Study And Application Of Liquid-phase Microextraction On The Analysis In Biological Samples

With the rapid development of the analysis techniques in recent years,the traditional sample pretreatment method couldn't satisfy with the high sensitivity and specificity of the advanced analysis instrument.Many scholars have recently focused on the novel liquid-phase microextraction(LPME)for sample pretreatment,which can provide a high analyte enrichment and excellent sample clean-up with the advantages of low-cost,time-saving,and environmentally friendly.This technique has been successfully used in food safety,environmental monitoring,and bio-pharmaceuticals.In this paper,LPME combined with high performance liquid chromatography(HPLC)are applied for a study and test analysis on the pretreatment of the biological samples.This study consists of three parts.1.Four glucocorticoids(GCs)in animal-derived food are analyzed with dispersive liquid-phase microextraction based on solidified floating organic drop(SFOD-LPME)and HPLC.Objective:To analyze four GCs in animal-derived food by SFOD-LPME combination HPLC.Method:1-Undecanol is taken as the extraction agent to propose SFOD-LPME pretreatment methods for four GCs,including prednisone(PDN),betamethasone(BT),dexamethasone(DX),and cortisone acetate(CSA),several important parameters that influenced the extraction efficiency of SFOD-LPME were investigated and optimized.Result:The optimal extraction factors are under the conditions below:extraction solvent and its volume:40?l 1-undecanol,dispersant and its volume:1200?l THF,the volume of sample solution 15 ml,salt addition:4.0g,pH=7.0,extraction reaction time:15 min,stirring rate:2200 rpm,temperature:35?,and the enrichment factors were 142-276.The calibration graph exhibited linearity over the range of 1.2-200.0 ng/ml for the four analytes,with a reasonable linearity(r~2?0.9990),and the detection limits was 0.39-0.46 ng/ml(0.078-0.23?g/kg),and good spiked recoveries of over 81.5%-114.3%were obtained.Conclusion:A simple,sensitive method based on SFOD-LPME in conjunction with HPLC was successfully applied for GCs extraction and determination in animal-derived food with good reproducibility and high sensitivity.2.The androgen in human urine are analyzed with SFOD-LPME and HPLC.Objective:To analyze the androgen in human urine by the combination of SFOD-LPME and HPLC.Method:The sample firstly enzymatic hydrolyzed through?-Glucuronidase,then the 1-dodecanol is chosen as the extraction solvent to establish the extraction system of SFOD-LPME for the separation,purification,extraction and concentration of androgen in human urine,these androgen including Androstenedione(AD),Testosterone(T),Dehydroepiandrosterone(DHEA),Dihydrotestosterone(DHT),several crucial factors that influenced the extraction efficiency of SFOD-LPME were optimized.Result:The best experimental conditions are listed as below:extraction solvent:20?l 1-dodecanol,dispersant and its volume:200?l methanol,sample solution volume:8 ml,and the pH=7.0,salt addition:2.5 g.The enrichment factors were 241~305 under the optimized conditions.The corresponding linear range for the four analyties exhibited a good linearity(5~200 ng/ml for AD,T and DHEA,100~3000 ng/ml for DHT),the correlation coefficient were varied from0.9978~0.9995,and the detection of limits was 1.28~27.6 ng/ml.The recoveries of real sample spiked were obtained over 82.5~112.4%.The levels of AD(38.5±22.1ng/ml)and T(51.4±23.8 ng/ml)in urine of polycystic ovary syndrome patient were significantly higher than the healthy(P<0.05),there were no differences of the level of DHEA(9.08±2.40 ng/ml)and DHT(496±30.4 ng/ml)between the different groups(P>0.05).Conclusion:This method shows good concentration effect and full purification with well reproducibility and operability,it could be expected to determine the androgen in the female urine for the prevent,diagnosis and prognosis of polycystic ovary syndrome(PCOS).3.Three-phase solvent bar liquid phase microextraction(TPSB-LPME)followed by HPLC are applied for the determination of sarcosine in human urine.Objective:To analyze the sarcosine(SAR)in human urine by using the method of TPSB-LPME combine with HPLC.Method:The method of TPSB-LPME is utilized for purification and enrichment of SAR after derivatization with4-Dimethylarminoazobenzene-4'-sulfonyl Chloride(Dabsyl-Cl)in human urine,and the effective parameters on the extraction efficiency were investigated and optimized.Result:The optimal extraction factors are under the conditions below:extraction solvent:toluene,dispersant and its volume:200?l THF,donor phase:pH=2.0,acceptor phase:40?l of carbonate bicarbonate buffer solution,pH=10.20,salt addition:0.5 g,extraction reaction time:35 min,stirring rate:840 rpm,temperature:35?.The calibration graph exhibited linearity from 0.05 to 25?mol/l for the SAR,with a well linearity(r~2=0.9990),and the detection limits was 0.026?mol/l,and the enrichment factor was as high as 168.The recoveries were in the range of90.5%~93.6%in real urine sample.The level of SAR in urine in PCa group(7.07-14.3?mol/l)was significant higher than the healthy group(2.11-4.51?mol/l)with statistical significance(P<0.01).Conclusion:The proposed method exhibits high sensitivity and high enrichment factor as well as good precision with a simple setup,it has been developed for direct evaluation of SAR after derivatization in urine,and it could provide a great support to the clinical research of SAR in the early diagnosis of prostate cancer(PCa).