Development Of HPLC Method Of5-fluorouracil In Plasma Of Patients With Colorectal Carcinoma: Application To Pharmacokinetics And Steady State Concentration Monitoring

Objective:A simple, rapid and sensitive HPLC method wasdeveloped and validated for determination of5-Fluorouracil (5-FU) in plasmaof patients with colorectal carcinoma: application to pharmacokinetics andsteady state concentration monitoring.Methods:1. Plasma sample was extractedusing a simple liquid-liquid extraction with ammonium acetate buffer (PH3.5;0.01M) and ethyl acetate/isopropanol (85:15, v/v). A high-performance liquidchromatography (HPLC) method was developed and validated for thedetermination of5-Fluorouracil (5-FU) in plasma of patients with colorectalcarcinoma.2. The drug concentration of5-FU in plasma of patients withcolorectal carcinoma and pharmacokinetics parameters were calculated bypharmacokinetic software DAS2.0.3. The drug plasma concentration at steadystate (Css) of5-FU was determined and analyzed with statistical analysismethod.Results:1. HPLC system: The determination of5-FU in plasma samplewas carried out by HPLC system consists of Dionex ultimate3000seriesincluding pump (LPG-3400SD), UV-vis detector (VWD-3100), auto injector(WPS-3000) and column oven (TCC-3000). Separation was performed on areverse phase C18column (Inertsil ODS-SP;4.6×250mm,5μm particle size,made in Japan) with a guard column (Phenomenex C18,4.0mm×3.0mm,5μmparticle size, made in USA) using a mobile phase of acetonitrile-ammonium acetate buffer (pH3.5;0.01M)(2.5:97.5v/v) at the flow rate of0.8ml/min. Thecolumn temperature was maintained at25oC. The UV detection wavelengthwas265nmand the sample volume was20μl.2. Establishment and validationmethods:The method is specific, less interference peaks.Standard curve waslinear between0.01-5μg/ml and10-100μg/ml for plasma sample,the linearrelationship is good,R>0.999.The limit of quantification was10ng/ml.Theintra-and inter-day precision was below10%(RSD).The accuracy ranged from85.24to104.14%.Extraction recovery ranged from87.55%to95.26%.Retention times was less than7min.3. The pharmacokinetic parameters of10cases of colorectal cancer after intravenous bolus injection: For the former5patients, the value range of AUC, MRT, t1/2, V, CL and Cmaxwere504.31-748.85mg/L*min,7.25-18.02min,5.41-23.56min,4.45-26.68L/m2,0.53-0.79L/min/m2and29.47-46.94mg/L, respectively. For the latter5patients, the value range of AUC, MRT, t1/2, V, CL and Cmax were12.28-75.17mg/L*min,21.23-33.02min,34.23-79.46min,383.81-3094.81L/m2,3.52-26.99L/min/m2and0.44-1.60mg/L, respectively.4. The drug plasmaconcentration at steady state (Css) of5-FU: The Cssrange of the5patients (P1to P5) was2.655-56.995mg/L, other5patient was0.019-1.324mg/L, whichthe Cssvalues of the former5patients was significantly higher than those of thelatter5patients （P<0.05）.Conclusion:1. The HPLC analysis methoddeveloped and validated in this study was simple, rapid, high sensitivity, whichhas been successfully applied in5-FU clinic pharmacokinetics study and plasma5-FU concentrations at steady state (Css) determination.2. Thepharmacokinetics and Cssof5-FU in patients with colorectal cancer ischaracterized by a large inter-patient variability. Therefore, to increasetherapeutic response and reduce toxicity, we should optimize5-FU dose byinvestigating pharmacokinetic behavior to obtain ideal Cssfor each patient inclinical practice.