Application Of On-line Solid Phase Extraction Coupled To High Performance Liquid Chromatography In The Analysis Of Food And Environmental Samples

In recent years,with the rapid development of our society and economy,the quantity and variety of food are increasing.The phenomenon of hormone residue in milk is common,which is a serious threat to human health.Antibiotic is one of the most common pollutants in water environment,and the abuse and misuse of antibiotics induces the production of multi-drug resistant bacteria,which causes great harm to human health and aquatic ecosystem.Hence,it is imperative to develop a rapid and effective method for the detection of hormones in milk and antibiotics in water.This experiment combined on-line solid phase extraction with high performance liquid chromatography,and established methods for the determination of five hormones in milk and two antibiotics in water respectively.The experimental results are satisfactory and have guiding significance for the practical work of sanitary inspection.PART 1 Simultaneous determination of five hormones in milk by on-line solid phase extraction coupled to high performance liquid chromategraphyObjective: An on-line solid phase extraction coupled to high performance liquid chromatography with UV detection(SPE/HPLC-UV)method was established for the simultaneous determination of five hormones(i.e.prednisolone,dexamethasone,testosterone,methyltestosterone,megestrol acetate)in milk.Method: Milk samples were precipitated by acetonitrile and centrifuged,then the supernatant was injected.The right pump of the double gradient high performance liquid chromatography system was used as the enrichment pump,and the methanol solution with a concentration of 15% was used as mobile phase.The sample was enriched onto the PEP solid phase extraction column,and the impurities flowed out from the liquid pipeline.Then the left pump was used as the analytical pump,and methanol-water as gradient elution mobile phase,then the analysts were transferred from on-line solid phase extraction column to analytical column through column switching valve to be separated.The analytes were detected by a UV detector with switchable wavelength,and quantified by external standard method.Results: After the optimization of liquid chromatography conditions and on-line enrichment conditions,the calibration range of five analytes was 0.05 ?g/mL~25.0 ?g/mL with correlation coefficients(r)remains 0.9998.Limits of detection(LODs)were 0.008 ?g/mL?0.009 ?g/mL?0.009 ?g/mL?0.013 ?g/mL and 0.012 ?g/mL(S/N=3).Limits of quantitation(LOQs)were 0.026 ?g/mL?0.031 ?g/mL?0.029 ?g/mL?0.043 ?g/mL and 0.040 ?g/mL respectively(S/N=10).The average recoveries of five hormones ranged from 75.98% to 115.75%.And the RSD of intra-day and inter-day assays were less than 5.44%,and the sample was stable in 24 hours at room temperature.Conclusion: By using an on-line SPE purification technique,this method significantly simplified sample pretreatment,reduced the use of organic reagents,improved the reproducibility and efficiency of the analysis.The results of methodology verification were satisfactory,and this method was successfully applied to the determination of hormones in milk.PART 2 Simultaneous determination of Clarithromycin and Roxithro-mcin in urban river by on-line solid phase extraction coupled to high performance liquid chromatographyObjective: To establish a method for the simultaneous determination of clarithromycin and roxithromycin in urban river with on-line solid phase extraction coupled to high performance liquid chromatography with UV detection(SPE/HPLC-UV).Method: The water sample was filtered by filter membrane of 0.22 ?m and injected into the chromatographic system directly.The left pump of the system was used as the enrichment pump,and the acetonitrile solution with a concentration of 10% was used as mobile phase.The sample was enriched onto the PEP solid phase extraction column,and the impurities flowed out from the liquid pipeline.After purified and enriched,the right pump was used as the analytical pump,and acetonitrile-phosphate as mobile phase,then the analysts were transferred from on-line solid phase extraction column to analytical column through column switching valve to be separated.The analytes were detected by a UV detector with the detection wavelength was 210 nm,and quantified by external standard method.Results: After the optimization of liquid chromatography conditions and on-line enrichment conditions,the calibration range of the two analytes was 0.50~50.0 ?g/mL and the correlation coefficient were 0.9999 and 0.9998 respectively.Limits of detection(LODs)were: 0.24 ?g/mL for clarithromycin and 0.10 ?g/mL for roxithromycin;Limits of quantitation(LOQs)were: 0.80 ?g/mL for clarithromycin and 0.34 ?g/mL for roxithromycin.The RSDs and recoveries are in the range of 1.70%~4.14% and 94.38%~120.98%,and the sample is stable in 24 hours at room temperature.Conclusion: In this study,a novel method based on on-line solid phase extraction(SPE)coupled to high performance liquid chromatography(HPLC)was established for the simultaneous separation and determination of clarithromycin and roxithromycin in urban river.The present method provides the advantages of good specificity,rapid analysis,high automation and reagents saving.