The 2021 CCTV March 15 Party reported that farmers in Qingxian County, Hebei Province, secretly mixed clenbuterol into the feed in order to increase meat yield and profits during the sheep raising process. According to incomplete statistics, there have been dozens of clenbuterol-related incidents and notifications in my country in the past ten years, and meat and its products containing "clenbuterol" can be detected almost every year. "Clenbuterol" is mainly a class of substances called adrenoceptor agonists, including clenbuterol, ractopamine, albuterol, phenylethanolamine A and other substances. When used in the breeding process, it can promote the metabolism of animal mechanisms and reduce Skeletal muscle fat, increase lean meat rate and improve meat color and luster. Long-term consumption of livestock and poultry meat with residual clenbuterol will cause various adverse reactions, such as arrhythmia, allergic papules, nausea and vomiting, when the drug accumulates in the body to a certain extent. etc. In severe cases, it may induce coronary heart disease, hypertension, and even malignant tumors. Our country has explicitly banned the use of any clenbuterol in livestock feeding.

Clenbuterol remains in hair for a long time and can still be detected after discontinuation of use for more than 12 weeks. Accordingly, the Ministry of Agriculture and Rural Affairs has formulated technical standards for detecting clenbuterol using animal hair samples. In September 2022, the Ministry of Agriculture and Rural Affairs officially issued an announcement, releasing and starting to implement the "Determination of Clenbuterol, Ractopamine, Salbutamol and Phenylethanolamine A in Animal Hair" detection method, which is used to detect clenbuterol residues in animal hair. . High-performance liquid chromatography-tandem triple quadrupole mass spectrometry for the determination of clenbuterol has the advantages of high sensitivity, easy operation, automated analysis, time saving, good reproducibility, high efficiency and cost-effectiveness, and low false positive rate. With reference to the above standards, this application center has established a rapid and highly sensitive analytical method for the determination of "clenbuterol" residues in animal hair using ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry, providing effective technical support for food safety testing.

Keywords: LC-MS/MS, food, breeding, animal hair, clenbuterol, Ministry of Agriculture and Rural Affairs Announcement No. 600

Experimental part

instrument

Table 1 Liquid chromatography tandem triple quadrupole mass spectrometer

Table 2 Detection parameters of liquid chromatography tandem triple quadrupole mass spectrometer

Reagents and standards

Reagents: mass spectrometry grade formic acid (CNW), chromatography grade methanol (Merck), chromatography grade acetonitrile (Merck); sodium citrate, sodium chloride, sodium acetate (Sinopharm Group); ammonia water (CNW); sodium dodecyl sulfonate (Aladdin)

Pure water: 18.2 MΩ·cm deionized water (25℃);

Standard products: Clenbuterol, ractopamine, salbutamol and phenylethanolamine A are provided by customers; internal standards clenbuterol-D9, ractopamine-D6, salbutamol-D3 and phenylethanolamine A-D3 are purchased from Alta.

Sample preparation

Referring to Announcement No. 600 of the Ministry of Agriculture and Rural Affairs, the specific pre-processing steps are as follows:

Sample Preparation:

Select the hair from the back of the animal, cut it at the root, and save it for later use. Take an appropriate amount of hair and put it into a beaker, add sodium dodecyl sulfate solution, and ultrasonically clean it in an ultrasonic cleaner for 30 minutes, wash the hair with water, dry it in a 40 ℃ electric drying oven, and make it into powder after drying.

Sample preparation:

Weigh the sample into a centrifuge tube, add the mixed internal standard working solution and 0.1 mol/L hydrochloric acid, vortex mix, hydrolyze in a 60 ℃ water bath for 4 h, centrifuge at 7000 r/min for 10 min, and take the supernatant for use. The solid phase extraction column is pre-washed with a small amount of methanol and water in turn, and all the reserve liquid is passed through the column, rinsed with a small amount of water and methanol in turn, drained, eluted with 5% ammonia methanol solvent, collected the eluate, blown dry with nitrogen at 50 ℃, dissolved with 0.1% formic acid acetonitrile solution, filtered through a 0.22 um filter membrane, and used for liquid chromatography-tandem mass spectrometry determination.

Test Results

Sensitivity

Accurately weigh an appropriate amount of the mixed standard working solution and the mixed internal standard working solution, dissolve them in 0.1% formic acid acetonitrile and quantitatively dilute them into a solution with a concentration of 0.50 ng/mL, and test the signal-to-noise ratio (peak-to-peak) of each compound.

Table 1 Signal-to-noise ratio of each compound

Figure 2 Signal-to-noise ratio results of each compound

Sample test results

The test results of the animal hair samples after pre-treatment in 2.6 are shown below.

Table 2 Test results of animal hair samples

*ND=Not Detected

Table 3 Test results of animal hair samples

Spike recovery results

10 uL, 25 uL, and 50 uL of the 100 ng/mL mixed standard working solution were added to the samples, and the samples were processed according to the pretreatment method in 3.6. The test results are as follows. The recovery rates of salbutamol spiked were 86.20%-103.11%, the recovery rates of ractopamine spiked were 80.64%-117.28%, the recovery rates of clenbuterol spiked were 81.93%-109.23%, and the recovery rates of phenylethanolamine A spiked were 71.83%-98.40%.

Table 4 Test results of the recovery rate of each compound

Conclusion

This article uses LC-MS/MS to detect the contents of clenbuterol, ractopamine, salbutamol and phenylethanolamine A in animal hair, and examines the linearity, sensitivity, stability and spike recovery rate of the method. The linear coefficient of each compound is greater than 0.998; the signal-to-noise ratio (peak-to-peak) of the 0.5 ng/mL standard material solution is greater than 27; the standard solution has a concentration of 1 ng/mL, 2 ng/mL, 5 ng/mL and 10 ng/mL. After 6 consecutive injections at mL, the retention time RSD did not exceed 0.16%, and the relative peak area RSD was less than 10%; the spike recovery rate of each compound at the 100 ng/mL concentration level was between 80% and 120%. The above results show that this method has high sensitivity and good precision, and meets the requirements of the Ministry of Agriculture and Rural Affairs for the determination of clenbuterol, ractopamine, salbutamol and phenylethanolamine A in animal hair.

appendix

Equipment and consumables solutions

1. EXPEC 5210 configuration details

2.Standard products