1200 lc ms system

To determine the concentration of intact ASO in tissues, samples were minced, weighed into individual wells in a 96-well plate, and then 500 μL homogenization buffer was added to those corresponding wells. Control tissue homogenate for curves was made by weighing control mouse liver and adding homogenization buffer at a 9:1 ratio. 500-μL aliquots were pipetted into a 96-well plate, and the appropriate amounts of calibration standards were spiked in the corresponding wells. Wells containing samples and calibration standards then had internal standard and approximately 0.25-cm³ granite beads added. The plates were then extracted via a liquid-liquid extraction with ammonium hydroxide and phenol:chloroform:isoamyl alcohol (25:24:1). The aqueous layer was then further processed via solid-phase extraction with a Strata X plate. Eluates had a final pass through a protein precipitation plate before being dried down under nitrogen at 50°C. Dried samples were reconstituted in 140 μL water containing 100 μM EDTA. These samples were injected into an Agilent 1200 LCMS system consisting of a 1200 binary pump, 1200 isocratic pump, variable wavelength UV detector, a column oven, an autosampler, and a single quadrupole mass spectrometer (Agilent, Wilmington, DE, USA).