Zongyi Wang

Screening and determination of melamine residues in tissue and body fluid samples

Melamine is a chemical product that was sporadically mixed into animal feeds to boost protein content. Excessive melamine in animal feed can induce renal failure and even death in animals. The residue of melamine in edible animal products also threatens human health. Currently, there is no real-time and high throughput method to detect residual melamine in animal tissues. Successful development of such methods is very important for fast and on-site screening of melamine residue in animal tissues to eliminate the potential threat to human health. Here we demonstrate the detection of residual melamine from swine and chicken tissues and body fluids using indirect competitive enzyme-linked immunosorbent assay (ELISA) method. A detection sensitivity of 0.5 microg mL(-1) and a limit of detection of 0.05 microg mL(-1) were achieved with this method. A gas chromatography-mass spectrometry (GC-MS) method was also developed to act as a confirmatory and quantitative procedure for the ELISA results. The limits of quantitation (LOQ) of were 0.01 microg g(-1) and 0.005 microg mL(-1) for tissues and body fluids, respectively. The two methods showed good agreement (r(2)>0.992). The method developed was performed on samples of tissues from chickens fed with melamine-spiked feed.

Molecularly-imprinted microspheres for selective extraction and determination of melamine in milk and feed using gas chromatography-mass spectrometry.

Molecularly-imprinted polymers in the form of microspheres were synthesized using the dispersion polymerization protocol; cyromazine was used as dummy template, while methacrylic acid, ethylene glycol dimethacrylate and acetonitrile (MeCN) were used as functional monomer, cross-linker, and porogen, respectively. When compared with the non-imprinted polymer, the molecularly-imprinted polymers (MIPs) showed outstanding affinity toward melamine in MeCN with a maximum binding concentration (B(max)) of 53.20 nmol mg(-1) MIPs, imprinting effect of 4.6, and a dissociation constant (K(d)) of 90.45 microM. After optimization of the molecularly-imprinted solid-phase extraction conditions, a new method was developed to determine the melamine in milk and feed with gas chromatography-mass spectrometry. The performance of this method has been evaluated in the tainted milk and feed in terms of recovery, precision, linearity, the limit of detection (LOD) and limit of quantitation (LOQ). Recovery ranged in samples from 93.1 to 101.3% with intra-day and inter-day relative standard deviation values below 5.34%. The LOD and LOQ of melamine in milk and feed were 0.01 microg mL(-1) (microg g(-1)) and 0.05 microg mL(-1) (microg g(-1)), respectively.

Deposition of Melamine in Eggs from Laying Hens Exposed to Melamine Contaminated Feed

The deposition profile of melamine was studied in eggs obtained from laying hens fed melamine contaminated feed. A total of 180 laying hens were divided into five groups and were fed diets spiked with 0, 5, 25, 50, or 100 mg of melamine per kg of feed. Eggs collected on days 1, 3, 5, 7, 9, 11, 13, and 15 were analyzed by a gas chromatography-mass spectrometry method, which was fully validated for melamine analysis prior to use. For each treatment group, the melamine level in the eggs was similar from day 1 to day 15 (P > 0.05), suggesting that laying hens did not accumulate melamine for later deposition in eggs. The average melamine concentrations in eggs were 0.00 (below limit of detection), 0.16, 0.47, 0.84, and 1.48 mg/kg for the 0, 5, 25, 50, and 100 mg/kg treatment groups, respectively, which demonstrated an apparent dose-response relationship, and a safety threshold of 164 mg/kg melamine in the feed of laying hen was estimated when a maximum tolerance level of 2.5 mg/kg melamine in egg was adopted. These results provide a scientific basis for the risk assessment of melamine in feeds fed to laying hens.

HPLC–MS analysis of iodotyrosines produced by sample hydrolysis: A simple method for monitoring iodinated casein in feed premixes

A new and simple HPLC-MS method was developed for monitoring iodinated casein in feed premixes. In this method, feed premixes were hydrolyzed, and the iodotyrosines thus released were analyzed. Sample pretreatment included precipitation of transition metals ions with Na(2)S, hydrolysis with sodium hydroxide, and cleaning up with an Oasis SAX cartridge. Gradient elution was carried out on a C(18) column with water (containing 0.1% formic acid) and acetonitrile. Ion detection was performed using ESI positive SIM at m/z 262, 308, 388, and 434. Iodinated casein levels were monitored by qualitative analysis of the iodotyrosines released upon sample hydrolysis and by quantifying the 3,5-diiodotyrosine released. The validation data demonstrated that the method was selective and sensitive (<or=0.2mgg(-1)) for iodinated casein and had acceptable accuracy (recoveries: 81.3-106.7%) and precision (RSD: 1.7-16.0%).