Immunoassay tests are used in clinical and forensic toxicology laboratories to determine illicit drug use in biological samples. Therefore, this study aims to optimize the cutoff concentrations of DOA I Plus in the blood and compare the LC-MS/MS results. 680 authentic forensic whole blood specimens with Randox Evidence DOA I Plus array were screened for drug of abuse and confirmed by LC-MS/MS. Regarding the manufacturer recommended threshold values, 139 out of 680 authentic blood samples were positive for one or more analytes, while 541 were negative. Nearly all of the 139 positive blood samples confirmed by LC-MS/MS were true positive for one or more analytes, while 522 of the 541 negative blood samples were true negative. The overall sensitivity and the specificity were 87.8% and 99.6%, respectively. THC was considered in detail, and a receiver operator characteristic curve analysis was performed to determine the optimum cutoff for THC, as it accounts for 78% of all positive results according to the manufacturer's recommended thresholds. The optimal threshold value for THC was determined at a concentration of 23 ng/mL, while these values for other parameters were defined as recommended by the manufacturer.
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