Tqms mass spectrometer

The assay was conducted at 37°C in single time point mode (60 min incubation) in triplicate. Test compounds or procaine were tested at a final concentration of 1 μM in either 2.5% DMSO / 97.5% mouse plasma (Bioreclamation MSEPLNAHP-C57-M; pooled filtered plasma from male C57BL/6 mice; sodium heparin as anticoagulant; pH adjusted to 7.4 with 2N HCl on day of use) or 2.5% DMSO / 97.5% PBS (pH = 7.4 PBS: 136.9 mM NaCl; 2.68 mM KCl; 8.1 mM Na₂HPO₄; 1.47 mM KH₂PO₄; 0.9 mM CaCl₂; 0.49 mM MgCl₂). Samples were analyzed with a Waters^® Aquity UPLC (BEH C18 1.7 μm, 2.1 × 50 mm column) in tandem with Waters^® TQ MS mass spectrometer. 5 μl of sample was injected with a 3 min linear 5% to 95% acetonitrile/water gradient in 0.1% formic acid with a 0.5 ml/min flow rate. Compound electrospray mode, capillary voltages, cone voltages, MRMs (one per compound) and quantization were optimized and determined using Waters^® QuanOptimize software with propafenone as an internal standard. Standard curves (six concentrations from 0–1000 nM) were generated with room temperature PBS or plasma. In order to identify plasma-dependent stability effects, stability was expressed as % compound remaining (PBS). The reference compound procaine typically was 100% stable in PBS but was > 95% degraded in plasma.