Bruker Daltonics
Application Note # LCMS-49
Melamine and Cyanuric Acid Detection
in 5 Minutes using LCMS
Since September 2008, 294,000 infants and young children
suffered urinary problems due to the contamination of
melamine in infant milk powder and were hospitalized.
This hospitalization was required to treat the symptoms
caused by the ingestion of melamine (1) contaminated infant
formula and related dairy products. Previously in 2007, pet
food, animal feed wheat gluten, and other protein-based
foods were found to contain residues of melamine and its
degradation product cyanuric acid (2).
Introduction
There is a requirement for a fast and accurate LC-MSMS
method capable of detecting melamine in various matrixes
at concentrations determined by food authorities. For
example, the Food and Drug Administration has set a limit
of 1 ppm for melamine in infant formula and 2.5 ppm for
other foods. There have been many methods developed for
the determination of melamine and cyanuric acid residues
in infant formula and meat products. LC-MSMS methods
are superior to GC-MS methods in that they have an easy
sample preparation and do not require sample derivatisation.
The high-capacity ion-trap mass spectrometer (HCTultra)
is a highly robust system capable of operating in full scan,
MSn and MRM modes. The option to use the full scan
mode aids in the identification of unknown compounds with
NH2
N
H 2N
OH
N
N
1
N
NH2
HO
N
N
OH
2
sensitivity superior compared with triple-quadrupole mass
spectrometer technology. The HCTultra has a compact
footprint in addition to advanced ease of operation, including
manual optimisation of MRM parameters. Here we report
a recently developed method for the extraction, detection,
and quantitation of melamine and cyanuric acid residues
using high-capacity ion-trap (HCTultra) mass spectrometry
in association with the Acclaim Mixed-Mode WAX-1 column
from Dionex.
Experimental conditions and settings
All organic solvents were HPLC grade purchased from
Honeywell Burdick and Jackson (Morristown, NJ, USA).
Melamine and cyanuric acid standards were purchased
from Sigma Aldrich (St. Louis, MO, USA). 18 MΩ water was
obtained from a Milli-Q™ (Millipore corporation, Billerica,
MA, USA) purification system.
Table 1: HPLC gradient
Instruments employed
Time (min)
%A
%B
0.0
90
10
Calibration standards
2.0
50
50
Standards were prepared from a stock solution of melamine
(1 mg/mL) and cyanuric acid (1 mg/mL) with serial dilutions
performed to prepare standards from 0.1 to 1000 ng/mL.
Stock solutions were made in methanol:water (1:1), whilst
serial dilutions were made in acetonitrile:water (1:4).
2.5
90
10
5.0
90
10
HCTultra, Bruker Daltonics
UltiMate 3000, Dionex
Full scan mass spectrometer parameters
Sample preparation
Infant milk power was extracted in water:acetonitrile (1:4) by
sonication (30 min). The resulting extract was centrifuged
at 10,000 rpm for 10 minutes, followed by the supernatant
being removed and recentriduged at 12,000 rpm for 15
minutes. The resulting extract was diluted as required for
HPLC analysis. Spiked samples were prepared in the same
manner with melamine and cyanuric acid added prior to
extraction at the required concentration.
A high-capacity ion trap LC/MSn system (HCTultra)
from Bruker Daltonik GmbH (Bremen, Germany) was
equipped with the standard ESI ion source. Full scan data
was acquired in UltraScan mode between m/z 50-200,
in positive mode for melamine, and negative mode for
cyanuric acid, and with a fixed accumulation time of 20ms.
The use of run-time segments facilitated the polarity
swapping.
Experimental results
Chromatographic conditions
Full Scan results
Chromatographic separations were carried out using a
Dionex UltiMate 3000 using an Acclaim Mixed-Mode
WAX-1 (Dionex, Sunnyvale, CA, USA) column (2.1 x 150
mm, 5µm) maintained at 30 °C. For the simultaneous
detection of melamine and cyanuric acid the mobile phase
consisted of water / 0.1% formic acid (A) and acetonitrile
(B) gradient at a flow rate of 300 µL/min (table 1). 10 µL
injections of each standard and sample were made.
Figure 1: Blue trace: positive EIC of m/z 127 (melamine)
Green trace: negative EIC of m/z 128 (cyanuric acid)
It is possible to detect both melamine and cyanuric acid in
full scan mode in under five minutes (Figure 1).
MRM results (standards)
A calibrations curve ranging from 1 to 1000ppb is shown for
melamine in Figure 2. The limit of detection was found to be
0.05 ppb which resulted in a signal to noise of 50:1 (Figure
3). Melamine was spiked into infant milk formula (1 ppm and
2.5 ppm) and extracted as per the conditions above. Despite
the low detection limits, these concentrations were chosen
on the basis of infant milk contamination recommendations.
Less than 5 minutes for the detection of melamine and cyanuric acid
Intens.
x10 6
2.5
2.0
1.5
1.0
0.5
0.0
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
Time [min]
Melamine calibration curve
Figure 2: Calibration curve of MRM
for melamine (1 – 1000 ppb)
MRM results (contaminated infant milk powder)
Conclusions
Two contaminated infant milk powders from China were
analysed by the method described. One sample was
found to contain 17 ppm melamine, whilst the second
sample contained 9 ppm melamine, both well over the
recommended concentration for infant milk formula.
It is demonstrated that melamine is conveniently detected at
very low concentrations (0.05 ppb). Using the High Capacity
Ion Trap Mass Spectrometer HCTultra, it is possible to
detect and quantify melamine at concentrations relevant to
food authorities in less than 5 minutes.
MRM mass spectrometer parameters
MRM data for melamine was acquired using the UltraScan
mode with monoisotopic isolation to achieve high
selectivity. The acquisition software was configured for
pseudo MRM mode with the following settings:
Melamine:
Isolated mass: 127.1 Da
Isolation width: 0.6 Da
Amplitude:
0.6 V
Melamine limit of detection is 0.05 ppb
Intens.
6000
4000
2000
0
Authors
Leith J. Fremlin and Matthias Pelzing
Bruker Daltonics Division, Australia.
([email protected] and
[email protected])
For research use only. Not for use in diagnostic procedures.
1.0 1.2 1.4 1.6 1.8 2.0 2.2
Time [min]
Figure 3: positive EIC trace of m/z 85.3 & 68.5 for 0.05ppb injection
of melamine.
Keywords:
Instrumentation & Software
LC-MS Detection
HCTultra
Melamine
Compass QuantAnalysis
MRM
Bruker Daltonics Inc.
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Fax +1 (978) 667-5993
[email protected]
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