The Study On Steroid Hormones Detection Technology In Raw Milk And Dairy

Milk and dairy products are one of the most popular foods in people's life, due to the rich in protein, vitamins, calcium and other essential nutrients. So its quality and safety issues are very important. A large number of dairy products quality and safety accidents, not only undermined interests of the consumers and society, but also caused huge economic losses of dairy farmers. However, Sheng Yuan milk powder led to the occurrence of infant sexual precocity events in 2013 which let people understand the level of hormones in milk powder is an important factor. Steroid hormone exists as a trace material in the human body, with physiological functions, lack and excess will affect people's health. Using high performance liquid chromatography(HPLC) and ultra high performance liquid chromatography tandem mass spectrometry(UPLC-QTOF-MS) analysis the residue levels of steroid hormone residues in raw milk and dairy products. The main research contents and results are as follows:(1)An efficient method for determination of 4 estrogens(17?-estradiol,estriol,estrone and diethylstilbestrol)with HPLC in milk power and fresh milk was studied. The samples were dissolved in water. Acetonitrile was used to deposit the proteins and Qu ECh ERS method was used to extract 4 estrogens. The fats were removed by hexane.4 estrogens were separated in C18 chromatographic column, using acetonitrile and water as the mobile phase, UV-detector was used at 230 nm. The detection limits(S/N=3) of 17?-estradiol, estriol, estrone and diethylstilbestrol in milk powder and fresh milk were 0.03 mg/kg,0.026 mg/kg,0.03mg/kg,0.012 mg/kg and 0.024 mg/kg,0.021 mg/kg,0.025 mg/kg,0.01 mg/kg. The limit of quantity(S/N=10) in milk powder and fresh milk were 0.1 mg/kg,0.09 mg/kg,0.1 mg/kg,0.04mg/kg and 0.07 mg/kg,0.07 mg/kg,0.08 mg/kg,0.03mg/kg. The recoveries were 81%~97%,the RSD were 3.2%~8.7%.(2)The extraction and purification were optimized by Qu ECh ERS method. The mass spectra were easily influenced by the matrix effect, so the extraction and purification conditions were very important. In this study, the extraction results of methanol, acetonitrile,ethyl acetate and acetone were compared. The results showed that the extraction effect wasthe best, the recovery rate was the highest, and the protein in milk could be effectively precipitated. The effects of three fillers PSA, GCB and acidic alumina were discussed on recovery and matrix effects of samples. Finally, anhydrous magnesium sulfate 900 mg,PSA150 mg, acidic alumina 200 mg were determined which could get better experimental results.(3)13 kinds of steroid hormones(metandienone, boldenone, testosterone etc.) were analyzed with UPLC-QTOF-MS after extraction and cleanup with the optimized Qu ECh ERS method of extraction and purification of steroid hormones in samples of raw milk The limits of detection for the raw milk with this method(S/N=3) was in range of 0.07?g/kg to0.51?g/kg and the limits of quantification(S/N=10) covered the ranges from 0.23?g/kg to1.7?g/kg. With matrix external standard method, the substances presented recoveries over the range80.2 %-99.7%. Qualitative analysis was also done in the MS/MS mode and each debris structure of 13 steroid hormones was achieved.(4)The analysis of steroid hormone levels in raw milk and dairy products, we collected 30 raw goat milk samples and raw cow milk samples in 4 seasons from Shandong province,Sichuan province and Shanxi province and 30 raw buffalo milk samples from Guangxi province in 2013, 2014 and 2015. Using the method of section(3) to analyze the levels of steroid hormones. Among them, the progesterone was widely detected in raw milk, the detection rate was 90.2% and the contents ranged from ND to 9.06?g/kg.The detection rate of raw goat milk was 74.2% and the average content range was from ND to 6.36?g/kg. Estrone was detected in raw milk, the detection rate was lower than 40%, the contents ranged from ND to 4.13?g/kg and estrone was not detected in raw goat milk. Other steroid hormones were not detected. In the seasonal difference, the content of progesterone and estrone in raw milk in winter and summer was slightly higher than that in spring and autumn, and the content of progesterone in different provinces was about 1?g/kg. We selected four kinds of common brands milk and six kinds of brand milk powder on the market. The milk was analyzed by using the method of section 3. The milk powder was managed with the method of section 1.The progesterone was detected in the milk, but the content of progesterone was significantly lower than that of raw milk. No steroids were detected in milk powder.