Analytical method validation of amphetamine and methamphetamine in blood by LC-MS/MS and its application to real samples

The use of illicit sedative, narcotic, and stimulant substances classified as psychoactive drugs is increasingly prevalent both globally and within our country. In addition to preventing the use of such substances, accurately identifying their presence in individuals, both qualitatively and quantitatively, is of critical importance. In this context, the demand for rapid, reliable, and scientifically grounded toxicological analysis methods is steadily growing. Forensic sciences aim to meet this need by closely following the latest scientific and technological developments and conducting studies to obtain highly accurate and precise results. Judicial authorities require definitive and trustworthy evidence to clarify incidents and ensure fair rulings. This study encompasses the development and validation of an analytical method based on solid phase extraction (SPE) and liquid chromatography–tandem mass spectrometry (LC-MS/MS) for the detection of amphetamine and methamphetamine in blood samples. Obtaining reliable and valid results in toxicological analyses is of critical importance in forensic sciences, and the conformity of developed methods to international validation criteria is a fundamental requirement. The method's selectivity was evaluated through analyses of blank blood samples, and no interference was observed for amphetamine and methamphetamine. The linear range was established between 1–200 ng/mL, with correlation coefficients of 0.996 for amphetamine and 0.992 for methamphetamine. The limits of detection (LOD) were calculated as 0.46 ng/mL for amphetamine and 0.31 ng/mL for methamphetamine; the limits of quantification (LOQ) were 0.95 ng/mL and 0.65 ng/mL, respectively. Repeatability of the method was assessed through intra-day and inter-day analyses, with %RSD values found to be below 15%. Recovery rates ranged from 93.28% to 111.87% for amphetamine and from 105.15% to 115.17% for methamphetamine. Stability studies conducted at +4 °C using an autosampler over 7 days and long-term storage at −20 °C for 6 months revealed deviation rates of less than 10%. All validation parameters obtained fall within the acceptable ranges defined by international guidelines for toxicological method validation. Accordingly, the developed method has been demonstrated to be valid for the reliable detection and quantification of amphetamine and methamphetamine in blood samples. The validated method was successfully applied to real forensic cases and is expected to contribute significantly to the elucidation of forensic investigations.