| With the development of feed industry, the demand for feed grease is gradually increasing. Theproblems of protein anabolic hormones residue are becoming increasingly prominent. Protein anabolichormones play an important role in promoting animal growth and improving feed conversion rate.However, the residue is also harmful to the health of people. Recent studies have shown that residualprotein anabolic hormones in feed grease could be assimilated into animal tissue, and then are absorbedby the human body. Long-term abuse of protein anabolic hormones not only might raise the risk ofcontacting disease such as cancer, as well as neonatal malformation, but also have negative effects onthe adolescents’ growth and development. However, at present, there is no about detection method ofprotein anabolic hormone in feed grease. Therefore, in order to confirm the current condition ofanabolic hormones residues in feed grease and provide basic data for evaluation of feed oil safety, weestablished a highly sensitive detection method and used it to detect the hormones residue of samplesfrom different feed companies in Shandong province in this study.In this study, the protein anabolic hormones (including Testosterone, Progesterone, TestosteronePropionate,17-methyltestosterone, Boldenone, Trenbolone， Nandrolone， Dehydrotestosterone,Nandrolone17-propionate，Metandienone) were detected based on LC-MS-MS. The extraction reagentselection, degreasing solvent and solid phase extraction conditions were mainly tested because the fatcontent was high in the sample and difficult to be extracted. In order to improve the precision andaccuracy, the parameters of LC-MS-MS were studied and optimizated. In addition, we also determinedthe limit of detection (LOD) and limit of quantification(LOQ), and assayed the recovery rate, linearrelationship and the relative standard deviation (RSD) of this method. The best experimental conditionsand the sample testing results are as follows:1. Pretreatment：The sample was dissolved in acetonitrile and frozen at-18℃.The freezed-lipidwas filtrated and removed. Adding the hexane and sodium hydroxide produced saponification reaction.Above steps were repeated2times. The hexane solution was harvested and was purified by CN-E solidphase extraction, and then was eluted by Ethyl acetate and n-hexane（1+3）. The final solution wasobtained and analyzed by LC-MS-MS.2. Detection with LC-MS-MS：Agilent Eclipse Plus C18Column was selected. The target hormonwas separated by gradient elution method with Acetonitrile plus0.2%Formic acid water as the mobilephase. The flow rate was0.25mL/min. Multi-reaction monitoring (MRM) mode was used for signalacquisition of the anabolic hormone standard solution. The positive polarity mode and ESI ion sourcewere selected in the process of scanning. Ten different kinds of hormones can be completely separatedwithin15minutes.3. Assessment of detection method：Under the range of1.0-200ng/ml, the calibration curvesshowed good linearity with correlation coefficient (r) of>0.99. The LOD is1ng/ml and the LOQ is 50ng/ml. The RSD is not more than15%. These ten kinds of hormones are easily dissolved in fats, andare difficult to extract resulting in a recovery rate between55%-82%.4. The results of detecting sample: The132samples from113feed companies locating in28citiesand counties of Shandong province (including Jining, Tai’an, Laiwu, Weifang, Dongying, Linyi etc.)were tested with LC-MS-MS. The grease included in those samples is from more than90oil productionfactories. We found that nine of ten kinds of hormone residues were not found. The levels ofprogesterone were above the limit of detection in11batches of sample, but were below the limit ofquantification.In conclusion, these results showed that the detection method of protein anabolic hormones basedon LC-MS-MS is not only suitable for detection of the residue drug in feed grease, but also couldeffectively avoid false positiveresults results, therefore, have the very good practicability in the foodsafety controls. |
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