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Determination of melamine in feed by high performance liquid

chromatography coupled mass spectrometry

Article in Bulletin of the Veterinary Institute in Pulawy · March 2010

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Bull Vet Inst Pulawy 54, 543-547, 2010

DETERMINATION OF MELAMINE IN FEED

BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
COUPLED MASS SPECTROMETRY
TOMASZ ŚNIEGOCKI, BARTOSZ SELL, ANDRZEJ POSYNIAK, AND JAN ŻMUDZKI

Department of Pharmacology and Toxicology,

National Veterinary Research Institute, 24-100 Pulawy, Poland
sniego@piwet.pulawy.pl

Received for publication March 9, 2010

Abstract
A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed for the determination of
melamine in feed samples. After a preliminary dissolution in acetonitryle and 0.05 M phosphate buffer, the obtained extract was
sonicated for 10 min and supernatant was collected. The supernatant was cleaned up on SCX solid phase extraction cartridges.
Melamine was determined, using electrospray ionisation in the positive mode, followed by separation on the carbon column Thermo
Hypercarb. The whole procedure was validated for the identification and determination purposes. Feed samples were spiked with
melamine solution at levels corresponding to 0.5–1.0–2.5-10.0 mg/kg. At the studied levels, trueness ranged between 78.0% and
82.3% and within-laboratory reproducibility expressed as a relative standard deviation was lower than 15%. The limit of detection
was estimated at the level of 0.58 mg and limit of quantitation was 0.65 mg/kg.

Key words: feed, melamine, validation, HPLC-MS/MS.

