A Review of Methods For Detecting Melamine in Food Samples: Critical Reviews in Analytical Chemistry

Critical Reviews in Analytical Chemistry
ISSN: 1040-8347 (Print) 1547-6510 (Online) Journal homepage: http://www.tandfonline.com/loi/batc20
A review of methods for detecting melamine in

food samples
Yang Lu, Yinqiang Xia, Guozhen Liu, Mingfei Pan, Mengjuan Li, Nanju Alice
Lee & Shuo Wang
To cite this article: Yang Lu, Yinqiang Xia, Guozhen Liu, Mingfei Pan, Mengjuan Li, Nanju Alice
Lee & Shuo Wang (2016): A review of methods for detecting melamine in food samples, Critical
Reviews in Analytical Chemistry, DOI: 10.1080/10408347.2016.1176889
To link to this article: http://dx.doi.org/10.1080/10408347.2016.1176889
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A review of methods for detecting melamine in food samples
Yang Lu 1 , Yinqiang Xia 1a , Guozhen Liu 2,3 , Mingfei Pan 1 , Mengjuan Li 4 , Nanju
Alice Lee 5, * , Shuo Wang 1,*
1
Key Laboratory of Food Nutrition and Safety, Ministry of Education of China, Tianjin
University of Science and Technology, Tianjin 300457, China

