Journal of Analytical Sciences, Methods and Instrumentation, 2012, 2, 68-73 Published Online June 2012 (
Organic Solvent-Free and Simple Method for Determining
Cyromazine and Its Metabolite, Melamine, in Cow’s Milk
Naoto Furusawa
Graduate School of Human Life Science, Osaka City University, Osaka, Japan.
Received April 12th, 2012; revised May 3rd, 2012; accepted May 20th, 2012
This paper described an organic solvent-free, rapid, simple, and space-saving method of sample preparation followed by
HPLC coupled photo-diode array (PDA) detector for simultaneous quantification of cyromazine (CYR) and its decy-
cropropylated metabolite, melamine (MEL), in milk. The HPLC-PDA was performed on an Inertsil® HILIC column
with an isocratic aqueous mobile phase. Analytes were extracted from the sample using water, and purified by Mono-
Spin®-C18, a centrifugal monolithic silica spin mini-columns, and quantified within 20 min. The method, performed
under 100% aqueous conditions, obtained average recoveries for CYR and MEL in the range of 93.2% - 99.1% with
relative standard deviations 2.8%. The quantitation limits were 8.5 ng/mL for CYR and 10 ng/mL for MEL, respec-
tively. No organic solvents were used at any stage of the analysis.
Keywords: Cyromazine; Melamine; HPLC-PDA; Milk: Organic Solvent-Free
1. Introduction
Cyromazine (CYR) is a triazine insect growth regulator
used as an insecticide. In veterinary medicine, CYR is
used as an ectoparasiticide and is added to animal feed to
prevent fly from the manure, so as to improve the hy-
giene control of animal housing environments. CYR can
be decycropropylated to melamine (MEL) (Figure 1) in
plants and food-producing animals. The biotransformed
MEL has been isolated from laying hen, goat, sheep, cow,
and their products [1].
The 2008 Chinese milk scandal broke as infants con-
sumed formula containing MEL were falling sick with
urinary calculus and kidney damage [2] and the mela-
mine adulteration of a variety of food products has in-
stantly become a global problem. MEL, a nitrogen-rich
organic chemical, is added to milk to boost the protein
levels, making them appear higher than they actually are.
Chronic exposure may lead to reproductive damage, or
bladder or kidney stones, which can lead to bladder can-
cer [3-7].
On October, 2008, FDA issued its Interim Safety and
Risk Assessment of MEL in Food for Humans in consid-
eration of the potential public health concerns from
In applying an additional 10-fold safety factor and us-
ing the TDI (Tolerable Daily Intake) of 0.63 mg/kg
b.w./day, the safety/risk assessment concluded that a
maximum tolerance levels of MEL below 2.5 ppm in
foods other than infant formula do not raise public health
concerns [8].
In response the global MEL problem, the United Na-
tions food standards body, the Codex Alimentarius
Commission, announced formal international limits for
melamine allowed in food and animal feed during its
meeting on July, 2010. The maximum amount of MEL
allowed in foods and animal feed is 2.5 mg/kg, equal to
the FDA limit. The international maximum levels help
governments differentiate between low levels of un-
avoidable melamine occurrence that do not cause health
problems, and deliberate adulteration—thereby protect-
ing public health without unnecessary impediments to
international trades [9].
Milk contains a good balance of protein, fat and car-
bohydrate, is an indispensable food because it is inex-
pensive and readily available. It becomes a raw material
of every processed food.
Figure 1. Molecular structures of cyromazine and its decy-
cropropylated metabolite (melamine).
Copyright © 2012 SciRes. JASMI
Organic Solvent-Free and Simple Method for Determining Cyromazine and Its Metabolite,
Melamine, in Cow’s Milk
To ensure that milk for human consumption is resi-
due-free of CYR, the Codex has set a maximum residue
limit (MRL) for CYR in milk at 10 ng/mL [10]. Strict
and rapid monitoring the presences of CYR and MEL in
milk is, therefore, an important means of guaranteeing
the international food safety.
