HPLC-MS/MS法测定晚期乳腺癌患者血浆中FCN-437c浓度及临床应用

Determination of FCN-437c in human plasma for patients with advanced breast cancer by HPLC-MS/MS and its clinical application

目的:建立测定人血浆中FCN-437c药物浓度的HPLC-MS/MS方法,并用于FCN-437c的Ⅰ期临床研究。方法:血浆经蛋白沉淀处理后,采用HPLC-MS/MS法进样测定。色谱柱为YMC Triart PFP柱(50 mm×2.1 mm, 5μm),以0.5%甲酸水溶液(含5 mmol·L^(-1)乙酸铵,A)-乙腈(B)为流动相,梯度洗脱,流速为0.5 mL·min^(-1),柱温为35℃,进样量为2μL,进样器温度为10℃。质谱采用ESI+,MRM模式。检测离子反应对为m/z 549.4→449.5(FCN-437c)和m/z 552.5→449.0(FCN-437-D3,氘代内标),雾化气压力276 kPa,辅助气压力207 kPa,去簇电压100 V,碰撞室出口电压25 V。结果:人血浆中FCN-437c的线性范围为5~1 000 ng·mL^(-1)(r=0.999 0),定量限为5 ng·mL^(-1),批内、批间精密度分别小于2.0%和4.1%,平均回收率为104.0%(FCN-437c)、78.6%(FCN-437-D3),内标归一化基质因子为100%~102%。FCN-437c储备液4℃放置202 d, FCN-437c及FCN-437-D3工作液室温放置24 h,血浆样品室温放置20 h、冻融四循环、-20℃放置134 d、-80℃放置662 d,样品处理后自动进样器放置24 h,全血样品室温放置4 h均稳定。应用此方法检测了受试者口服FCN-437c后血浆药物浓度,ISR样品测试结果为97.0%与初测值的偏差在±20%以内。血浆样本稀释10倍后准确度为102.0%~108.0%。FCN-437c连续给药与单次给药相比,R_(AUC0-24)和R_(Cmax)的累积比为1.33倍与1.59倍。结论:本方法简便、准确、耐用、专属性强,可满足人血浆中FCN-437c的定量分析的要求。

Objective:To establish an HPLC-MS/MS method for the determination of FCN-437c in human plasma and its application to the phase I clinical study of FCN-437c.Methods:Following protein precipitation,plasma was injected and measured using HPLC-MS/MS method.The analytes were separated on a YMC Triart PFP column(50 mm×2.1 mm,5μm)using 0.5%formic acid(containing 5 mmol·L^(-1)of ammonium acetate,A)and acetonitrile(B)as the mobile phase with gradient elution at the flow rate of 0.5 mL·min^(-1),the column temperature was set at 35℃,the injection amount was 2μL,and the injector temperature was 10℃.MS detection was performed with multiple reaction monitoring(MRM)mode using positive electrospray ionization.The ion transitions were m/z 549.4→449.4 for FCN-437c and m/z 552.3→449.3 for FCN-437-D3,respectively.Other mass spectrometry parameters were TEM,500℃,GS1,276 kPa,GS2,207 kPa,DP,100 V,CXP,25 V.Results:The linear range of FCN-437c in human plasma was 5-1000 ng·mL^(-1)(r=0.9990).The lower limit of quantification was 5 ng·mL^(-1).The intra-batch and inter-batch precisions were less than 2.0%and 4.1%,respectively.The average recovery was 104.0%(FCN-437c),78.6%(FCN-437-D3),and the internal standard normalized matrix factor was 100%-102%.The stock solution of FCN-437c was stable at 4℃for 202 d,the working solution of FCN-437c and internal standard were stable at room temperature for 24 h.FCN-437c in human plasma was investigated to be stable at room temperature for 20 h,four cycles of freeze-thaw,-20℃for 134 d,-80℃for 662 d,as well as for 24 h in the autosampler after treatment.The whole blood samples were stable at room temperature for 4 h.This method was applied to the determination FCN-437c in human plasma,and the deviation between the test results and the initial values of 97.0%ISR samples was within±20%.The accuracy was 102.0%-108.0%after 10-fold dilution of plasma samples.The cumulative ratio of R_(AUC0-24)and R_(Cmax) was 1.33 times and 1.59 times when FCN-437c was administered continuously compared with single administration.Conclusion:This method is simple,accurate,robust and specific,which can meet the requirements of quantitative analysis of FCN-437c in human plasma and also can be used to determine FCN-473c in human plasma of the phase I clinical study.