Downloaded by [Library Services City University London] at 17:49 18 April 2016
2
Key Laboratory of Pesticide and Chemical Biology of Ministry of Education, College of
Chemistry, Central China Normal University, Wuhan 430079, PR China
3
ARC centre of Excellence in Nanoscale Biophotonics (CNBP), Department of Physics and
Astronomy, Macquarie University, North Ryde 2109, Australia
4
College of Textile & Clothing, Jiangnan University, Wuxi 214122, China
5
School of Chemical Engineering, Faculty of Engineering, University of New South Wales,
Sydney, NSW 2052, Australia
a
Current address: School of Chemical Engineering, Tianjin University
*
Corresponding author: Tel.: +61-2- 9385 4363, E-mail: alice.lee@unsw.edu.au Tel: +86-22-
60601430, E-mail: s.wang@tust.edu.cn
Abstract
Melamine is a synthetic chemical used in the manufacture of resins, pigments, and
superplasticisers. Human beings can be exposed to melamine through various sources such as
migration from related products into foods, pesticide contamination, and illegal addition to
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foods. Toxicity studies suggest that prolonged consumption of melamine could lead to the
formation of kidney stones or even death. Therefore, reliable and accurate detection methods are
essential to prevent human exposure to melamine. Sample preparation is of critical importance,
since it could directly affect the performance of analytical methods. Some methods for the
detection of melamine include instrumental analysis, immunoassays, and sensor methods. In this
paper, we have summarised the state-of-the-art methods used for food sample preparation as well
as the various detection techniques available for melamine. Combinations of multiple techniques
and new materials used in the detection of melamine have also been reviewed. Finally, future
perspectives on the applications of microfluidic devices have also been provided.
Keywords
melamine, instrumental analysis, immunoassay, biosensors, chemosensors
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Introduction
Melamine is a chemical that is produced in high volumes and is used in the manufacture of
plastics, textile finishes, paints, flame retardants, and permanent-press fabrics (Sugita et al.,
1990). Human beings can be exposed to melamine from a variety of sources. For example,
dinnerware typically contains some melamine-related products. Melamine may migrate in trace
amounts from these products to the food and beverages served in such dinnerware (Bradley et
al., 2005). Melamine contamination in food may also arise from the metabolites of cyromazine,
which is an insecticide widely used on vegetable crops for inhibiting the moulting process (Graf,
1993; Keiding, 1999). Additionally, owing to its high nitrogen concentration and low price,
melamine has been massively added into foods, especially milk products, since 2007. As a result,
thousands of humans and animals consume the contaminated foods, leading to various illnesses
(Ehling et al., 2007; Yang et al., 2009).
Prolonged consumption of melamine could cause the accumulation of kidney stones, which
are formed by the reaction of melamine and cyanuric acid. This may eventually lead to kidney
failure and even death (Brown et al., 2007; Puschner et al., 2007). Besides, melamine has also
been linked to decrease in fetal weight, increase in skeletal variation, inhibition of cell growth,
and change in cytomorphology in a concentration-dependent manner (Kim et al., 2011; Wang Z.
et al., 2011).
High exposure risks and health effects of melamine have caused great public concern.
Government institutions have developed regulations to limit melamine residues in food and have
set maximum residue levels (MRL) for melamine in various food products (China net, 2008;
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Food and Environmental Hygiene Department, 2008). The Codex Alimentarius Commission set
the MRL of melamine at 1×103 µg kg1 in infant powdered formula and 2.5×103 µg kg1 in other
foods (World Health Organization, 2010).
In addition to the above regulations, several monitoring and quantification methods based on
instrumental analysis, immunoassays, and sensor methods have been developed. Each of these
methods has its own advantages and limitations. For example, while instrumental analysis is
highly accurate, the need for bulky equipment disallows its use in in-field detection. In addition,
highly trained operators and costly laboratories are required, which may also limit its usage in
some developing countries. To meet these drawbacks, recently, sensor methods have been
developed. While these methods are advantageous for trace analysis and have benefits such as
cost-efficiency, low sample volume requirements, sensitivity, and portability, the stability of the
results still needs to be improved in some cases before these methods can be put to practical use
in the commercial market.
Tittlemier (2009), Sun F (2010) and Liu (2012) have summarised various methods for
melamine detection, including instrumental analysis methods, enzyme-linked immunosorbent
assays (ELISAs), and sensor methods. However, the state-of-the-art sensor methods have not
been included in their reviews. Rovina and Siddiquee (2015) have also not reviewed the sample
preparation techniques in their report. Similarly, in reviews by Domingo (2014) and Li Y (2015),
discussions have been limited to only some detection techniques such as vibrational spectroscopy
analysis, chemometrics analysis, and sensor methods.
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To draw a whole picture on the detection techniques available for melamine, in this review,
we comprehensively summarise and compare the sample preparation methods and the main
detection techniques for monitoring melamine in foodstuffs.
Sample preparation
Separation and purification are the two main procedures used in the sample preparation for
analytical measurements of melamine. The choice of sample preparation method could directly
affect the precision and accuracy of the analytical methods. In addition, interference from other
elements in the matrix such as fats, pigments, sugar, and salt can affect the detection
performance and cause false positives (Lee and Kennedy, 2007). Thus, appropriate sample
preparation is often required to extract the analyte out of the samples and remove the interfering
components. For example, food samples such as milk, soya powder, biscuits, and pet foods
typically contain fat globules, casein micelles, solubilised lactose, whey proteins, and some
minerals (Karbiwnyk et al., 2008; Tittlemier et al., 2009). During the preparation of these
samples, protein and fat have to be removed prior to extraction by centrifugation or prior to the
addition of trichloroacetic acid, methylene chloride, dichloromethane, or hexane (Andersen a et
al., 2008; Karbiwnyk et al., 2008; Tittlemier et al., 2009). Following this, solvent extraction (SE)
is usually utilised and sometimes followed by solid phase extraction (SPE) for further
purification.
SE is an efficient and highly selective separation and purification method based on the relative
solubilities of substances in two different immiscible liquids. Melamine is a small polar
compound that can hydrolyse under both acidic and alkaline conditions (Sun H et al., 2010).
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Therefore, polar solutions such as methanol/water, acetonitrile/water, diethylamine/water,
cyanuric acid, acetic acid, hydrochloric acid, and carboxylic acid (Sun H et al., 2010; Yu et al.,
2010; Araújo et al., 2012) are generally used for extraction. Similarly, SPE is another frequently
used method for on-line and off-line separation and pre-concentration in the trace analysis of
food products (Mashhadizadeh et al., 2014; Rezaee et al., 2014). This technique exhibits rapid
response, has high efficiency, high reproducibility, and low or zero organic-solvent consumption
(Oleschuk et al., 2000). As melamine is positively charged under acidic conditions, cation
exchange SPEs (strong cation exchange sorbents, polystyrene cation exchange, mixed-mode
anion-exchange, etc.) are usually used for extracting melamine, especially in milk-related
samples. More recently, dispersive micro solid phase extraction (DMSPE) has also been reported
as a method for extracting melamine from samples before instrumental detection (Chen et al.,
2015). This method is superior to SPE since it has low cost (USD 0.3), is rapid (3 min), and
exhibits high sensitivity and repeatability (Chen et al., 2015).
In addition to the methods described above, a growing number of new sorbent materials with
high selectivity and high adsorption capacity such as molecular imprinting polymers (MIPs) and
antibodies have been applied for the fabrication of chromatographic columns. For example, the
immuno-affinity chromatography column (IAC) is a unique purification column that is based on
the interactions of antibodies/antigens. This technique may offer high selectivity, resolution, and
capacity for extracting substances at low concentrations in large volumes of crude samples. In a
study by Zhong and co-workers (2011), IAC was prepared using a mixture of polyclonal anti-
melamine antibodies and cyanogen bromide activated sepharose 4B gel. The prepared IAC and
SPE (SCX cartridge) were optimised to extract melamine from food samples. This was followed
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by liquid chromatography (LC) detection. In this study, the IAC method exhibited better