In response to the recent expansion in the internal food
trade, the development of international harmonized
methods to determine chemical residues in foods is es-
sential to guarantee equitable international trade in these
foods and protect health for consumers. Whether in in-
dustrial nations or developing countries, an internal har-
monized method for residue monitoring in foods is ur-
gently-needed. The optimal harmonized method for the
routine monitoring chemicals such as melamine in foods
must be simple, small scale, economical in time and cost,
and must cause no harm to the environment and humans.
The Food Safety and Inspection Service (FSIS) of the
United States Department of Agriculture (USDA) have
been provided a test method based on high-performance
liquid chromatography (HPLC) for analyzing CYR and
MEL in animal tissues in its Chemistry Laboratory
Guidebook which contains test methods used by FSIS
Laboratories to support the Agency’s inspection program,
ensuring that meat, poultry, and egg products are safe,
wholesome and accurately labeled [11]. Previous tech-
niques based on high-performance liquid chromatogra-
phy (HPLC) for simultaneous determination of CYR and
MEL in animal-derived foods [12-16] has crucial draw-
backs: 1) all of methods consume organic solvents in the
instrumental analyses as well as for extraction and
de-proteinization in sample preparation. The risk associ-
ated with these solvents extends beyond direct implica-
tions to human health by affecting the ecosystem in
which we all reside. Furthermore, the disposal of waste
organic solvents through incineration has steadily in-
creasing over the past decade and costs large amounts of
money. Thus, eliminating the use of organic solvents and
reagents is an important goal in terms of protecting the
environment, human health, and the economy [17,18]. 2)
most of the recent methods are based on LC-MS/MS.
The facility is available are limited to part of industrial
nations because these are hugely expensive, and the
methodologies use complex and specific. These are un-
available in a lot of laboratories for routine analysis, par-
ticularly in developing countries. No optimal method that
satisfies the aforementioned requirements has yet been
This article describes an ultrasafe, idiotproof, and in-
expensive method to strictly monitor CYR and MEL
residues in milk using a 100% aqueous solution in sam-
ple preparation and HPLC separation without organic
solvent consumption.
2. Experimental
2.1. Reagents
Standards of cyromazine (CYR) and melamine (MEL)
and other chemicals were purchased from Wako Pure
Chem. Ltd. (Osaka, Japan). Distilled water was of HPLC
grade. 0.5 moL/L 1-octanesulfonic acid sodium salt solu-
tion (low UV type) was of ion-pair chromatography
grade. These standards and chemicals were greater than
99% purity.
2.2. Apparatus
The following apparatuses were used in the sample
preparation: handheld ultrasonic-homogenizer (model
HOM-100, 2 mm ID probe, Iwaki Glass Co., Ltd., Funa-
bashi, Japan); micro-centrifuge (Biofuge® fresco, Kendo
Lab. Products, Hanau, Germany); two types of Mono-
Spin® as centrifugal monolithic silica spin mini-columns
(sample throughput volume 300 μL), MonoSpin-C18
(octadecyl and non-polar functional group) and Mono-
Spin-SCX (bonded propyl benzene sulfone acid combin-
ing both strong cation and non-polarity) (GL Sciences,
Inc., Tokyo, Japan). The following types of non-polar
sorbent columns (5 μm d
p, 150 × 4.6 mm) for HPLC
were used: Wakosil 5TMS (C1) (Wako); Mightysil®
RP-4GP (C4) (Kanto Chemical Co., Inc., Tokyo, Japan);
Mightysil® RP-18 GP Aqua (C18) (Kanto); Inertsil®
ODS-4 (C18) (GL Science); Inertsil® HILIC (alkyl diol)
(GL Science) (Table 1).
The HPLC system, used for method development, in-
cluded a model PU-980 pump and DG-980-50-degasser
(Jasco Corp., Tokyo, Japan) equipped with a model CO-
810 column oven (Thosoh Corp., Tokyo, Japan), as well
as a model SPD-M10A VP photodiode-array (PDA) de-
tector (Shimadzu Scientific Instruments, Kyoto, Japan).
