Determination of Methadone and Tramadol in Vitreous Humor Specimens: Experimental Design to Optimize Dispersive Liquid-Liquid Microextraction Before Ultrahigh Performance Liquid Chromatography Analysis

Background: Drug abuse is spreading rapidly all over the world. Methadone and tramadol are among not only the most abused opioids but also important from the forensic point of view. Therefore, we need to devise a simple and sensitive method for the sample preparation and identification of abused drugs in postmortem specimens.Methods: A simple and rapid dispersive liquid-liquid microextraction (DLLME) technique coupled with ultrahigh performance liquid chromatography (UHPLC) was developed for the extraction and analysis of methadone and tramadol from postmortem vitreous humor samples. Different parameters affecting the extraction recovery, such as the type and volume of extraction and dispersion solvents, pH value, sensitivity, and specificity, were optimized and studied. Results: Under optimized conditions, the recovery ranges were 82.3%-89.6% and 85.4%-87.1% for methadone and tramadol, respectively. The linear range was 25-100 ng/mL for both methadone and tramadol with a correlation coefficient (R2) of more than 0.98. Limit of detection (LOD) and limit of quantification (LOQ) were 3 and 8 ng/mL for methadone and 6 and 16 ng/mL for tramadol. The accuracy level of the methods for methadone and tramadol detection were 99.4%-100% and 99.7%-99.9%, respectively. The method was specific enough for the qualitative and quantitative determination of methadone and tramadol.Conclusion: The obtained results showed that DLLME combined with UHPLC is a fast and straightforward method for determining methadone and tramadol in postmortem vitreous humor specimens.