| Objectives:1.Sensitive and simple LC-MS/MS methods were established for determination of 5-fluorouracil(5-FU),paclitaxel(PTX)and docetaxel(DTX)in human plasma,respectively.2.NPIA was used to measure the concentration of 5-FU,PTX and DTX in human plasma,respectively.3.To compare the difference and correlation of the results of the concentration of 5-FU,PTX and DTX between the determinations of LC-MS/MS and NPIA,respectively.Methods:1.LC-MS/MS method:5-FU:5-FU was separated on a Diamonsil C18 column(150 mm × 4.6 mm,5 ?m),eluted with the mobile phase of 0.05%ammonia:methanol(2:98,v/v)with a flow rate of 0.3 mL/min at 30? with a thermostated column oven.The plasma sample was extracted by liquid-liquid extraction and was injected 10 ?L to analyze by HPLC system.PTX and DTX:PTX and DTX both were separated on a Diamonsil C18 column(150 mm × 4.6 mm,5 ?m),eluted with mobile phase of 0.1%formic acid:acetonitrile(40:60,v/v)with a flow rate of 0.6 mL/min at 30? with a thermostated column oven.The plasma sample both were extracted with protein precipitation extraction and was injected 10 ?L to analyze by HPLC system.2.NPIA method:5-FU,PTX and DTX plasma samples were treated based on a certain operating procedures(using exclusive kits)and then were injected into the CS-600B automated biochemical analyzer for analysis.3.Statistical analysis:(1)With LC-MS/MS results as X-axis and NPIA results as Y-axis,a linear regression model was established and a regression analyses was performed with SPSS.17.0 for the evaluation of correlation.(2)The data of the 5-FU,PTX and DTX samples were analyzed by Bland-Altman software to evaluate the consistency,respectively.Results:1.The LC-MS/MS methods were successfully established with high specificity.There were no endogenous interferences for determination of 5-FU,PTX and DTX in blank or human plasma.Method validation of 5-FU,PTX and DTX,the matrix effects were 104.40,107.18%and 112.57%,respectively.The mean recoveries were 80.61%,99.41%and 95.76%,respectively.The range of inter-day and intra-day accuracy and precision were 88.2%-109.4%,100.1%-115.7%and 95.1%-100.2%,RSD%were 3.95-13.69,2.49-9.66 and 2,88-11.9,respectively.5-FU,PTX and DTX had a good linearity ranging from 10-800 ng/mL,10-200 ng/mL and 10-2000 ng/mL respectively with R2>0.99.5-FU,PTX and DTX plasma samples with good stability,met the determination requirements.2.The linear regression equation for 5-FU,PTX and DTX were y=1.019x-8.746(R=0.939,p<0.001),y=0.839x+17.975(R=0.878,p<0.001)and y=1.105x+10.003(R=0.965,p<0.001),respectively.A good linear correlation relationship was obtained between LC-MS/MS and NPIA.3.A mean of 273.82 ng/mL,43.56 ng/mL and 378.1 ng/mL of 5-FU,PTX and DTX were determined by LC-MS/MS,while 270.18 ng/mL,54.49 ng/mL and 427.7ng/mL for NPIA.As shown in the plot,the 95%CI of-132.5?125.2 ng/mL,-11.6-33.4 ng/mL and-231.7?331.1 ng/mL respectively were observed for 5-FU,PTX and DTX,and most of the data was in the 95%CI.The data of methods comparison showed the LC-MS/MS was closely related to NPIA method.Conclusions:1.LC-MS/MS methods for determining 5-FU,PTX and DTX in human plasma were sensitive,accurate,stable and reproductive.It was suitable for monitoring 5-FU,PTX and DTX.2.NPIA method for determination 5-FU,PTX and DTX in human plasma was convenient,rapid,sensitive,automated and small sample size.It was suitable for routine monitoring concentration of 5-FU,PTX and DTX.3.The data of methods comparison showed that the LC-MS/MS was closely related to NPIA method.With the validation of NPIA,the application of 5-FU,PTX and DTX testing in clinical practice may gain wider acceptance for individualizing patient 5-FU,PTX and DTX dosing. |
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