Table 1 lists the particle physical specifications.
2.3. HPLC Operating Conditions
The analytical column was an Inertsil® HILIC (150 × 4.6
mm, 5 μm dp) column using a 0.05 moL/L 1-octane-
sulfonic acid mobile phase at a flow rate of 1.0 mL/min
at 40˚C. PDA detector was operated at 190 - 300 nm: the
monitoring wavelength was adjusted to 206 nm which
represents an average maximum for the analytes. The
injection volume was 20 μL.
2.4. Preparation of Stock Standards and
Working Mixed Solutions
Stock standard solutions of CYR and MEL were pre-
pared by dissolving each of CYR and MEL in water to a
concentration of 10,000 ng/mL. Working mixed standard
solutions of these two compounds were prepared by
Copyright © 2012 SciRes. JASMI
Organic Solvent-Free and Simple Method for Determining Cyromazine and Its Metabolite,
Melamine, in Cow’s Milk
Copyright © 2012 SciRes. JASMI
suitably diluting the stock solutions with water. These
solutions were kept in a refrigerator (5˚C).
2.5. Preparation of Calibration Standards and
Quality Control Samples
For method validation studies, calibration standards and
quality control samples (QCs), terms defined in the FDA
guideline [22], were prepared by spiking appropriate
aliquots of the mixed standard solution in blank milk
samples. Calibration standards were used to construct
calibration curves from which the concentrations of ana-
lytes in unknown monitoring samples are determined
practically. QCs used to evaluate the performance of the
proposed method. In this study, the standards were pre-
pared in the range of 20 - 1000 ng/mL for CYR and 20 -
3000 ng/mL, respectively. Three QC levels (QC1 = 30
ng/mL for CYR and 50 ng for MEL; QC2 = 100 ng/mL
for both analytes; QC3 = 500 ng/mL for CYR and 250
ng/mL for MEL) were prepared.
2.6. Sample Preparation
An accurate 0.1 mL sample was taken into a micro-cen-
trifuge tube and homogenized with 0.6 mL of water with
a handheld ultrasonic-homogenizer for 30 s. After being
homogenized, the capped tube was centrifuged at 10,000
g for 5 min. A 0.1mL of supernatant liquid was poured to
a MonoSpin-C18 and centrifuged at 3500 g for 1 min.
The eluate was injected into the HPLC system.
2.7. Method Validation
The performance of the developed method was validated
in terms of some parameters from the international guide-
lines for bio-analytical procedure [19-23]. The quality
parameters established were linearity, accuracy, precision,
sensitivity, specificity, selectivity, robustness, system
3. Results and Discussion
3.1. Sample Preparation
The ultrasonic-homogenization enabled the satisfactory
extraction of CYR and MEL from a milk sample with a
100% water. The author used two types of centrifugal
monolithic silica spin mini-columns, MonoSpins
(MonoSpin-C18 and-SCX), for the further cleanup tech-
nique. The spin mini-column is a monolithic SPE column
excellent for the small volume sample with easy and
quick operation by centrifuge [24]. A 100% water was
used as the eluent and the recoveries of CYR and MEL
from these mini-columns were compared. Since the re-
coveries of the target compounds from the column vary
with centrifugal acceleration (rotary speed), this study
was also investigated an optimal acceleration ( 1000 g)
to recovery CYR and MEL from the spin mini-columns.
A 100 μL portion of a mixed standard solution contain-
ing 0.5 μg of each compound was applied to the spin
mini-column. The centrifugal time was standardized at 1
min. MonoSpin-C18 gave satisfactory recoveries ( 96%)
and repeatabilities (RSD 2%, n = 3) for CYR and MEL
when the centrifugal acceleration was 3500 g for 1 min.
The MonoSpin-C18 was therefore used a centrifugal
monolithic silica spin mini-column. The present proce-
dure can realize small-scale extractions and easy purifi-
cations of CYR and MEL in a short time while com-
pletely eliminating the consumption of organic solvents.
