Measurement of ASO exposure in brain tissues with LC-MS

Standards for ASO exposure were prepared by spiking matrix made from mouse brain tissue with known quantities of ASO. Mouse brain matrix was prepared by homogenizing brain tissue in lysis buffer containing 2.5% IGEPAL, 0.5 M NaCl, 5 mM EDTA, 50 mM tris (pH 8.0), and proteinase K (250 μg/ml) with a motor-driven Dounce homogenizer (Thomas Scientific, 3431D76) on ice. Brain samples were weighed and homogenized the same way as the standards. An internal standard (a different oligonucleotide) was added to all the standards and samples at a concentration of 25 μg/g of brain tissue.

ASOs were extracted from brain lysates using the Clarity OTX solid phase extraction kit (Phenomenex), following the manufacturer’s protocol. LC-MS analyses were performed using a Q-Executive mass spectrometer (Thermo Fisher Scientific) coupled with a Vanquish HPLC system (Thermo Fisher Scientific). Three microliters of the reconstituted extraction solution was injected onto Waters ACQUITY UPLC Oligonucleotide BEH C18 Column (130 Å,1.7 μm, 2.1 × 50 mm) at 80°C using mobile phase A (200 mM hexafluoroisopropanol and 8 mM triethylamine) and mobile phase B (methanol) at a flow rate of 0.3 ml/min. The LC gradients measured by mobile phase B were 0 to 2 min (18%), 2.0 to 6 min (80%), 6.0 to 6.5 min (90%), 8 to 9 min (18%), and 9 to 12.0 min (18%). The effluent from the LC column was introduced into the ion source of the Q-Exactive mass spectrometer operated in negative mode at a spray voltage of 2.57 kV, with sheath and auxiliary gasses set at 45 and 15 arbitrary units, respectively. ASO and the positive control were detected by targeted single ion monitor m/z (mass/charge ratio) scan mass analysis by Orbitrap detector with negative ion mode. Xcalibur software (version 4.0.27 10, Thermo Fisher Scientific) was used for data capture and to calculate peak areas and peak area ratios of analytes (ASO and the positive control) to internal standard. ASO contents in brain samples were interpolated against the standard curve and were normalized to the internal standard.