purification efficacy over the SPE procedure. It could be reused for up to 30 cycles and showed
high binding capacity for melamine with recovery exceeding 96%. Besides the antibodies, MIPs
constitute another promising material for absorbing melamine (Yan et al., 2012; He et al., 2014).
They can selectively bind targets that exhibit affinity towards them and are therefore, referred to
as synthetic receptors (Bui and Haupt, 2010). MIPs containing cyromazine as a template have
been used for melamine enrichment from milk and feed food, prior to subjecting the samples to
gas chromatography-mass spectrometry (GC-MS) and LC-ultraviolet (UV) detection (Yan et al.,
2012). Compared to IAC, MIP-based columns are more stable and easier to prepare (Hu et al.,
2013). Recently, Zhu and co-workers (2015) prepared MIP stir bars (MIP-SB) for sample
extraction. These stir bars were able to absorb melamine from the solution in 50 min. After
extraction, melamine was removed from the stir bar by sonication in methanol/acetic acid (9:1,
v/v), dried, and re-dissolved in methanol, before being subjected to high performance liquid
chromatography (HPLC)-UV analysis.
Hollow-fibre microdialysis (MD) sampling is another rapid and miniaturised sample
preparation technique, particularly suitable for complex matrices. It has been developed as a pre-
concentration method for use before various types of instrumental analyses measurements (Li et
al., 2009). In the first step of this method, zirconia hollow fibres are immersed in the sample.
These fibres absorb melamine by forming hydrogen bonding between Zr-OH and H2N- (Li et al.,
2009). After extraction, the fibres are removed from the sample, dried, and immersed in N,O-
bis(trimethylsilyl)trifluroacetamide to derive the target analyte before GC-MS analysis. The
derived fibre can be reused with the same extraction efficiency (Li et al., 2009). On the other
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hand, electromembrane extraction (EME) is a relatively novel clean-up and pre-concentration
technique, in which the extraction is achieved in lesser time than with MD. Driven by an
electrical potential difference, the charged analytes can separate from the sample solution into an
acceptor solution through a supported liquid membrane. Using this method, melamine can be
extracted from dairy products within 15 min before the HPLC analysis (Fashi et al., 2015).
Xu and co-workers (2010) proposed a sample preparation method involving strong magnetic
cation exchange resins, which were prepared using styrene, divinylbenzene, acetyl sulfonate, and
hydrophobic Fe3O4 magnetite. During the extraction procedure, melamine was adsorbed onto the
resins from the sample matrix through ion-exchange interactions. After the resins were separated
from the matrix under an appropriate magnetic field, melamine was collected and subjected to
detection by LC-MS/MS.
Methods for melamine detection
To identify and quantify melamine in various samples, a wide range of methods including
instrumental analysis, immunoassays, and sensors, have been reported. The detection limit and
detection time for the various methods are summarised in Table 1. Instrumental analysis is
widely used in various analytical chemistry laboratories with high accuracy and sensitivity. On
the other hand, immunoassays such as ELISA are more suitable for sample screening. A decade
ago, sensors received widespread attention, stemming from a desire to miniaturise detection
tools. Sensor-based methods provide higher sensitivity than instrumental analysis methods. In
addition, sensor-based methods are cheaper, more rapid, and do not require trained personnel.
They combine the advantages of immunoassays and signal transducers to provide suitable
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solutions for real-time and in situ detection. In this section, the various methods available for
monitoring melamine and future trends in their development are discussed in detail.
Instrumental analyses
So far, food samples, including soya milk powder, milk powder, soya powder, cookies, biscuits,
and dog food, have been analysed to detect the presence of melamine (Ibáñez et al., 2009; Yu et
al., 2010). To identify and quantity the melamine present in the samples, various instrumental
analyses approaches, including GC-MS, LC-MS-MS, HPLC, capillary electrophoresis (CE), and
surface-enhanced Raman spectroscopy (SERS) (Table 2), have been used owing to their
reliability and accuracy.
GC-MS and GC-MS/MS
GC is the most important analytical technique for determining low-molecular-weight organic
chemicals. MS can directly detect substances or their fragments. The combination of GC and MS
has been demonstrated to exhibit high sensitivity and selectivity in broad applications related to
substance quantification (Hübschmann, 2009). Owing to the high boiling point and polarity of
melamine, derivatization with three methyl silanised reagents is usually essential prior to
subjecting the samples to GC-MS (Xia et al., 2009; Miao et al., 2010; Zhao et al., 2010; Pan et
al., 2013). The reagents used include N,O-bis(trimethylsilyl)trifluoroacetamide,
bis(trimethylsilyl) trifluoroacetamide, and bis(trimethylsilyl)trifluoroacetamide containing 1%
trimethylchlorosilane.
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In 2009, Xia and others developed a sensitive method for detecting melamine in egg samples
using GC-MS and ultra-performance liquid chromatography coupled with tandem mass
spectrometry (UPLC-MS/MS). In the GC-MS analysis, melamine was derivatised with N,O-
bis(trimethylsilyl)trifluoroacetamide and detected under the electron impact mode. The Varian
GC3800 system equipped with a VF-5 ms quartz capillary column and a triple quadrupole
spectrometer were utilised for GC separation and peak identification, respectively. The detection
limit was 10 µg L1. However, since melamine contains three amino groups, incomplete
derivatization could cause poor reproducibility of the results. In addition, the application of GC-
MS is limited by the need for expensive derivatizing reagents and time-consuming reactions. To
overcome these limitations, Xu and co-workers (2009) investigated the use of a separation
column system consisting of a DB-5ms coupled with an Innowax for direct determination of
melamine using GC-MS. The isotope internal standard fortified sample was extracted using 1%
trichloroacetic acid aqueous solution and defatted using a 2.2% lead acetate solution. After the
purification of the cation exchange cartridge, the extract was injected into a GC-MS column for
analysis. To further improve the detection limit, studies were conducted on the sample extraction
procedures. Li and co-workers (2009) prepared zirconia hollow fibres to enrich melamine in milk
samples, prior to GC-MS analysis. Briefly, the fibres were immersed in the samples for a certain
duration and were subsequently removed from the samples and dried. Melamine was then
directly derived and injected into the instrument. This method has been proved to be a rapid and
solvent-saving method with a detection limit of 1 µg L1.
HPLC
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HPLC, which is a technique used for the separation of the components of a mixture, is more
attractive than GC for detecting melamine, since it does not require preliminary derivatization
(Sun H et al., 2010). Venkatasami (2010) proposed an HPLC-UV method involving the Kromasil
C18 column. An advantage of this method was that expensive acetonitrile was not required in the
mobile phase. In addition, detection limits as low as 0.1 µg L1 were achieved. Similar studies
were also conducted for screening milk and plant-derived protein powders by Ding (2008) and
Sun H (2010). Recently, Zhang and co-workers (2014) prepared a strong cation-exchange
restricted access material (sil-g-p(SPM/EDMA)-g-pGMMA) by atom transfer radical
polymerization for the online SPE/HPLC method. During the analysis, melamine was eluted
from the sil-g-p(SPM/EDMA)-g-pGMMA column and analysed on the hydrophilic interaction
chromatography (HILIC) column using 0.1 M ammonium acetate (pH 8.0)/acetonitrile (15/85,
v/v), as the eluent. As mentioned above, HPLC-UV has been highlighted as a method for
monitoring melamine in food samples owing to its ability to provide high accuracy, good
reproducibility, and sensitivity. However, since UV detection could suffer from interference by
compounds that absorb in the same spectral region as melamine, complex sample extraction and
purification procedures are essential for applying the HPLC-UV method (Lutter et al., 2011). To
improve the performance of the HPLC analysis, Finete and co-workers (2015) proposed an
HPLC method with fluorescence detection (HPLC-Fluo). Melamine was detected in milk
samples at its natural fluorescence emission wavelength (250/365 nm) within 15 min, including
the retention time of melamine and the time required for acquiring the entire chromatogram.
LC-MS and LC-MS/MS
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LC-MS and LC-MS/MS are widely used as highly specific and sensitive determination
techniques. During analysis by these techniques, it is difficult to achieve sufficient retention in a
C18 column, in order to separate highly polar basic compounds such as melamine (Hernández et
al., 2002). To improve retention, ion-exchange LC and ion-pair reversed-phase LC are
recommended because they can stabilise the electrospray process and keep the interface clean
