Reversed-phase high-performance liquid chromatography method for simultaneous analysis of two liposome-formulated short interfering RNA duplexes

Abstract

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 385 mM hexafluoro-2-propanol, 14.5 mM triethylamine, and 5% methanol (mobile phase A) and 385 mM hexafluoro-2-propanol, 14.5 mM triethylamine, and 90% methanol (mobile phase B). The column used was an XBridge C18 column (50 × 2.1 mm i.d., 2.5 μm particle size), and separation was performed at 60 °C. Quantitation was achieved with ultraviolet (UV) detection at 260 nm. Linearity was established for the single strands of both siRNA duplexes for concentrations ranging from 10 to 110 μg/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.