Clenbuterol detection high performance liquid chromatography (Clenbuterol detection method)
First, how to detect lean meat?
At present, there are four main methods for detecting clenbuterol, namely high performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), capillary zone electrophoresis (CE), and immunoassay (IA). In China, combined with its own actual situation, in 2001 the Ministry of Agriculture first organized the determination of the standard of Clenbuterol in feed. The standard selection identified two: high performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS). The HPLC method is used as a semi-positive method for detecting clenbuterol. The minimum detection limit is 0.05μg/kg. The advantage is that the detection accuracy is high and the false positive rate is low. The disadvantage is that the detection process is cumbersome, the detection time is long, and the cost is expensive. The instrument is difficult to operate and expensive. The advantage of the GC-MS method is that it can qualitatively and quantitatively analyze a specific residue in the presence of multiple residues. Compared with the HPLC method, the GC-MS method has higher detection sensitivity and lower false positive rate. GC-MS has been legally confirmed as a confirmatory method for detecting clenbuterol. The disadvantages of the GC-MS method are similar to those of HPLC.
Currently, Randox Laboratories litd in the UK and R-biopharm in Germany have developed ELISA kits for the detection of clenbuterol in meat and feed.
Second, the reagents and test strips required for the detection of lean meat
(1) Reagents and materials All reagents below are analytically pure reagents unless otherwise specified; water is secondary water in accordance with GB/T6682.
1 Clenbuterol control substance: Clenbuterol hydrochloride should not be less than 98.5%
2 hydrochloric acid 3 anhydrous methanol 4 sodium hydroxide 5 potassium dihydrogen phosphate 6 phosphoric acid 7 hydrochloric acid solution Take hydrochloric acid 4.2ml, add water to 1000ml, that is.
8o. 5mol/L potassium dihydrogen phosphate solution Take 68g of potassium dihydrogen phosphate, add 800ml of water to dissolve and adjust the pH value to 3. with phosphoric acid. O, diluted with water to 1000ml, that is.
90.05 mol potassium dihydrogen phosphate solution Take potassium dihydrogen phosphate 6.88, add 800 ml of water to dissolve and adjust the pH to 3.0 with phosphoric acid, and dilute with water to 1000 mi.
a. Sodium hydroxide solution Take sodium hydroxide 48, add water to dissolve into 100mi, that is.
b. Clenbuterol reference substance stock solution (1mg/mi) Accurately weigh 28.3mg Clenbuterol control crystals into 25ml volumetric flask, dissolve with methanol and dilute to 25ml, save below 20~C, valid period 1 year.
10 Clenbuterol reference working solution (10ttg/mi) Take lmg/mi stock solution o. 5ml to 50mi volumetric flask, add water to 50mi, 2 ~ 8 ~ C to save, valid for 1 month.
(11) Clenbuterol Hydrochloride Control Solution (100ng/m1) Take 1 (ug/m1 working solution o.5ml to: 50mi volumetric flask to 50mi, 2~8~C, valid for 1 month.
(12) Clenbuterol Hydrochloride Detection Kit (2 to 8~C refrigerator)
(13) Solid phase extraction column C, s: 100mglml carbon content ≥ 17%
(2) Instruments and equipment
STI500 high performance liquid chromatography
VI2010 Smart Chromatography Software
UV501 variable light UV detector
Agilent 1100 Diode Array Detector
8725i manual injection valve, VI-11 manual injection valve
VertexC18 4.6*250, 5um high performance liquid chromatography column 1 enzyme-linked immunoreactivity reader 2 analytical balance accuracy 0.00001g
3 balance accuracy 0.01g
4 refrigerated centrifuge 550mi with centrifuge tube 6 homogenizer 7 micro oscillator 8 cyclotron 9 micro pipette single channel 20uL, 50ul, 100uL; multi-channel 50 ~ 250uL,
10 dry heat concentrator 1 air compressor
(3) Determination step 1 Preparation of sample (urine)
Take 1 ml of test urine, centrifuge at 3000r/min for 10 minutes, and use the supernatant as a test material, and directly measure it by enzyme-linked immunosorbent assay.
1 ml of blank urine was taken and centrifuged at 3000 r/min for 10 min. The supernatant was used as a blank sample and directly determined by enzyme-linked immunosorbent assay.