(Sancho et al., 2005). Sancho and co-workers (2005) investigated an ion-pair LC with MS/MS
(ESI) for detecting cyromazine and melamine. The method took around 12 min of
chromatographic run time and exhibited a detection limit of 10 µg L1. A similar method was
also applied for analysing melamine in milk powder, baking powder, biscuits, soya-based foods,
and fruit juice by Ibáñez (2009). In 2013, Khedr proposed an LC-MS/MS method involving a
combination of two extraction columns (C18 Sep-Pak and silica gel LiChroprep RP-2 columns)
and an analytical column (Agilent Eclipse XDB-C8 column). Using this method, different
analytes could be simultaneously separated based on their hydrophilicity. After separation,
melamine was detected by positive triple quad mass spectrometry (TQ-MS) using an acidic
mobile phase with a limit of detection of 1.2 ×105 µg L1.
Additionally, in order to improve the practical applicability of the detection methods for real
sample monitoring, the simultaneous analysis of melamine and five other residues in infant
formula was also demonstrated by ultra-high-pressure liquid chromatography (UPLC) coupled
with electrospray ionization tandem mass spectrometry (UPLC-ESI-MS-MS) using the multiple
reaction monitoring (MRM) mode and UPLC quadrupole time-of-flight mass spectrometry
(UPLC-QTOF-MS) (Zhan et al., 2013; Chen et al., 2015; Meng et al., 2015).
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Capillary electrophoresis
CE is an efficient detection method with many advantages such as cost-effectiveness, high
separation speed, high theoretical plate number, and low sample and reagent consumption (Tsai
et al., 2009). On the other hand, the most common problem with CE is its low detection
sensitivity, which is caused by the small injection volume and small inner diameter of the
capillary (Klampfl et al., 2009; Lv et al., 2011).
Capillary zone electrophoresis (CZE), capillary electrochromatography (CEC), micellar
electrokinetic capillary chromatography (MEKC), and sweeping-MEKC coupled with UV-
detector, fluorescence detector, and MS have also been applied for the determination of
melamine (Yan et al., 2009; Chen and Yan, 2009; Xia et al., 2010). Meng et al. (2009) proposed a
method based on CZE-UV detection for quantifying melamine in wheat protein, chicken, fish,
pork, milk, milk powder, and eggs. The sample preparation took around 50 min including
deproteinization and SPE purification. In the CZE-UV analysis, the capillary was conditioned
with 0.1 M NaOH, water, buffer, and running buffer for 30 min. Subsequently, the prepared
sample was injected into the instrument for analysis. Between runs, the capillary was cleaned by
sequentially flushing with 0.1 M NaOH, water, and buffer. The detection limit was in the range
of 250-500 µg L1. Huang and co-workers (2012) developed the CEC method coupled either
with UV or MS for detecting melamine and its by-products in milk. The detection limits for four
melamine derivatives in the CEC-UV method were in the range of 600-2180 µg L−1, which was
much higher than the detection limits of the CEC-MS method, which were in the range of 2.2-
19.4 µg L−1. To improve sensitivity, sweeping and stacking on-line concentration techniques
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were utilised (Tsai et al., 2009; Wang and Chen, 2009). Wang and Chen (2009) successfully
developed a capillary electrophoretic method with K+ as the leading ion and tris+ as the
terminating ion to analyse aromatic amines including melamine in food samples. With an inline-
coupled transient isotachophoretic stacking technique, the UV detection sensitivity was improved
by 200 times. Another CE method coupled with the pH-mediated stacking technique was
demonstrated by Kong (2014). Very low concentrations of melamine (2.552 µg L1) could be
detected by this technique. In addition, in a study by Tsai et al. (2009), field amplified sample
stacking (FASS) and sweeping-MEKC techniques were investigated and compared for melamine
analysis. During detection, the matrix effect was more prominent in the FASS technique
compared to the sweeping technique, which indicated that sweeping-MEKC was more suitable
for analysing melamine in real samples. The detection limit achieved by the sweeping-MEKC
method was 10.9 µg L1. Most recently, Lu and co-workers (2015) proposed an assay method for
detecting melamine in milk powder and liquid milk by CZE after electrokinetic supercharging.
Under optimal conditions, the sensitivity was enhanced 2285 times compared to normal
hydrodynamic injection and a detection limit of 0.7 µg L1 was achieved. In addition, the sample
volume was less than 3% of the capillary volume.
SERS
SERS is a sensing technique based on laser spectroscopy and localised plasmonic
enhancement of metal nanostructures (Liu et al., 2014). This technique is capable of providing
high sensitivity with 103-106 fold enhancement in the Raman scattering signal intensities. This
method is suitable for label-free detection of chemical and biochemical contaminants (Nie and
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Emory 1997; Fan et al., 2014). In recent years, many SERS methods for monitoring melamine
have been developed with different types of nanomaterial substrates used for signal
enhancement. The various nanomaterial substrates include spherical gold nanoparticles (AuNPs),
silver nanoparticles (AgNPs), gold nanorods, and silver nanowires (Lee et al., 2010; Wen et al.,
2011; Yazgan et al., 2012; Zhao et al., 2013; Shi et al., 2014). The enhancement in the signal is
believed to be caused by either the electromagnetic (EM) mechanism (Kahl et al., 2000) or the
charge-transfer (CT) (Kambhampati et al., 1998) mechanism. Mecker (2012) developed a SERS
method with AuNPs for detecting melamine in lactose, povidone, wheat bran, wheat gluten,
whey protein, milk powder, and infant formula. They found that the Raman signal associated
with melamine could be increased 105 times when AuNPs were used. Jiang and co-workers
(2011) utilised nanosilver to carry aptamers (ssDNA). When melamine was present in the
samples, the T-bases of ssDNA and melamine formed a number of hydrogen bonds and produced
stable AgssDNA-melamine conjugates. The assay sensitivity was further improved to as low as
1×105 µg L1 by the AgssDNA catalytic-resonance scattering (RS) method (Jiang and others
2011). Some nano-materials have also been utilised for sample pretreatment for the SERS
method. Shi and others (2014) fabricated a disordered silver nanowire membrane by a
solvothermal polyol process. The membrane was advantageous for both SPE and SERS, owing
to good Raman performance. In the analysis, melamine was first concentrated on the membrane
using the flow-through method, following which it was dried and analysed using SERS
detection. Under optimal conditions, the detection limit for melamine could reach 1 µg L1. Li X
(2015) demonstrated that immunological separation can be combined with SERS for detecting
melamine in milk. Using the principle of antigen-antibody binding, melamine was extracted from
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milk using the converted protein G-anti-melamine antibody complex. The anti-melamine
antibody and melamine were then eluted from the complex and deposited onto the silver dendrite
SERS-active substrate for monitoring. The detection limit was 99.54 µg L1 and the total analysis
time was under 20 min.
Immunoassays
Since the 1970s, immunoassays have been widely used as immunochemical methods for trace
residue monitoring (Engvall and Perlman, 1971; Lee et al., 2001). Immunoassay methods are
based on the principles of antibody-antigen interactions (Lee and Kennedy, 2007). Compared to
the instrumental techniques, immunoassays offer high sensitivity and specificity, which are
achieved by using specific antibodies. They also possess several advantages such as small
sample volumes, low operational costs, simplified methodologies, possibility of parallel sample
processing, and fewer extraction steps (Lee and Kennedy, 2007).
The main components that are used in the immunoassay methods are antibodies and
conjugates (Lee and Kennedy, 2007). The reporters used for synthesising conjugates could be
enzyme (Cao B et al., 2013), colloidal gold (Le et al., 2013), silver nanoparticle (Wang et al.,
2015), quantum dot (Wang et al., 2012), fluorescent dyes (Desfosses et al., 1986) and
upconversion material etc. (Hu et al., 2015). For producing antibodies against small molecules
such as melamine, haptens are designed. The potential immune-dominant epitopes of the analyte
are preserved, in addition to their electronic and geometric features (Lee and Kennedy, 2007).
The conjugation sites of haptens and proteins and the chemical functionality at the sites could
also influence the production of antibodies (Hammock and Mumma, 1980). For melamine,
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haptens have been designed using one of the amine groups of melamine and have been
conjugated with the proteins as immunogens (Table 3). The linkers for the conjugation could be
3-carboxybenzaldehyde (Li et al., 2014), 4-aminobenzoic acid (Cao B et al., 2013), succinic
anhydride (Cao B et al., 2013), glutaraldehyde (Zhou et al., 2012), succinic anhydride (Liu et al.,
2010), 6-aminocaproic acid, and 3-mercaptopropionic acid (Lei et al., 2010). Alternatively, the
commercial product, 6-hydrazinyl-1,3,5-triazine-2,4-diamine, has also been utilised as the

