Abstract:

【Background】 Blood product for feeds is a kind of unconventional animal-derived feed material. It is made through coagulating the blood of livestock or poultry, cooking at high temperature, pressing out juice, drying and grinding. However, due to the existence of illegal use of β- agonists, the use of blood product from blood containing β-agonists may become a potential source of harm to human health. In order to reduce the safety risk, it is necessary to study the methods of β-agonists in blood product for feeds. The detection methods of β-agonists include enzyme-linked immunosorbent assay (ELISA), high performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC/MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). At present, most of the methods or standards are aimed at commercial finished feed or animal-derived food, however there is a lack of relevant research on the detection technology of β-agonists in blood product for feeds. 【Objective】 In order to study and monitor the status of β-agonists in blood product for feeds, a method of LC-MS/MS combined with solid phase extraction (SPE) was developed for the determination of 18 β-agonists in blood product for feeds.【Method】2 g (accurate to 0.01 g) blood product sample was weighed in 50 mL centrifugal tube, and then 20 mL ammonium acetate extract (pH=5.2) and 50 mL beta-glucuronidase/arylsulfatase were added accurately. The eddies were mixed evenly hydrolyzed overnight at 37 (＞16 hours), then centrifuged for 5 min at 8 000 r/min, the supernatant was transferred to another centrifugal tube, and 0.5 mL 30% perchloric acid solution was added. After vortex mixing for 30 seconds and centrifugation for 5 minutes at 8 000r/min, supernatant was reserved. PCX solid phase extraction column was activated with 3 mL methanol and 3 mL water in turn. The supernatant was load and washed by 3 mL water and 3 mL methanol, then drained, eluted by 3 mL 5% ammonia methanol solution. The eluent was blown to near dry by nitrogen at 50 °C, dissolved by 1.0 mL 0.1% formic acid water + acetonitrile solution (95+5) and filtered through 0.22 μm filter membrane, then detected by Waters TQ liquid chromatography tandem mass spectrometer. The column ACQUITY UPLC BEH C18 (100 mm, 2.1 mm, 1.7 μm) was used as analysis column; acetonitrile and 0.1% formic acid solution were used as mobile phase for gradient elution. The ionization modes of mass spectrometry were electron spray ion source, positive ion detection method and multi reaction monitoring (HRM), the spray voltage was 3.5 kV, the dissolvent temperature was 480 °C, the source temperature was 150 °C, flow rate of the dissolvent gas was 600L·h ^-1, and flow rate of the cone gas was 5 L·h ^-1. The dissolvent gas, cone gas and collision gas were all high purity nitrogen gas.【Result】18 β-agonists showed a good linear relationship between 5 and 100 μg·L ^-1, with correlation coefficients ranging from 0.99 to 0.999. The average recovery of blood powder, plasma protein powder and globulin powder was 65.1%-110% at the levels of 5, 10 and 50 μg·kg ^-1, and the relative standard deviation below 15%. The coefficient of variation between batches was less than 20%. The detection limit was less than 5 ng·g ^-1.【Conclusion】The results of recovery, precision and actual samples showed that the method was suitable for monitoring β-agonists in blood products for feed.

Key words: β-agonists, LC-MS/MS, blood products for feeds