Assessing the usefulness of solid-phase microextraction (SPME) for methadone and its metabolites analysis in urine samples using HPLC/SPME fibre tips

Solid-phase microextraction (SPME) has been introduced as a novel, simple and single- step technique of extracting methadone (MDN), 2-ethylidene-1,5-dimethyl-3,3- diphenylpyrrolidine (EDDP) and 2-ethyl-5-methyl-3,3-diphenyl-1-pyrroline (EMDP) from urine samples. In this study, direct immersion SPME followed by high-performance liquid chromatography with ultraviolet detection (HPLC-UV) was developed to identify methadone and its metabolites. The separation was performed on a Gemini C18 (octadecyl carbon chain) analytical column (150 x 2.0 mm, 5 µm) and detected by an ultraviolet (UV) detector at 210 nm. The factors influencing the SPME procedure, such as the sample’s pH, fibre coating type, salt addition and desorption solvent type were optimised. The best conditions were obtained with a C18 coating at a pH of 11, NaCl 15% and ethyl acetate. The recoveries of MDN, EDDP and EMDP under optimum conditions were 90.8%, 89.3% and 86.5 %, respectively. The calibration curves for urine samples showed good linearity under optimum conditions (R² range 0.9983–0.9988) in a concentration range of 0.025–4 µg/mL for the analytes, using hydrocodone as the internal standard. All samples were analyzed using validated method. The selectivity of this method was evaluated and provided clean chromatograms with no interference in the analysis. Finally, the results show that the novel SPME fibre tips have relatively high extraction efficiency for methadone and its metabolites.