immunogen (Wang Q. et al., 2011). In addition, the antibodies generated could be polyclonal,
monoclonal, or single-chain Fv antibodies (Liu et al., 2010; Huang and Lu, 2011; Cao B et al.,
2013; Li et al., 2013). Inhibition assays with monoclonal antibodies (mAb) usually provide
sensitivities as high as 0.01 µg kg1 in buffer (Zhou et al., 2012). In the recent years, some
melamine ELISA kits have been commercialized (Kim et al., 2008). For instance, in Kim’s
report, Beacon melamine plate kit has been proved to be a rapid and reliable method to quantify
melamine with a higher sensitivity over HPLC instrumentation (Kim et al., 2008).
ELISAs also carry their own limitations. Compared to instrumental analysis, ELISA requires
longer development time for new analytes (Lee and Kennedy, 2007). A new instrumental method
can be developed and validated in weeks or even months. The development of an ELISA method
involves 1) immunogen preparation, 2) antibody generation, and 3) assay development,
optimisation, and validation (Lee and Kennedy, 2007). An ELISA method is also unsuitable for
multi-residue analysis, as it is usually only capable of detecting structurally related compounds.
Additionally, the experimental procedures involve multiple steps and have to be conducted in a
laboratory. Therefore, ELISA is unsuitable for efficient and cost-effective on-site analysis
(Charles et al., 2001).
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Lateral flow immunoassay (LFIA) is another well-established type of immunoassay which is
ease of use and very suitable for field use application (O’Farrell, 2009). Some publications
have established LFIA with the colloidal gold and selenium nanoparticles labels for detecting
melamine in milk, milk powder, animal feed and animal origin (Table 4; Le et al., 2013; Wang
Z. et al., 2014; Zhong et al., 2016). The sensitivity of LFIA is normally lower than 96-microwell
based ELISA, however, in Le’s report, the LOD of melamine have reduced to 0.26 µg L1 (Le et
al., 2013). Most recently, another encouraging improvement for LFIA has been reported by
Zhong and co-workers (2016). In the fabrication of LFIA, an enhancement pad with the
assembled anti-blocking protein AuNPs were introduced for signal amplification (Zhong et al.,
2016). Once the anti-melamine antibody AuNPs were bound with coating antigen, the anti-
blocking protein AuNPs could further labeling the anti-melamine antibody AuNPs and
strengthen the colour intensity (Zhong et al., 2016).
Biosensors
A biosensor is a compact analytical device consisting of a biological receptor and a
physicochemical transducer (Palchetti and Mascini, 2008). The development of biosensors has
become increasingly important and they are used in various applications, including
environmental monitoring, medical diagnostics, health care, and industrial processes (Eggins,
1996). Compared to other methods that require additional processing steps and operation by
highly trained personnel, biosensors can be operated by non-trained operators. In addition, they
allow for rapid analysis and offer the potential for real-time monitoring and portability (Daniels
and Pourmand, 2007; Choi et al., 2011; French et al., 2011). Currently, a vast amount of research
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is being conducted on the use of sensor systems for quantifying trace elements. Based on the
transducers used, these sensor systems can be classified as electrochemical biosensors, optical
biosensors, piezoelectric biosensors, and thermal biosensors. Among them, electrochemical and
optical biosensors are the most frequently used types for detecting melamine (Table 5).
Electrochemical biosensors
The detection of an analyte by electrochemical biosensors is based on the measurement of
electrochemical signals (Daniels and Pourmand, 2007). Electrochemical transducers employ
potentiometric, amperometric, conductometric, and impedance measurements. They are portable,
low cost, robust, and simple to operate; moreover, they can be easily scaled down and used in
turbid media with optically absorbent and fluorescent compounds (Grieshaber et al., 2008;
Bezbaruah and Kalita, 2010). The receptors could consist of polyclonal antibodies, monoclonal
antibodies, aptamers, and MIPs. MIP-based electrochemical biosensors have been applied for the
detection of several small molecular targets, including melamine (Liang et al., 2009; Liu et al.,
2011; Tian et al., 2013). Liu and others (2011) reported the development of an electrochemical
sensor with an MIP-modified glassy carbon electrode for monitoring melamine. The sensor
exhibited a detection limit of 45.5 µg L1.
Ion sensitive field effect transistor (ISFET), also known as enzyme field effect transistor
(ENFET), is a metal/oxide/semiconductor (MOS) field-effect transistor constructed with a
selectively permeable polymer layer (Dzyadevych et al., 2006; Mohanty and Kougianos, 2006).
It has emerged as a powerful electronic sensor for quantifying chemical and biochemical
analytes. On an ion-sensitive surface, the interactions between the ions and the semiconductor
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could manifest as changes in the surface electrical potential, as a result of which the interactions
can be measured. (Monǒík et al., 2012). In 2013, Tian and Regonda compared two
electrochemical sensors containing functionalised Si-nanowire transistors (SiNW FETs)
constructed with antibodies as receptors for monitoring melamine. The assay, which was based
on the competitive format, offered a much lower detection limit (2.52103 µg L1) than a direct
format assay (252.24 µg L1) (Tian et al., 2013). However, there were no further reports
demonstrating the quantification of analytes in real samples using these SiNW FETs-based
sensors with competitive format.
Optical biosensors
Optical transducer systems could be either labelled (light absorption, fluorescence, or
chemiluminescence) or label-free detection systems (Gauglitz and Proll, 2008). In fluorescence-
based detection, either an antigen or a recognition element is labelled with fluorescent tags. The
fluorescence intensity is measured to quantify the analyte with extremely high sensitivities
(Goldman et al., 2002; Moerner et al., 2007; Ehrentreich-Forster et al., 2008). In contrast, in the
case of label-free detection, target molecules are not labelled and the signal is generated by the
binding between recognition element and analyte. This method is relatively easy to perform and
quantify. In the label-free detection method, surface plasmon resonance (SPR) sensors are
commonly used. These sensors measure the changes in the refractive index that occur in the
electromagnetic wave field supported by the sensor (Homola, 2008). Based on the principle of
total internal reflection fluorescence and immunoreactions, some planar waveguide fluorescence
biosensors constructed with polyclonal or monoclonal antibodies have been developed (Wu et
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al., 2013; Guo H et al., 2014; Lu et al., 2014). High affinity monoclonal antibodies may be
obtained during purification. Therefore, biosensors with monoclonal antibodies offer higher
sensitivities than the other types of sensors. Another promising fluorescence-based biosensor
based on MIP-modified quantum dots (QDs) was reported by Huy (2014). The principle of this
fluorescence-based biosensor has been described in the fluorescence chemosensor section later in
the manuscript. The main disadvantage of this method is the high cross-reactivity between
melamine and clenbuterol, which is caused by the low selectivity of the synthesised MIPs.
Chemosensors
A chemosensor is a device containing a recognition element and a physical signal transducing
element. In the recognition step, the analyte interacts selectively with the receptor elements.
Chemosensors only contain recognition elements that interact chemically with the analytes
(Table 6). This distinguishes chemosensors from the biosensors described previously. As the
generation of the biosensing receptors requires various complicated and time-consuming steps,
chemosensors are much easier to fabricate and are more cost-efficient.
Fluorescence chemosensors
Fluorescence refers to the emission of light upon the relaxation of molecules from the singlet
excited state to the singlet ground state (So and Dong, 2002). As the fluorophores exhibit specific
excitation and emission wavelength, fluorescence-based sensors usually have low detection
limits, high sensitivity, and high specificity. The fluorescence emission of AuNPs was caused by
the radiative recombination of conduction Fermi level sp-electrons and d-band holes (Shen et al.,
2006). Significant advantages of these AuNP-based florescence sensors include their operational
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simplicity and rapidity of the tests. Under acidic conditions, melamine can conjugate with the
AuNPs to form AuNP-melamine aggregates through electrostatic interactions. These interactions
result in significant enhancement in the fluorescence intensity of AuNPs. Based on this
enhancement in fluorescence intensity, some sensors was developed (Xiang et al., 2011;
Vasimalai and Abraham, 2013). QDs, which exhibit broad absorption, narrow emission
wavelengths, and size-tunable photoluminescence spectra, can also be utilised for fabricating
‘turn-on’ sensors (addition of the analytes would regenerate the fluorescence of the system) for
melamine (Liu et al., 2012; Wang et al., 2012). In acidic solutions, the amine groups of melamine
can provide electrons to bind with QDs and passivate the thioglycolic acid-capped QDs, which
restores fluorescence (Wang et al., 2012).
Förster resonance energy transfer (FRET) is a type of non-radiative dipole--dipole coupling
interaction with the electronic energy transferring from a donor to an acceptor. The detection
performance of FRET-based sensors depends on the following three factors: 1) relative
orientations between the donor and acceptor, 2) distance between the donor and acceptor, and 3)
spectral overlap between the emission spectrum of the donor and the absorption spectrum of the
acceptor. Many organic dyes and QDs such as 5-carboxyfluorescein have been successfully used
as efficient FRET donors. In addition, AuNPs have been considered as promising acceptor
candidates for FRET systems over the last several years. Some turn-on FRET-based sensors such
as AuNPs and L-cysteine-capped CdS QD systems have been demonstrated for melamine
detection. Melamine can bind to the surface of the AuNPs through amine groups and inhibit
quenching (Cao X et al., 2013).
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However, since a specific distance between the QDs and AuNPs is required to ensure valid
interactions during the FRET process, complicated surface modification processes for the AuNPs
or QDs are essential for developing FRET-based sensors. This makes the detection process time-
consuming and further impacts its practicality.
Colourimetric chemosensors
Melamine causes the aggregation of nanoparticles or functionalised nanoparticles, and the
signal changes can be easily observed by the naked eye. Based on this principle, some
colourimetric sensors, also known as ‘turn-off’ sensors, have been demonstrated for detecting
melamine (Ping et al., 2012; Guan et al., 2013). The major advantage of metallic nanoparticle-
based turn-off assays is their portability, simplicity of operation, and low-cost. Qi and co-workers
(2010) further demonstrated a detection mode using thymine-modified AuNPs, in which thymine
formed triple H-bonds with melamine, leading to the aggregation of AuNPs. However, the
detection limit of this assay was only 2.5224×106 µg L1 (Qi et al., 2010). A method with a high
