Development and Validation of a HPLC-UV Method for Methadone Hydrochloride Quantification in a New Oral Solution with Preservatives to be Implemented in Physicochemical Stability Studies.

Purpose: The Pharmacy Service of the Infanta Leonor University Hospital acquires, compounds, distributes and dispenses more than 3,000 liters of methadone oral solution to Drug Addiction Patients Centers per year. Our purpose is to develop and validate an improved high performance liquid chromatography (HPLC) method to quantify methadone hydrochloride in a new oral solution with methylhydroxybenzoate (methylparaben) and propylhydroxybenzoate (propylparaben) to be implemented in physicochemical stability studies that allow to provide more information and even to increase the beyond-use date. Methods: : A HPLC-Agilent® 1,100 equipment, comprising a quaternary pump and an ultraviolet diode-array-detector (DAD) was used. An analytical method development and validation was completed. The curve was constructed from methadone working concentrations of 75%-125% (7.5, 9.0, 10.0, 11.0 and 12.5 mg/mL) to assess the linear relationship between the concentration of the analyte and the obtained areas. Precision and accuracy were calculated. Detection and quantification limit (LD, LQ) were estimated using the EURACHEM method. Forced-degradation studies were also performed. Results: : Chromatographic conditions were: flow rate 1.6 mL/min; mobile phase 55% acetonitrile and 45% sodium phosphate 25 mM (pH=10); injection volume was 5 µL. The column was a Waters-XTerra TM RP18, maintained at 40 o C. DAD was λ=254 nm. Retention times for methadone, methylparaben and propylparaben were 4.34, 0.70 and 0.88 minutes respectively.The method was linear (y=284.3x-97.8, r=0.997). Instrumental precision was 0.33% for standards (n=10); intra-assay precision 0.53% (n=6) and inter-assay precision 1.95% (n=12). The relative standard deviation percentage for accuracy was 1.28%. The recovery percentage was 101.5 ± 1.5%. LQ and LD were 2.18 mg/mL and 2.0 mg/mL respectively. The most destabilizing conditions were oxidizing and alkaline. The chromatograms confirmed no interference with the methadone signal. Conclusions: : The HPLC method has proved to be valid and reproducible for methadone quantification in a new oral solution with methylparaben and propylparaben. This assay is a rapid, simple and reliable technique that can be used in daily analysis and physicochemical stability studies.