2 ml of blank urine was taken, centrifuged at 3000 r/min for 10 min, and 1.0 ml of the supernatant was added with 10 ug/L of clenbuterol control solution 20/H. The sample was added as a 2 ug/l blank and directly measured by an enzyme-linked immunosorbent assay.
2 sample preparation (liver)
Take about 100g of fresh or frozen melted blank or test pig liver, homogenize with 10000r/rain for 1-2min with homogenizer, make it evenly paste, and store it in a refrigerator below 20~C.
Take the test liver sample after homogenization, as a test material.
A blank liver sample after homogenization was taken as a blank sample.
A blank liver sample (1 soil 0.05) after homogenization was added to add 20 tzL of 10 ng/ml of the Clenbuterol control solution as a 2 ng/g blank addition sample.
a: extraction (liver) take (1 士 o.05) z sample, placed in a 50ml centrifuge tube; add hydrochloric acid solution 5.0mi, vortex mixing, medium-speed oscillation 1.5h; at 10-15~C to 4000r Centrifuge at /mill for 15 min; transfer the supernatant to another 50 ml centrifuge tube, add 300 g of sodium hydroxide solution, mix and shake for 15 min; add 0.5 mol/l potassium dihydrogen phosphate solution to 4. 0 ml, mix, 2 - 8 ~ C placed overnight; take the above solution at 10 ~ 15 ~ C at 4000r / 111113 or higher speed for 15min, the supernatant was used.
b. Purification (liver) C, the solid phase extraction column was pre-washed with anhydrous methanol 3mi, 0.05mol/L potassium dihydrogen phosphate solution 2mi, and the column bed was not dried; the spare supernatant was all passed through the column; 0.05mol/ 2ml of potassium dihydrogen phosphate solution was rinsed, squeezed, and all eluent was discarded; eluted with anhydrous methanol 2mi, the flow rate was controlled below 0.5ml/mm, squeezed out, and the eluate was collected; 50~60~ The eluate was slowly dried with nitrogen or air at C; the residue was dissolved in 1.0 ml of water, and centrifuged at a speed of 4000 r/mill or more for 15 min, and the supernatant was used as a sample solution for enzyme-linked immunoassay.
3 determination a. The room temperature should be controlled at 19~30~C.
b. Take the kit placed at 19-30 ° C for 1-2 h, calculate the number of required enzyme slats according to at least two wells of each standard solution and sample solution, and insert into the frame. Add 100-tL of antibody diluted 100-fold with buffer solution to each well, seal the plate with a sealing membrane, and incubate for 30 min at room temperature.
c Pour out the liquid in the well, and pour the enzyme plate on the absorbent paper to make no residual liquid in the hole. Add 250 pL of water to the micro-L of the enzyme-linked plate with a multi-channel pipette, and then pour out the liquid in the well, then invert the enzyme-linked plate on the absorbent paper, and repeat the operation for 3 times.
d. The standard solution, the blank sample, the blank addition sample and the test material were each taken up to 20 uL, respectively, and placed in the center of different micropores, and the enzyme conjugate 100~I diluted with the buffer solution was added to each well. Mix on a micro-oscillator, seal with a sealing film, and incubate for 30 min at room temperature.
e. The liquid in the well was poured out, and the enzyme-linked plate was inverted on a blotting paper, so that there was no residual liquid in the well, and the plate was repeatedly washed three times.
f. 100 uL of the substrate diluted 10 times with the substrate buffer solution was added using a multichannel pipette, and incubated at room temperature for 15 min in the dark.
g. Use a multi-channel pipette to add 100 uL of stop solution to each well.
h. The enzyme-linked plate was placed in a microplate reader within 1 h, and the absorbance value was measured at a wavelength of 450 nm.
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(4) Calculation The results were analyzed by data analysis software, and the standard curve was drawn, and the content of clenbuterol in the sample was obtained.
(5) Sensitivity and recovery The average detection limit of the method is 0.1 mg/Kg.
The method has a pig urine recovery rate ranging from 60% to 120% at a concentration level of 1 ug/kg, and a pig liver recovery rate ranging from 40% to 120%.
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