| Using solid phase extraction technique under the gradient elution mode and through optimizing the chromatographic condition for tetracyclines(TCs) and analyzing its influencing factors,a novel method for simultaneous determination of residues of tetracycline, chlortetracycline, oxytetracycline, doxycycline, demeclocycline, methacycline and minocycline in milk was developed by reversed phase high performance liquid chromatography1.The optimization of the chromatograph condition of TCs The optimal condition for the chromatograph was found through the research of the influential factors such as the property, the proportionment, the acidity, the flow rate and the determining wavelength on the separate degree of TCs. Under above condition, TCs were quickly separated on a Sunfire C18 column (250 mm×4.6 mm, 5μm i.d) using methanol-acetonitrile-0.01mol/L oxalic acid at the flow rate of 1.0mL/min and detected at the wavelength of 355 nm .2. The foundation of the gradient elution mode and the study on its influencing factors Analysis of gradient elution condition and its influential factors was determinated. The results indicated that the direct ratio between elution time and separate degree, and the reverse connection between gradient and separate degree, and the best separation were attained by changing the concentration of organic phase and the curve shape.3. The application of solid phase extraction technology to determine the seven TCs residues in milk Taking into account the ingredient complexity in milk, the conventional pretreating approaches of samples about TCs were improved by the solutions of Mellvaine-EDTA-chloroactic acid with pH 4.0, and the best condition of solid phase extraction was established using 30% methanol for lotion with 8 ml and methanol-acetic acid ester for washing solution at 1.0mL/min. In the pretreatment procedure,the extraction of the seven TCs was carried out by using solid phase extraction (SPE) with C18 column to clean up the extract. The optimum pretreatment conditions were obtained by optimizing the conditions of the extraction and cleaning up which widened the linearity range of seven tetracyclines, and the well linear relation in the concentration range of 20μg/kg~100μg/kg was achieved. Average recoveries obtained by this pretreatment procedure ranged from 78.66% to106% and determinated 0.02μg/mL of limit of detection (LOD).The results showed that the method under the gradient elution was more sensitive, quicker and lower detection limit than that of the isocratic elution, which quickly separated seven tetracyclines in 18 minutes. The LOD was 20μg/kg for TCs that were below maximum residue limits (MRLs), which were regulated by some Europe countries and USA. Recoveries of TCs from samples were more than 78% and RSDs were less than 5.76%. Therefore, this method can be used to constantly monitor and can provide further technical dates for LC-MS corroboration. |
