一个磷酸肌酸钠新杂质的结构鉴定及含量测定方法

Structural identification and content determination of a new related impurity in phosphocreatine disodium

目的鉴定肌酐-三氯氧磷缩合法生产的磷酸肌酸钠中一个新发现的杂质(1),并建立其含量测定方法。方法通过核磁共振、ESI高分辨质谱、X单晶衍射等光谱学方法解析该杂质1的结构;采用反相高效液相色谱法测定含量,使用C_(18)色谱柱,以乙腈-质量分数为0.2%的磷酸二氢钠水溶液(含质量分数为0.1%的四丁基氢氧化铵,磷酸或氨试液调节p H值至6.6)(体积比为20∶80)为流动相,检测波长:210 nm。结果杂质1鉴定为具焦磷酰结构的新化合物。反相高效液相色谱法的线性为0.25~100 mg·L^(-1);加样回收率,在磷酸肌酸钠粗品测定中为99.8%(RSD=1.1%,n=6),在精制品测定中为98.6%(RSD=1.6%,n=6)。结论对化学合成法来源的磷酸肌酸钠,可以用HPLC法来测定其中杂质1的含量。

Objective To separate and characterize a new impurity from crude phosphocreatine disodium that was manufactured by a Creatinine-Phosphorus oxychloride method,and to establish a method for its content determination. Methods The structure of the new impurity was elucidated via spectroscopic methods including 1D-and 2D-NMR spectra,and high-resolution ESI mass spectrometry,as well as single crystal Xray diffraction. Reversed phase high performance liquid chromatography（ RP-HPLC） was chosen for the content determination of the impurity. The chromatographic seperation was performed on a C_（18） column in an isocratic mode using a mixture of acetonitrile and water phase（ V ∶ V = 80） as the mobile phase with UV detection at 210 nm. The water phase containing 0. 2% sodium dihydrogen phosphate and 0. 1%tetrabutylammonium hydroxide was adjusted to pH 6. 6 with phosphoric acid or ammonia solution. Results The impurity was identified as a new compound（ 1） bearing a pyrophosphoryl group. The linear range for 1 was 0. 25-100 mg·L^（-1）. The recoveries were 99. 8%（ RSD = 1. 1%,n = 6） in the crude phosphocreatine disodium,and 98. 6%（ RSD = 1. 6%,n = 6） in the refined products. Conclusions The established RP-HPLC method is practical and applicable to control the content of 1 in phosphocreatine disodium products manufactured by chemical synthesis.