Alnylam Pharmaceuticals, Cambridge, MA 02142, USA.
Anal Biochem. 2010 Jun 1;401(1):61-7. doi: 10.1016/j.ab.2010.02.012. Epub 2010 Feb 13.
Gene silencing induced by short interfering RNA (siRNA) has proven to be useful in genomic research and has great potential for therapeutic applications; however, siRNAs are not readily bioavailable. Cationic liposomes offer effective protection of drug product from nucleases and enable distribution to desired target organs. The amount of siRNA in the formulation must be determined accurately. We have developed a stability-indicating, ion-pair, reversed-phase high-performance liquid chromatography method to separate and accurately quantitate two siRNA duplexes in a liposome without sample pretreatment. The gradient mobile phase system consisted of 385mM hexafluoro-2-propanol, 14.5mM triethylamine, and 5% methanol (mobile phase A) and 385mM hexafluoro-2-propanol, 14.5mM triethylamine, and 90% methanol (mobile phase B). The column used was an XBridge C18 column (50x2.1mm i.d., 2.5microm particle size), and separation was performed at 60 degrees C. Quantitation was achieved with ultraviolet (UV) detection at 260nm. Linearity was established for the single strands of both siRNA duplexes for concentrations ranging from 10 to 110microg/ml. Accuracy of the method was determined by replicate analysis (n=5) at four concentrations (R(2)>0.996 and relative standard deviations [RSDs] of 1-4%). The use of an ion-pairing reagent that is compatible with mass spectrometry detection makes this method amenable to liquid chromatography-mass spectrometry (LC-MS) impurity profiling.
小干扰 RNA（siRNA）诱导的基因沉默已被证明在基因组研究中非常有用，并且在治疗应用方面具有巨大的潜力；然而，siRNA 不易生物利用。阳离子脂质体可有效保护药物产品免受核酸酶的侵害，并使药物分布到所需的靶器官。制剂中 siRNA 的含量必须准确确定。我们已经开发了一种稳定指示的、离子对、反相高效液相色谱方法，无需样品预处理即可分离和准确定量脂质体中的两种 siRNA 双链。梯度流动相系统由 385mM 六氟-2-丙醇、14.5mM 三乙胺和 5%甲醇（流动相 A）和 385mM 六氟-2-丙醇、14.5mM 三乙胺和 90%甲醇（流动相 B）组成。所用的柱是 XBridge C18 柱（50x2.1mm id，2.5μm 粒径），在 60°C 下进行分离。定量采用 260nm 处的紫外（UV）检测。两种 siRNA 双链的单链在 10 至 110μg/ml 的浓度范围内均建立了线性关系。通过在四个浓度（R（2）>0.996，相对标准偏差[RSD]为 1-4%）下重复分析（n=5）确定方法的准确性。使用与质谱检测兼容的离子配对试剂使该方法适用于液相色谱-质谱（LC-MS）杂质分析。