In 2007, a pet food recall was initiated by the
Menu Foods and other pet food manufacturers, which
had found that their products were contaminated and
caused serious illnesses or even deaths in some of the
animals (16, 17, 24). In March 2007, the US Food and
Drug Administration reported the presence of melamine
in the pet food, in samples of white granular wheat
gluten imported from a single source in China, Xuzhou
Anying Biologic Technology (21), and later on in other
vegetable proteins imported from China (22), as well as
in kidneys and urine from affected animals (18). Many
tests revealed melamine (MEL), a triazine based
chemical substance, to be present in food and feed as an
adulterant (6, 22). The presented investigations show,
that some imported pet food ingredients, like wheat
gluten or rice protein, containing wheat flour with
melamine were added to illegally increase protein
content in these products (8). Investigators discovered
that waste material from the pet food manufacturing
process contaminated with melamine (MEL) had been
added to chicken feed and also found that contaminated
wheat gluten had been used in the manufacture of
aquaculture’s feeds (13). In September 2008, the
European Union imposed special conditions governing
about feed or food products (with limit for melamine at
the level 2.5 mg/kg) with a high protein content
imported from China (2) to protect European
community.
The aim of the presented study was to develop
and validate a confirmation high performance liquid
chromatography mass spectrometry (LC–MS/MS)
method for the determination of melamine in feed. This
paper presents a robust, efficient, and simple analysis of
melamine.
Material and Methods
Reagents. Melamine (CAS: 108-78-1) standard
was obtained from Sigma-Aldrich Chemical Company
(USA). Acetonitrile, methanol, and formic acid were
obtained from J.T. Baker (Germany). Sodium
dihydrogen phosphate was obtained from POCH
(Poland). Water was purified using Milli-Q system.
Strata X-C 33 µm Polymeric Strong Cation 500 mg/6 ml
extraction columns were purchased from Phenomenex
(Germany).
Standard solutions. Stock standard solutions
(1 mg/mL), prepared by weighing 10.0 ±0.1 mg of
standard substances and dissolving in 10 ml of mixture
acetonitryle: water (1/1; v/v), were stable for 6 month,
when stored at the temperature below -18°C in an amber
glass. Working standard solutions, prepared by diluting
suitable aliquot of stock standard solutions in mobile
phase, were stable for 1 month, when stored at
temperature between 2-8°C in amber glass.
544
Samples. The animals feed samples (soya,
corn, calf feed, chicken feed, pigs feed) were obtained
from a livestock farm.
Extraction and clean up. An aliquot of 5.0
±0.1 g of homogenised sample was weighed into a 50 ml
plastic centrifuge tube coated with teflon and extracted
for 1 min with 15 ml of acetonitryle and 30 ml of 0.05
M phosphate buffer, pH 7.0, using vortex mixer. The
obtained suspension mixture was put into ultrasonic-
bath for 10 min, centrifuged at 3,500 rpm in room
temperature for 20 min and the supernatant was
collected. SPE cartridges were conditioned with 10 ml
of methanol and 10 ml of 0.05 M phosphate buffer, pH
7.0. The extract was loaded to SPE cartridges, rinsed
with 10 ml of fresh re-distilled water, 5 ml of methanol,
and dried for 5 min under vacuum. Melamine was eluted
twice with 4 ml of alkaline acetonitryle (acetonitryle:
ammonia solution 25% in water (95/5; v/v)). After each
portion of alkaline acetonitryle, it was necessary to wait
for 1 min to better condition cartridges. The combined
eluat was evaporated to dryness at temperature below
50°C under nitrogen. The dry residue was dissolved in
0.5 ml of LC mobile phase and transferred to a vial for
analysis.
Liquid chromatography-mass spectrometry.
The LC-MS/MS system consisted of an Agilent Series
1200 HPLC system (Agilent Technologies, Germany)
connected to a PE Sciex API 4000 triple quadrupole
mass spectrometer (PE Sciex, Canada) in electrospray
positive ionisation mode. To achieve the maximum
sensitivity, the mass spectrometry parameters including
ionisation mode, the capillary voltage, cone voltage,
source temperature, and desolvation gas temperature,
desolvation gas and cone gas flow, the pressure of the
collision chamber, and the collision energy were first
optimised by direct flow infusion of standard solution.
The results indicated that the positive ion mode was
more favourable than the negative. The chromatography
was performed with a Thermo Hypercarb 100 mm x 2.1
mm x 5 m (Thermo, USA), with a Hypercarb
precolumn 3 m x 2 mm x 4 mm (Thermo, USA). The
mobile phase was composed by two solutions: A (formic
acid in water 0.1% (v/v)) and B (acetonitryle) in a
Table 1
Validation report for feed
Parameters
Compound
Linear regression equation (y=mx+b)
Correlation coefficient
Linearity (working range) mg/kg
Limit of detection (LOD) mg/kg
Limit of quantification (LOQ) mg/kg
Level of spiked samples feed mg/kg
Recovery, %
Repeatability %
Reproducibility,%
Uncertainty combined (uc)
expanded (U)
coverage factor (k)
gradient mode, which started with 99% of A and 1% of
B; from 0 to 3 min the concentration of B was raised to
90% and remained for 8 min. Finally, after 9 min, the B
concentration was decreased to 1%. The column was
operated at 40°C and a flow rate of 0.2 mL/min. The MS
detector was operated in the electrospray mode, and data
were acquired in multiple reaction monitoring mode:
m/z 127→68 and 127→85 for melamine. The source
block temperature was set to 250°C and the electrospray
capillary voltage to 4.5 kV.
Validation. Standard calibration curve was
prepared by injection of standard solutions on five levels
and working range was established. Feed samples were
spiked with melamine at levels of 0.5, 1.0, 2.5, and 10,0
mg/kg in order to evaluate matrix-matched calibration
curves. Precision (repeatability and reproducibility) of
the assay was determined as the coefficient of variation
calculated for four concentrations levels. Recoveries
were calculated by comparing peak areas of spiked
samples to corresponding standards. Limit of detection
(LOD) and limit of quantification (LOQ) were also
evaluated.
Results
Due to the ionic character of melamine, it is
difficult to obtain sufficient retention time on an
octadecyl and many others reverse phase columns.
Initial trials carried out on C8 or C18 HPLC columns
with a variety of eluent conditions showed that retention
time on these columns was too short (Fig. 1A) and led to
matrix interferences. In our method, the Thermo
Hypercarb column was used, that allow increasing the
analyte retention time (Fig. 1B) and reducing, or
eliminating ionisation enhancement and/or matrix
interference. In Fig. 2 typical ion chromatograms of a
blank feed sample, blank feed sample spiked with
melamine, and noncompliant sample are shown. In the
prepared procedure, the retention time was about 7.6
min using the gradient mode elution on the Hypercarb
column with the mobile phase consisting at start of 0.1%
formic acid in water: acetonitryle (99:1; v/v).
Results
Melamine
y=0.9817x+0.0181
0.9997
0.5-10
0.58
0.65
0.5 1.0 2.5 10
81.9 78.0 78.5 82.3
15.0 7.8 13.4 10.7
16.8 11.2 15.3 11.4
0.15
2
1.00±0.3
 545

A B

Fig. 1. LC chromatograms illustrating retention of melamine using: (A) C18 analytical column; and (B) Thermo
Hypercarb column.

