The Separation of L Citrulline & L Arginine Using Isocratic HPLC Methodology Discussion 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). 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 © 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 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 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 -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-4 -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 ≥ 99%)
and L-arginine (purity ≥ 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 ≥ 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 −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 −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 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 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 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 × 4.6 mm, 80 Å, 5 m (Phenomenex, Torrance, CA), and Gemini C18 , 250 × 4.6 mm,
110 Å, 3 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 g/mL for
L-citrulline and 0.1–500 g/mL for L-arginine. The regression
equation was calculated in the form of = + , where
is the concentration and is the peak area of compounds.
Linearity was established by the coefficient of determination
( 2 ). 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 g/mL; Level
2: 60 g/mL; Level 3: 150 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 (
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 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
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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 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.
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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 m, and (b) Gemini C18 , 3 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
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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…
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