Hi all, I've completed a microsome stability assay for 16 novel compounds, and for about half of them, the calculated half-life is negative. During the experiment, I withdraw sample at t=0, 5, 10, 20, and 30 min and crash into ACN with internal standard. I then spin the plate down, collect the supernatant, and analyze the analyte peaks by LCMS/MS.

When I generate the LN percent remaining based on the analyte peak:internal standard AUC ratios, the value at t=30 min is higher than at t=0 minutes, meaning the compound isn't getting degraded/metabolized but also that there is also more compound present at the t=30 min time point. I then plot the LN percent remaining against time (min) to generate a slope, which is almost always positive, so when I divide ln(2) by my gradient (-slope), the value ends up being negative, which means my rate of intrinsic clearance is also negative.

Does anyone have any sort of explanation?

Thanks!