二维液相色谱法检测注射用核糖核酸Ⅰ/Ⅱ/Ⅲ中苯酚残留量

目的：采用二维液相色谱法检测注射用核糖核酸Ⅰ/Ⅱ/Ⅲ中苯酚的残留量，并对方法进行验证。方法：一维色谱采用ACQUITY UPLC BEH200SEC（150 mm×4.6 mm，1.7 μm；填料：亚乙基桥杂化二醇包膜颗粒）色谱柱，流动相为0.1 mol·L^-1 磷酸二氢钠溶液（pH 7.0），流速0.2 mL·min^-1，检测波长267 nm，柱温30 ℃；二维色谱采用Inertsil ODS SP（4.6 mm×250 mm，5 μm；填料：高纯硅胶）色谱柱，以ZORBAXSB-Aq 保护柱（4.6 mm×20 mm，5 μm；填料：经二异丙基修饰的硅氧烷键合硅胶）作为富集柱，流动相为25% 甲醇，流速1.0 mL·min^-1，检测波长267 nm，柱温30 ℃。检测核糖核酸（RNA）样品分别为来自健康猪肝脏的核糖核酸Ⅰ，来源于牛胰脏的核糖核酸Ⅱ，以及来源于猪脾脏的核糖核酸Ⅲ。结果：苯酚质量浓度在0.246 6~2.219 4 μg·mL^-1 范围内与峰面积呈良好线性（r=0.999 7），回收率在99.7%~100.3%；企业A的注射用核糖核酸Ⅰ和企业D 的注射用核糖核酸Ⅲ样品中均未检出苯酚，企业B 的注射用核糖核酸Ⅰ样品中苯酚残留量为0.002%，企业C 的注射用核糖核酸Ⅱ样品中苯酚残留量为0.008%。结论：本实验建立的二维液相色谱方法能用于注射用核糖核酸Ⅰ/Ⅱ/Ⅲ中苯酚残留量的检测。

Objective: To determine the residual phenol content in ribonucleic acid Ⅰ/Ⅱ/Ⅲ for injection by twodimensional liquid chromatography,and to validate the method. Methods: In the first dimensional chromatography, an ACQUITY UPLC BEH200 SEC(150 mm×4.6 mm,1.7 μm;column packing:ethylene bridged hybrid diol coated particles)column was used as stationary phase and 0.1 mol·L^-1 sodium dihydrogen phosphate(pH 7.0)as the mobile phase;the flow rate was 0.2 mL·min^-1;the column temperature was maintained at 30℃ with an ultraviolet detection of 267 nm for analysis. The target peak from the first dimensional LC was trapped in ZORBAX SB-Aq column(4.6 mm×20 mm,5 μm;column packing:silicone bonded silica gel modified by diisopropyl)and then was separated from pump of the second dimensional chromatography. An Inertsil ODS SP column (4.6 mm×250 mm,5 μm;column packing:high purity silica gel)was used in the second dimensional chromatography,with 25% methanol as the mobile phase,the flow rate was 1.0 mL·min^-1,the column temperature was maintained at 30℃ with an ultraviolet detection of 267 nm for analysis. Ribonucleic acid Ⅰ from pig liver, ribonucleic acid Ⅱ from bovine pancreas and ribonucleic acid Ⅲ from porcine pancreas were detected. Results: The calibration curve of phenol was linear in the concentration range of 0.246 6-2.219 4 μg·mL^-1(r=0.999 7). The recoveries were 99.7%-100.3%. The phenol was not detectable in ribonucleic acid Ⅰ for injection produced by enterprise A and ribonucleic acid Ⅲ for injection produced by enterprise D. The phenol in ribonucleic acid Ⅰ for injection produced by enterprise B is 0.002%,and the phenol in ribonucleic acid Ⅱ for injection produced by enterprise C is 0.008%. Conclusion: The established two-dimensional liquid chromatographic method can be used for the determination of residual phenol content in ribonucleic acid Ⅰ/Ⅱ/Ⅲ for injection.

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