sensitivity of 0.08 µg L1 was reported by Wu (2011). In their method, the intramolecular
hydrogen-bonding of pyrocatechol-3,5-disodiumsulfonate (PD) reducer was interrupted by the
presence of melamine, which consequently resulted in a change in the colour of the AuNPs from
green to yellow.
Another method for detecting melamine was based on the interruption of melamine during the
formation of metal nanoparticles (Cao et al., 2010a; Ma et al., 2011; Wang et al., 2015). Cao
(2010a) used 3,5-dihydroxybenzoic acid as a reducer for generating AuNPs. Upon the addition
of melamine, 3,5-dihydroxybenzoic acid interacted with melamine through strong hydrogen-
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bonding interactions and thereby, interrupted the formation of AuNPs. The colour change in the
AuNPs could be observed and quantified. In addition, the presence of melamine also caused
changes in the absorption ratio during the formation of AgNPs (Ma et al., 2011; Wang et al.,
2015). The adsorption of melamine onto the surface of the AgNPs resulted in the aggregation of
adjacent AgNPs with detectable decreases in the absorption signal. This decrease was used for
monitoring melamine. A detection limit of 1.2612×106 µg L1 was achieved for this method
(Wang et al., 2015). The advantages of AgNPs over AuNPs of the same size include higher
extinction coefficients and lower cost of preparation, in the former case (Lee et al., 2007).
Electroluminescence chemosensors
Electroluminescence is a type of luminescence that results from an electric current passed
through a substance. Compared with spectroscopic techniques, this method does not require the
use of external light sources and exhibits improved signal-to-noise ratios. So far, some
electroluminescence sensors based on Ru(bpy)32+ coupled with nanospheres, nanotubes, or
graphene have been reported for the detection of melamine (Liu et al., 2011; Cao H et al., 2013;
Zhou et al., 2014). It may be noted that the sensitivities of these sensors are dramatically
improved over that of the sensors not modified by nanomaterials (Guo et al., 2011). Liu and
others (2011) investigated the performance of Ru(bpy)32+ adsorbed onto bare and single-wall
carbon nanotube-modified GC electrodes, in the determination of melamine. The
electroluminescence intensity of the single-wall carbon nanotube-modified GC electrodes was
much stronger than that of the bare GC electrode in the presence of Ru(bpy)32+. This was mostly
attributed to the fact that single-wall carbon nanotubes could accelerate the electron transfer
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reaction by causing an increase in the active surface area of the electrode surface. Additionally,
the interactions between Ru(bpy)32+ and melamine were confirmed by further enhancement in
signal intensity observed after the addition of melamine. The detection limit (1.2612×105 µg
L1) was reduced to 1/1000th of the value without modification (0.012612 µg L1) of single-wall
carbon nanotubes. Mechanisms for the interactions between Ru(bpy)32+ and melamine were
proposed by Guo and others (2011) and Cao H and others (2013). In the report by Cao H (2013),
a sensor with mesoporous SiO2 nanospheres/Ru(bpy)32+/Nafion-modified electrodes was
fabricated and utilised for the analysis of melamine. In the electroluminescence reactions,
melamine was believed to first react with Ru(bpy)33, generating MEL(-H)· and Ru(bpy)32+. The
active MEL(-H)· radical further interacted with Ru(bpy)33+ to generate Ru(bpy)32+ in the excited
state, leading to emissions at 620 nm.
Electrochemical chemosensors
The mechanism by which electrochemical chemosensors detect melamine could be related to
the hydrogen bonding interaction between melamine and the sensor surface modified molecules,
the oxidation of non-electroactive melamine monomers to electroactive dimers, or the
electrochemical reduction of melamine copper chloride ionic pairs formed by the dissolution of
copper electrodes in a medium containing melamine and chloride (Cao et al., 2010b; Guo, Xu et
al., 2014). In a study by Guo and Xu et al. (2014), an ordered mesoporous carbon and Nafion-
modified glassy carbon electrode was successfully fabricated and utilised for the detection of
melamine in milk samples. The detection was based on the oxidation of melamine from a single
molecule state to an electroactive dimer state via the formation of an azo group. During
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differential pulse voltammetry (DPV) measurements, the modified electrode showed more stable
and sensitive performance towards melamine compared to the multiwall carbon nanotube-
modified glassy carbon electrode. As a result, the modified electrode was suitable for melamine
determination at concentrations as low as 0.3 µg L1. Other strategies for determining melamine
involve the investigation of interactions between melamine and the modified electrodes with
electrochemical indicators (Li Y et al., 2015). In a report by Cao (2009), oligonucleotides were
first self-assembled on a gold electrode. [Fe(CN)6]3−/4− was then used as an electrochemical probe
in the modified electrode and the interactions between melamine and the oligonucleotides were
investigated. The results of electrochemical impedance spectroscopy (EIS), cyclic voltammetry
(CV), and DPV studies showed that melamine interacted with d(T)20 through electrostatic
interactions and hydrogen-bonding, resulting in the reduction in electron-transfer resistance. The
proposed method was successfully developed and optimised for monitoring melamine and a
detection limit of 1.21 µg L1 was achieved. 3,4-Dihydroxyphenylacetic acid (DOPAC) has also
been utilised as an electrochemical probe for recognising melamine via hydrogen bonding (Cao
et al., 2010b). The hydrogen bonding interactions inhibited the electroactivity of the phenolic-
OH group and decreased the redox peak currents of DOPAC, which was exploited for monitoring
melamine.
Trends in the detection methods for melamine
In attempts to miniaturise the detection systems, microfluidic systems have been studied as
promising analytical methods. In these systems, the sample pretreatment, separation, and
detection steps are integrated on a single chip. In addition, they offer a long list of attractive
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advantages over instrumental analysis techniques including low cost, low reagent consumption,
short separation time, and high separation efficiency. For example, milk could be directly
injected into a microfluidic device coupled with a UV detector for pretreatment and melamine
could then be automatically detected within 75 s (Zhai et al., 2010). In recent years,
nanomaterials, microfabrication technology, and 3D printing have gradually become enablers for
the microfluidic systems. A multiplex bead-based microfluidic system has been developed for the
simultaneous detection of food-borne pathogenic bacteria. In this system, the detection procedure
takes less than 30 min (Jin et al., 2009). Thus, the integration of different detection methods (e.g.,
sensors, SERS, and immunoassays) with microfluidics offers great potential for fast, sensitive,
and automatic melamine detection. Yazdi and White (2012) established a highly automated opto-
fluidic SERS microsystem by integrating a porous microfluidic concentration and detection zone
with a micromixer. The on-chip micromixer enabled the device to be portable and circumvented
the need for human involvement during sample preparation. Silica microspheres were used to
concentrate the AgNPs and adsorbed analytes, thus significantly enhancing the SERS signal. The
device has been successfully employed for the on-site detection of melamine with a detection
limit of 63 µg L1.
Conclusions
Monitoring melamine residues in foods is critical, in view of the series of negative health
effects that exposure to melamine poses in humans. In this review, sample preparation methods
and three categories of detection methods for monitoring melamine, namely instrumental
analysis, immunoassays, and sensor methods, were thoroughly summarised and discussed.
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Depending on the food matrix and the detection purpose, the analytical instruments employed for
melamine quantification may vary. Highly technical instrumentations such as GC-MS and LC-
MS/MS rely on data treatment and require trained operators. LC-MS/MS, in particular, is more
sensitive and reliable than HPLC-UV and GC-MS, owing to the quantification of melamine on
the fragment level. On the other hand, GC/MS is more cost-efficient and simpler to apply and
maintain than LC-MS/MS. However, since melamine is a highly polar basic molecule,
derivatization is essential for detection by GC-MS. Melamine has three amino groups, which
could easily cause incomplete derivatization and lead to poor reproducibility during quantitative
analysis. Additionally, the need for expensive derivative reagents and the amount of time
consumed during the reaction could also possibly limit the implementation of GC-MS. CE is
superior to the above techniques, since it is more efficient in terms of sample separation and
utilises lower amounts of solvent and sample. However, it suffers from low sensitivity and
reproducibility. The combination of CE with MS as a detector could be a solution to overcome
these disadvantages. While the recently developed SERS method is simpler and faster, its most
challenging problems include signal weakness and the need for further data processing. Overall,
although a number of on-line and off-line sample preparation methods have been developed to
simplify the analysis procedures, compared to the immunoassay and sensor methods, the sample
preparation methods still remain much more complicated and time-consuming. In addition, the
high-operating costs, bulky and costly equipment, and requirement for highly trained operators
limit their applications in developing countries and for on-site detection. Recent trends in the
detection methods have focused on miniaturization, high-throughput performance, on-line and
cost-effective operation, and high sensitivity and selectivity. Therefore, sensors are now widely
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used for trace analysis, as they offer several advantages such as low sample volume, high
sensitivity, and suitability for portable use. Among the biosensors, electrochemical and optical
biosensors using antibodies, MIPs, or aptamers as receptors are most widely used. Chemosensors
have also been developed and are generally based on the aggregation of nanoparticles or
functionalised nanoparticles in the presence of melamine. While the fabrication cost of
chemosensors is much lower than that of biosensors, their usage might be limited by poor
selectivity. On the other hand, microfluidic systems, which combine the sample pretreatment and
detection procedures on a single chip, offer advantages such as automation and miniaturization.
The incorporation of new materials in the microfluidic systems and their combination with other
detection methods are increasingly being explored in the analytical field.
Acknowledgments
The authors gratefully acknowledge the financial support from the Scientific Research
Foundation for the Returned Overseas Chinese Scholars from State Education Ministry, the
Natural Science Foundation of China (31501566, 21575045), the self-determined research funds
of CCNU (CCNU15A02015), and the funding from MQ University MQRDG and ARC CNBP
CE (CE140100003).
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Table 1 Comparison of the detection methods for melamine
Detection
Detection Time per
Detection methods limits (µg kg1 Reference
sample (min)
or µg L1)
0.5 30 Tzing and Ding,

