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Author; Jiang Wei, Fang Xiaoming, Tang Yifeng, Pang Guofang
Objective To establish a method for the determination of melengestrol acetate, megestrol acetate and chlormadinone acetate residues in fat. Methods chromatographic column: Kromasil C 18(250mm×4.6mm, 5 μ m); Mobile phase: acetonitrile-water (65: 35); Flow rate: 1.0 ml/min; Column temperature: 35℃; Detection wavelength: 292nm；; Results The average recovery rate of samples was 86.7% ~ 965438 0.8%, and the relative standard deviation was 4.5% ~ 6.0%. The average recovery rate of samples was 865438 0.4% ~ 95.0%, and the relative standard deviation was 3.7% ~ 7.65438 0%. Conclusion The method is simple and accurate, and can be used for the determination of the residual amounts of medroxyprogesterone acetate, megestrol acetate and chlormadinone acetate in fat.
High performance liquid chromatography; Fat; Medroxyprogesterone acetate; Megestrol acetate; chlormadinone acetate
Determination of Progesterone in Beef and Pork Fat by HPLC
Objective To establish a method for the determination of medroxyprogesterone acetate, chlormadinone acetate and megestrol acetate in beef and pork fat. Methods The chromatographic column was c 18(250mm×4.6mm, 5μm) and the mobile phase was acetonitrile-water (65:35). The flow rate is 65438 0.0 ml/min; The column temperature is 35℃; The detection wavelength was 292nm. Results The average recovery rate in the laboratory was 86.7% ~ 965438 0.8%, and the relative standard deviation was 4.5%~6.0%. The recovery rate of inter-laboratory standard addition is 865438 0.4% ~ 95.0%, and the relative standard deviation is 3.7% ~ 7.65438 0%. The LOQ is 10 μ g/kg. Conclusion This method is simple and sensitive. It is suitable for the determination of melengestrol acetate, chlormadinone acetate and megestrol acetate in beef and pork fat.
Keywords high performance liquid chromatography; Fat; Melinoprogesterone acetate; Chlormadinone acetate; Megestrol acetate
Megestrol acetate, chlormadinone acetate and megestrol acetate are synthetic drugs for promoting pregnancy, which have obvious effects of promoting pregnancy and antiandrogen, and can inhibit ovulation. Progesterone can inhibit the release of pituitary gonadotropin to some extent. Animal experiments show that it can increase the stillbirth rate and teratogenesis, and its side effects are: (1) nausea, dizziness and burnout; (2) Breakthrough bleeding; (3) Taking it during pregnancy will increase the masculinity of female offspring. Progesterone drugs can enhance the deposition of substances in the body, improve production performance, and quickly produce significant and direct economic benefits, so they are very attractive to producers. Progesterone drugs (non-therapeutic use) have been used in animal husbandry for a long time, but long-term consumption of meat food containing hormones, even if the content is small, will obviously affect the hormone balance in the body, and there is a risk of cancer, resulting in abnormal development of children and other hazards. Therefore, it is necessary to develop a method that can detect progesterone residues.
There are many methods to detect progesterone, such as liquid chromatography/mass spectrometry (LC/MS) [1], gas chromatography/mass spectrometry (GC/MS) [2,3] and liquid chromatography [4-6]. In this paper, high performance liquid chromatography (HPLC) was used to detect progesterone in cattle and pig fat.
1 reagents and instruments
1. 1 Reagents: The standard reagents such as melengestrol acetate, chlormadinone acetate, Megestrol acetate (Sigama Company), acetonitrile (HPLC grade), methanol (HPLC grade) and ethyl acetate (HPLC grade) are analytically pure. Water is made by Milli-Q purification system (Millipore Company).
(1) Standard stock solution: 1000 μ g/ml. Accurately weigh 0.050g of melengestrol acetate, chlormadinone acetate and megestrol acetate into a 50ml volumetric flask, and use methanol to fix the volume. Stored at 4℃, it can be used for one year.
(2) Mix the intermediate solution I: 100μ g/ml, respectively absorb 100ml of standard stock solutions of melengestrol acetate, chlormadinone acetate and megestrol acetate in 100ml volumetric flask, and make constant volume with methanol. Stored at 4℃, it can be used for one year.
(3) Mix intermediate solution II: 1.0μ g/ml, put 1.00ml mixed intermediate solution I in a 100ml volumetric flask, and use methanol to make constant volume. It can be stored at 4℃ for half a year.
(4) mixing standard working solution: respectively absorb 10, 20, 40, 80 and 100μl mixed intermediate solution II, and add it to 980, 970, 950, 9 10 and 890μl acetonitrile-water (65:35). The mixed standard working fluids with concentrations of 0.0 10μg/ml, 0.020μg/ml, 0.040μg/ml, 0.080μg/ml and 0. 10μg/ml were obtained for the liquid chromatography determination in the same day.
1.2 instrument waters liquid chromatography system, 5 10 pump body, 486 ultraviolet-visible light detector, Emprower pro chromatography software. Organic drying associates (Jnc). ); Solid phase extraction unit (supelco); Cryogenic centrifuge (Eppendorf, Germany); Vortex mixer (XW-80A, Instrument Factory of Shanghai Medical University); CN solid phase extraction column (3ml, Waters Company).
