Detection of melamine in milk

Nai-Yuan Liu
Chem 4101
What Is Melamine?
IUPAC name:1,3,5-Triazine2,4,6-triamine
Uses: a very durable
thermosetting plastic used in
Formica, and melamine foam,
a polymeric cleaning product.
High nitrogen content:
contains 66% nitogen by
Outbreak in China, 2008:A
company induce melamine
into milk causing death of
several infants.
More Information
According to WHO, the tolerable daily intake (TDI)
of melamine is 0.2 mg per kg of body mass.
According to the regulation in China, the amount
lower than 2.5ppm in milk product is permitted.
Ingesting excessive melamine leads to kidney
lithiasis and makes it more possible to have
malignant tumor inside kidney.
High nitrogen content sometimes misleads the
detection of protein in food product by the
commonly used standard test.
Analytical Problem
Company X induce melamine in their milk product
and its amount exceeds the permitted standard.
Importance of solving problem:
Finding an easy, efficient and reproductive way to
be a good food control process.
Choice of Technique: HPLC/UV
Good reproductivity
Not labor intensive
Low detection limit
Simple method for preparing liquid milk
Other Techniques Can be Used
Alternative method
fast, high N, good LOD,
sensitivity, low sample
volume, very small sample
Low reproducibility, sample
Soft ionization, good LOD,
high sensitivity
Multiply charged ions,
Adequate sensitivity, good
stability, good
Need dirvatization,
multipled charge ions,
expensive, labor intensive
More sensitive than UV-vis
Undetectable, need
Dionex UltiMate® 3000 HPLC system (1200 USD): consisted of an HPG-3400A
pump, WPS-3000TSL autosampler, TCC-3000 thermostated column compartment, and VWD-3400RS UVVIS detector.
HPLC:Chromatographic conditions
WCX-1 Method
Guard column
Acclaim Mixed WCX-1, 5 μm, 4.3 × 10 mm
Analytical column
Acclaim Mixed-Mode WCX-1, 5 μm, 4.6 ×
250 mm
Column temp.
30 °C
Mobile phase
NH4Ac buffer (10 mM, pH 4.3)/CH3CN (8:2,
Flow rate
1.0 mL/min
Inject vol.
20 μL
UV detection
Absorbance at 240 nm
Method limit detection
0.028 μg/mL
Measure approximately 10mL of Samples into 15mL centrifuge tubes, dilute it with 1 mL dilute HAc
(3%, v/v), and store it at 4 °C for at least 30 min.
Prepare spiked samples adding 40 μL of the stock
standard solution of melamine to the 15-mL
centrifuge tubes together with the measured sample,
and then following the step above.
Run both samples and spiked samples through UVVIS UltiMate® 3000 HPLC system.
Detect at 240nm.
Data Analysis
Compare the samples to spiked sample results to
determine the melamine peak.
Calculate the area of the peak to determine the
amount of melamine.
Compare the amount of melamine in milk samples to
the permitted amount.
Example Result
Figure 1. Comparison
between samples and
spike samples. Upper
is the sample and
lower line is the spiked
Figure 2. Samples
containing melamine.
Upper line is for
sample A. Lower
line is for sample B
Sample A
Sample B
Quantitative detection of melamine can be done
fast and easy by using HPLC/UV.
This method can be commonly used as a standard
test of melamine to avoid deadly milk product goes
into market.
A Fast Method for Determination of Melamine in Liquid Milk and Milk Powder by
HPLC With UV Detection by Lang Li, Ji Luo, Qun Xu, Jeff Rohrer, and Scott Lefferts
A rapid, acetonitrile-free, HPLC method for determination of melamine in infant
formula Gopalakrishnan Venkatasami, John R. Sowa Jr. ∗Department of Chemistry
and Biochemistry, Seton Hall University
Rapid Determination of Melamine in Liquid Milk and Milk Powder by HPLC on the
Acclaim Mixed-Mode WCX-1 Column with UV Detection
Determination of Melamine in Food by SPE and CZE with UV Detection, Liang Meng,
Guijun Shen, Xiaolan Hou, Lili Wang, College of Environmental and Chemical
Engineering, Dalian University, 116622 Dalian, People’s Republic of China
Detection of Melamine in Milk Products by Surface Desorption Atmospheric Pressure
Chemical Ionization Mass Spectrometry Shuiping Yang, JianhuaDing, Jian Zheng, Bin
Hu, Jianqiang Li, Huanwen, Chen, Zhiquan, Zhou, and Xiaolin Qiao.

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