DETECTION OF MELAMINE IN MILK

Nai-Yuan Liu Chem 4101 12/13/2011

What Is Melamine?

    IUPAC name:1,3,5-Triazine 2,4,6-triamine Uses: a very durable thermosetting plastic used in Formica, and melamine foam, a polymeric cleaning product.

High nitrogen content: contains 66% nitogen by mass 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

  Hypothesis: 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  Efficient  Cheap  Low detection limit  Simple method for preparing liquid milk

Other Techniques Can be Used

Alternative method

CE ESl/MS GC/MS Fluorescence

Advantage

fast, high N, good LOD, sensitivity, low sample volume, very small sample value Soft ionization, good LOD, high sensitivity

Disadvantage

Low reproducibility, sample recovery Multiply charged ions, expensive Adequate sensitivity, good stability, good reproductivity More sensitive than UV-vis Need dirvatization, multipled charge ions, expensive, labor intensive Undetectable, need reagent

Instrumental

  Dionex UltiMate® 3000 HPLC system (1200 USD): consisted of an HPG-3400A pump, WPS-3000TSL autosampler, TCC-3000 thermostated column compartment, and VWD-3400RS UV VIS detector.

HPLC:Chromatographic conditions Guard column

WCX-1 Method

Acclaim Mixed WCX-1, 5 μ m, 4.3 × 10 mm Analytical column Column temp.

Mobile phase Flow rate Inject vol.

UV detection Method limit detection Acclaim Mixed-Mode WCX-1, 5 μ m, 4.6 × 250 mm 30 ° C NH4Ac buffer (10 mM, pH 4.3)/CH3CN (8:2,

v/v)

1.0 mL/min 20 μ L Absorbance at 240 nm 0.028 μ g/mL

Procedure

    Measure approximately 10mL of Samples into 15 mL 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 and then following the step above.

μ L of the stock standard solution of melamine to the 15-mL centrifuge tubes together with the measured sample, Run both samples and spiked samples through UV VIS 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.

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

Detected (μ g/mL)

1.8

22

Conclusion

 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.

Reference

    

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.