A Sensitive LC-MS/MS Assay for the Quantification of Methadone and its Metabolites in Dried Blood Spots: Comparison With Plasma

Introduction: Methadone, a synthetic narcotic, is widely used both in adults and children for pain control and as a replacement drug in opioid use disorder to prevent craving and withdrawal. To support clinical pharmacokinetic trials in neonates, infants, and children, the authors developed and validated a novel, automated, highly sensitive liquid chromatography–electrospray–tandem mass spectrometry ionization (LC-ESI-MS/MS) method for the quantification of methadone and its metabolites, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) and 2-ethyl-5-methyl-3,3-diphenylpyraline (EMDP), in samples collected as dried blood spots. Methods: Blood was spiked with different concentrations of methadone, EDDP, and EMDP, and blood drops were applied to filter paper cards. Punches of 6.4 mm were removed from the cards, and 600 µL of protein precipitation solution (methanol/0.2M ZnSO 4 , 7:3, vol/vol) containing the internal standards (methadone-d 9 and EDDP-d 5 ) at a concentration of 1 mcg/L was added. The extracts were analyzed using LC-ESI-MS/MS in combination with online extraction. The mass spectrometer was run in the positive multiple reaction monitoring mode, and the total run time was 3.2 minutes. Results: For the dried blood spots, the assay has a lower limit of quantification of 0.1 mcg/L for methadone, EDDP, and EMDP. The range of reliable response for methadone for the ion transition m/z = 310.2→265.1 was 0.1–100 mcg/L and for the ion transition m/z = 310.2→223.1 5–1000 mcg/L. For EDDP, on the range of reliable response for the ion transition, m/z = 278.2→234.3 was 0.1–100 mcg/L and for the ion transition m/z = 278.2→186.1 5–1000 mcg/L. The calibration range for EMDP was 0.1–100 mcg/L. Accuracy (85%–115%) and imprecision (<15%) met predefined acceptance criteria. Discussion: This assay allows for the measurement of small volume blood samples without the need for an intravenous blood draw, and thus, it is suitable for pharmacokinetics studies and therapeutic drug monitoring in pediatric patients.