 104 9736 11320 9541 068 9594 033






(1) (2)

minus50050100150200250300350400450

(mAU

)

1 2 3 4 5 6 7 8 9 100(min)

(a)

(2)(1)

minus500

0

500

1000

1500

2000

2500

(mAU

)

1 2 3 4 5 6 7 8 9 100(min)

(b)

(2)(1)

1 2 3 4 5 6 7 8 9 100(min)

minus50050100150200250300350400

(mAU

)

(c)

(1)(2)

1 2 3 4 5 6 7 8 9 100(min)

minus500

0

500

1000

1500

2000

2500

(mAU

)

(d)

(1)(2)

minus20020406080100120140160180

(mAU

)

1 2 3 4 5 6 7 8 9 100(min)

(e)

(1) (2)

minus500

0

500

1000

1500

2000

2500

(mAU

)

1 2 3 4 5 6 7 8 9 100(min)

(f)

(1)

(2)

1 2 3 4 5 6 7 8 9 100(min)

minus50050100150200250300350400

(mAU

)

(g)

(1)(2)

minus500

0

500

1000

1500

2000

2500

(mAU

)

1 2 3 4 5 6 7 8 9 100(min)

(h)








Rind 4502 2499



Rind 1661 1603



4 Conclusion


3P04) in







Acknowledgments


References






































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls








(1198772)






L-citrulline100 10194 10194 17060 6124 10207 14630 3102 10338 100

L-arginine100 9987 9987 19660 5933 9888 17630 3102 10341 117



L-citrulline150 10125 123 10042 10960 10207 146 10344 10120 11644 203 11622 037

L-arginine150 10201 056 10383 20560 9626 104 9736 11320 9541 068 9594 033






(1) (2)

minus50050100150200250300350400450

(mAU

)

1 2 3 4 5 6 7 8 9 100(min)

(a)

(2)(1)

minus500

0

500

1000

1500

2000

2500

(mAU

)

1 2 3 4 5 6 7 8 9 100(min)

(b)

(2)(1)

1 2 3 4 5 6 7 8 9 100(min)

minus50050100150200250300350400

(mAU

)

(c)

(1)(2)

1 2 3 4 5 6 7 8 9 100(min)

minus500

0

500

1000

1500

2000

2500

(mAU

)

(d)

(1)(2)

minus20020406080100120140160180

(mAU

)

1 2 3 4 5 6 7 8 9 100(min)

(e)

(1) (2)

minus500

0

500

1000

1500

2000

2500

(mAU

)

1 2 3 4 5 6 7 8 9 100(min)

(f)

(1)

(2)

1 2 3 4 5 6 7 8 9 100(min)

minus50050100150200250300350400

(mAU

)

(g)

(1)(2)

minus500

0

500

1000

1500

2000

2500

(mAU

)

1 2 3 4 5 6 7 8 9 100(min)

(h)








Rind 4502 2499



Rind 1661 1603



4 Conclusion


3P04) in







Acknowledgments


References






































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls





(1) (2)

minus50050100150200250300350400450

(mAU

)

1 2 3 4 5 6 7 8 9 100(min)

(a)

(2)(1)

minus500

0

500

1000

1500

2000

2500

(mAU

)

1 2 3 4 5 6 7 8 9 100(min)

(b)

(2)(1)

1 2 3 4 5 6 7 8 9 100(min)

minus50050100150200250300350400

(mAU

)

(c)

(1)(2)

1 2 3 4 5 6 7 8 9 100(min)

minus500

0

500

1000

1500

2000

2500

(mAU

)

(d)

(1)(2)

minus20020406080100120140160180

(mAU

)

1 2 3 4 5 6 7 8 9 100(min)

(e)

(1) (2)

minus500

0

500

1000

1500

2000

2500

(mAU

)

1 2 3 4 5 6 7 8 9 100(min)

(f)

(1)

(2)

1 2 3 4 5 6 7 8 9 100(min)

minus50050100150200250300350400

(mAU

)

(g)

(1)(2)

minus500

0

500

1000

1500

2000

2500

(mAU

)

1 2 3 4 5 6 7 8 9 100(min)

(h)








Rind 4502 2499



Rind 1661 1603



4 Conclusion


3P04) in







Acknowledgments


References






































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls








Rind 4502 2499



Rind 1661 1603



4 Conclusion


3P04) in







Acknowledgments


References






































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls


































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls









Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls




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