For the analysis of urinary free cortisol (UFC), liquid chromatography (LC), LC-mass spectrometry (LC-MS), and liquid chromatography-tandem MS (LC-MS/MS) offer better specificity and accuracy of result than immunoassay-based methods (1). The high specificity of LC-MS/MS provides simplification of sample preparation, reduced costs, increased sample throughput, and a low rate of sample interference. Published approaches to LC-MS/MS analysis of UFC use either sample extraction (1) or direct sample injection (2). The direct methods reduce the labor requirements and decrease the potential for human error. We have developed a direct sample injection method similar to one published by Nassar et al. (2). The primary changes in the proposed method are the use of a guard cartridge for trapping cortisol as part of an online purification strategy and the use of step gradient rather than isocratic LC conditions. Both electrospray ionization (ESI) and atmospheric pressure ionization (APCI) are well suited for LC-MS/MS. We used APCI because preliminary experiments demonstrated a two- to threefold increase in sensitivity compared with ESI. Better sensitivity for cortisol with an APCI interface compared with ESI can be explained by the poor ionization of cortisol in solution. Cortisol and ammonium formate were purchased from Sigma, and d4-cortisol was purchased from Cambridge Isotope Laboratories. Methanol (HPLC grade) was purchased from Fisher Scientific. Patient samples were analyzed for cortisol within 1–3 days after collection. All studies with samples from humans were approved by the Institutional Review Board of the University of Utah. Sample preparation for the method was performed as follows. A 500-μL aliquot of centrifuged urine sample and 100 μL of the working internal standard (IS; d4-cortisol, 0.5 mg/L) were added to autosampler vials, and the vials were vortex-mixed. A PE series 200 HPLC system (Perkin-Elmer Analytical Instruments) was equipped with a Luna C18 column …