Each year in the U.S., approximately 7-8 million patients with non-traumatic chest pain visit hospital emergency departments (ED) for medical evaluation. It is estimated that approximately 2-5% of these patients are experiencing acute cardiac ischemia, but due to the shortcomings of current test methods, they are incorrectly diagnosed and discharged without appropriate treatment provided, thus leading to poor patient outcome and potential medical malpractice litigation.The goals of this research were to evaluate plasma samples for potential biomarker(s) of acute cardiac ischemia prior to heart tissue necrosis, and to ultimately develop a rapid method for detection of the potential biomarker(s) in human plasma. Initial experiments were performed using the mouse model, with subsequent evaluations on human plasma samples using high performance liquid chromatographic ultraviolet detection (HPLC-UV). The final phase of this research involved the development of a rapid luminometer test method (An HPLC-UV detection method was developed and utilized for inosine, hypoxanthine and other adenosine triphosphate (ATP) catabolic by-products in Krebs-Henseleit (Krebs) buffer solution, with analysis on perfusate samples from isolated mouse hearts undergoing 20 min acute global ischemia. The HPLC-UV method was modified for subsequent use on human plasma samples, obtained from hospital emergency department (ED) patients presenting with non-traumatic chest pain (potential acute cardiac ischemia) and from healthy normal individuals. The HPLC-UV (component quantification) and HPLC-MS (component identification) test methods utilized C18 column technology, mobile phases consisting of aqueous trifluoroacetic acid (0.05% TFA in deionized water pH 2.2, v/v) and methanol gradient to achieve component separation, with both utilizing simple sample preparations (e.g. direct injection of Krebs perfusate samples and centrifugal membrane filtration on plasma samples).Results of the animal experiments using isolated mouse hearts undergoing 20 min acute global ischemia demonstrated significant levels of endogenous inosine effluxed from the heart tissue, indicating its use as a potential candidate biomarker of acute cardiac ischemia. The HPLC results from human plasma representing ED non-traumatic chest pain patients demonstrated elevated levels of inosine (hypoxanthine precursor) and significant levels of hypoxanthine, which provided additional support for the use of these candidate biomarker(s) as a potential diagnostic tool for the initial acute cardiac ischemic event, prior to heart tissue necrosis.The final phase of this research focused on the development of a rapid, simple and sensitive chemiluminescence test method. Using a microplate luminometer with direct injectors and continuous mixing, the measurement of inosine and hypoxanthine in human plasma was achieved for healthy normal individuals and on patients with confirmed acute MI, with an analysis time of less than 5 minutes. The utility of this rapid luminescence technique would be the potential use at point-of-care (POC) services (e.g. hospital clinical laboratory or emergency medical services) as part of the initial ED treatment protocol on patients presenting with non-traumatic chest pain and signs/symptoms of acute myocardial ischemia or acute MI.