The procedure resulted in high recoveries and repeat-
3.2. Optimum HPLC Conditions
In order to achieve the separation with a 100% aqueous
mobile phase, this study tested five types of non-polar
sorbent columns. Table 1 lists the physical and chemical
specifications. The author used 0.05 moL/L 1-octane-
sulfonic acid as the isocratic aqueous mobile phase and
examined column temperatures 25˚C, flow rates 0.5
Table 1. Physical/chemical specifications of the reversed-phase columnsa used and chromatographic CYR and MEL separa-
tions obtained under the HPLC conditions examinedb.
Symbol Silica (type) Trade name
Pore diameter
Pore volume
Surface area
Carbon load
A C1 Wakosil 5TMS 12 1.0 300 4 Not separated (NS)
B C4 Mightysil RP-4 GP 12.5 1.1 350 4 NS
C C18 Mightysil RP-18 GP Aqua 13.5 0.9 270 15 NS
D C18 Inertsil ODS-4 10 1.05 450 11 NS
E Alkyl diol Inertsil HILIC 10 1.05 450 20 Completely separated
a150 × 4.6 mm; dp = 5 μm: data supplied from manufactures; bMobile phase of water; column temperatures 25˚C; flow-rates 0.5 mL/min; HPLC retention
times 15 min; cNot separated: between MEL and the interfering milk extract.
Organic Solvent-Free and Simple Method for Determining Cyromazine and Its Metabolite,
Melamine, in Cow’s Milk
mL/min, and HPLC retention times 15 min. Because
the HPLC separations were performed serially, the time
per run was critical for routine residue monitoring. The
short run time not only increased sample throughout for
analysis but also affected the method-development time.
The seven columns were compared with regard to the
separation among CYR, MEL, and their interfering peaks
obtained upon injection of equal amounts. The chroma-
tographic separations within the conditions ranges ex-
amined are also presented in Table 1.
Columns A-D had difficulty separating MEL and the
interfering milk extract throughout the examined condi-
tion ranges. An ideal chromatogram with complete sepa-
rations of CYR, MEL, and interfering peaks, and their
short retention times was obtained by a Column E and a
0.05 mol/L 1-octanesulfonic acid mobile phase with a
column temperature of 40˚C and a flow rate of 1.0
mL/min. The HPLC system achieved optimal separation
< 6.5 min without the need for a gradient system to im-
prove the separation. The ion-pair action of 1-octane-
sulfonic acid mobile phase used here was necessary to
obtain the findings described.
Figure 2 displays that the resulting chromatograms
were free of interfering compounds for quantitation and
identification of CYR and MEL by HPLC, with PDA
detector set at 206 nm (giving an average maximum for
CYR and MEL). This figure demonstrates that the pre-
sent method can provide the quantitation and identifica-
tion of CYR and MEL.
3.3. Method Validation
3.3.1. Main Quality Parameters
Table 2 summarizes the values obtained for the main
parameters. The accuracy and precision are well within
the international method acceptance criteria [19,21,23].
The QL for CYR (9 ng/mL) was lower than the MRL of
10 ng/mL [10]. The other validation findings are as fol-
3.3.2. Specificity and Selectivity
The application of the proposed procedure to 10 blank
milk samples from different species (Holstein-Friesian
and Jersey) demonstrated that no interference peak was
presented around the retention times for CYR and MEL
in any of the sample examined.
The present HPLC-PDA system easily confirmed the
peak identity of target compound. CYR and MEL were
identified in a milk sample by their retention times and
absorption spectra. The CYR and MEL spectra obtained
from the milk sample were practically identical to those
of the standards. Because of the complete separations and
the high absorbance of CYR and MEL, PDA detection at
trace levels is fully available. It is, therefore, instructive
to demonstrate purification effectiveness of the sample
preparation. The system did not require the use of MS,
which is very expensive and is not available in a lot of
laboratories for routine analysis, particularly in develop-
ing countries.
