Abstract The ractopamine in the sample was extracted with alkaline methanol, purified by an SLA solid phase extraction column, concentrated to a constant volume with a 2% acetic acid solution, separated and measured by high performance liquid chromatography-fluorescence detection after passing through the membrane; Column type Waters Symmetry C18 column, 250mm × 4.6mm (id), particle size 5μm; mobile phase is sodium pentane sulfonate solution: acetonitrile (volume ratio 80:20); excitation wavelength is 226nm, emission wavelength is 306nm; flow rate is 1ml / min; The injection volume is 50μl; the detection limit of ractopamine in feed is 0.5mg / kg; the determination of ractopamine has a good linear relationship in the range of 0.02 ～ 0.50μg / ml, the average recovery rate is more than 85.1%, and the RSD is less than 7.5%; The method is simple and sensitive, and can be used for the determination of ractopamine in feed. Keywords ractopamine; residue; feed; high performance liquid chromatography CLC number S816.7 Lake dopamine is a phenylethanolamine β2-adrenergic stimulant, which can promote protein deposition in animals and inhibit fat deposition, and has a nutritional "redistribution effect" [1,2]. Therefore, it is used as a feed additive to increase the lean meat rate of porcine ketone bodies. However, after feeding animals with ractopamine-containing feed, it will cause varying degrees of residue in muscles and tissues. Improper use will lead to varying degrees of poisoning in consumers. In order to protect the health and safety of the people and the healthy and sustainable development of animal husbandry, China has explicitly included it in the list of banned drugs. However, the illegal use of ractopamine in feed or rearing has already appeared and is spreading. Because ractopamine is a new type of β2-adrenergic stimulant, the current detection methods and detection standards are few [3,4], especially in the country, there has not been any literature report. Therefore, it is of great significance to explore the detection method of ractopamine in feed. We compared the effects of extraction agents, extraction methods, solid-phase extraction columns, extraction reagents, and flow relative to the extraction effect. On this basis, we conducted a systematic investigation of the HPLC measurement method, that is, linear range, repeatability test, stability test, addition Standard recovery rate, etc., finally determined a set of ideal detection conditions. 1 Experimental part 1.1 Instruments and reagents Waters 2695 high performance liquid chromatograph; Waters W-474 fluorescence detector; PE LS-45 fluorescence spectrophotometer; KQ-500 ultrasonic oscillator; Sigma 2k15 refrigerated centrifuge; BüCHI B-490 rotary evaporator; MS2 minishaker vortex Mixer; METTLER AG-285 electronic balance; nitrogen blower; SLA solid phase extraction column (500mg / 5ml), provided by Hangzhou Fuyu Technology Service Co., Ltd. The reference product of ractopamine; acetonitrile for high performance liquid chromatography is chromatographically pure, US TEDIA; water is ultrapure water; sodium pentane sulfonate is chromatographic grade; other reagents are analytically pure. 1.2 Chromatographic conditions Column type Waters Symmetry C18 column, 4.6mm × 250mm (id), particle size 5μm; mobile phase is sodium pentane sulfonate solution (take 800ml water, add 20ml glacial acetic acid and 0.87g sodium pentane sulfonate): acetonitrile (Volume ratio 80:20); excitation wavelength is 226nm, emission wavelength is 306nm; flow rate is 1ml / min; injection volume is 50μl. 1.3 Drawing the standard curve Precisely weigh 25mg of ractopamine reference substance into a 100ml brown measuring flask, dissolve in methanol and dilute to volume, shake well to make ractopamine stock solution. Precisely measure 1ml of ractopamine stock solution, place it in a 50ml measuring flask, and dilute with methanol to the mark to obtain ractopamine working solution. Accurately absorb a certain amount of standard working solution, dilute with 2% acetic acid solution, make volume to a concentration of 0.02μg / ml, Standard solutions of 0.04μg / ml, 0.1μg / ml, 0.2μg / ml, 0.5μg / ml were tested by HPLC. 1.4 Sample extraction and purification Weigh 3.0g of feed, add 25ml of 2% ammonia / methanol solution accurately, vortex for 1min, oscillate for 10min, centrifuge at 5 000r / min for 5min, take 10ml of supernatant into the heart bottle, and spin to dry at 50 ℃. Dissolve the chicken heart bottle with 2mL of methanol and 2ml of n-hexane, repeat once, and discard the upper layer; add 2ml of n-hexane, repeat once, separate methanol into a 10ml centrifuge tube, and blow dry with nitrogen at 45 ° C. Dissolve with 5ml of ethyl acetate, add a small amount of anhydrous sodium sulfate, and centrifuge at 5 000r / min for 3min. The washing solution was passed through an SLA solid phase extraction column (first rinsed with 5 ml of ethyl acetate), and the column was washed with 3 ml of 50% acetonitrile / ethyl acetate solution, and then eluted and collected with 5 ml of 50% methanol / ethyl acetate solution. . Make up to 1ml with 2% acetic acid solution, pass through the membrane and put on the machine. 