Tetracycline class (Tetracyclines, TCs) is a kind of broad spectrum antibiotics produced by Streptomyces with the basic frame structure including their tetracene. It mainly includes Tetracycline (TC), Oxytetracycline (OTC), Chlortetracyline (CTC) and so on. It has been widely used in the treatment of edible animal disease and medicated premix for the prevention and treatment of animal diseases.
Tetracycline class antibiotics are instable both in acidic and alkaline conditions. They contain many hydroxyl groups, enolic hydroxyl and carbonyl group, which can form insoluble chelation with a variety of metal ions under neutral condition. Such as form insoluble calcium or magnesium salts with calcium or magnesium ions, form red complex with iron ion and form yellow complex compound with aluminum ions.
European Union, Japan and the United States set the maximum residue limits of animal tissues and milk in order to effectively prevent the abuse of tetracycline drugs, which stipulated the maximum residue limits in animal tissues is 0.1 mg/mL.
Reagents and materials
Methanol, acetonitrile, formic acid are chromatography pure; Citric acid, disodium hydrogen phosphate, sodium hydroxide and ethylenediamine tetraacetic acid disodium are analytically pure;
Citric acid solution: 0.1 mol/L, dissolve 21.01 g citric acid in water and constant volume to 1L;
Disodium hydrogen phosphate solution: 0.2 mol/L, dissolve 28.41g disodium hydrogen phosphate (Na2HPO4•H2O) with water and constant volume to 1 L;
Mcllvaine buffer solution: Mixture 1 L citric acid (0.1 mol/L) with 625 mL disodium hydrogen phosphate (0.2 mol/L), adjust the pH value to 4.0±0.05 with sodium hydroxide;
Na2-EDTA-Mcllvaine buffer solution: 0.1 mol/L, dissolve 60.5 g ethylenediamine tetraacetic acid disodium into 1625 mL Mcllvaine buffer solution shaking for future use;
5% methanol aqueous solution;
Methanol-acetic acid ethyl ester (1:9, v/v);
Acycline hydrochloride is no less than 97%;
Cleanert® PEP-2 cartridge: 60mg / 3mL
1) Sample extracts
Put 5.0 g (accurate to 0.01 g) sample to the centrifuge tube of 50 mL and then 20 mL,20 mL,10 mL 0.1 mol/L EDTA Mcllivaine buffer solution, respectively. Vortex mixed the sample for 1 min under 1000 rpm, supersonic extraction for 10 min and then centrifuged for 5 min under 3000 rpm. Merge the supernatant (control the volume of the supernatant no more than 50 mL) and constant volume to 50 mL. Mixed the sample and centrifuged for 10 min under 5000 rpm. Filter the sample with rapid filters for future purification.
2) Sample purification
Condition the PEP-2 cartridge with 5 mL methyl alcohol and 5 mL activated water. Take 10 mL extracting solution to pass the SPE cartridge with the speed of 1drop/s. Wash the cartridge with 5 mL water and 5 mL 5% methanol aqueous solution after all the extracting solution outflow and discard all the effluent. Reduced pressure and swab off the cartridge for 5 min and finally elution with 10 mL methyl alcohol+ ethyl acetate (1:9, v/v). Dry the eluent with N2 under 40 °C and then dissolve solution residue with 1 mL methyl alcohol solution (3:7, v/v). Filtered with 0.45 μm filter membrane for future examination.
AB SCIEX API 4500 HPLC-MS
HPLC Column:Durashell C18, 2.5 μm; 100 Å; 2.1 ´ 50 mm
Mobile phase: A-0.3% formic acid aqueous solution, B-0.3% formic acid acetonitrile
Column temperature: 30 °C
Sample size: 5 μL
Ion source: electrospray ion source
Scanning mode: positive ion scanning
Electrospray voltage: 5500 V
Atomization gas pressure: 65 psi
CUR: 12 psi
Aux Gas Pressure: 60 psi
Ion source temperature: 550 °C
Acquisition methods: multiple reactions monitoring (MRM)
Q1 and Q3 are resolution ratio for the unit
Qualitative ion pair, quantitative ion pair, cluster voltage and collision voltage
Table 1 HPLC gradient elution conditions
Table 2 Spectrometry conditions of tetracycline drugs
Results and Discussion
Table 3 illustrates that when using solid phase extraction combined with liquid chromatography - tandem mass spectrometry method to detection three kinds of tetracycline drugs, the sample amount is 10 μg/kg, 50 μg/kg, 200 μg/kg, the recovery rate is 90%-105%, which can meet the requirements for the detection. Fig.1-3 showed the clean-up with Cleanert PEP-2 SPE cartridge and detection with Durashell C18 HPLC column can make better separation to the 3 kinds of tetracyclines drugs. And each material peak shape is good, the retention time and the retention time is stable.
Table 3 Tetracycline drugs standard addition recovery experiment results (n = 3)
Recovery rate /%
We established the LC-MS/MS method for the detection of 3 kinds of tetracycline drugs. The samples with the add amount of 10 μg/kg, 50 μg/kg and 200 μg/kg were established and the results indicated that the recovery rate is 90%-105%, which meet the requirement of national standard. Solid phase extraction method is stable and HPLC Column has good reproducibility, so this method can be used to detect tetracycline drugs residues in food of animal origin.