GC-MS/MS
2010
GC-MS 0.6 35 Zhao et al., 2010
HPLC-UV 6 30 Tan et al., 2012
LC-MS 4 12 Tittlemeier et al.,

2009
LC-MS/MS 5 23 Yu et al., 2010
CE-UV 9.2 13 Tsai et al., 2009
SERS 0.01 7 Jiang et al., 2011
ELISA 0.01 3 Zhou et al., 2012
LFIA 0.26 3 Le et al., 2013
Electrochemical biosensor 1.2 0.5 Cao et al., 2009
Vasimalai and
Fluorescence chemosensor 1.26×106 5
Abraham, 2013
Electrochemiluminescence
1.3×105 22 Zhou et al., 2014
chemosensor
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Table 2. The determination of melamine using instrumental analysis
Classificatio LOD (µg kg1 or µg

Sample Reference
n L1)
GC-MS milk 1 Li et al., 2009
GC-MS milk, milk powder 10 Xu et al., 2009

10 (in chicken and

GC-MS chicken, tilapia, egg, milk tilapia); 5 (in milk Zhu et al., 2009
and egg)
soya milk powder, milk powder,

GC-MS soya powder, cookies and biscuits, 150 (in buffer) Araújo, 2012
dog food
GC-MS/MS egg 10 Xia et al., 2009
crab legs 180 Truzzi et al.,

GC-MS
2014
animal feed 0.6 Zhao et al.,

GC-MS
2010
GC-MS milk, milk powder, yoghurt 25 Pan et al., 2013
2 (in milk); 8 (in

GC-MS/MS milk, milk powder, cheese milk powder and Xu et al., 2015
cheese)
milk powder 0.5 Tzing and

GC-MS/MS
Ding, 2010
GC-MS milk, feed 10 Li et al., 2010
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muscles and livers of swine,

HPLC-UV bovine, sheep and chicken, kidneys 10 Yu et al., 2010
of swine, bovine and sheep, milk
powder
wheat gluten, chicken feed, cakes,

HPLC-UV 1000 Lin et al., 2008
noodle
HPLC-UV liquid milk and milk powder 6 Tan et al., 2012

Rima et al.,
HPLC--UV fish 63
2009
Venkatasami
HPLC-UV infant formula 100 and Sowa Jr,
2010
milk powder, baking powder, milk-

10 (in liquid
chocolate biscuit, milk-chocolate Ibáñez et al.,
LC-MS samples); 100 (in
egg, biscuit, dry pasta (wheat), 2009
solid samples)
fruit juice, milk blends
LC-MS infant formula 4 Tittlemeier et

al., 2009
muscles and livers of swine,

LC-MS/MS bovine, sheep and chicken, kidneys 5 Yu et al., 2010
of swine, bovine and sheep, milk
powder
chard 10 Sancho et al.,

LC--MS/MS
2005
CE-UV milk 163 Lv et al., 2011
9.2 (in buffer); 10.9

CE-UV - Tsai et al., 2009
(in infant formula)
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milk 47 Chen and Yan,

CE-UV
2009
Huang et al.,
CE-UV milk powder 600
2012
CE-UV liquid milk, yogurt, whole milk 10 Yan et al., 2009

powder, fish feed, fish
Huang et al.,
CE-MS milk powder 19.4

2012
wheat gluten, chicken feed, cakes,

SERS 33 Lin et al., 2008
noodle
SERS - 100 Du et al., 2010
SERS milk powder 200 Lee et al., 2010
Jiang et al.,
SERS - 0.01
2011
SERS - 12.612 Li et al., 2011
1.26 (in buffer); Chen and Liu,

SERS raw milk
6.31 (in milk) 2012
Zhao et al.,
SERS milk powder 2.4
2013
Kumar et al.,
SERS - 25.2
2013
Giovannozzi et
SERS milk 170
al., 2014
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Guo, Cheng et
SERS milk 1000
al., 2014
630 (in water); 1260 Wang G. et al.,

SERS milk
(in milk) 2014
SERS - 1.26 Yi et al., 2014
-Denotes for no data shown.

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Table 3. Determination of melamine using ELISA
Detection
Hapten (for
limits (µg
Antibody generation of Sample Reference
kg1 or µg
immunogen)
L1)
polyAb
milk 1 Li et al., 2014
mAb - milk 3.88 Wu et al., 2013
milk, milk
mAb powder, chicken 0.1 Cao et al., 2013
feed, pig feed
Trapiella-
milk infant
Ab - 0.15 Alfonso et al.,
formula
2013
single-chain Fv milk, milk Huang and Lu,

-
Ab powder 2011
milk, milk
polyAb 9.3 Wang et al., 2011
powder
mAb milk powder 50 Sun et al., 2013
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mAb milk, pet food 0.01 Zhou et al., 2012
chicken, pork,
polyAb 1.8 Liu et al., 2010
beef, mutton
-Denotes for no data shown.

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Table 4. Determination of melamine using LFIA
Detection limits
Antibody Reporter Sample (µg kg1 or µg Reference
L1)
50 (in raw milk);

2000 (in milk
raw milk, milk drinks, drinks, yogurt,

yogurt, condensed milk, condensed milk,
mAb colloidal gold Li et al., 2011
cheese, animal feed, cheese, and
milk powder animal feed);
1000 (in milk
powder)
pork, chicken, beef,

mAb colloidal gold 0.26 Le et al., 2013
mutton, milk
150 (in milk);

selenium milk, milk powder, 1000 (in milk Wang Z. et al,
mAb
nanoparticle animal feed powder); 800 (in 2014
animal feed)
mAb colloidal gold milk 1.4 Zhong et al., 2016
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Table 5 Biosensors for monitoring melamine
LODs (µg
Recognition 1 Referenc
Classification Principle Device fabrication kg or µg
element e
L1)
melamine
functionalized Si
Tian et
electrochemic nanowire transistors
direct Ab 252.24 al.,
al via 11-
2013
(triethoxysilyl)undecan
al linker
BSA-SM2
functionalized Si
Tian et
electrochemic competitiv nanowire transistors
Ab 2.52103 al.,
al e via 11-
2013
(triethoxysilyl)undecan
al linker
electrochemical sensor
based on a poly(para- Liu et
electrochemic
direct MIP aminobenzoic acid) 45.4 al.,
al
film modified glassy 2011
carbon electrode
Liang
electrochemic
direct MIP potentiometric sensor 201.8 et al.,
al
2009
oligonucleotides film Cao et

electrochemic oligonucleotid
direct modified gold 1.2 al.,
al es
electrodes 2009
Wu et
competitiv polyAb immobilized
optical polyAb 2104 al.,
e SPR sensor chip
2013
optical inhibition mAb melamine-BSA 6.6 (in Guo H

functionalized planar PBS); 10.2 et al.,
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waveguide (in milk) 2014

fluorescence
immunosensor
6-hydrazino-1,3,5-
Fodey
competitiv triazine-2,4-diamine
optical polyAb <500 et al.,
e functionalized sensor
2011
chip
Xing et
aptamer modified 4.29 (in

optical direct aptamer al.,
AuNPs milk)
2013
Yun et
aptamer modified 1500 (by
AuNPs naked eyes)
2014
500 (by UV-

