1. Detecting Melamine in the Food Supply Michael Parks CHEM 4101, Fall 2009 12/04/09

2. Melamine Melamine-tainted food caused pet deaths in the US in 2007 and human deaths in China in 2008 Melamine masquerades as protein in some quality control tests Many tests for melamine are not cost effective to use repeatedly

3. Melamine Problem Hypothesis: A cost effective, specific way to detect melamine can be used to keep foods containing toxic levels of melamine out of the food supply. Key Points: Must detect melamine in food specifically, especially as distinct from protein Must be inexpensive and quick enough to use repeatedly as a part of the quality control process for foods Must be versatile enough to work on a range of food products

4. Toxic Levels of Melamine FDA defined as 0.063 mg melamine/kg-body weight per day [1] Ingesting excessive melamine results in formation of crystals in kidneys, causing kidney failure Similar composition to protein under certain analytical methods, making it tricky to detect in food

5. Capillary Zone Electrophoresis Pros:  Useful at relevant range  Recovery of sample unnecessary  Per sample cost very low  Quick results

6. Instrumentation Beckman P/ACE MDQ 32 Karat 7.0 software $36,000 Instrumentation/Solution Parameters [4] :  Buffer: 20 mM NaH 2 PO 4  pH: 2.4 (best separation of peaks)  Capillary: Fused silica, effective length: 40 cm  Detector: UV detector, 206 nm (adjustable)  25 °C  LOD: 0.01 ppm  LOQ: 0.05 ppm  Linear Range: 0.05-1 ppm  s/n: 3  Relative migration time stdev: 0.36% intraday  Relative peak area stdev: 1.67% intraday

7. Other possible methods ProsCons Voltammetry [2] Fastest results Low detection limit (9.6 × 10−9 M) Lengthy setup process for each sample GC/MS [3] Very precise qualitative results Longer time to perform test More difficult to interpret quantity HPLC [5] Low detection limit (1.7 ppb) Non-destructive Higher standard deviation of results

8. Procedure Prepare melamine standard solution (2 ppm) Homogenize the food sample mixed with 1 ml 10% TCA and 2 mL chloroform Centrifuge, filter, and extract the supernatant Prep the capillaries by running DI water, then NaOH, then NaH 2 PO 4 buffer through the capillaries Split sample into control and spike group Spike half of sample with melamine standard solution Run each sample through Beckman P/ACE MDQ Detect ions at 206 nm

9. Data Analysis Compare spiked sample to control sample results to determine melamine peak Calculate area under melamine peak Attribute difference in area to 2 ppm melamine from standard Calculate control melamine content from that attribution

10. Example results [4] Top line: spiked food sample, bottom: control

11. Conclusions Detection of melamine can be done economically with Capillary Zone Electrophoresis This testing method will allow testing to be done before melamine can enter the food supply

12. References [1] Update: Interim Safety and Risk Assessment of Melamine and its Analogues in Food for Humans, United States Food and Drug Administration, 11/28/08 [2] Qian Caoa et al., Electrochemical determination of melamine using oligonucleotides modified gold electrodes, Talanta, Volume 80, Issue 2, 15 December 2009, p. 484-488 [3] GC-MS Method for Screening and Confirmation of Melamine and Related Analogs, United States Food and Drug Administration, 5/7/07 [4] Na Yan, et al., Determination of Melamine in Dairy Products, Fish Feed, and Fish by Capillary Zone Electrophoresis With Diode Array Detection, National Key Laboratory of Applied Organic Chemistry and Department of Chemistry, Lanzhou University [5] Michael S. Filigenzi, et al., The determination of melamine in muscle tissue by liquid chromatography/tandem mass spectrometry, Rapid Communications in Mass Spectrometry, Volume 21, Issue 24, p. 4027-4032