Fig. 2. Typical chromatograms of: A - a blank feed sample; B - a blank feed sample spiked with melamine at the level
of corresponding to 2.5 mg/kg; C - noncompliant sample at the level corresponding to 74 mg/kg.
546
The single peak in chromatogram of feed
sample spiked with melamine at the level of 1 mg/kg
(Fig. 2B) and no peaks in blank feed samples at the
melamine retention time (Fig. 2A) indicated that there
were no interferences. The use of polymeric strong
cation SPE cartridges was found suitable for clean up
and enabled to achieve the best recoveries, which
oscillated about 80%. Confirmation of the identity was
performed by the LC-MS/MS in electrospray positive
ionisation mode by a multiple reaction monitoring with
ion transitions as shown in Table 1. The satisfaction
results of the limit of detection (LOD) and the limit of
quantification (LOQ) were obtained.
Discussion
Animal feeds are very complex and difficult to
analyse matrixes. The composition is very often variable
or unknown. Cereals, oil seeds, fat, citrus pulp, and
many others ingredients are common in feed and
additionally some vitamins, minerals, and preservatives
can be added (11).
Several methods for the determination of
melamine in animal feeds and their ingredients, based on
GC and LC chromatography, were described (3, 4, 7, 9-
12, 19). With classical detectors, like UV, it is hard to
find acceptable clean-up method for feed because of low
maximum absorbance wavelengths, very complex and
difficult to analyse matrix. It is possible to determine
melamine on GC/MS, which is relatively cheaper, but
the clean-up procedure and necessary derivatisation with
three-methyl silanised reagent (TMS) process are more
time-consuming. Additionally, the derivatisation step
can cause many problems. Melamine with three NH2
groups in the structure can form three derivative
products: di-, mono-, tri-TMS-melamine, (4, 10, 25).
Due to the incomplete derivatisation, the GC/MS can
have worse reproducibility and bigger uncertainty than
LC/MS/MS analysis. In our opinion, LC/MS/MS is the
most compatible instrument to detect melamine in feeds.
Separation of melamine by LC/MS/MS on
different columns like: Synergi fusion-RP (SF) (11),
Waters Acquity UPLC, BEH HILIC Silica, Waters (1),
Acclaim® Mixed-Mode WAX-1 (15) has been
described. In many others methods, the retention time is
often similar or close to void time. However, the use of
these kinds of columns did not allow separating analyte
from matrix with good results. In our method, Thermo
Hypercarb column was used to increase analyte
retention and reduce or eliminate matrix interferences
(Fig. 2).
For the extraction purpose, a wide variety of
different solvent mixes have been used. The mostly used
extraction solvent in pet food and muscle tissue was
acetonitrile/water (20, 23), or acetonitrile/water/
diethylamine mixture (4, 5, 10). For much more
complex matrix samples such as fish feed,
acetonitrile/trichloroacetic acid aqueous solution or lead
acetate/trichloroacetic acid aqueous solution were used
(12). In our procedure, the extraction of the feed samples
with mixture of acetonitryle and phosphate buffer was
proposed. In the first step, the denaturation of proteins
was carried out by acetonitryle, and by adding phosphate
buffer the melamine was extracted from biological
martrices at satisfactory recovery level.
In the described procedures with clean-up step
using solid phase extraction (1, 20, 23, 25), most
commonly cartridges used for purification were strong
cation exchange cartridges like, SCX columns (20, 23,
25). In the presented method, the clean-up using SPE
cartridges with polymeric strong cation exchange
sorbent – Phenomenex Strata X-C was most effective.
This clean up step allows removing many water-soluble
substances from feed matrix, which can cause ion
suppression. The application of solid phase extraction
gives better results than using only liquid-liquid
extraction clean up step.
The elaborated method was applied to feed
samples from monitoring and border control
programmes in Poland analysed by the National
Veterinary Research Institute in Pulawy. In our
laboratory, around 100 feed samples (corn and wheat
gluten, rice protein, feed concentrate, therapeutic feed)
were analysed using this method. Four of them were
contaminated (Fig. 2C). The presence of melamine in
positive samples was confirmed by acquiring two MRM
transitions and the accomplishment of their ion intensity
ratio.
References
1. Andersen W.C., Turnipseed S.B., Karbiwnyk C.M., Clark
S.B., Madson M.R., Gieseker C.M., Miller R.A., Rummel
N.G., Reimschuessel R.: Determination and confirmation
of melamine residues in catfish, trout, tilapia, salmon, and
shrimp by liquid chromatography with tandem mass
spectrometry. J Agric Food Chem 2008, 56, 4340–4347.
2. COMMISSION DECISION of 14 October 2008 imposing
special conditions governing the import of products
containing milk or milk products originating in or
consigned from China, and repealing Commission
Decision 2008/757/EC.
3. Ehling S., Tefera S., Ho I. P.: High-performance liquid
chromatographic method for the simultaneous detection
of the adulteration of cereal flours with melamine and
related triazine by-products ammeline, ammelide, and
cyanuric acid. Food Addit Contam Part A Chemistry,
Analysis, Control, Exposure & Risk Assessment 2007,
24, 1319–1325.
4. Ferro G.L, Marchis D., Mauro C., Palmegiano P., Amato
G., Poma Genin E., Abete M.C.: Determination of
melamine in feed: Validation of a gas chromatography–
mass spectrometry method according to 2004/882/CE
regulation. Squadrone Food Control 2010, 21, 714–718.
5. Filigenzi M.S., Puschner B., Aston L.S., Poppenga R.H.:
Diagnostic determination of melamine and related
compounds in kidney tissue by liquid
chromatography/tandem mass spectrometry. J Agric Food
Chem 2008, 56, 7593-7599.
6. Hanson D.J.: FDA will test more food ingredients.
Chemical & Engineering News. April 30, 2007, 85, 22.
7. Heller D. N., Nochetto C. B.: Simultaneous determination
and confirmation of melamine and cyanuric acid in
animal feed by zwitterionic hydrophilic interaction

