LC-MS法结合天然同位素校正和^(1)H NMR相对定量法2种方法测定氘代新药甲苯磺酸多纳非尼的氘代率

Determination of deuterium incorporation of new drug donafenib tosilate by two methods of LC-MS with natural isotope abundance correction and^(1)H NMR relative quantitative method

测定氘代抗肿瘤新药甲苯磺酸多纳非尼中氘同位素杂质(包括非氘代和不完全氘代杂质)的含量,并计算氘原子百分比,即氘代率。建立液相色谱-质谱(LC-MS)法通过选择离子扫描模式测定各氘同位素杂质及氘代药物峰面积,并根据ChemDraw软件给出的天然同位素分布对峰面积进行校正,消除^(13)C、^(37)Cl等天然同位素干扰,通过校正后的峰面积计算各氘同位素杂质的相对含量,继而计算药物氘代率;并采用非氘代杂质对照品对该方法下天然同位素分布、氘同位素杂质与氘代药物的摩尔相对响应因子、基质效应等进行验证。另采用^(1)H定量核磁共振波谱技术(^(1)H qNMR),以δ8.63的总芳香^(1)H峰和δ2.79的氘同位素杂质残留^(1)H峰为定量峰,采用氘代二甲基亚砜-重水(10∶1)混合溶剂,改变活泼氢对残留^(1)H的偶合裂分,使其以单峰的形式参与定量,提高方法灵敏度,并优化采集参数,测定^(1)H原子百分比,继而计算药物氘代率。结果表明,在建立的LC-MS方法下,天然同位素化合物相对丰度的实测值与理论值的相对偏差均小于15%;氘同位素杂质与氘代药物的摩尔响应因子基本一致;方法无基质效应。建立的^(1)H qNMR方法定量限低至0.1%。将建立的2种方法用于3批商业化样品的测定,测定结果基本一致。建立的LC-MS法结合天然同位素校正和^(1)H NMR相对定量法2种方法均准确、快速、灵敏,且无需准确称量,无需任何对照品,可用于甲苯磺酸多纳非尼氘代率的测定,也为其他氘代药物氘代率的测定提供了新思路。

An LC-MS method with natural isotope abundance correction and a^(1)H NMR relative quantitative method were established to determine the deuterium incorporation of donafenib tosilate,a new deuterated drug molecule.First,the peak areas of isotopic impurities(non-deuterated and incompletely deuterated impurities)and deuterated drug were recorded through the single ion monitoring(SIM)mode of the established LC-MS method and then corrected in terms of the natural isotope abundance offered by ChemDraw soft,removing the nature isotope interference from^(13)Cl,^(37)Cl,etc.The corrected areas were subsequently used to calculate mo1%of isotopologues(D_(0),D_(1),D_(2),D_(3))and Atom%D,namely,deuterium incorporation.In addition,a^(1)H qNMR experiment was conducted with the aromatic proton atδ8.63 and the residual proton of isotopic impurities atδ2.79 as quantitative peaks.The mixture of DMSO-d_(6)and D,O(10:1)was employed as the solvent to change the spin-coupling between the residual proton and active hydrogen so that the residual proton could be measured as the single peak,and the sensitivity was greatly improved.The acquisition parameters were also optimized,and Atom%^(1)H and the deuterium incorporation were then calculated.The two methods were applied to samples of three commercial batches,and the testing results were almost consistent.Both methods proved accurate,sensitive,fast and independent of standard substances and accurate weighing,which could be applied to the determination of the deuterium incorporation of donafenib tosilate and provide a reference for other deuterated drugs.