Overcoming a false-positive mechanism in RapidFire MRM-based high throughput screening

False-positives plague high-throughput screening in general and are costly as they consume resource and time to resolve. Methods that can rapidly identify such compounds at the initial screen are therefore of great value. Advances in mass spectrometry have led to the ability to screen inhibitors in drug discovery applications by direct detection of an enzyme reaction product. The technique is free from some of the artefacts that trouble classical assays such as fluorescence interference. Its direct nature negates the need for coupling enzymes and hence is simpler with fewer opportunities for artefacts. Despite its myriad advantages, we report here a mechanism for false-positive hits which has not been reported in the literature. Further we have developed a pipeline for detecting these false-positive hits and suggest a method to mitigate against them.