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Effective separation and sensitive determination

of cyanuric acid, melamine and cyromazine
in environmental water by reversed phase
high‐performance liquid chromatography
a a a a
Hanwen Sun , Xiaolan Qin , Xusheng Ge & Lixin Wang
a
College of Chemistry and Environmental Science , Hebei University , Key Laboratory of
Analytical Science and Technology of Hebei Province, Baoding 071002, China
Published online: 26 Mar 2011.

To cite this article: Hanwen Sun , Xiaolan Qin , Xusheng Ge & Lixin Wang (2011) Effective separation and sensitive
determination of cyanuric acid, melamine and cyromazine in environmental water by reversed phase high‐performance liquid
chromatography, Environmental Technology, 32:3, 317-323, DOI: 10.1080/09593330.2010.499543

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 Environmental Technology
Vol. 32, No. 3, February 2011, 317–323

Effective separation and sensitive determination of cyanuric acid, melamine and cyromazine
in environmental water by reversed phase high-performance liquid chromatography
Hanwen Sun*, Xiaolan Qin, Xusheng Ge and Lixin Wang
College of Chemistry and Environmental Science, Hebei University, Key Laboratory of Analytical Science and Technology of
Hebei Province, Baoding 071002, China
(Received 22 November 2009; Accepted 1 June 2010 )
Taylor and Francis

10.1080/09593330.2010.499543

A new method for the simultaneous determination of cyanuric acid (CA), melamine (MM) and cyromazine (CM) in
Downloaded by [University of Saskatchewan Library] at 15:35 30 January 2015

different water samples was developed by using reversed phase high-performance liquid chromatography diode-array
detection (RP-HPLC-DAD). The conditions of HPLC were investigated and optimized. A KROMASIL ® C18 column
(250 × 4.6 mm ID, 5 µm) was used for the RP-HPLC using gradient elution with a mobile phase composed of 0.1
mM KH2PO4-K2HPO4 buffer solution (pH = 7.3) and methanol (75:25, v/v). The conditions for HPLC were
investigated and optimized. Under optimal conditions, the linearity was satisfactory in the range of 0.04–10.00 µg/
mL with a correlation coefficient of 0.999, and the method limits of detection (LODs) of the proposed method were
0.02 µg/mL for CA and CM, and 0.01 µg/mL for MM. The recoveries were: 96.0–116.0% with relative standard
deviations (RSDs) of 0.9–5.7% for CA, 94.3–115.0% with RSDs of 1.5–5.3% for MM, and 91.0–112.0% with RSDs
(n = 6) of 1.0–4.9% for CM. The proposed method can permit the detection of CA and CM at levels as low as 0.07
µg/mL and MM at levels as low as 0.03 µg/mL in environmental water samples.
Keywords: high-performance liquid chromatography; cyanuric acid; melamine; cyromazine; environmental water

1. Introduction to food and environmental analysis by liquid chroma-

Cyromazine (CM, N-cyclopropyl-1,3,5-triazine-2,4,6-
triamine) is a triazine pesticide used for fly control in
crop production and animal feed, by inhibiting insect
growth. In recent years, it has been detected in food and
the environment in the countries where it had been used,
and has caused the potential environmental and human
health problems [1]. Cyromazine can be metabolized
via dealkylation reactions in both plants and animals to
form melamine (MM) and cyanuric acid (CA) [2,3].
Both MM and CA are important industrial chemicals,
and they widely exist in habitation and industrial
districts such as in swimming water, fisheries, the
production of melamine–formaldehyde resins, scouring
powders and so on. Melamine and cyanuric acid, when
co-ingested form an insoluble precipitate in kidney
tubules that is of sufficient severity to cause renal fail-
ure via physical blockage [4].
We have reviewed the research methodologies used
for the analysis of MM and related analogues [5]. In
this review, the release process of these compounds
from melamine resin, fertilizer, herbicide and insecti-
cide products into feed and foods was generalized,
including thermal degradation, hydrolysis, biodegrada-
tion and photocatalysis, and special attention was given
tography (LC) and liquid chromatography–tandem
mass spectrometry (LC–MS/MS). Recently, Tittlemier
also reviewed methods for the analysis of melamine
and related compounds in foods [6]. A series of LC
methods have been used for the determination of MM
in different feed and food samples. There were a few
reports for the determination of CM [7,8] and CA in pet
food and water by HPLC [9,10] and CA in urine by
LC–MS [11] as well as in fish by LC–MS/MS [12].
For the simultaneous determination of CM and MM,
several LC methods have been reported [13,14]. An
LC–ESI (electrospray ionization)–MS/MS method was
developed for analysis of chard samples, with a low
limit of quantification (LOQ) value [15]. Recently, an
LC–MS/MS method was established for the determina-
tion of CM and MM residues in milk and dairy products
[16]. For the simultaneous determination of MM and
CA, a series of methods have been reported for sample
analysis, including LC for cereal flours [17], LC–MS/
MS for foods [18], milk [19], animal feed [20] and
infant formula [19,21] as well as isotope-dilution–LC–
MS for catfish, pork, chicken and pet food [22].
Recently, Muñiz-Valencia et al. [23] utilized LC with
Luna cyano and Synergi fusion RP (reversed phase)

