Project Summary/Abstract Drug screening in toxicology cases is performed mainly using immunoassays, gas- chromatography mass spectrometry (GC-MS), and liquid chromatography-mass spectrometry (LC-MS). Immunoassays lack the sensitivity, multiplexing capability, and flexibility to respond to the rise of potent synthetic drugs including fentanyl analogs and synthetic cannabinoids. GC- MS requires extensive sample preparation including derivatization reactions to achieve adequate performance. LC-MS, despite being a mature technology, is still not used by most labs for drug screening due to is high cost and complexity. We therefore see a both a technical and market need for better drug screening technologies that combine the simplicity and automation of immunoassays with the high sensitivity and specificity of mass spectrometry. In this Fast-Track (combined phase I and phase II) proposal, we seek to develop a mass spectrometry-based drug screening system utilizing paper spray mass spectrometry. The outcomes of this project will include two disposable cartridge devices which perform sample collection, storage, extraction, and ionization for MS detection. In addition to the disposable devices, we will also develop a simple manually operated and a second automated mass spectrometer interface which will enable analysis of the sample analysis cartridge on commercially available mass spectrometers. Finally, we will create instrument control and data analysis software package to provide a complete, end- to-end solution for drug screening. In phase I of this proposal, a prototype for the disposable paper spray analysis cartridge as well as the manual mass spectrometry interface will be developed and tested. If the performance of the analysis cartridge and interface are adequate for screening a representative set of synthetic drugs, we will proceed to phase II. In the second phase, we will improve the sample analysis cartridge, develop a sample collection device which will enable robust collection of a known volume of blood from an unspecified initial volume, develop the automated MS interface, develop needed control and data analysis software, and test the performance of the approach on both high resolution and low resolution mass spectrometers.