The Pretreatment And Analysis Of Active Components From Biological Samples

Biological samples such as urine, blood and herbal drugs are verycomplex materials. The analysis of active components would be easilyinterfered, because of many types and low level concentration of the analytes.Though, in some cases the samples were directly used for analysis only with asimple treatment, an unsatisfactory analytical sensitivity and accuracy wasobtained due to the existing of interference. And the limited improvablespace of detection sensitivity depend on instrument analysis has stimulated thedevelopment of sample enrichment technology with the goal of increasing theanalytical sensitivity. The focus of the present study is to exploit new samplepretreatment method for the purification of the analytes. By removing theinterfering substance, supply the analyte a favorable detection conditions so asto improve the method sensitivity. It is reported that in the whole analyticalprocess, sample pretreatment cost61%time and introduce30%error. It hasbeen a key issue that has restricted the improvement of analytical speed,sensitivity and accuracy. Therefore, sample separation and purification prior toanalysis becomes the focus of analytical chemist, and the studies on this fieldhave been increasing year by year.The purpose of our study is to adopt modern analytical means, such asselectively coprecipitation combinding with derivatizaition method,novel-hollow fiber centrifugal ultrafiltration technique and specificityadsorption separation technique to the analysis of target components fromurine, blood and herbal drugs with complex matrix. We choose somecomponents with special character and hard to analysis with traditionalmethod as our main research, for instance ibandronate with high polarity, highwater-solubility and poor chromatography performance; hesperidin which ishigh polarity and hardly dissolved in organic solvent, phenothiazine drugs which are hardly treat with solid phase extraction and so on. These techniquesprovide new, simple, utility and reliable purify means in biological samples.PART1Development and application of LC-MS/MS method to determineIbandronate in human urineObjective: To establish a LC-MS/MS method for the analysis ofibandronate in human urine and be applied to study the relationship betweenvitamin D3with the adsorption of ibandronate.Methods: Ibandronate was isolated from the biological matrix bycoprecipitated with CaCl2and K2HPO4in basic conditions. Then after a LLEthe analytes were derivatized with trimethylsilydiazomethane prior toseparation on a reversed-phase column and detection on a quadrupole-linearion trap mass spectrometer (API3200) equipped with an ESI source. Thetransitions of376.1/114.2for and m/z379.1/61.2were acquired to monitoribandronate and d3-ibandronate derivatives.Results: The calibration curves exhibited excellent linearity(r>0.99)between1and200ng·mL-1in human urine. The lower limit of quantitation(LLOQ) of ibandronate was1ng·mL-1. Ibandronate was recovered>86.5%with the intra-and inter-day RSDs were less than15%.Conclusion: The proposed method is selective and accurate with highsensitivity and can be used to the study of the relationship between vitamin Dwith the adsorption of ibandronate. And more the method can be used toevaluate the effects of coexisting medications on the adsorption ofibandronate. PART2Research and application of HFUF-CF technique in analysis ofmicromolecule active components in herbal drugs (1) Separation and determination of hesperidin in Huoxiang ZhengqiWater by hollow fiber ultrafiltration-HPLC methodObjective: To develop a simple hollow fiber based centrifugeultrafiltration pretreatment procedure for the analysis of active componentswith high polarity in Chinese traditional and herbal drugs.Methods: The procedure combined with HPLC was applied to thedetermination of hesperidin in huoxiang zhengqi water. Sample solutions werepurified by our patent hollow fiber centrifuge ultrafiltration device. Under theeffect of the centrifgual force, micromolecules were removed from solutionsamples, thus it increase the service life of the column. The accuracy and therepeatability of the method have also been improved. The separation wascarried out on a Promosil C18column (150mm×4.6mm, i.d.5μm) with V(methanol):V (0.5%acetic acid solution)=35:65as the mobile phase at aflow rate of1.0mL/min. The detector wavelength was283nm and the columntemperature was30℃.Results: A good linear relation was obtained in the range of4.69μg·mL-1~150μg·mL-1(r=0.9997) and the average recovery was100.1%with theRSD of2.4%.Conclusion: This method is simple, rapid and accurate, it provides asimple and cheap ultrafiltration means for the analysis of the polarcomponents in Chinese traditional and herbal drugs. (2) Direct-injection HPLC method for the simultaneous determination ofhonokiol and magnolol in Huoxiang Zhengqi ShuiObjective: To develop a simple sample pretreatment method for thedetermination of magnolol and honokiol in Huoxiang Zhengqi water by hollow fiber ultrafitration-HPLC.Methods: Under the effect of the centrifgual force, micromoleculeswere removed from solution samples, thus it increase the service life of thecolumn. The accuracy and the repeatability of the method have also beenimproved. The separation was carried out on a Diamonsil C18column(150mm×4.6mm, i.d.5μm) with V (methanol):V (acetonitrile):V (0.5%acetic acid solution)=44:22:34as the mobile phase at a flow rate of1.0mL/min. The detector wavelength was294nm and the column temperaturewas30℃.Results: It had good linear relationship between the peak areas ofHonokiol and Magnolol and their concentrations at6.40~205μg·mL-1、3.15~101μg·mL-1(r=0.9999), respectively. The method recovery was over92.6%with relative standard deviation less than3.0%. The averagerecovery of Honokiol was97.7%with RSD being3.0%, and that ofMagnolol was96.8%with RSD being2.8%.Conclusion: This method is simple, rapid and accurate, it provides asimple and cheap ultrafiltration means for the pretreatment of Chinesetraditional drug of liquid preparation and can be used for quality control ofHuoxiang Zhengqi Water. It provides a simple and cheap ultrafiltration meansfor the analysis of the molecular components in herbal drugs. PATR3A novel hollow fiber based SPME-UPLC/MS/MS platform for thesimultaneous quantitation of5antipsychotics in human whole blood andurineObjective: To develop a sensitive, specific and rapid hollow fiberadsorbing extraction, UPLC-MS/MS method for the simultaneousdetermination of5antipsychotics in human whole blood and urine. Methods: A12cm hollow fiber was placed into1mL1:1diluted wholeblood or10mL urine sample (adjust pH to9-10), extract20min inultrasonication bathe or by shaken. Then the analytes were desorpted20minby ultrasonication in methanol (1mL). A volume of5μL was injected onto aUPLC-MS/MS system. The separation was achieved using a UPLC HSS C18column (1.7μm,2.1×50mm) in gradient conditions at0.5mL·min-1.Results: The calibration curves were linear over the investigatedconcentration range:12.5pg·mL-1~10ng·mL-1(in human urine) and25pg·mL-1~10ng·mL-1(in whole blood), with all correlation coefficients higherthan0.99. The intra-and inter-day RSD were no more than15%. Accuracywas good with the percent deviation of the observed mean values from thenominal value within±15%.Conclusion: The proposed method is simple, rapid with high sensitivityand broader liner range and suitable for the analysis of trace of the targetanalytes with complex matrix such as human urine and whole blood. Itprovides a reliable tool for the forensic science.