*Corresponding author. Email: hanwen@hbu.edu.cn

ISSN 0959-3330 print/ISSN 1479-487X online

© 2011 Taylor & Francis
DOI: 10.1080/09593330.2010.499543
http://www.informaworld.com
 318 H. Sun et al.
columns to detect MM and its derivatives in rice
concentrates. Filigenzi et al. [24] reported an LC–atmo-
spheric-pressure chemical ionization–MS/MS method
for analysis of MM and CA and ammelide in kidney
tissue. In these methods, different LC separation
columns were used, but without C18 columns because
of the strong polarity of CA, MM and CM.
Environmental sample analysis of the three
compounds is essential for examining their transference
and pollution to environment. Liquid chromatography
with UV detection was used for CA analysis in environ-
mental and swimming-pool water with detection limits
of 0.02–0.07 µg/mL [25,26]. The MS and LC–MS
methods were utilized for the determination of CA in
urine with detection limits of 0.13 µg/mL and 0.1 µg/
Downloaded by [University of Saskatchewan Library] at 15:35 30 January 2015
mL, respectively [11,27]. The LC–UV and GC–MS
methods have been utilized for the detection of CM and
MM residues in soil [28]. Generally, LC is more attrac-
tive than GC because no preliminary derivatization
procedures are required, and LC–MS/MS methods have
high sensitivity, but with high cost. Considering their
strong polarity and different solvencies, effective sepa-
ration of CA, MM and CM is more difficult. Therefore,
it is of critical importance to develop a simple, sensitive
and simultaneous determination method.
To our knowledge, there is no report on the simulta-
neous determination of CA, MM and CM. The design of
an appropriate LC separation column, corresponding
mobile phase and gradient procedure is an important
factor for effective separation and simultaneous detec-
tion of the three compounds. The purpose of this paper
is to develop an LC–diode-array detection method for
effective separation and simultaneous determination of
CA, MM and CM. This approach utilized a KROMA-
SIL® C18 column and gradient elution with a mobile
phase composed of KH2PO4-K2HPO4 buffer solution and
methanol to achieve the baseline separation in simple LC
mode. To assess its applicability, the proposed method
was applied for the analysis of different water samples.
2. Experimental
2.1. Chemicals and reagents
Melamine, cyromazine and cyanuric acid were purchased
from Kermel Chemical Reagents Development Centre
(Tianjin, China). All the reagents were of analytical
grade. Double distilled water was obtained from quartz
distillation apparatus.
The mobile phase consisted of 0.1 mM KH2PO4-
K2HPO4 (2.3:7.7) buffer solution (pH 7.3) and methanol
(75:25,v/v). The buffer solution was filtered through a
0.45 um microporous membrane of mixed cellulose
ester. An individual stock standard solution, 1000 µg/
mL, was prepared by dissolving CA, MM and CM in
the buffer solution, respectively. These stock solutions
were stable for at least one month if stored at 4 °C. A
fresh working standard solution was prepared daily by
diluting the stock solutions with the buffer solution,
then was used for different studies. The methanol was
filtered through a 0.22 µm microporous membrane of
polyvinylidene fluoride before use.
2.2. Instrumentation
The chromatographic system consisted of a Shimadzu
HPLC system equipped with an LC-10A Multisolvent
Delivery System, a DGU-12A online degasser, an SCL-
10Avp gradient controller, a CTO-10Avp column
thermostat, and a multi-wavelength SPD-M10Avp
photodiode-array detector (DAD) covering the range
190–800 nm, which was interfaced to a computer for
data acquisition using a CLASS-VP workstation
(Shimadzu, Kyoto, Japan). A KROMASIL® C18 column
(250 × 4.6 mm ID, 5 µm, AKZO NOBEL, Bohus,
Sweden) and a six-port valve with a 20 µL sample loop
injector were also used. Ultraviolet data were measured
with a UV-265 spectrophotometer (Shimadzu, Kyoto,
Japan).
2.3. HPLC analysis
The separation of CA, MM and CM was carried out
using a KROMASIL® C18 analytical column and a
mobile phase composed of 0.1 mM KH2PO4-K2HPO4
(2.3:7.7) buffer solution (pH 7.3) and methanol
(75:25,v/v). The flow rate of the mobile phase was set
at 0.5 mL/min. A 20 µL volume of sample solution was
injected in the column, and then eluted in gradient with
0% of methanol in the mobile phase from 0 to 3 min,
20% from 4 to 16 min, and 0% from 18 to 20 min. There
was a re-equilibration time (about 20 min) after each
procedure. The elutions were monitored at 204 nm for
MM and CM, and at 214 nm for CA. The linear equa-
tions for the relationship between peak areas of CA,
MM and CM and their concentration were determined
by least-squares method. Quantification was carried out
by using matrix-matched standards calibration. Each of
the water samples was filtered through a 0.45 µm
microporous membrane of mixed cellulose ester and
subjected to analysis by HPLC.
3. Results and discussion
3.1. Selection of the detection wavelength
To choose a suitable detection wavelength, CA, MM
and CM were dissolved separately in phosphate buffer
solution; CM was also dissolved in phosphate-buffer–
methanol (80:20,v/v) solution which was used as a
mobile phase for eluting CM. Then the solutions were
 Environmental Technology 319

