Application Of Liquid-phase Microextraction For Analysis Of A₂ Components Of Teicoplanin In Human Plasma

Objective:In this paper,several liquid phase microextraction（LPME）methods were established as pretreatment methods for the extraction and enrichment of 5 A₂ components of teicoplanin in human plasma and combined with high performance liquid chromatography（HPLC）for the determination of teicoplanin to improve the detection sensitivity.Methods:（1）In the first part of the paper,a reverse micelle mediated dispersive liquid-liquid microextraction（RM-DLLME）combined with high performance liquid chromatography ultraviolet detector（HPLC-UV）was proposed and applied for enrichment and determination of 5 A₂ components of teicoplanin in human plasma.The reverse micelle solution of cetylpyridinium chloride（CPC）/n-hexanol was used as the extraction solvent for the separation,extraction and enrichment of the teicoplanin in plasma sample.All factors affecting the extraction efficiencies of the target analytes were investigated and optimized.The developed method was validated under the optimum conditions,and the mechanism of RM-DLLME was analysed and explored.（2）In the second part of this paper,a method of fuctionalized liquid film solvent bar microextraction（FLF-SBME）was proposed and combined with HPLC for concentration and determination of 5 A₂ components of teicoplanin in human plasma.In this method,tri-n-octylmethyl ammonium chloride（TOMAC）was used for the modification of PP-HF and the extraction parameters were investigated.Under the optimum conditions,the methodology research was conducted.Moreover,the extraction mechanism was discussed and described.（3）In the third part of the paper,a solvent bar miroextraction based on reverse micelle（RM-SBME）was proposed for extraction and concentration of teicoplanin in human plasma.The target analytes in sample phase were extracted and enriched to the reverse micelle solution fixed in the pores of the hollow fiber wall,and then eluted into desorption solution.All factors affecting the extraction efficiencies of the target annalytes were optimized.Under the optimum conditions,the developed method was validated and combined with HPLC for determination of teicoplanin in human plasma and the extraction mechanism was clarified.Results:In the first RM-DLLME experiment,CPC/n-hexanol reverse micelle solution（15.0mmol/L）was used as the extraction solvent.The p H and volume of sample phase was 11.0and 7.0 m L,respectively.The extraction was performed with hand-shaking for 40 s and the phase sepration was carried out by centrifugation at 4000 rpm for 2 min.Under the optimum conditions,the 5 A₂components of teicoplanin achieved effective enrichment with the enrichment factors of 228-347 and obtained good linearity in the range of0.8-100.0μg/m L with correlation coefficients higher than 0.99.The limits of detection（LODs）were ranged between 0.5-3.0μg/m L and the low limits of quantification（LLOQs）were ranged between 0.8-5.0μg/m L.Relative standard deviation（RSDs）values of intra and inter-day precisions were lower than 10.6%and the average RE%of intra and inter-day were in the range of-4.3%-10.7%and-2.5%-11.3%,respectively.In the second FLF-SBME experiment,the aqueous solution of TOMAC（5.0 mmol/L）was employed to functionalize PP-HF.The p H and volume of sample phase were 8.0 and7.0 m L,respectively.The extraction was performe by magnetic stirring at 1500 rpm for 90min.Under the optimum conditions,the 5 A₂components of teicoplanin achieved effective enrichment with the enrichment factors of 36-59 and obtained good linearity in the range of 1.0-110.0μg/m L with correlation coefficients higher than 0.99.The LODs were ranged between 0.5-3.0μg/m L and the LLOQs were ranged between 1.0-3.0μg/m.The RSDs values of intra and inter-day precisions were lower than 12.2%and the average RE%of intra and inter-day were in the range of-8.5%-9.6%and-5.5%-10.3%,respectively.In the third RM-SBME experiment,TOMAC/n-pentanol reverse micelle solution（6.0mmol/L）was fixed on the wall pores of PP-HF as extraction solvent.The p H and volume of sample phase were 7.0 and 3.0 m L,respectively.The extraction was performe by magnetic stirring at 1500 rpm for 90 min.Under the optimum conditions,the 5 A₂components of teicoplanin achieved effective enrichment with the enrichment factors of30-99 and obtained good linearity in the range of 0.5-110.0μg/m L with correlation coefficients higher than 0.99.The LODs were ranged between 0.3-1.0μg/m L and the LLOQs were ranged between 0.5-3.0μg/m.The RSDs values of intra and inter-day precisions were lower than 11.3%and the average RE%of intra and inter-day were in the range of-9.4%-7.7%and-9.2%-9.1%,respectively.Conclusions:In this study,three microextraction technologies（RM-DLLME,FLF-SBME and RM-SBME）combined with HPLC-UV was developed for simultaneous extraction,enrichment and determination of 5 components of teicoplanin(TA_2-1,TA_2-2,TA_2-3,TA_2-4,and TA_2-5)in human plasma samples,which significantly improved the detection sensitivity of HPLC-UV for teicoplanin and accurately detected the 5 A₂components of teicoplanin in human plasma.In addition,the three methods all have potential to be used for analysis of the trace compounds with high hydrophilicity in complex biological samples.