Determination of Melamine Residue
in Milk Powder and Egg Using Agilent
SampliQ Polymer SCX Solid Phase
Extraction and the Agilent 1200 Series
HPLC/UV
Abstract
This method was developed for the determination of melamine in milk powder and
egg. Solid phase extraction (SPE) and HPLC/UV are used consistent with the Chinese
regulatory method. The sample preparation is performed using a polymeric mixed
mode strong cation exchange resin. The separation and detection are performed by
HPLC/UV. The limit of detection is 10 µg/kg. Linear calibration curves were obtained
over the calibration range of 1 to 20 mg/kg. Overall recoveries range from 84 to
96 percent, with RSD values below 3.0 percent.
Authors
Chen-Hao Zhai and Yun Zou
Agilent Technologies Co. Ltd.
412 Ying Lun Road
Waigaoqiao Free Trade Zone
Shanghai, 200131
China
Carol Ball
Agilent Technologies, Inc.
200 Regency Forest Drive, Suite 330
Cary, NC 27511
USA
Application Note
Food Safety
2
Introduction
Melamine made headlines in September 2008 because it was
found to be the contaminant responsible for the deaths of
several infants and making many more sick. Melamine is used
as an adulterant in milk and milk products because it causes
a false positive value in a protein content measurement. The
presence of melamine in foods is strictly regulated world-
wide. Because milk is used in many other products for human
and animal consumption, melamine is now detected in other
food products in China and many other countries. The analy-
sis for melamine is proceeding in many labs all over the
world.
The Chinese government released a government regulation
(GB/T 22388-2008) that established analytical methods for
melamine in raw milk and dairy products and set a maximum
residue limit (MRL) for melamine in food of 2 mg/kg. This
application note describes the implementation and optimiza-
tion of the solid phase extraction (SPE) method used for milk
powder and egg described in GB/T 22388-2008.
Experimental
Reagents and Chemicals
Melamine was obtained from Sigma-Aldrich (Shanghai,
China). A 1 mg/kg melamine stock solution was prepared in
methanol and kept in the freezer (–4 °C). Appropriate dilu-
tions using mobile phase generated working solutions that
were prepared fresh daily.
Sample Extraction for Milk Powder or Egg
For egg, homogenize, place in a clean, sealed container, and
store in the freezer at –4 °C.
For homogenized egg and milk powder, weigh 2.0 ± 0.01 g of
sample and add to a 50-mL centrifuge tube. Add 15 mL of
1% trichloroacetic acid in water and 5 mL of acetonitrile, then
cap. Sonicate for 10 minutes and then place samples on a
vertical shaker for 10 minutes. Centrifuge the samples for
10 minutes at 4,000 rpm. Wet a filter paper with 1% trichloro-
acetic acid in water, then filter the supernatant into a 25.0-mL
volumetric flask and bring to volume with 1% trichloroacetic
acid in water. Transfer a 5.0 mL aliquot of the extract into a
glass tube, and then add 5.0 mL purified water. Mix thorough-
ly on a vortex mixer.
SPE Purification
Agilent SampliQ SCX SPE cartridges (p/n 5982-3236, 3 mL, 60
mg) were used to clean up sample extracts. All SPE steps,
including conditioning, sample load, wash, and the final elu-
tion, are performed without vacuum with a flow rate between
0.5 and 1 mL/min. For the drying step, vacuum is applied to
quickly dry the cartridges. The procedure used for the SPE
extraction is shown in Figure 1. Cartridges were conditioned
with 3 mL of methanol, then 5 mL of water. A 10-mL prepared
sample solution (equivalent to a 0.4-g sample) was passed
through the cartridge. After the sample effused completely,
the cartridge was washed with 3 mL of water and 3 mL of
methanol. The entire effluent was discarded. The cartridge
was dried under negative pressure below 2.0 kPa for 3 min-
utes. Finally, the cartridge was eluted with 6 mL of 5% ammo-
nium hydroxide in methanol. The eluent was collected and
dried under nitrogen at 45 °C. The resulting residue was
resuspended and made to a constant volume of 1 mL using
the mobile phase. Then the residue was filtered through a
0.45-µm filter membrane (p/n 5185-5836) and analyzed.
Figure 1. Melamine SPE procedure.
Sample Load:
10 mL sample solution
Condition:
3 mL methanol and 5 mL water
Wash 1:
3 mL water
Wash 2:
3 mL methanol
Elute:
6 mL 5% ammonium hydroxide in methanol
Dry under N , reconstitute residue with
mobile phase
Dry cartridge under vacuum
3 minutes
2
HPLC Conditions
Samples were analyzed on an Agilent 1200 Series HPLC with a diode array
detector.
Column: Agilent ZORBAX SB-C8
5 µm, 250 mm × 4.6 mm id (p/n 880975-906)
Flow rate: 1.0 mL/min
Column temperature: 40 °C
Detector wavelength: 240 nm
Injection volume: 20 µL
Mobile phase: Acetonitrile-Buffer (15:85)
Buffer: 10 mmol/L citric acid and 10 mmol/L sodium
octanesulfonate solution with a pH 3.0
Chromatography: Isocratic