Reversed-phase high-performance liquid chromatography method for simultaneous analysis of two liposome-formulated short interfering RNA duplexes.
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.
Murugaiah
Veeravagu
V
Alnylam Pharmaceuticals, Cambridge, MA 02142, USA. mmurugaiah@alnylam.com
Zedalis
William
W
Lavine
Gary
G
Charisse
Klaus
K
Manoharan
Muthiah
M
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D056148:Chromatography, Reverse-Phase / Q000379:methods*; D016547:Kinesins / Q000235:genetics; D008081:Liposomes / Q000737:chemistry*; D034741:RNA, Small Interfering / Q000032:analysis / Q000737:chemistry*; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics
D008081:Liposomes; D034741:RNA, Small Interfering; D042461:Vascular Endothelial Growth Factor A; D016547:Kinesins