HPLC-MS/MS法在检测慢性粒细胞白血病患者帕纳替尼血药浓度中的应用

Application of HPLC-MS/MS in the Detection of Blood Concentration of Ponatinib in Patients with Chronic Myelogenous Leukemia

目的建立测定慢性粒细胞白血病(chronic myelogenous leukemia, CML)患者血浆帕纳替尼浓度的高效液相色谱-串联质谱法(HPLC-MS/MS),并应用于帕纳替尼血药浓度日常监测,为帕纳替尼合理使用提供实验室依据。方法采用含内标(帕纳替尼-d8)的甲醇对患者血浆进行沉淀蛋白处理。色谱柱为Ultimate XB-C18,柱温60℃,流动相为甲醇相(0.1%甲酸)和水相(0.1%甲酸和2 mmol/L乙酸铵),梯度洗脱。质谱检测方式为电喷雾离子阱正离子模式,MRM扫描,同时监测帕纳替尼m/z 533.1>260.1和帕纳替尼-d8 m/z 541.2>260.1。采用内标法定量,以帕纳替尼和帕纳替尼-d8峰面积比为定量依据,计算血浆帕纳替尼浓度,并分别对其线性、专属性、精密度、准确度及稳定性进行考察。结果帕纳替尼浓度在(1~250) ng/ml范围内与峰面积线性关系良好,Y=0.019 3X+0.028 4(r=0.999 1)。专属性较好,血浆中的内源性物质不干扰帕纳替尼及其内标的测定;日内及日间RSD均小于5%;相对回收率为100.91%~105.18%;室温放置稳定性及冻融稳定性RSD均小于5%。结论该文建立的HPLC-MS/MS法,前处理简单,特异度及灵敏度高,能准确、快速地检测血浆帕纳替尼浓度,适合慢性粒细胞白血病患者帕纳替尼血药浓度的日常监测。

Objective To develop a HPLC-MS/MS method for determination of Ponatinib in CML patient, and make it used in clinic trial providing a laboratory evidence for clinical rational use of Ponatinib. Methods Patients plasma protein was precipitated by methanol containing internal standard(ponatinib-d8). The separation was performed on a Ultimate XB-C18 column with a mobile phase of water(containing 2 mmol/L ammonium acetate and 0.1% formic acid) and methanol(containing 0.1% formic acid). The way of eluting was gradient,and electrospray ion trop positive ion mode and MRM scan were used to simultameously monitor palatinib m/z 533.1>260.1 and Palatinib-d8 m/z 541.2>260.1. The internal standard method was used for quantitative analysis. Ponatiniband Ponatinib-d8 peak area ratio were used to calculate the plasma Ponatinib concentration and the performance verification(linearity, specificity, precision, accuracy and stability) was also investigated. Results The standard curve of Ponatinib was linear over the range of 1 ~ 250 ng/ml, Y=0.019 3 X + 0.028 4(r=0.999 1). The endogenous substances in plasma did not interfere with the determination of Ponatinib and Ponatinib-d8. The average recovery was among the range of 100.91%~105.18%, and RSD of intra-and inter-day validation were less than 5%. RSD of room temperature stability and freezethaw stability were less than 5%.Conlusion This HPLC-MS/MS method had the advantages of simple pretreatment, high specificity and sensitivity, and could accurately and quickly detect the concentration of plasma Ponatinib, which was suitable for daily monitoring of plasma Ponatinib concentration in patients with chronic myelogenous leukemia.