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Immunoafnity-Based Solid Phase Extraction

for the Determination of Melamine in Animal-Derived Foods
Followed by LC
Yong Ben Zhong

Li Jing Zhang

Hui Cai Zhang

Ju Xiang Liu

Jian Ping Wang
Received: 9 November 2010 / Revised: 24 February 2011 / Accepted: 18 March 2011
Springer-Verlag 2011
Abstract An immunoafnity chromatography (IAC) col-
umn was prepared and optimized to purify melamine in ani-
mal-derived foods followed by determination with a liquid
chromatography method. The column showed high binding
capacity of 1,250 ng for melamine with recovery higher than
96%, and the column could be renewed and repeatedly used
for at least 30 cycles. The extracts of chicken, egg and milk
samples were directly loaded onto IAC column for purica-
tion, and the analyte retained on the column was eluted with
4 mL of modied Tris buffer without carryover. The puri-
cation effect was satisfactory and better than that of the con-
ventional solid phase extraction. Recoveries from the
melamine-fortied blank samples were between 82.5 and
101.4% with coefcient of variation (CV) lower than 10.6%.
Keywords Column liquid chromatography
Immunoafnity chromatography Animal-derived food
Melamine (1,3,5-triazine-2,4,6-triamine, MA) is a kind of
industrial chemical that is used to manufacture durable
plastic tableware and ame retardants. Studies have shown
that high (2.5 mg kg
) and continuous dietary exposure to
MA can cause the formation of bladder stone and increase
the incidence of urinary bladder tumor in male rats [1].
Therefore, MA has not been approved for use as an
ingredient of animal feeds or food, though it is a nitrogen-
rich compound. However, it can be added illegally into
feeds or food to fraudulently increase the apparent protein
content. In 2007, thousands of illnesses and deaths of pets
in America were proven to be caused by MA-contaminated
pet food. In 2008, continuous consumption of MA-con-
taminated milk and infant milk powder caused renal stone
in thousands of children in China.
In addition to the illicit adulteration, MA can be
metabolized from a triazine drug, cyromazine (N-cyclo-
propyl-1,3,5-triazine-2,4,6-triamine). Cyromazine is an
insect growth regulator that has been licensed as a feed
additive to prevent ies from hatching in manure [2, 3].
The Joint FAO/WHO Meetings on Pesticide Residues
(JMPR) have shown that cyromazine is incompletely
metabolized in vivo and about 7% of the metabolites is
melamine. Furthermore, cyromazine can also be decom-
pounded to release melamine through photolysis [4].
Due to the potential harmful effects of MA to consum-
ers, the regulatory agencies of the USA and China have set
a maximum permit limit of 2.5 mg kg
for MA in raw
milk. However, no maximum residue level has been set in
other animal-derived products. Therefore, it is important to
monitor the residue of MA in animal-derived foods. As of
now, many papers have reported on the determination of
MA in various foods [514]. In those reports, MA was
extracted and puried with conventional methods, such as
liquidliquid extraction, solid phase extraction or molecu-
larly imprinted method. However, these extraction methods
required a large amount of organic solvents and disposable
Yong Ben Zhong and Li Jing Zhang contributed equally to this paper.
Y. B. Zhong H. C. Zhang J. X. Liu J. P. Wang (&)
College of Animal Science and Technology,
Agricultural University of Hebei,
Baoding 071000, Hebei, China
e-mail: wjpcau@yahoo.com.cn
L. J. Zhang
College of Life Sciences, Agricultural University of Hebei,
Baoding 071000, Hebei, China
1 3
DOI 10.1007/s10337-011-2012-8
cartridges, and usually involved a few steps that were time-
consuming, wasteful and inconvenient.
Immunoafnity chromatography (IAC) can be a good
alternative technique, which provides a simple and selec-
tive extraction procedure and reduces the use of organic
solvent. This technique has been used for purication of
b-agonists [15, 16], zeranol [17], quinolones and sulfona-
mides [18], and avermectins [19] in animal tissues.
Therefore, the objective of the present study was to prepare
an IAC column to purify MA in chicken, egg and milk
samples followed by determination with a high perfor-
mance liquid chromatography (HPLC) method.
