Study And Application Of HF-LPME As A Sample Pretreatment Method In Trace Matter Analysis

Due to the content of analytes are usually low, and the sample matrix isrelatively complex with many interferents, that we only use higher sensitivitydetermination method to improve the sensitivity for analyzing trace amountanalytes in biological samples, environmental samples and drugs is not well. Ifthe better pretreatment methods are added, the interference of the matrixcomponents to chromatographic system is not only reduced, the sensitivity butalso accuracy of the determination method are increased greatly. Thus, toestablish a better pretreatment method which is simple, quick, and with highsensitivity that is the key of the study in this paper.Such as Liquid-liquid extraction （LLE） and SPE, the traditionalpretreatment methods for separation and purification are tedious,time-consuming, labor-intensive, and harmful organic solvents, which makethese method application limited. Nowadays, miniaturized techniques suchas solid phase microextraction （SPME） and liquid phase microextraction（LPME） have been reported hot topics in field of pharmaceutical analysis. Thesuccess of SPME lies with its simplicity, solvent-free characteristics. However,it also suffers from some problems such as sample carry-over and relativelyhigh cost. While, single-drop liquid-phase microextraction （SD-LPME） isnot entirely satisfactory, because the organic solvent drop is easily lost fromthe needle tip of the syringe during extraction, especially when samples arestirred vigorously to speed up the extraction process. Thus, it is very necessaryto establish a simple, low cost, high selectivity pretreatment method foranalyzing trace compounds in complex samples.In recent years, hollow fiber liquid phase microextraction （HF-LPME） isdeveloped rapidly. Compared with other pretreatment methods, it can providea high analytes preconcentration and excellent sample clean-up, with the advantages that the fiber is disposable after use due to its low cost. Therefore,HF-LPME has been used as pretreatment methods for analyzing somecompounds in relatively simple samples. However, it is still a try that useHF-LPME to analyze some compounds in complex samples.Estrogens or with active estrogen substances can simulate theendogenous estrogen physiological, biochemical role and with antagonistendogenous estrogen effect. So, they are called environmental estrogens. Ifthese environmental estrogens are in the human body, they can interfere thesynthesis, release, transportation, metabolism of estrogen so that it causes theendocrine system function disorder, can damage the stability of theenvironmental body and even can lead to reproductive disorders, growthunusual, metabolic disorder and the risk of cancer. Thus, the relationshipbetween these environmental estrogens and some disease mechanism has beencaused the medical attention. Estrogen in the milk is a kind of environmentalestrogens. In recent years, the changes in the way of raising cattle and theincrease of milk consumption that makes the level of estrogen in milk higher.Due to these estrogens are not easy to degrade in human body, the potentialharm to human health are getting more and more serious. Estrogens in milkare mainly including estrone, estradiol, estriol. While, estradiol is about40%in the total, so it is very important to determinate estadiol in milk. If we useHPLC-UV to analyze estradiol in milk, the level of LOD is only10-6that doesnot meet the content analysis. However, MS detector with high sensitivity isexpensive and not popular, so in this paper we establish a method whichcombined HF-LPME and HPLC-UV to analyze estradiol in milk. In theprocedure, we have investigated the extraction solvents, extraction time,extraction temperature, stirring speed, ion strength. Under the optimizedconditions, the enrichment factor is about200, so the determination level ofHPLC-UV is as high as10-9. This method makes the sensitivity higher and issuitable for the analysis of estradiol in milk.It is reported that the level of estrone, estradiol and their metabolites isclosely related to the mechanism of breast cancer and endometrial cancer. While, these contents that lead to there is no method to determine them inbiological