Simultaneous Determination of Melamine, Ammelide, Ammeline, and Cyanuric Acid in Milk and Milk Products by Gas Chromatography-tandem Mass Spectrometry

Objective To develop an analytical method for simultaneously qualitative and quantitative determination of melamine and triazine-related by-products including ammelide, ammeline, and cyanuric acid in milk and milk products by gas chromatography- tandem mass spectrometry （GC-MS/MS）. Methods Melamine and triazine-related by-products namely ammelide, ammeline and cyanuric acid in the samples were extracted in a solvent mixture of diethylamine, water, and acetonitrile （10：40：50, V/V/V）. After centrifugation, an aliquot of the supernatant was evaporated to dryness under a gentle stream of nitrogen gas, and then melamine and triazine-related by-products were derivatized using BSTFA with 1% TMCS. The derivatives of melamine and its analogues were determined by gas chromatography/tandem mass spectrometry using multiple reactional monitoring （MRM） with 2, 6-Diamino-4-chloropyrimidine （DACP） being used as an internal standard. Results The linear detectable ranges were from 0.004 mg/kg to 1.6 mg/kg for melamine, ammelide, ammeline, and cyanuric acid with a correlation coefficient no less than 0.999. The recovery rates of the tour compounds in spiked blank milk powder at concentrations 0.5, 1, 2 mg/kg were between 61.4%-117.2%, and the relative standard deviation was no more than 11.5% （n=6）. The detection limits of melamine, ammelide, ammeline and cyanuric acid in milk powder were 0.002 mg/kg with a ratio of signal to noise of 3. Conclusion This GC-MS/MS method for simultaneous determination of melamine, ammelide, ammeline, and cyanuric acid in milk and milk products is sensitive and specific.

ANGPTL3 negatively regulates IL-1β-induced NF-κB activation by inhibiting the IL1R1-associated signaling complex assembly

Interleukin-1β(IL-1β)-induced signaling is one of the most important pathways in regulating inflammation and immunity.The assembly of the receptor complex,consisting of the ligand IL-1β,the IL-1 receptor(IL-1R)type 1(IL1R1),and the IL-1R accessory protein(IL1RAP),initiates this signaling.However,how the IL1R1-associated complex is regulated remains elusive.Angiopoietin like 3(ANGPTL3),a key inhibitor of plasma triglyceride clearance,is mainly expressed in the liver and exists in both intracellular and extracellular secreted forms.Currently,ANGPTL3 has emerged as a highly promising drug target for hypertriglyceridemia and associated cardiovascular diseases.However,most studies have focused on the secreted form of ANGPTL3,while its intracellular role is still largely unknown.Here,we report that intracellular ANGPTL3 acts as a negative regulator of IL-1β-triggered signaling.Overexpression of ANGPTL3 inhibited IL-1β-induced NF-κB activation and the transcription of inflammatory genes in HepG2,THP1,and HEK293T cells,while knockdown or knockout of ANGPTL3 resulted in opposite effects.Mechanistically,ANGPTL3 interacted with IL1R1 and IL1RAP through its intracellular C-terminal fibrinogen-like domain and disrupted the assembly of the IL1R1-associated complex.Taken together,our study reveals a novel role for ANGPTL3 in inflammation,whereby it inhibits the physiological interaction between IL1R1 and IL1RAP to maintain immune tolerance and homeostasis in the liver.