The standard of MA was obtained from the China Institute
of Veterinary Drug Control (Beijing, China). CNBr acti-
vated Sepharose 4B was purchased from Sigma (St. Louis,
MO, USA). Other chemical reagents were all of analytical
grade from Beijing chemical company (Beijing, China).
Standard stock solution (1 mg mL
) and working solu-
tions of MA (25, 50, 100, 200, 400 ng mL
) were pre-
pared with methanol/water (8/2, v/v) and stored at 4 C.
PBS (pH 7.2) was prepared by dissolving 0.2 g KH
0.2 g KCl, 1.15 g Na
HPO4 and 8.0 g NaCl in 1,000 mL
of water. Modied Tris buffer (0.1 mol L
, pH 8.0) was
prepared by dissolving 6.05 g Tris, 16.7 g NaCl and
1.2 mL HCl in 500 mL of water. The IAC eluent was
prepared by mixing 10 mL of modied Tris buffer and
90 mL of methanol. HPLC mobile phase was a mixture of
solution A and solution B. Solution A was HPLC grade
acetonitrile. Solution B was prepared by dissolving 2.02 g
sodium 1-heptanesulfonate and 2.1 g citric acid in
1,000 mL deionized water. Then, 100 mL of solution A
and 900 mL of solution B were mixed, ltered through a
membrane of 0.2 lm and sparged before use. SCX (strong
cation exchange) extraction cartridges were from Waters.
IAC Column Preparation and Optimization
The polyclonal antibody against MA was prepared in our
laboratory as described elsewhere [20]. The IAC column
was prepared as in a previous report [15]. Briey, about 1 g
CNBr activated Sepharose 4B powder was swallowed with
200 mL 1 mM HCl (swallowed in 4 mL gel) and equili-
brated with NaHCO
solution (0.1 M, pH 8.4). The gel was
then suspended in the antibody solution (20 mg antibody in
10 mL of NaHCO
, 0.1 M, pH 8.4) and stirred gently at
4 C for 24 h in a magnetic blender (78-1 Type, Leqing-
lecheng Zhejiang, China). After washing with 100 mL
PBS, the mixture was resuspended in 20 mL TrisHCl
buffer (0.1 M, pH 8.0) and stirred gently at 4 C for 2 h to
cap the uncoupled groups. The gel was washed with
100 mL of acetate buffer (0.1 M, pH 4.0) and 100 mL of
TrisHCl buffer (0.1 M, pH 8.0) in turn for three cycles,
and then equilibrated with 20 mL PBS. Finally, the gel of
1 mL bed volume was transferred to a glass column
(10 9 0.8 mm, i.d.) and stored in PBS at 4 C.
The column capacity of each newly prepared column
was evaluated by successive loading of a known concen-
tration of standard solution (diluted with PBS) onto the
column until the concentration of eluate reached the
loading concentration. The column was washed with
20 mL PBS and 20 mL deionized water in turn. Then the
column was eluted with 4 mL IAC eluent. The eluate was
evaporated to dryness and the dry residue was redissolved
in 1 mL mobile phase. After passing through a 0.22-lm
membrane lter, the ltrate was analyzed with the followed
HPLC method. The column was regenerated by equili-
brating with 20 mL water and 20 mL PBS, and stored in
PBS at 4 C when not in use. The recovery (loading 50% of
the column capacity) and recycle performance were eval-
uated on three different columns.
Sample Purication by IAC
An amount of 2.0 g homogenized sample (chicken, egg
and raw milk) and 20 mL of trichloroacetic acid (10 g L
were added into a 50 mL polypropylene centrifuge tube.
The mixture was extracted for 10 min under ultrasonic
assistance. The tube was then centrifuged for 5 min at
4,000 rpm to obtain the sample extract. About 10 mL of
the extract was diluted vefold with PBS and loaded onto
IAC column. The IAC column was preconditioned with
20 mL PBS immediately before use. The subsequent pro-
cedures of washing, eluting and concentrating and column
regeneration were the same as described above. Some
blank samples obtained from the controlled slaughter-
houses and farms known to be free from melamine were
used to evaluate the method recovery. Some real samples
(20 chicken samples, 20 eggs and 40 raw milk samples)
purchased from several markets of China were puried by
IAC column and determined by HPLC.