sample are low. If using HPLC-MS, it is not satisfied for the requestof the analysis. The study of the relationship between the level estroneestradiol and their metabolites and the mechanism of breast cancer andendometrial cancer is limited. Thus, it is a key problem to develop a methodwith high sensitivity to study estrogens and their metabolites.This subjest has been combined highly sensitive UPLC/MS analysismethod with dansyl chloride derivative, improving the ssensitivity of theestrogens analysis method, meanwhile with the function of enrichment whichis the HF-LPME sample pretreatment technology. We developed a methodwith high sensitivity which was successfully used to analyze eight estrogensand their metabolites in urine samples. The detection sensitivity of this methodwas up to10-14, together with the MRM detection mode, there were not onlyless disturbance, high sensitivity, but also shorter analysis time. The testresults of20cases endometrial cancer patients and20cases of healthyvolunteers showed that the difference contents of five analytes in totalbetween endometrial cancer and healthy people. That difference is helpful tostudy the relationship between the level estrone estradiol and their metabolitesand the mechanism of breast cancer and endometrial cancer. The developedmethod has provided a reliable way for clinical estrogen and their metabolitesresearch.The application of HF-LPME in the analysis of estrogens and theirmetabolites in biological samples has improved the sensitivity of thedetermination method that suggests it can be widely used in future. Such assome illegal drugs in biological samples these compounds can also bepretreated by HF-LPME. While, methamphetamine is a kind of thesecompounds. Simulants like ephedrine （E） and pseudoephedrine （PE） are pairsof diastereoisomeric sympathomimetic amines that have human centralnervous system stimulating properties. Moreover, they are included in thedoping list of pharmacological forbidden substances indicated by the medicalcommission of the international Olympic committee. Correspondingly, there is an increasing concern in their misuse and abuse, prompting researchers toinvestigate the abuse liability of E and PE in their own right. Otherwise, PEand E is not approved by the FAA （Federal Aviation Administration） providedthey are combined with an antihistamine, because the adverse reactionsexposed by them increased gradually. So the determination of PE and E inairman urine is helpful to ensure the safe medication of them.Thus, a simple and sensitive method for analyzing PE in urine sample byHF-LPME has been developed. In this study, combining separation,purification and concentration in a single step, HF-LPME has been emergedideal clean-up performance. It is necessary to point out here that enrichmentfactor of HF-LPME is as high as220, so that this method was shown highsensitivity. Combined with HPLC, the detection sensitivity is up to10-9and ithas been applied for the determination of PE in urine after oral administration10h; When combined with HPLC-MS, the detection sensitivity is up to10-12and it has been applied for the determination of PE in urine after oraladministration72h. It would be conduced to the study for PE control and hasbeen provide a new method to effectively monitor the medication use andrational drug use for special groups. The developed method has also beenapplied for the determination of ephedrine and pseudoephedrine in urine afteroral administration5h. Furthermore, the proposed method could provided anapproach for E and PE control, and has been provide a new method toeffectively monitor the medication use and rational drug use for specialgroups.PART1Study and Analysis of Estrogens and Estrogen Metabolites byHF-LPME1Determination of trace estradiol in milk by HF-LPME prior to highperformance liquid chromatographyObjective: To establish a method that was used to determinate traceestradiol in milk by miniaturized hollow fiber assisted liquid–phase microextraction （HF–LPME） and high performance liquid chromatography（HPLC）.Method: The experimental parameters of microextraction werecontrolled and optimized: the extraction solvent only was100μL n-octylalcohol, the extraction procedure was finished at a stiring rate of600rpm