separation and detection of CA, MM and CM. So phos-

phate–methanol solution was selected as the mobile
phase.

3.3. Optimization of phosphate buffer pH

The dissociation constants, pKa, are 8.0 for MM, 7.2 for
CM and 6.9 for CA [8,9]. Generally, for compounds
with lower pKa values, the dissociation ratio is higher
and acidity is stronger. The pH of phosphate buffer influ-
ences ionization of the analytes. The effect of pH of
phosphate buffer on the peak area of the three analytes
is shown in Figure 2. When the pH was 7.3, a high sensi-
tivity for the three analytes was achieved, avoiding CA
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keto-enol tautomerism (at pH 6.8–7.1) [11]. So pH 7.3

Figure 1. UV spectrogram of the analytes in phosphate- was selected as the optimal acidity condition.
buffered solution (1, 2, 3) and phosphate-buffered solution Figure 2. Effect of different pH of phosphate buffer on the peak area of CA, MM and CM.

with methanol (8:2) (4).

3.4. Optimization of the concentration of phosphate
scanned by a UV-265 spectrophotometer from 200 to buffer
300 nm, as shown in Figure 1. The maximum absorp-
The effect of the concentration of phosphate buffer
tion wavelength was 214 nm for CA, 204 nm for MM,
on the three analytes was investigated, as shown in
and 206 nm for CM in two kinds of solvent without
Figure 3. It was observed that with increasing concen-
solvent effect. So the analyte was monitored at 204 nm
tration of phosphate buffer the peak area for the analyte
for MM and CM, and at 214 nm for CA.
Figure 1. UV spectrogram of the analytes in phosphate-buffered solution (1, 2, 3) and phosphate-buffered solution with methanol (8:2) (4). increased slightly, but the possibility of jamming the
column would be increased. So 0.1 mM was selected
3.2. Selection of separation column and mobile for further tests.
Figure 3. Effect of concentration of phosphate buffer on the peak area of CA, MM and CM.