Sample Purication by SPE
The sample was extracted as described above. Then,
10 mL of sample extract was loaded onto SCX cartridge
for purication (The cartridge was preconditioned with
3 mL water and 3 mL methanol). Then, the cartridge was
washed with 3 mL methanol, 1 mL 0.3% acetic acid and
3 mL water in turn. The analyte was eluted with 3 mL 5%
ammonia methanol and the eluant was evaporated to
Y. B. Zhong et al.
1 3
dryness. Finally, the dry residue was dissolved in 1.0 mL
of mobile phase and the solution was ltered with a
0.22 lm membrane lter for HPLC analysis.
HPLC Analysis
The HPLC systems consisted of Shimadzu LC-6AD liquid
chromatography, SPD-20A UV detector and a C18 column
(200 9 4.6 mm, i.d.) with a data acquisition software of
LC solution (Shimadzu, Japan). The operation conditions
were as follows: injection volume, 20 lL; detection
wavelength, 240 nm; ow rate, 0.8 mL min
; total run-
ning time, 15 min. Qualitative analysis was performed by
comparing the retention time of chromatogram peak of the
sample with that of MA standard. Quantication was cal-
culated according to the chromatogram peak area of the
sample and that of MA standard. The limit of detection
(LOD) was calculated as the minimum concentration of
MA that could be detected without the interference of
baseline noise (S/N of 3:1). The limit of quantication
(LOQ) was assessed as the minimum quantity of MA that
could be quantied (S/N of 10:1).
Results and Discussions
Optimization of the IAC Column
The IAC procedure is a simple technique based on the
specic antigenantibody interaction to extract the analyte
of interest. In our previous report, the obtained antibody
was specic for MA [20]. Therefore, the antibody was used
to prepare the IAC column. This is the rst report
describing the preparation of an IAC column for purica-
tion of MA in animal-derived products. For optimization of
an IAC column, the evaluation of washing and elution
conditions is necessary because these conditions have
strong inuences on the purication effect and desorption
of the antigenantibody binding and, therefore, analyte
The rst important step in the IAC procedure is to nd
an appropriate eluent to elute the analyte retained on the
column, and the ideal eluent should enable analyte recov-
ery in the lowest eluent volume with no damage to the
antibody. In previous reports, various organic solvents or a
mixture of organic solvent and buffer was used to elute the
analyte on the IAC column. During our experiments, 100%
methanol, 5% ammonia methanol and the modied Tris
buffer were all used to elute the analyte. When using
methanol as eluent, only about 60% of the analyte was
eluted. When using 5% ammoniac methanol, the eluent
volume was found to be at least 14 mL and the recoveries
were about 80%. Furthermore, the column capacity was
reduced to about 100 ng after ve cycles, indicating that
maybe 5% ammonia damaged the antibody. However,
4 mL modied Tris buffer could elute all MA retained in
the IAC column with no carryover (Fig. 1). Three newly
prepared IAC columns were then tested for their capacity
to retain MA. The column capacity was 1,250 20 ng and
the recoveries were all higher than 96%. The three columns
were used successively for 30 cycles with column capacity
remaining above 790 ng, indicating that the eluent caused
almost no damage to the antibody.
MA in the samples can be selectively captured by the
specic antibody immobilized on the column, but the
impurities in the sample matrix may also be retained
because of the non-specic binding of the gel. These
impurities can be removed by an appropriate washing pro-
cedure. In this study, the blank extracts containing 100 ng
MA were loaded onto IAC columns and the columns were
washed with 20 mL of PBS and 20 mL water in turn. The
recoveries were in the range of 9398%, indicating that this
washing procedure does not inuence the IAC recovery.
More importantly, there was no impurity peak around the
retention time of MA in the chromatogram of the blank
sample, indicating that the washing procedure could almost
remove the matrix impurities completely (Fig. 2).