at25℃for1h. A Kromasil C18column was used. The mobile phase consisted ofmethanol and water （80:20, v/v）, the wave length was280nm and flow ratewas1.0mL·min-1. The direct injection volume was20μL.Results: The enrichment factor of estradiol was200. Under theoptimized experimental conditions, standard curve is linear with linearregression correlation coefficient typically greater than0.9985. The lowerlimit of quantitation for estradiol is0.571ng·mL-1, with good repeatability andreproducibility. The recovery were96.4%、98.7%、98.1%, respectively.Conclusion: The developed method has been analyzed trace estradiol inmilk sample, and the detection sensitivity of this method was up to10-10. Itwas proved that this process is a simple, low-cost and accurate method, whichis suitable for the determination of trace estradiol in milk. 2Analysis the relationship between Endogenous Eight Endogenous Estrogensin Urine Sample and the risk of human endometrial cancer with HF-LPME byUPLC/MSObjective: To analyze the difference between the level of E1, E2, andtheir metabolites in endometrial cancer patients urine and that in healthypeople urine by HF-LPME-UPLC/MS.Methods: Firstly, a hydrolysis step was included by NaOH solvent, thepH was adjusted to3.0. Under the optimized extraction conditions, theextraction solvent only was100μL n-octyl alcohol, the extraction procedurewas finished at at500rpm at25℃for1hour. In order to simultaneouslytransfer analytes both in the acceptor phase and membrane phase into a clean and dry polytef insert tube,100μL methanol was then slowly flushed throughthe lumen. The entire elution solution was evaporated to dryness at90℃undernitrogen gas. Subsequently,100μL of sodium bicarbonate buffer （pH at9.0）and100μL of dansylchloride solution (1mg·mL^-1in acetone) were added tothe dry sample. After vortexing, the sample was heated at60℃for5minutesand dansylchloride derivatives. The conditions of UPLC/MS: Briefly,separation was achieved with the usage of Waters ACQUITY UPLCTMsystem （Waters, UK） consisting of ACQUITY UPLC binary solvent managerand ACQUITY UPLCTM sample manager. Analytes were analysed with anAQUITY UPLC BEH C18（1.7μm;2.1mm×50mm） column, with a mobilephase consisting of mobile phase A:99.9%Acetonitrile,0.1%Formic acid andmobile phase B:99.9%H2O,0.1%Formic acid at a flow rate of0.5mL·min^-1and a temperature of30C. The gradient programme was as follows:0min–160%A,6min–92%A,7⁸min–60%A. An injection volume of20μL wasused. We ascertained the precursor ions and product ions for use in MRM. Theelectrospray interface （ESI） was employed for good sensitivity, andfragmentation was obtained. In order to obtain the higher signal for bothprecursor ions and product ions, we optimized the mass spectrometricparameters. Under the product ion scan mode, the most intensive product ionsof E1, E2,2-OHE1,16α-OHE1,2-OHE2,4-OHE2,2-MeOE1,2-MeOE2and ISwere m/z171.1from m/z504.2, m/z171.0from m/z506.1, m/z170.1fromm/z753.1, m/z171.2from m/z520.0, m/z170.0from m/z755.1, m/z170.0from m/z755.1, m/z171.0from m/z534.2, m/z171.0from m/z536.0and m/z171.3from m/z530.3.Results: The enrichment factor of eight estrogens and estrogenmetabolites were all about180. The linear range for eight estrogens andestrogen metabolites was the same from0.01ng·mL^-1to1.0ng·mL^-1（R washigher than0.9914）. The LOD and LOQ were0.5×10^-4and2×10^-4ng·mL^-1,respectively.Conclusion: This subject has been combined highly sensitive UPLC/MSanalysis method with dansyl chloride derivative, improving the sensitivity of the estrogens analysis method, meanwhile with the function of enrichmentwhich is the HF-LPME sample pretreatment technology. We developed amethod with high sensitivity which was successfully used to analyze eightestrogens and their metabolites in urine samples. The detection sensitivity ofthis method was up to10^-14. The test results of20cases endometrial cancerpatients and20cases of healthy volunteers showed that the difference contentsof five analytes in total between endometrial cancer and healthy people. Thatdifference is helpful to study the relationship between the level estroneestradiol and their metabolites and the mechanism of breast cancer andendometrial cancer. The developed method has provided a reliable way forclinical estrogen and