A full method validation should be performed for any analytical method whether new or based upon literature [29] as this ensures that the method developed is reproducible, stable, sensitive, robust, suitable and reliable for its application in pharmaceutical analysis.
The ICH recommends evaluating linearity in the range 80–120% [25]. In our case, a margin of 75–125% was chosen and the method demonstrated good linearity over the range assayed. The method was repeatable. Intra- and inter-assay precision %RSD were < 10% indicating that the assay method was reproducible across days. The accuracy of an analytical procedure expresses the closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value and the value found [25]. The small percentage of difference between the nominal and found concentration of the standards demonstrated that the assay is accurate enough for its application. The mean recovery values obtained were 100.0% and 98.5% respectively. Higher recovery indicates an efficient extraction procedure and higher sensitivity and accuracy of the analytical method.
Following the specifications for methadone hydrochloride in oral solution described in the USP, Spanish Pharmacopoeia and Spanish National Formulary [2, 4, 5], it contains no less than 90.0% and no more than 110.0% of methadone hydrochloride. Consequently, one of the objectives of our method was to quantify methadone near 100% concentration.
When the method is defined as a content assessment analysis, where we will always work in ranges distant from the minimum detectable or quantifiable quantity using the equipment, it is not necessary to determine LQ and LD. However, they were calculated, on the one hand to demonstrate this situation, and on the other hand, to have a more complete validation because it allows a better understanding of the analytical method, and knowing the minimum analyte amounts that can be quantified, can be useful for other applications. The method has been shown to be capable of detecting and quantifying at the value of 0.02%, so the method not only quantifies perfectly in the limit of 90–110% indicated by the pharmacopoeias, but also is capable of quantifying impurities or degradation products that could be present in 0.02% of the final sample analyzed, in our case, the new oral solution.
The ability of the assay to detect methadone decomposition was demostrated by stressing a methadone sample in forced-degradation studies which showed that the most destabilizing stress conditions were the oxidizing and alkaline conditions. The peaks obtained did not interfere with methadone. Therefore, we can infer that under pH conditions according to the specifications (< 6.5), the pH variation within said limits will not produce any product that interferes with methadone quantification. Thus, it has been demonstrated that our method is suitable for the detection and quantification of methadone hydrochloride in the presence of degradation products. It is important to highlight that methadone solution as final pharmaceutical product will never occur in these extreme conditions, so it is unncessary to characterize and quantify the peaks found in the chromatograms.
On the other hand, to understand the chromatographic conditions chosen for this study, it is first necessary to study the characteristics of the methods described in the literature, and secondly, to know the methadone hydrochloride behavior in order to improve the conditions described. In this sense, at the beginning of the method design, we studied methadone characteristics, and based on this, we tested different conditions to choose the more suitable ones.
The methadone pKa is 8.3, so a high pH environment was needed in which we make sure that methadone is totally deprotonated. It was necessary to choose a suitable column and a mobile phase to work at a high pH values. For this reason, the column chosen was the Waters-XTerraTM RP18, which in addition to being resistant (it allows working at pH up to 12), allowed us to avoid the tailing factor, obtain short retention times and achieve a good resolution of the methadone peak, in addition to being sufficiently effective in avoiding possible interference between methylparaben, propylparaben and methadone (Fig. 1). The XTerraRP (First-Generation-Hybrid-Filler) columns, combine the best properties of silica and polymer bonded phases with patented hybrid-particle-technology, which replaces one in three silanols with a methyl group. The result is a mechanically strong particle that can be used for high pH separations; as a consequence the charge and peak shapes of the basic compounds are improved.
Our wavelength is the one collected inside the UV-DAD and in USP method, where comparing with 274 nm, a higher absorption was observed. Regarding the mobile phase, different percentages were tested until 45–55% was reached, where the peaks were perfectly separated and resolved in < 5 min.
The evaluation of robustness was considered not necessary. Variations in the flow rate or temperature will not put the resolution between paraben and methadone at risk, as the resolution between them is close to 20. Nevertheless, the pH of the mobile phase is a critical parameter due to the ionization of methadone and the stability of the stationary phase. According to the pKa of methadone and the column manufacturer´s directions, pH must be adjusted between 9.3 and 11. Below 9.3 methadone peak will show lower retention time and tailing, and above 11 the shelf life of the column is diminished.
In this study we needed an analytical method to quantify methadone hydrochloride in a new oral solution with preservatives, not previously analyzed in the literature. Comparing our results with those found in other HPLC methods developed in different methadone formulations, we can affirm that our method is efficient, rapid, simple, capable of quantifying methadone without interference from preservatives and better than the methods already described whose disadvantages have been highlighted.
The method reported in this assay can also be used to carry out physicochemical stability studies in which the possible methadone degradation can be detected in different conservation environments over a period of time.