2 determination steps
2. 1 sample preparation and preservation
2. 1. 1 Preparation of samples About 5g of adipose tissue of pigs or cattle was cut into small pieces, then put into a funnel with glass wool at the bottom, put into a 150ml beaker, and put the beaker into a microwave oven. According to the amount of boiled fat, heat it with maximum power for 30 ~ 60s. If the fat does not melt, heat it repeatedly for 30 ~ 60s after an interval of 30 ~ 60s until the liquid fat flows out and drops it into the beaker through the funnel. Put the boiled fat oil into the sample bottle, seal it and mark it.
2. 1.2 Preservation of samples The boiled fatty oil samples were frozen at-18℃.
2.2 extract and weigh the boiled fat oil 2.00 0.01g, put it in a 50ml centrifugal tube with a plug, add 5ml acetonitrile, keep the temperature in a water bath at 60℃ for 3 37minutes to melt the solid fat, vortex and oscillate 1min, and centrifuge at -5℃ for 7 minutes (centrifugal force1/kloc-) Add 2×2ml of n-hexane into the combined acetonitrile extract, swirl for 65438 0 minutes, centrifuge at -5℃ for 5min (centrifugal force is 1 160g), and discard the n-hexane layer. The acetonitrile extract was dried at 60℃ with a nitrogen dryer, and the residue was saponified.
2.3 saponification: add 4ml of n-hexane, 1ml 0. 1mol/L sodium hydroxide solution and 0.5ml 1.0mol/L magnesium chloride solution to the residue (item 2.2) in turn, mix them evenly in a vortex mixer 10s, and keep the temperature in a water bath at 60℃. Add 4ml of n-hexane to the precipitate, mix well, heat in a water bath at 60℃ for 15 minutes, centrifuge at -5℃ for 5 minutes (centrifugal force 1 160g), and combine the supernatants. Blow dry at 60℃ with a nitrogen blower, dissolve the residue with 1.0ml n-hexane and purify it.
2.4 Purification Pour the saponified sample solution (item 2.3) into the CN column pretreated with 5ml of ethyl acetate and 6ml of n-hexane, wet the glass tube with 2× 1ml of n-hexane, and pour the washing liquid into the CN column. After the circulation of the sample liquid, the column was washed with 5ml of n-hexane and 6 ml of n-hexane solution containing 5% ethyl acetate in turn, and then the vacuum pump was started to discharge the liquid in the column, and the suction was kept for 2 minutes. Finally, it was eluted with 3.5 ml of 20% ethyl acetate in n-hexane, and the eluent was collected in a 15ml glass test tube and dried at 60℃ with a nitrogen dryer. The residue was dissolved in 990μl acetonitrile-water (65:35) by vortex, and after standing for 65438 0.5 minutes, 65438 00μ l 0.2% hydrochloric acid solution was added, mixed evenly and determined by liquid chromatography.
2.5 determination
2.5. 1 HPLC conditions: Kromasil C 18 column (250mm×4.6mm, 5 microns); Pre-column: C 18 column (12.5mm×4.6mm, 5 microns); Mobile phase: acetonitrile-water (65: 35); Flow rate: 1.0 ml/min; Column temperature: 35℃; Detection wavelength: 292nm；; Sample volume: 40 microliters
2.5.2 Determination by liquid chromatography According to the contents of three kinds of progesterone in the sample solution, select a standard working solution with similar concentration, and the response values of three kinds of progesterone in the standard working solution and the sample solution should be within the linear range of instrument detection. Insert standard working solution and sample solution into the sample for determination. See figure 1 for the chromatogram of the standard under the above chromatographic conditions.
Fig. 1 chromatograms of megestrol acetate, chlormadinone acetate and megestrol acetate (100ng/ml) 1- megestrol acetate, 2- chlormadinone acetate and 3- megestrol acetate.
Determination of progesterone residues in pig and bovine fat by high performance liquid chromatography.
3 Results and discussion
3. 1 linear range The concentration ranges of melengstrone acetate, chlormadinone acetate and megestrol acetate are 0.0 10 ~ 0. 10μ g/ml, and there is a good linear relationship between the peak area and the concentration, and the correlation coefficient (γ2) is greater than 0.998.
3.2 Recovery rate, precision and limit of quantitative detection In this experiment, two kinds of fats (cow fat and pig fat) were taken as samples. Add an appropriate amount of standard to 2.0g sample, so that the addition amounts are equivalent to 10, 20 and 50μg/kg, and take 24 samples at each addition level (take 12 samples for each fat). Fig. 2 is a chromatogram of a blank fat sample and an added sample (10μg/kg). Table 1 shows the indoor recovery rate and accuracy of the external standard method for the determination of medroxyprogesterone acetate, chlormadinone acetate and megestrol acetate. Table 2 shows the results of inter-room recovery and accuracy in 8 laboratories. According to the recovery rate test, the lowest concentration that can be reliably determined can determine the quantitative detection limit (LOQ), which is 10μg/kg, which meets the detection requirements of residue limit.
(1)
(2)
(3)
(4)
Fig. 2 Chromatogram of blank fat sample and added sample (10g/kg) A: blank sample of bovine fat; B: blank sample of pig fat; C: beef fat addition sample; D: pig fat addition sample
Table 1 indoor recovery rate and accuracy results (n=24 for each addition)
Table 2 Inter-laboratory recovery and accuracy results of 8 laboratories (n=32 for each addition)
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Determination of Progesterone Residues in Pig and Bovine Fat by Liquid Chromatography: Thought Report Network
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