Reagents, reference standards and materials.
It has been used methadone hydrochloride purchased from Laboratorios Dr. Esteve S.A. (Barcelona, Spain). As preservatives, methylparaben and propylparaben, acquired from the Fagron Iberica (Terrassa, Spain) were used. Pure water was obtained from Grifols laboratory (Barcelona, Spain). All of them were of Pharmacopoeia grade.
In the mobile phase we used acetonitrile HPLC grade, purchased from VWR Prolabo Chemicals (Fontenay-Sous-Bois, France). Phosphoric acid, sodium hydroxide (>99%), hydrochloric acid and hydrogen peroxide were supplied from Panreac (Barcelona, Spain) and Milli-Q water. All reagents and solvents were of analytical grade.
HPLC analyses were performed on a qualified and calibrated chromatography system, Agilent-Technologies 1,100 series (Madrid, Spain) comprising a quaternary gradient pump, an ultraviolet photodiode-array detector (UV-DAD), a 100-vial programmable autosampler, a column oven compartment, an automatic injector and a software controller.
We have used a Waters-XTerraTMRP18 (3.5µm;4.6x100mm) column. The column temperature was maintained at 40oC. The mobile phase consisted of acetonitrile as the organic phase (55%) and sodium phosphate 25mM (adjusted to pH=10) as the aqueous phase (45%). The flow rate was 1.6mL/min. The injection volume was 5µL for each chromatographic analysis. The UV-DAD was set at λ = 254nm.
Validation of the HPLC method.
The methods and their acceptance criteria were established on the basis of the International Conference on Harmonization (ICH) guidelines Q2 (R1).
A standard solution (100mL) of methadone 50mg/mL was prepared from which, by means of serial dilutions, a total of 20 calibration standards were obtained for the linearity test (four replicates for each concentration level). The curve was constructed from methadone working concentrations of 75%-125% (7.5, 9.0, 10.0, 11.0 and 12.5mg/mL) to assess the linear relationship between the concentration of the analyte and the obtained areas. Once the regression equation was obtained, an analysis of variance (ANOVA) was performed.
Instrumental precision (repeatibility), intra-assay precision and inter-assay precision (intermediate precision) were measured. For instrumental precision, a standard solution (10mL) of methadone 10mg/mL was prepared by the same analyst on a single day and consecutively analyzed ten times to check the repeatability of the method and to assess the dispersion degree among the series of measurements obtained. For intra-assay precision, six standards of methadone solution 10mg/mL were prepared and analyzed. Inter-assay precision was also performed in another six standards of 10mL of methadone solution 10mg/mL which were prepared on a second day by different analysts, obtaining a total of 12 samples.
The accuracy of the method was determined through spike recovery of the methadone solution with a preservative matrix, diluted within the range used for final sample measurements, and within the range of the corresponding calibration curves. Afterwards, three 10mL replicates of three concentration levels 7.5, 10 and 12.5mg/mL were prepared by serial dilutions from 100mL of a 50mg/ml stock solution. The recovery percentage and relative standard deviation percentage (%RSD) were calculated. The maximum aceptable levels were 10%.
Detection and quantification limit.
Detection limit (LD) and quantification limit (LQ) in case of instrumental method, can be estimated using various equations. However, in this assay, it was decided to use an experimental method, following EURACHEM recommendations which consisted of preparing a series of samples with decreasing amounts of analyte and analyzing each of them six consecutive times, representing %RSD of the precision against the concentration of each sample. For this purpose, we prepared a battery of serial dilutions from a stock solution of methadone 20mg/mL. The concentrations were 2, 0.2, 0.02, 0.002 and 0.0002mg/mL. Six replicates of each concentration were prepared, from which the area and retention times were obtained. Normally, a precision criterion of %RSD of 10% is set at the LQ although up to 20% can be accepted, depending on the characteristics of the method. Both were also expressed as a percentage of the theoretical concentration.
The methadone hydrochloride 10mg/ml oral solution was subjected to the following denaturing conditions to determine the capacity of the HPLC method in order to detect any possible degradation products produced during storage: in acid (0.1M HCl at 25oC), in base (0.1M NaOH at 25oC) and in oxidation (3% H2O2 at 25oC). For this, 0.1mL of methadone 10mg/mL was diluted in 1mL of each denaturing reagent and they were kept in contact for 1 hour until analysis. Then, following the same chromatographic conditions, elution cycles of 60 minutes were programmed, and the test was carried out eight times for each stress condition. Peak purity was also calculated using the Agilent-ChemStation software tool based on the similarity factor.