The Study Of Pretreatment Methods Of Biological Samples In The Clinical And The Significance Of Clinical Application Of Developed Methods

Therapeutic drug monitoring(TDM),as an important pathway of realizing individualized treatment of patients,ensures the safety and efficacy in clinical medication,as well as supports the development of precision medicine.The analysis of biological sample of clinical patient is one of the most significant ways in TDM.All the biological samples,whether from clinical patients or from the healthy,contain a large number of endogenous macromolecule,which easily cause matrix effects interfering the analysis of targets.Thus,it is usually needed to pretreat sample with an appropriate method to separation the target from biological matrix,reducing the impact of matrix effects.Nowadays,the frequently used preparation methods are protein precipitation(PPT),liquid-liquid extraction(LLE),solid phase extraction(SPE)and so on,these methods usually need blank plasma from healthy volunteer to perform the method validation,however,there is remarkable difference in the sample condition between the sick and the normal.Usually the condition of normal sample is relatively stable and the variation of matrix effect is little,whereas the condition of sample of patient shows significantly inter-individual difference because of the influence of disease,leading to significant difference of matrix effect,thus,it is difficult to guarantee the accuracy of the analytical results of clinical sample.Previous research has shown that the fat,protein level,colloid osmotic pressure,et al.are easily affected by disease state,demonstrating potential variability,while these factors impact exactly the condition of protein precipitation and extraction efficiency,hence,when using PPT,LLE or SPE to analyze the biological samples from patients,even if the addition of precipitator or extraction agent is precisely same,the recovery of target will still be different,influencing on the veracity of the results.In addition,traditional preparation methods only detect a single index,such as total drug concentration(Ct)or free drug concentration(Cf),while Ct or Cf are susceptible to the effects of sample time,duration of taking medicine,food,et al.These factors could be controlled in common study of biological sample,the determined Ct or Cf could reflect the medication in body accurately.While it is difficult to control these factors in clinical study or routine monitoring strictly,resulting in inveracious reflection of drug level in vivo.Meanwhile,there are some other defects in traditional preparation methods,such as complex manipulation,time-consuming process and so on,reducing the accuracy of results and the efficiency of analysis.Due to the traditional preparation methods without considering the specificity of clinical sample,the analytical results often bring about confusion to clinical study and TDM,even impact on the effect of individualized treatment.Therefore,to develop a kind of preparation method,simple,accurate,appropriate and without influence of disease,is th-e key factor for accurate result,proper detection index and the universality of method.A good preparation method can provide a reliable analysis platform for clinical routine testing and the study pharmacodynamic actions,ensuring the safety and efficacy of medicine.PART 1: A Novel Pretreatment Method for Biological Sample Combining Blood Specimen Collection and Treatment.Objective : A simple,effective,processless pretreatment method of biological sample was devised by combining sealing technique with hollow fiber centrifugal ultra-filtration(HFCF-UF),guaranteeing the validity of the analytical result of clinical sample.Methods:We used antiviral drug of adefovir and tenofovir as model drugs and developed a Fully Enclosed Hollow Fiber Centrifugal Ultra-Filtration(FE-HFCF-UF)method combining sealing technique with direct injection technique.A total of 0.6 mL whole blood sample entered into tube under negative pressure through the blood lances.Shake the devise gently after blood collection.Then,the anticoagulant on the wall of tube and fiber would dissolve into blood to get anticoagulant blood sample.After simple centrifugation at 3500 r/min for 15 min at 25 °C,about 50 ?L ultrafiltrate in the lumen of the hollow fiber,after deriving,20 ?L aliquot was introduced for HPLC analysis;Chromatographic conditions:Separations were performed on an Phenomenex C18 column(4.6 mm×250 mm,5 ?m);Liquid chromatography was performed using 10 mM potassium dihydrogen phosphate(pH=6.0)containing 2 mM tetrabutylammonium hydroxide and acetonitrile(88:12,v/v)for adefovir and the same potassium dihydrogen phosphate and acetonitrile(90:10,v/v)for tenofovir,the flow rate was 1.0 mL/min.The column temperature was set at 30 °C,the fluorescence detection wavelength for adefovir were 309 nm for excitation and 425 nm for emission,and the fluorescence detection wavelength for tenofovir were 254 nm for excitation and 425 nm for emission.Results: The results demonstrated that the developed FE-HFCF-UF method achieved several advantages including higher precision,favorable sensitivity and satisfactory recovery.The inter-and intra-day precisions(RSD%)of the method were lower than 4.1%,and the extraction recoveries could reach 100%.Conclusions: The operation of novel method is briefness,combines blood sample collection and preparation,saves manual operation,obtains ultrafiltrate with micromolecule from blood directly by one simple step of centrifugate,moreover,the prepare calibration curve can be prepared by standard solution,avoiding impact from disease state.Hence,the novel technology improve the simplicity,accuracy and sensitivity of analysis.In addition,simple operation and sealing system improve the biosafety in the process of bioanalysis.With these highly practical and desirable characteristics,the proposed method may become a feasible platform in bioanalysis,and provides a fresh idea for improving biosafety of analytical method.PART 2: The Determination of Plasma Protein Binding of Aminophylline and the Study of the Relationship between Plasma Protein Binding and PharmacodynamicObjective : To determine the plasma protein binding(PPB)of aminophylline(AYP)from clinical patients based on the preparation method of hollow fiber centrifugal ultra-filtration(HFCF-UF).Meanwhile,to study the influence factors of PPB combined with the information of clinical patients.Methods:The free concentration(Cf)of AYP was analyzed by HFCF-UF,then,the release reagent was added to make AYP released from drug-protein complexes,analyzing total concentration(Ct)of AYP,and PPB was calculated according to a formula.Chromatographic conditions:The separation of AYP was performed on a Diamonsil C18 column(150 mm×4.6mm,5 ?m,Dikma,China)under an isolate elution with a mixture of methanol-10 mmol ammonium acetate(15:85,v/v)and the flow rate was 1.0 mL/min.The injection volume was 20 ?L and the detection of AYP was carried out at the wavelength of 275 nm.Results: The PPB of 80 plasma samples from 80 patients were detected,the results showed that the range of PPB was 8% to 98.1%.We found that the gender,physical condition,disease profile and combined medication had influence on PPB of AYP.Conclusions: To monitor the PPB of clinical patients taking APY is necessary,perfects medical information of APY,improves the safety and effectiveness of the APY.