Yun et
aptamer modified Vis
AuNPs spectrometer
2014
)
Huy et
optical direct MIP MIP-CdTe QDs 75 al.,
2014
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Table 6 Chemosensors for monitoring melamine
LODs (µg
Classification Device fabrication kg1 or µg Reference
L1)
Guo, Xu et al.,
electrochemical ordered mesoporous carbon 0.3
2014
electrochemical electrochemistry (DOPAC) 0.4 Cao et al., 2010
electrochemical method/copper de Araujo and

electrochemical 0.11
electrode Paixão, 2014
3-amino-5-mercapto-1,2,4-triazole Vasimalai and

fluorescence 1.26×106
capped AuNPs Abraham, 2013
oligonucleotide-stabilized silver
fluorescence 1.26 Han et al., 2012
nanoclusters
fluorescence citric acid anion-stabilized AuNPs 0.0769 Xiang et al., 2011
mercaptopropionic acid capped

fluorescence 0.63 Liu et al., 2012
CuInS2 quantum dots
Nascimento et al.,
fluorescence Triton X-114 800
2015
fluorescence citrate-stabilized AuNPs 0.18 Cao et al., 2014a
fluorescence acid-capped CdS quantum dots 0.13 Wang et al., 2012
fluorescence PDA liposome-based colorimetry 500 Lee et al., 2011
FRET CdTe@SiO2 and AuNPs system 113.14 Gao et al., 2012
AuNPs and L-cysteine-capped CdS

FRET 17 Cao X et al., 2013
QDs system
dual hairpin-like molecular beacon

FRET based on coralyne-adenosine 0.63 Wang et al., 2014
interaction
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colorimetric chitosan-stabilized AuNPs 6 Guan et al., 2013
in-situ formation of silver

colorimetric nanoparticles (AgNPs) through 1.26 Wang et al., 2015
modified Tollens process
colorimetric dopamine-stabilized AgNPs 10.09 Ma et al., 2011
colorimetric label-free AgNPs 292.6 Ping et al., 2012
colorimetric unmodified AuNPs 151.34 Guo et al., 2010

polythymine-modified AuNPs
colorimetric 2.52 Qi et al., 2010
(visualization and light scattering)
colorimetric nonaggregation-based AuNPs 0.1 Cao et al., 2010
AuNPs-based UV--visible
colorimetric 100 Han and Li, 2010
spectrometry
AuNPs-based UV--visible
colorimetric ~25 Chi et al., 2010
spectrometry
Fe3O4 NPs-based UV--visible

colorimetric 2500 Ding et al., 2010
spectrophotometry
immunochromatographic strip test

colorimetric with AuNP detection using 50 Li et al., 2011
spectrophotometer
magnetic relaxation switch assay

colorimetric 252.24 Shen et al., 2014
with functionalized Fe/Fe3O4 NPs
p-DNA modified GNPs (dynamic

colorimetric 2.0 Wu et al., 2014
light scattering)
reusable evanescent wave fiber-

colorimetric 5.14 Hao et al., 2014
optic biosensor
colorimetric ZnSe QDs 0.11 Cao et al., 2014b
colorimetric AuNPs-based colorimetry 231 Ping et al., 2012
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3-mercaptopriopionic acid-
colorimetric 400 Cai et al., 2014
functionalized gold nanoparticles
colorimetric detection/cysteamine-
colorimetric 400 Liang et al., 2011
modified AuNPs
colorimetric Mb-mediated AuNPs 701.22 Xin et al., 2015
colorimetric AuNPs 2.5 Ai et al., 2009
colorimetric AuNPs 292.6 Ping et al., 2012

colorimetric AuNPs 40 Wei et al., 2010
Ru(bpy)32+@SiO2/graphene/Nafion
electrochemiluminescence 1.3×105 Zhou et al., 2014
modified electrode
Ru(bpy)32+/mesoporous SiO2
electrochemiluminescence nanospheres/Nafion composite 17 Cao H et al., 2013
modified electrode
Ru(bpy)32+/single-wall carbon
electrochemiluminescence 45.4 Liu et al., 2011
nanotubes modified electrode
-Denotes for no data shown
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A Review of Methods For Detecting Melamine in Food Samples: Critical Reviews in Analytical Chemistry

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A review of methods for detecting melamine in

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A review of methods for detecting melamine in food samples

Alice Lee 5, * , Shuo Wang 1,*

University of Science and Technology, Tianjin 300457, China

Chemistry, Central China Normal University, Wuhan 430079, PR China

Astronomy, Macquarie University, North Ryde 2109, Australia

Sydney, NSW 2052, Australia

60601430, E-mail: s.wang@tust.edu.cn

Melamine is a synthetic chemical used in the manufacture of resins, pigments, and

essential to prevent human exposure to melamine. Sample preparation is of critical importance,

perspectives on the applications of microfluidic devices have also been provided.

melamine, instrumental analysis, immunoassay, biosensors, chemosensors

(Ehling et al., 2007; Yang et al., 2009).

and change in cytomorphology in a concentration-dependent manner (Kim et al., 2011; Wang Z.

foods (World Health Organization, 2010).

in the commercial market.

melamine detection, including instrumental analysis methods, enzyme-linked immunosorbent

analysis, chemometrics analysis, and sensor methods.

detection techniques for monitoring melamine in foodstuffs.

addition of trichloroacetic acid, methylene chloride, dichloromethane, or hexane (Andersen a et

solubilities of substances in two different immiscible liquids. Melamine is a small polar

Therefore, polar solutions such as methanol/water, acetonitrile/water, diethylamine/water,

exhibits high sensitivity and repeatability (Chen et al., 2015).

immuno-affinity chromatography column (IAC) is a unique purification column that is based on

chromatography (HPLC)-UV analysis.

Hollow-fibre microdialysis (MD) sampling is another rapid and miniaturised sample

bis(trimethylsilyl)trifluroacetamide to derive the target analyte before GC-MS analysis. The

hand, electromembrane extraction (EME) is a relatively novel clean-up and pre-concentration

Methods for melamine detection

reliability and accuracy.

GC-MS and GC-MS/MS

GC is the most important analytical technique for determining low-molecular-weight organic

al., 2013). The reagents used include N,O-bis(trimethylsilyl)trifluoroacetamide,

bis(trimethylsilyl) trifluoroacetamide, and bis(trimethylsilyl)trifluoroacetamide containing 1%

solvent-saving method with a detection limit of 1 µg L1.

restricted access material (sil-g-p(SPM/EDMA)-g-pGMMA) by atom transfer radical

from the sil-g-p(SPM/EDMA)-g-pGMMA column and analysed on the hydrophilic interaction

LC-MS and LC-MS/MS

techniques. During analysis by these techniques, it is difficult to achieve sufficient retention in a

al., 2002). To improve retention, ion-exchange LC and ion-pair reversed-phase LC are

analytes could be simultaneously separated based on their hydrophilicity. After separation,

mobile phase with a limit of detection of 1.2 ×105 µg L1.

formula was also demonstrated by ultra-high-pressure liquid chromatography (UPLC) coupled

CE is an efficient detection method with many advantages such as cost-effectiveness, high

capillary (Klampfl et al., 2009; Lv et al., 2011).

Capillary zone electrophoresis (CZE), capillary electrochromatography (CEC), micellar

electrokinetic capillary chromatography (MEKC), and sweeping-MEKC coupled with UV-

volume was less than 3% of the capillary volume.

SERS is a sensing technique based on laser spectroscopy and localised plasmonic

time was under 20 min.

processing, and fewer extraction steps (Lee and Kennedy, 2007).

commercial product, 6-hydrazinyl-1,3,5-triazine-2,4-diamine, has also been utilised as the

involves 1) immunogen preparation, 2) antibody generation, and 3) assay development,

multi-residue analysis, as it is usually only capable of detecting structurally related compounds.

(Charles et al., 2001).