chromatography and tandem mass spectrometry. Rapid
Commun Mass Spectrom 2008, 22, 3624-3632.
8. Hileman B.: Pressure for food safety reform. Chem Eng
News 2007, 85, 30.
9. Kim B., Perkins L B., Bushway R.J., Nesbit S., Fan T.,
Sheridan R., Greene V.: Determination of melamine in
pet food by enzyme immunoassay, high-performance
liquid chromatography with diode array detection, and
ultra-performance liquid chromatography with tandem
mass spectrometry. J AOAC 2008, 91, 408–413.
10. Litzau J., Mercer G., Mulligan K.: GC-MS screen for the
presence of melamine, ammeline, ammelide and cyanuric
acid (version 2.1),
http://www.fda.gov/cvm/GCMSMelamine.htm, 2007.
11. Muñiz-Valencia R., Ceballos-Magaña S. G., Rosales-
Martinez D., Gonzalo-Lumbreras R., Santos-Montes A.,
Cubedo-Fernandez-Trapiella A., Izquierdo-Hornillos
R.C.: Method development and validation for melamine
and its derivatives in rice concentrates by liquid
chromatography. Application to animal feed samples.
Anal Bioanal Chem 2008, 392, 523–531.
12. Na Yan, Lei Zhou, Zaifang Zhu, Xingguo Chen:
Determination of melamine in dairy products, fish feed,
and fish by capillary zone electrophoresis with diode
array detection. J Agric Food Chem 2009, 57, 807–811.
View publication stats
547
13. Richwine L., Doering C.: US says 2 companies recall
tainted feed additives Reuters 2007, Wed-May,
Washington, p.30.
14. Turnipseed S., Casey C., Nochetto C., Heller Da.N.:
Determination of melamine and cyanuric acid residues in
infant formula using LC-MS/MS FDA, October 2008,
LIB, Silver Spring, USA, No. 4421, Vol. 24.
15. Wang L., Henday S.M., Liu X., Tracy M., Schnute W. C.:
Simultaneous determination of melamine and cyanuric
acid using LC-MS with the Acclaim Mixed-Mode WAX-
1 Column and Mass Spectrometric Detection. Dionex
Corporation, Sunnyvale, USA, www.DIONEX.com.
16. www.avma.org/aa/menufoodsrecall/products.asp.
17. www.cnn.com/2007/US/03/30/pet.food.recall.ap/index.ht
ml.
18. www.fda.gov/bbs/topics/NEWS/2007/NEW01599.html.
19. www.fda.gov/cvm/GCMSMelamine.html.
20. www.fda.gov/cvm/GCMSscreen.html.
21. www.fda.gov/cvm/MenuFoodRecallFAQ.html.
22. www.fda.gov/oc/opacom/hottopics/petfood.html.
23. www.fsis.usda.gov/PDF/FERN_CHE_0003.pdf.
24. www.naturalbalanceinc.com/press_release.html.
25. Xiao-min Xu, Yi-ping Ren, Yun Zhu, Zeng-xuan Cai,
Jian-long Han, Bai-fen Huang, Yan Zhu: Direct
determination of melamine in dairy products by gas
chromatography/mass spectrometry with coupled column
separation. Anal Chim Acta 2009, 650, 39–43.