their metabolites research. PART2Application of HF-LPME to Analyze Ephedrine andPseudoephedrine in urine sample1HF-LPME combined with HPLC/LC-MS for the determination of tracepseudoephedrine in urine sampleObjective: A simple and sensitive method based on hollow fiber liquidphase microextraction （HF-LPME） has been developed that would be helpfulto get more information about athletes and airman who take medicineincluding pseudoephedrine （PE）.Methods: Extraction process was performed in a device that was mainlyconstructed by a glass vial and8cm hollow fiber. Firstly, PE was isolatedfrom10mL of urine sample （pH11.0） into organic phase （n-octyl alcohol）impregnated in the pores of hollow fiber, then into an acidic microlitersolution （pH2.8） as acceptor phase filled in the cavity of hollow fiber. The ionstrength is30%. The extraction procedure had been finished for1hour. Thechromatographic separation was performed on a KROMASIL C18column （150mm×4.6mm, particle size5μm）, and the column temperature was keptat30℃. The mobile phase was consisted of water and acetonitrile （92:8, v/v）containing10mmol·L-1KH2PO4and10mmol·L-1triethylamine （adjusted pHat4.0by H3PO4）, with a flow rate of1mL·min-1, the wave length was210nm,and the injection volume was10μL. The instrument was operated usingelectrospray ionized ation source in positive mode. Multiple reactionmonitoring （MRM）（m/z166.1-148.1for EP） mode was employed forquantitation.Results: Under the optimized conditions, an enrichment factor was ashigh as220along with a good sample clean-up process. The calibration curveexhibited linearity in the range of5.0×10-3⁰.75μg·mL-1with regressioncoefficient0.9957, and the LOD, based on a S/N of3, was2.0ng·mL-1obtained by HPLC-UV. The range of recovery was92.8～97.2%, and RSDwas lower than9.7%. The linear range of LC-MS method was2.0×10-3⁰.50μg·L-1and LOQ of that was1.0×10-3ng·mL-1.Conclusion: It is necessary to point out here that enrichment factor ofHF-LPME is as high as220, so that this method was shown high sensitivity.Combined with HPLC, the detection sensitivity is up to10-9and it has beenapplied for the determination of PE in urine after oral administration10h;When combined with HPLC-MS, the detection sensitivity is up to10-12and ithas been applied for the determination of PE in urine after oral administration72h. It would be conduced to the study for PE control and has been provide anew method to effectively monitor the medication use and rational drug usefor special groups. 2Hollow fiber-liquid phase microextraction for the determination of traceephedrine and pseudoephedrine in urine sampleObjective: A simple and sensitive method based on hollow fiber liquidphase microextraction （HF-LPME） has been developed, and it would be helpful to separate, purify and enrich ephedrine and pseudoephedrine inhuman urine sample.Methods: With HF-LPME device, the extraction conditions have beenoptimized: the organic phase impregnated in the pores of hollow fiber isn-octyl alcohol; the acceptor phase is acidic microliter solution （pH2.8）; theion strength is30%. At the room temperature, the extraction procedure hadbeen finished for1hour. The chromatographic separation was performed on aKROMASIL C18column （150mm×4.6mm, particle size5μm）, and thecolumn temperature was kept at30oC. The mobile phase was consisted ofwater and acetonitrile （96:4, v/v） containing10mmol·L^-1KH2PO4and10mmol·L^-1triethylamine （adjusted pH at4.0by H3PO4）, with a flow rate of1mL·min^-1, the wave length was210nm, and the injection volume was10μL.Results: The calibration curve was exhibited linearity in the range of0.01⁵μg·mL^-1with regression coefficient0.9982for ephedrine, in the rangeof5.0×10^-30.75μg·mL^-1with regression coefficient0.9978forpseudoephedrine, and the LOD, based on a S/N of3, were0.005,0.002μg·mL^-1, respectively.Conclusion: Under the optimized conditions, an enrichment factor wasboth about200along with a good sample clean-up process. The developedmethod has also been applied for the determination of ephedrine andpseudoephedrine in urine after oral administration5h. Furthermore, theproposed method could provided an approach for E and PE control, and hasbeen provide a new method to effectively monitor the medication use andrational drug use for special groups.