phase
A Zwitterionic HILIC® column and a Luna cyano 3.5. Optimization of methanol proportion in mobile
column were used for the separation of MM and CA, phase
respectively [22,23]. A Zorbax SCX column was used
for separation of MM and CM using an isocratic mobile The effects of different proportions (0–50%) of metha-
phase consisting of 25% methanol and 75% potassium nol in the mobile phase on the peak area and the reten-
phosphate buffer [28]. Because MM, CA and CM are tion time of CM, MM and CA in isocratic and
small and highly polar molecules, it is difficult to gradient elution programmes were investigated. For
achieve sufficient retention by using traditional C18 the isocratic elution programme, it was observed that
columns, and the ion-pair reagents could be used to the retention time for MM and CA decreased slightly
improve retention for MM, CA and CM [15].
In our work, the initial test for the separation of CA,
MM and CM was carried out using a KROMASIL®
C18 column and different ionic reagents to enhance the
retention of these polar compounds. Sodium dodecyl
sulphate, tetrabutyl ammonium bromide and phosphate
could be used as an ionic reagent, forming neutral ion
pairs with ionized MM, CA and CM. Considering the
phosphate buffer solution as the mobile phase for the
detection of CA, we attempted to utilize phosphate for
the separation of MM, CM and CA. The result showed
that CM could not be detected. When the phosphate–
acetonitrile solution was used, the effective separation
of the three analytes was achieved, but split peaks of the
three analytes were observed. To overcome this prob-
lem, as methanol has a better solvency than acetonitrile, Figure 2. Effect of different pH of phosphate buffer on the
methanol was used instead of acetonitrile to achieve the peak area of CA, MM and CM.
 320 H. Sun et al.

maintained at 4.4 min and 6.8 min, respectively, and

that for CM decreased from 17.8 min to 14.6 min.
Otherwise, the peak area of CM increased with increas-
ing proportion of methanol up to 30%, and thereafter
the peak area decreased markedly. The proportion of
methanol in the mobile phase was selected to be 25%,
with higher sensitivity for CM and MM, and complete
baseline separation of CA from an impurity. Based on
the above results the gradient elution programme for
eluting CA, MM and CM was designed as follows: 0–3
min without methanol for CA, 4–16 min with 25%
methanol for MM and CM, and 18–20 min without
methanol to return to the initial state.
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Figure 3. Effect of concentration of phosphate buffer on the 3.6. Validation of the method
peak area of CA, MM and CM. 3.6.1. Specificity
Under the following optimal conditions: a mobile phase
and, for CM, decreased markedly (from 41 to 13 min) of 0.1 mM KH2PO4-K2HPO4 (2.3:7.7) buffer solution
with the increase in the proportion of methanol from (pH 7.3) and methanol (75: 25), flow rate of 0.5 mL/
3% to 13%. If methanol was used, the sensitivity of min, column temperature of 33 °C, detection wave-
CA would decrease and CA would be eluted early, length of 214 nm for CA and 204 nm for MM and CM,
even in the dead time. This was unfavourable to the and using the gradient elution programme, the chro-
analysis of CA; even if the solvent contained metha- matograms of real water samples and spiked water
nol, the peaks of CA would overlap with those of samples for five kinds of environmental water were
MM, so no methanol must be used for eluting CA. In observed. The representational chromatograms are
contrast, the signal peak of CM could be observed shown in Figure 5.
only in the presence of methanol. Therefore the Chromatograms of CA, MM and CM in different samples: (a) unspiked swimming water (CA: 3.49 µg/mL); (b) unspiked plant wastewater (MM: 0.1 µg/mL); (c) fishery pond water spiked with 0.07 µg/mL CA, 0.04 µg/mL MM and 0.07 µg/mL CM.

The effective baseline separation of the analytes was

Figure 5.

isocratic elution programme with phosphate-buffer– observed in different water samples. No interfering
methanol as a mobile phase was unfavourable for the peaks were observed at the retention times of CA, MM
analysis of CA, MM and CM. and CM, respectively. Quantification was carried out by
For the gradient elution programme, the effects of using standard curve calibration.
different proportions of methanol in the mobile phase
on the retention time and the peak area of MM, CA and
CM were investigated, as shown in Figure 4. 3.6.2. Linearity and detection limit
With the proportion of methanol varying from Under the optimal conditions, the linearity for analysis
Figure 4. Left: Effect of different proportions of methanol in mobile phase on the peak area of CM. Right: retention time of CA (1), MM (2) and CM (3).

20% to 40%, the retention time for MM and CA was of spiked real samples was evaluated with the measured

Figure 4. Left: Effect of different proportions of methanol in mobile phase on the peak area of CM. Right: retention time of CA
(1), MM (2) and CM (3).
 Environmental Technology 321
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Figure 5. Chromatograms of CA, MM and CM in different samples: (a) unspiked swimming water (CA: 3.49 µg/mL); (b) unspiked
plant wastewater (MM: 0.1 µg/mL); (c) fishery pond water spiked with 0.07 µg/mL CA, 0.04 µg/mL MM and 0.07 µg/mL CM.

