Trace analysis of melamine in milk powder using surface enhanced Raman scattering spectroscopy

Abstract

Melamine (1, 3, 5-triazine-2, 4, 6-triamine) is an important organic chemical and widely used in industry. Because of its high nitrogen level (66% by mass), melamine has been used for illegal additives in pet food and milk products to produce an incorrectly high readout in the measurement of protein when traditional Kjeldahl method is performed to determine protein content based on total nitrogen. However, the currently used analytical methods for the detection melamine, such as high performance liquid chromatography-ultraviolet visible spectroscopy (HPLC-UV), high performance liquid chromatography-mass chromatography (HPLC-MS), gas chromatography-mass spectrometry (GC-MS), ultrasound-assisted extractive electrospray ionization mass spectrometry (EESI-MS), and enzyme-linked immunosorbent assay (ELISA), are laborious, high cost and time consuming. Therefore, it is still necessary to establish a rapid and feasible method for trace analysis of melamine in milk powder. In this study, a rapid and sensitive analytical method for detection of melamine was developed by using surface enhanced Raman scattering (SERS)-based microfluidic sensor. Firstly, we studied SERS spectra of melamine using silver nanoparticles (AgNPs) as SERS-active substrates. Compared with conventional Raman spectra, SERS displayed high sensitivity for the determination of melamine. SERS method showed a good linear relationship between Raman intensity and the concentration of melamine in the range of 1 ppm ~ 400 ppm. The detection limit of this method was estimated to 0.5 ppm, which is much lower than the safety limit of 2.5 ppm in the USA and EU, and 1 ppm for infant formula in China. Secondly, this method was used for the qualitative analysis of melamine in milk powder. We established a new method for extracting melamine from melamine-contaminated milk powder by using of acetonitrile and acetic acid as extraction solvents. The recovery of melamine was in the range of 86.9% ~ 106.2% by using acetonitrile as an extraction solvent, which is much better than the recovery obtained by using acetic acid as an extraction solvent. Therefore, acetonitrile was selected as an extraction solvent for further experiments. Under optimum conditions, SERS was able to rapidly detect as low as 0.1% melamine in milk powder. We also developed a microfluidic system coupled with a SERS detection for the trace analysis of melamine. Acetic acid was used as a solvent to detect melamine in the microfluidic chip. The detection position for SERS measurements was optimized. This SERS-based microfluidic sensor showed a good linear relationship between Raman signal and the concentration of melamine in the range of 1 ppm ~ 400 ppm with the detection limit of 0.5 ppm. In summary, a rapid and highly sensitive detection method has been developed by using SERS-based microfluidic sensor for the trace analysis of melamine. We also established a new method for extracting melamine from contaminated milk powder. By using acetonitrile as an extraction solvent, a satisfactory recovery could be actived. This SERS-based microfluidic sensor has the promising potential for on-site and real-time detection of melamine in raw milk, infant formula, and other milk products.