Study On Sensitive Determination Of Tetracycline Antibiotics And Their Derivatives By Isocratic HPLC Method

The high antimicrobial activity and relatively low cost of tetracycline antibiotics are responsible for their wide current use not only in the medical practice for treatment of illness,but also among the most extensively used growth promoters and therapeutic drugs in animal agriculture or in the food industry for growth promotion and disease preventing,increasing the period of storage of foodstuffs,or to prevent losses during transportation.While the incorrect use of antibiotics in veterinary practice may leave residues in edible tissues.This study is intended to establish a simple,sensitive and effective method for qualitative and quantitative detection of 8tetracycline antibiotics?minocycline?MINO?,4-epitetracycline?4-ETC?,oxytetracycline?O TC?,tetracycline?TC?,4-epi-chlortetracycline?4-ECTC?,chlortetracycline?CTC?,methacycline?MTC?and doxycycline?DC??in bovine milk,using an isocratic RP-HPLC method with UV detection.By comparing several different SPE column products that commonly used in pretreatment of TCAs and optimizing their extraction process,a high solid-phase extraction method with high recovery rate,simple process,and low price is used for milk sample detection.1.A new method for isocratic HPLC analysis of TCAs.Through the optimization and screening of mobile phase modifier concentration,mobile phase ratio,detection wavelength,flow rate,column temperature,and column chromato-graphy,an ultra-sensitive HPLC-UV method for simultaneous determination of eight tetracycline antibiotic residues in milk was developed successfully within 13 minutes.The sample to be tested enters the high performance liquid chromatographic system through a manual injector and the injection volume is 200?L.The chromatography was performed using a Waters XBridge C₁₈ column?150mm×4.6mm I.D.,3.5?m?with a innovative mobile phase:aqueous methanesulfonic acid?p H 2.5,3.5mmol/L?-methanol-acetonitrile?68:20:12,v:v:v?.The flow rate of mobile phase was kept constantly at 1.0 ml/min.UV detector set 350 nm with a run time of 13 min.2.Comparative study on pretreatment of tetracycline antibiotic residues in milk by different SPE columns.This article uses solid phase extraction technology to pretreat milk samples.Several reversed-phase SPE columns with good extraction effect on tetracycline antibiotics were selected,and the solid-phase extraction process was gradually optimized to compare the extraction effects of different SPE columns.The Oasis HLB solid phase extraction column was finally determined.The 0.1 mol/L Na₂EDTA-Mcllvaine buffer solution with a pH of 4.0±0.05 was used as the extract.The volume of each extract was 20 mL and the extraction was repeated twice.After sample loading,the tube was then washed with 1.0%formic acid solution and pure water respectively.Elution was performed with acetonitril and samples were dried under a stream of nitrogen.The evaluation results show that under the optimal conditions of high performance liquid chromatography,the calibration curves showed good linearity?R²?0.9990?for all analytes in the range of 20nmol/L⁴000nmol/L,and the limits of detection is as low as 0.8 nmol/L.The Oasis HLB solid phase extraction column was used to pretreat the milk samples.Average recoveries of the eight tetracyc line antibiotics from spiked milk samples were in the range of 63.53%¹14.04%,with their standard deviations less than 8.30%within a day and 14.81%between days.The analytical method was successfully applied to milk samples.This study established a simple and sensitive HPLC-UV method for the determination of eight tetracycline antibiotic residues in milk.Compared with other systems,the elution method is simple and the selectivity is improved.The innovative choice of methanesulfonic acid as a mobile p hase modifier added to the mobile phase system not only allows the analysis of basic compounds,but also has good compatibility with mass spectrometry detection.At the same time,it also provides new research highlights for the selection of suitable mobile phase modifiers in related HPLC studies.The actual sample detection shows that the method is easy to operate,fast in analysis,good in selectivity,and high in recovery.It has a very broad application prospect.