peak area of CA, MM and CM against their concentra-
tions. The obtained linear regression equations and
linearity range as well as correlation coefficients for the
determination of CA, MM and CM are listed in Table 1.
The limit of detection (LOD) was determined as the
sample concentration that produced a peak with a height
three times the level of the baseline noise, and the limit
of quantification (LOQ) was calculated as the sample
concentration that produced a peak with a height 10
times the ratio of signal to noise. The LOD of the
proposed method for CA, MM and CM was 0.02, 0.01
and 0.02 µg/mL, respectively, and their LOQs were
0.07, 0.03 and 0.07 µg/mL, which is lower than that of
HPLC methods for water analysis [25,26], and LC–MS
and MS methods for urine analysis [11,27].
It is indicated that the proposed method has good
linearity, and can permit the detection of CA and CM at
levels as low as 0.07 µg/mL and MM at levels as low as
0.03 µg/mL in environmental water.
3.7. Sample analysis
The proposed method was applied for the determination
of CA, MM and CM in swimming water, plant waste-
water, river water, domestic sewage and fishery pond
water. The recovery experiments for the three analytes
were carried out by adding their standard solution
(lowest concentration point nears LOQ value) in real
water samples followed by chromatographic analysis.
The test results are given in Table 2.
Cyanuric acid in the swimming water and MM in
the plant wastewater were found to be 3.49 µg/mL
and 0.1 µg/mL, respectively. The recoveries of CA for
all samples spiked with 0.07–4.0 µg/mL were in the

Table 1. The linear regression equation, correlation coefficient, linearity range, LOD and LOQ for CA, MM and CM.
Correlation Linear range LOD LOQ
Analyte Regression equation* coefficient (µg/mL) (µg/mL) (µg/mL)
CA A = 1.203 × 105C + 3.942 × 103 0.9999 0.07–10 0.02 0.07
MM A = 8.658 × 105C + 4.060 × 104 0.9997 0.04–10 0.01 0.03
CM A = 4.890 × 105C − 6.296 × 103 0.9998 0.07–10 0.02 0.07
*A is th peak area of standards based on three parallel measurements and C is the concentration (µg/mL) of standards for CA, MM and CM.
 322 H. Sun et al.

Table 2. Recovery and RSD of CA, MM and CM in real samples.

Content Added Found Recovery RSD
Sample Analyte (µg/mL) (µg/mL) (µg/mL) (%) n = 6 (%)
Swimming water CA 3.49 2 5.65 104 1.5
4 8.25 110 0.9
MM * 0.05 0.053 106 4.8
CM * 0.1 0.097 97.0 2.7
0.2 0.204 102 2.2
Plant wastewater CA 0 0.1 0.098 98.0 5.7
0.4 0.394 98.5 3.6
MM 0.1 0.1 0.198 98.0 2.5
0.4 0.481 95.3 2.6
CM * 0.1 0.091 91.0 2.1
Downloaded by [University of Saskatchewan Library] at 15:35 30 January 2015

0.2 0.205 105 1.0

River water CA * 0.1 0.097 97.0 4.9
0.2 0.192 96.0 2.7
MM * 0.07 0.080 114 3.6
0.14 0.148 106 2.7
CM * 0.08 0.074 92.2 4.4
0.16 0.152 95.1 1.7
Domestic sewage CA * 0.07 0.0806 115 3.4
0.28 0.285 102 2.2
MM * 0.07 0.069 94.3 4.9
0.28 0.322 115 1.5
CM * 0.04 0.037 92.5 2.8
0.08 0.090 112 2.5
Fishery pond water CA * 0.07 0.081 115 3.5
0.14 0.162 116 3.2
MM * 0.07 0.068 97.1 3.3
0.14 0.146 104 5.3
CM * 0.07 0.065 92.5 4.9
0.14 0.132 94.6 3.9
*not detected.

range of 96.0–116.0% with relative standard devia-
tions (RSDs) (n = 6) of 0.9–5.7%. The recoveries of
melamine for all samples spiked with 0.05–0.4 µg/mL
were in the range of 94.3–115.0% with RSDs (n = 6)
of 1.5–5.3%. The recoveries of cyromazine for all the
samples spiked with 0.04–0.2 µg/mL were in the
range of 91.0–112.0% with RSDs (n = 6) of 1.0–
4.9%.
simultaneous determination of CA, MM and CM at trace
levels in environmental waters.
Acknowledgement
This work was supported by the Science Foundation Educa-
tion Office of Hebei Province (B2008000583).
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