IAC Purication
The advantages of IAC are its simple sample preparation
and its good purication efcacy. In this study, the extracts
of chicken, egg and raw milk samples were directly loaded
onto the IAC column for purication. As shown in Figs. 2
and 3, there was no interfering peak around the MA peak in
the chromatograms of standard fortied samples, indicating
that the IAC column could purify MA in these samples
with good purication effect. For comparison of the puri-
cation effects of SPE and IAC, the MA fortied blank
samples were puried with a representative solid phase
extraction cartridge (SCX). As shown in Fig. 3, there were
some interfering peaks around the peak of MA in the
chromatogram of MA fortied milk, revealing that the
1 2 3 4 5
Eluent fraction (mL)


Fig. 1 Recoveries of MA in the 4 mL elution buffer
Immunoafnity-Based Solid Phase Extraction
1 3
purication efcacy of the IAC method was better than that
of conventional SPE procedure.
HPLC Determination
In this study, MA was determined with an HPLCUV
method. The mean retention time was 7.732 0.2 min.
Under the chromatographic conditions, good linearity and
coefcient were achieved. Regression analysis of the data
was obtained by running a series of working solutions. The
calibration curve was constructed by plotting the peak
areas (Y) of MA standard against concentrations (X). It was
found to be linear in the range of 25400 ng mL
with a
regression equation of Y = 313.14X ? 1,608.8 and a cor-
relation coefcient of 0.9994 (r
). The relative standard
deviation (RSD) based on the peak areas of replicate
injections was between 0.796 and 1.97%. The LOD and
LOQ for MA standard were 15 and 30 ng g
, respec-
tively. The extracts of blank samples were used to prepare
the matrix-matched MA solutions for determination of the
LOD and LOQ of MA in these samples. Results showed
that the sample matrixes had almost no inuence on the
determination of MA due to the good purication effect of
the IAC column. Therefore, the LOD and LOQ for MA in
these samples were according to those of MA standard.
Then, blank chicken, egg and milk samples fortied MA at
concentrations of 25, 50 and 100 ng g
, which was puried
with IAC and determined by HPLC. Intra- and inter-assay
recoveries were in the range of 82.5101.4% with coef-
cients of variation (CV) lower than 10.6% (Table 1). Fur-
thermore, the MA fortied blank samples puried by SCX
cartridges were also determined by the HPLC. Results
showed the recoveries were lower than that of the IAC
method (Table 1). Therefore, the IACHPLC method could
be used as a sensitive and accurate method to monitor the
residues of MA in animal-derived products.
Analysis of Real Samples
The 80 unknown samples were analyzed by the proposed
IACHPLC method. No egg and milk sample was deter-
mined as positive samples, but one chicken sample was
determined as containing MA residue at a level of
34 ng g
. This value is much lower than the maximum
permitted limit of 2.5 mg kg
for MA in raw milk and,
maybe, the residue is metabolized from cyromazine,
released from the coating of feeds, or from the illicit
adulteration of MA in feeds.
This paper rst reported the preparation of a novel
immunoafnity chromatography column for purication of
melamine in animal-derived foods. The IAC procedure
reduced the use of organic solvent and the column could be
reused for at least 30 recycles. The proposed IACHPLC
method achieved a good cleanup effect, high sensitivity
and satisfactory recovery. Therefore, the method could be
used as a practical tool for routine monitoring of the resi-
due of melamine in animal-origin foods.
Fig. 2 Chromatograms of a blank chicken, b fortied blank chicken
(100 ng g
), and c the real positive chicken (34 ng g
Fig. 3 Chromatograms of blank fortied milk (100 ng g
) puried
with a IAC and b SPE
Y. B. Zhong et al.
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Table 1 Recoveries of MA
from standard fortied blank
Sample Added
(ng g
Intra-assay of IAC Inter-assay of IAC SPE method
Chicken 25 92.7 5.1 90.2 6.8 80.6 7.4
50 95.0 6.5 91.4 7.4 86.4 6.3
100 101.4 4.0 97.1 5.3 90.7 7.2
Egg 25 86.0 6.3 82.5 9.2 79.8 8.5
50 91.1 4.9 89.4 8.2 84.9 6.9
100 97.3 8.8 99.2 9.4 89.0 7.1
Milk 25 84.7 9.2 89.8 10.6 77.4 9.2
50 93.0 6.7 98.5 7.4 89.5 9.0
100 93.6 8.2 99.1 7.8 92.4 7.7
Immunoafnity-Based Solid Phase Extraction
1 3