Waters HPLC (Separation module 2695) chromatographic system equipped with PDA-detector 2487 Xterra C18 (250 × 4.6 mm, 5 μL) thermostatic column compartment connected with Empower-3 software, consisting of pump, autosampler, and auto-injector. Shimadzu-(ATX 224)-digital weighing balance, BT ultra sonicator 48, digital systronic pH meter (802), and Millipore vacuum filter pump (XI 5522050) were used for the method development. A 0.22-μm Nylon filter of Merck Millipore was used for filtration.
Materials and reagents
The pharmaceutical grade working standards of dalfampridine were obtained as a gift from Enaltec Pharma. Research Pvt. Ltd (Ambernath, Mumbai, India). Dalfampridine tablet formulation (10 mg) was used of brand AMPYRA, marketed by Accorda Therapeutics Inc. The HPLC grade triethylamine, OPA, methanol, and ACN were procured from SD Fine Chem., Mumbai, India, for the present study. The Milli-Q water procured from Mumbai, India, was used for the analysis.
The buffer pH 3.0 and ACN in the ratio 60:40% v/v was used as a mobile phase. The flow rate was maintained at 0.5 mL/min. The column temperature was kept at 40 °C, and the detection was carried out at 262 nm with an injection volume of 5 μL.
Preparation of the mobile phase
One milliliter of triethylamine was added to 1000 mL Milli-Q water. The pH was adjusted to 3.0 ± 0.05 with OPA. The solution was filtered through a 0.45-μ membrane filter and was sonicated for 15 min to degas it. The buffer (0.1%) pH 3.0 and ACN in the ratio of 60:40% v/v was used as a mobile phase. Dalfampridine was separated and eluted in an isocratic program.
Preparation of standard solution (50 ppm)
Accurately weighed 50 mg of dalfampridine as working standard was transferred to a volumetric flask of 50 mL followed by 30 mL of methanol and sonicated. The cooled solution was adjusted up to the mark with methanol. Further 5 mL solution was diluted to 100 mL with diluents.
Preparation of sample solution (50 ppm)
Ten intact tablets of dalfampridine were transferred into a 100-mL volumetric flask. About 70 mL of methanol was added, and the resulting solution was sonicated for 25 min with intermittent shaking, then cooled, and diluted up to the mark with methanol. The resulting solution was then allowed to settle for 15 min and then the solution was centrifuged at 5000 RPM. Further, 5 mL of the supernatant solution was diluted to 100 mL with diluents and filtered through a 0.45-μ Nylon membrane syringe filter or equivalent.
Forced degradation study
Forced degradation studies were carried out on dalfampridine under several conditions as per ICH guidelines Q1A(R2) and Q1 B.
Photolytic degradation was performed by amusing samples under UV and white light for 1.2 million lux hours in a Petri plate for 7 days. The previously exposed sample (10 tablets) was weighed and diluted with methanol to 100 mL after sonication. Supernatant 5 mL solution was diluted to 100 mL with diluent.
The thermal stability of the drugs was calculated by keeping the sample in an oven at about 70 °C for 24 h. About 5 mL of this stressed solution was diluted with diluent.
Degradation under acidic condition was calculated by treating the stock solution with 5 mL of 5N HCl and refluxed at 60 °C for 2 h. The diluent is used for the dilution of the resulting solution.
Under alkaline conditions, degradation was studied by refluxing standard solution with 5 mL of 5N NaOH and refluxed at 60 °C for 2 h. The diluent is used for the dilution of the resulting solution.
The standard solution was subjected to oxidative degradation by refluxing with 5 mL of 30% v/v hydrogen peroxide (H2O2) solution at 60 °C for 2 h and then treated with diluent.
The chromatogram was studied according to the area of peak of drug and appearance of another/secondary peaks. Any change in area and appearance of secondary peaks will be considered as degradation.
The developed method was validated as per ICH Q2 (R1) and USFDA guidelines. The method was validated for the parameters such as linearity and range, specificity, accuracy, precision, robustness, ruggedness, limit of detection (LOD), and limit of quantitation (LOQ). The specificity was determined by injecting samples of blank, placebo (except dalfampridine), standard solution, and sample solution from the formulation.
According to the force degradation experiments and chromatographic analysis, it was concluded that dalfampridine resulted into minimal degradation in conditions such as acid (SF-2), base (SF-3), and peroxide (SF-4) and thermal and photolytic degrading conditions in the range of 1–8%. The higher degradation was found in base degrading condition up to 8.6%, and only one degradation product was found in peroxide degrading condition, which was eluted at a retention time (RT) of 4.9 min. The control sample (SF-1), i.e., the sample of unstressed condition, was also employed for analysis, which was used for comparison with results of forced degradation. The results of force degradation are tabulated in Table 6.