Abstract
Tetracyclines （TCs） are broad-spectrum antibiotics that are classified as natural or semi-synthetic. Natural TCs include chlortetracycline， tetracycline， and oxytetracycline， while semi-synthetic ones include doxycycline， minocycline， methacycline， and demeclocycline， among others. While TCs are widely used in the livestock， poultry， and aquaculture industries， their indiscriminate use detrimentally affects ecosystems， and residual TCs in animals can adversely affect human health. In this study， we developed an ultra-performance liquid chromatography-tandem mass spectrometry （UPLC-MS/MS） method for determining TCs， including minocycline， 4-epioxytetracycline， 4-epitetracycline， oxytetracycline， tetracycline， demeclocycline， 4-epichlortetracycline， chlortetracycline， methacycline， and doxycycline. The developed protocol was used to establish a method for qualitatively and quantitatively analyzing TCs in animal muscle tissue （pork， chicken， and fish meat）. To this end， we systematically optimized the mass spectrometry （MS） parameters， solid-phase extraction （SPE） cartridge， and extraction conditions of the method. Animal muscle-tissue samples were homogenized and extracted with 10 mL of 80% acetonitrile aqueous solution containing 0.2% formic acid. How the acetonitrile/formic acid ratio affects the TC-extraction efficiency was investigated using one-way analysis. The supernatant was purified using an Oasis PRiME HLB solid-phase-extraction cartridge， evaporated under flowing nitrogen， and redissolved. Two different C(18) UPLC columns were systematically evaluated， and the optimal UPLC conditions were established for the TCs. The Eclipse Plus C(18) column （100 mm×2.1 mm， 3.5 μm） was used for separation. The effects of mobile phases A （0.1% formic acid aqueous solution， 5 mmol/L ammonium acetate aqueous solution， and 5 mmol/L ammonium formate aqueous solution） and B （methanol or acetonitrile） on the separation and response values of the TCs were investigated. Optimal response values and peak shapes were obtained using 0.1% formic acid aqueous solution as mobile phase A and 0.1% formic acid acetonitrile solution as mobile phase B， at a flow rate of 0.2 mL/min and a sample injection volume of 5 μL. Gradient elution was performed as follows： 0-2.0 min， 5%B； 2.0-3.5 min， 5%B-15%B； 3.5-7.0 min， 15%B-20%B； 7.0-9.0 min， 20%B-65%B； 9.0-9.1 min， 65%B-90%B； 9.1-10.0 min， 90%B； 10.0-10.1 min， 90%B-5%B； 10.1-12 min， 5%B. The effect of the glass sample bottle on adsorption was also investigated. Both positive- and negative-ion modes were explored in the UPLC-MS/MS experiment to fully scan the parent ions. Positive mode was selected for electrospray ionization （ESI）. Two product ions that exhibit strong signals and minimal interference were selected for quantitative and qualitative ion analyses， with quantification performed using the external standard method. Tandem mass spectrometry （MS/MS） was performed in positive electrospray ionization （ESI(+)） and multiple reaction monitoring （MRM） modes. The following MS/MS parameters were used： capillary voltage， 0.5 kV； cone voltage （CV）， 30 V； ion-source temperature， 150 ℃； desolvation temperature， 300 ℃； desolvation gas flow， 800 L/h. Other instrument settings， such as the collision energy （CE） and collision gas flow， were also optimized. The TCs exhibited good linearities within the 1-500 ng/mL mass-concentration range， with all correlation coefficients （r(2)） above 0.994， and limits of detection and quantification （LODs and LOQs） of 0.10-0.15 and 0.20-0.50 μg/kg， respectively. The target analytes exhibited average recoveries of between 62.6% and 119.0% at three levels （1.0， 5.0， and 10.0 μg/kg）， with relative standard deviations （RSDs， n=7） ranging from 2.0% to 9.8%. The developed method was used to determine TCs in 60 animal muscle-tissue samples acquired from a fresh-food supermarket. Tetracycline was detected at a rate of 10% in 20 pork samples， while doxycycline was detected at a rate of 15% in 20 chicken samples， and oxytetracycline was detected at a rate of 5% in 20 fish-meat samples. Minocycline， 4-epioxytetracycline， 4-epitetracycline， tetracycline， demeclocycline， 4-epichlortetracycline， chlortetracycline， and methacycline were not detected. The developed method is simple to operate， highly sensitive， and can be used to precisely and accurately determine TCs. Accordingly， it is suitable for determining the abovementioned ten tetracycline antibiotics in animal muscle tissue.