3.3.3. R obu stness
Some HPLC parameters were performed using a spiked
(100 ng/mL of each compound) milk sample obtained
under the established procedure.
Changes of ±5% units of the flow rate (1.0 mL/min)
and the column temperature (40˚C) were determined.
The effect on the peak areas and the validations in the
retention times were evaluated. Changes of ±5% of the
flow rate and the column temperature had no effect on
the peak areas, whereas the variations in the retention
Figure 2. Chromatograms obtained from the HPLC system
for a blank milk sample (upper profile) and a milk sample
spiked with CYR (500 ng/mL) and MEL (250 ng/mL)
(lower profil e). The PDA detector was set at 206 nm. Peaks,
1 = MEL (retention time, Rt = 3.08 min); 2 = CYR (Rt = 6.23
Table 2. The main method validation data.
Paramer CYR MEL
Linearity (ra) 0.991 0.994
Accuracyb 93.2 - 97.8 94.5 - 99.1
Precisionc 2.8 2.4
Sensitivityd 8.5 10
ar is the correlation coefficient (P < 0.01): mean of three determinations
using spiked milk samples for calibration curves: ranges of concentrations
were 20 - 1000 ng/mL for CYR and 20 - 3000 ng/mL for MEL, respectively;
bAverage recoveries (%) from six replicates at three QC levels (QC1 = 30
ng/mL for CYR and 50 ng for MEL; QC2 = 100 ng/mL for both analytes;
QC3 = 500 ng/mL for CYR and 250 ng/mL for MEL); cValues are RSDs (%,
n = 6 of each level); dQuantitation limit (ng/mL), QL as the concentration of
analyte giving a signal-to-noise ratio > 10.
Copyright © 2012 SciRes. JASMI
Organic Solvent-Free and Simple Method for Determining Cyromazine and Its Metabolite,
Melamine, in Cow’s Milk
times were obtained with the flow rate and the column
temperature. Normal retention times for CYR and MEL
were 6.23 and 3.08 min, respectively. At +5% the flow
rate, the two retention times were decreased, ranging
between 1.6 and 5.7% and at –5%, the times were in-
creased ranging between 5.5 and 8.0%. By changing the
column temperature by +5%, decreasing retention times
obtained were 1.9% - 7.5%, however, no significant
variations were observed with –5%. During these studies,
all the target compounds were separated.
3.3.4. System Suitability
The system-suitability evaluation is an essential parameter
of HPLC determination, and it ascertains the strictness of
the system used. The suitability was evaluated as the
relative standard deviations of peak areas and retention
times calculated for 20 replicate injections of a spiked
milk sample (100 ng/mL of each compound). The values
for CYR and MEL were estimated to be <0.5% for peak
areas and <0.8% for retention times, respectively.
3.4. Cost and Time Performance
The total time and budget required for the analysis of a
single sample was < 20 min and approximately US $6.2
as of April 12, 2012, respectively. For sequential analy-
ses, a batch of 24 samples could be analyzed in 4 h.
These findings became term required for an international
harmonized residue analysis. The organic solvent-free
and short analytical time not only increased the sample
throughput for analysis but also positively affected the
cost and environmental/human impacts.
3.5. Application to Residue Monitoring in
Commercial Samples
Milk was purchased from a number of convenience stores
in Osaka, Japan, used as real milk samples and analyzed
using the proposed method. No samples contained de-
tectable concentrations of CYR and MEL. The resulting
chromatograms were free from interference.
4. Conclusion
An organic solvent-free method does not use organic
solvents for simultaneous determination of CYR and
MEL in milk has been successfully developed and vali-
dated. The present procedure provided an easy-to-use,
rapid, space-saving, and non-use of organic solvents and
resulted in high recovery and repeatability with consid-
erable saving of analysis time/cost. The procedure may
be proposed as an international harmonized analytical
method for the routine monitoring of CYR and MEL in
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Melamine, in Cow’s Milk
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