2 Results and discussion 2.1 Optimization of chromatographic conditions Because the maximum ultraviolet absorption wavelength of ractopamine is about 224nm, if this wavelength is used as the detection wavelength, the impurity interference is very large. Since ractopamine has fluorescence absorption, we use a fluorescence detector for detection. The results show that when the excitation wavelength is 226 nm and the emission wavelength is 306 nm, the absorption is strongest. The mobile phase is a certain ratio of sodium pentanesulfonate solution and acetonitrile. By adjusting the ratio, when the mobile phase is sodium pentanesulfonate solution: acetonitrile volume ratio is 80:20, the retention time of ractopamine is more suitable. , To avoid the interference of impurity peaks. 2.2 Linear relationship and linear range The standard curve will be drawn with ractopamine working solution according to the concentration from low to high, followed by high performance liquid chromatography analysis. According to the obtained peak area and the corresponding working fluid concentration, make a standard curve, and calculate the regression equation and correlation coefficient. The regression equation of ractopamine concentration and peak area is: y = 10 080 548x + 6 438, the correlation coefficient r = 0.999 9, the linear relationship is good in the range of concentration 0.02 ～ 1.0ug / ml, Figure 1 is the standard curve of ractopamine Figure. 2.3 Determination of purification conditions 2.3.1 Comparison of different solid phase extraction columns The solid-phase extraction columns used for β-stimulant purification include diatomaceous earth, C8, C18, silica gel, strong cation exchange column, and weak cation exchange column. In this paper, the SLA, SLH, C18, silica gel, and amine-based SPE purification Column for impurity removal and column recovery comparison, the results see Table 1 (addition amount is 5.0 mg / kg). After comparing the above effects, the SLA type SPE purification column was selected as the purification column for the determination of ractopamine in the feed. After the methanol extract of the sample was purified by the SLA column, the purification purpose could be better achieved, and the recovery rate of the column was above 90%. 2.3.2 Determination of solid phase extraction conditions 2ml of methanol and 2ml of n-hexane were used to dissolve the chicken heart bottle, the upper layer was discarded, and some impurities were removed by using the fat solubility of n-hexane, and good results were obtained. After blowing nitrogen to dryness, dissolve with 5ml of ethyl acetate, add a small amount of anhydrous sodium sulfate, remove trace water, and wash the solution through SLA solid phase extraction column. The column was washed with 50% acetonitrile / ethyl acetate solution. The recovery rate was higher when the washing solution was 3ml, and the interference of impurities was less. Then elute and collect with 50% methanol / ethyl acetate solution, first elute with 5ml of eluent; take another 5ml of eluent and repeat once, the same method does not find ractopamine, indicating that 5ml can be adsorbed on the column The ractopamine eluted completely. Therefore, the ideal amount of washing solution is 3ml of 50% acetonitrile / ethyl acetate solution, and the amount of eluent is 5ml of 50% methanol / ethyl acetate solution. Figure 2 is a high-performance liquid chromatogram of ractopamine reference substance, blank sample, and blank added sample. 2.4 Repeatability test Repeat the test for the feed supplemented with 0.5 mg / kg, 1.0 mg / kg and 5.0 mg / kg ractopamine. Take 5 parallel samples for each concentration, calculate the recovery rate and the relative standard deviation within the batch based on the measurement results, repeat the measurement 3 times, and calculate the relative standard deviation between batches. Table 2 is a list of recovery rates of ractopamine at 0.5 mg / kg, 1.0 mg / kg, and 5.0 mg / kg. The above data shows that the recovery rates of ractopamine at 0.05mg / kg, 1.0mg / kg and 5.0mg / kg are relatively stable. At the addition amount of 5.0 mg / kg, the relative coefficient of variation within the batch is 5.0, and the relative coefficient of variation between batches is ≤10.0. On the other hand, the relative coefficient of variation within the batch was ≤10.0 and the relative coefficient of variation between batches was ≤10.0 for the recovery rates at the addition amounts of 0.5 mg / kg and 1.0 mg / kg. In order to ensure the effectiveness of this method, it is determined that the coefficient of variation between parallel determinations of this method in the laboratory is not greater than 10%. 3 Conclusion This method accurately determined the content of the illegal drug ractopamine in the feed. Under the chromatographic conditions of this experiment, the components in the tested sample can be well separated, and the peak time is appropriate. Through the establishment of this method, it provides a technical guarantee for crackdown on the use of ractopamine. references 1 Zheng Chuntian. Research progress of β-stimulants to improve the carcass composition of livestock and poultry. China Feed, 1997 (10), 10 ~ 12 2 Ding Huanzhong, Chen Zhangliu, Yang Zhiling. Pharmacological action and application of nutritional redistribution agent ractopamine. Veterinary medicine and feed additives, 2002 (7) 18 ~ 20 3 Determination of ractopamine in animal tissues by liquid chromatography-fluorescence and liquid chromatography / tandem mass spectrometry.E.Shishani, SCChai.S.Jamokha et al.Analytica Chimica Acta 483,2003,137 ~ 145 4 Identification of ractopamine residues in tissue and urine samples at ultra-trace level using liquid hromatography-positive electrospraytandem mass spectrometry.JPAntignac, P.Marchand, BLBizec et al.J. Chromatogr.B774,2002,59 ~ 66 Firefighting supplies vchomy , https://www.jsvichen.com