Development and validation of stability-indicating RP-HPLC method for estimation of dalfampridine in bulk drug and tablet dosage form

In the current study, a simple, improved, precise, rapid, and accurate reverse phase liquid chromatographic method was produced for the estimation of dalfampridine in bulk and tablet dosage form which is a potassium channel blocker used for the treatment of multiple sclerosis (MS). The separation of dalfampridine was achieved isocratically on a C18 column (250 × 4.6 mm, 5 μm) using (0.1% v/v) buffer pH 3.0 ± 0.05 adjusted with diluted orthophosphoric acid (OPA) and acetonitrile (ACN) in the ratio of 60:40% (v/v) as a mobile phase, at a flow rate of 0.5 mL/min, and column temperature of 40 °C. HPLC grade methanol as diluents was used. Five microliters of the standard solution of the drug was injected, and the eluted analytes were detected at 262 nm. Dalfampridine was eluted at 4.5 min with a run time of 10 min. Linearity in the method was measured in the concentration range of 25–75 ppm with a correlation coefficient of 0.999. Limit of detection and limit of quantitation were found to be 0.711 μg/mL and 2.154 μg/mL, respectively. Dalfampridine was subjected for forced degradation stability study in conditions of thermal, acid, alkali, and oxidation and photo-degradation condition. The degradants were well resolved from the dalfampridine main peak. Validation of the developed method is carried as per USFDA and ICH guidelines. The results of the analysis prove that the method is simple, improved, precise, accurate, and rapid for estimating the content of dalfampridine in bulk drug and tablet dosage form and can be applied for routine analysis.


Background
The dalfampridine, known as 4-aminopyridin and chemically is 1,4-dihydropyridin-4-imine ( Fig. 1), is used mostly for the treatment of multiple sclerosis. Dalfampridine is considered a broad-spectrum in action. Pharmacologically, the drug is a potassium channel blocker and mostly lipophilic in nature, which binds favorably to the open state potassium channel in the central nervous system (CNS) [1,2]. Dalfampridine is rapidly and completely absorbed orally to attain relative bioavailability up to 96%. The excretion takes in unchanged form, mostly from urine (96%) [3]. The reported solubility of dalfampridine was found in water, methanol, acetonitrile (ACN), acetone, ethyl ether, and very soluble in ethanol. It was found slightly soluble in ligroin [4,5]. The stability of a drug in formulation refers to the ability of a particular formulation to maintain its specifications related to its identity, strength, quality, and purity [6]. Degradation studies over the drug can be calculated by exposing the drug in extremes pH conditions (acidic or basic), oxidative reactions, ecstatic temperature, UV, and dry heat to an extent of 5-20% [7,8]. For analysis of a drug and its substances, sensitive methods such as LC/MS and GC/MS are preferred but are expensive. The HPLC is found to be the most reliable and cost-effective [9,10]. The methods reported by the use of reverse phase high-performance liquid chromatography (RP-HPLC) mostly involve the gradient mode of analysis, which makes analysis complex [11]. Hence, the current work aims to develop an accurate, specific, stability-indicating, isocratic method for the estimation of dalfampridine in bulk and tablet form.

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.

Chromatographic conditions
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
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.

Thermal degradation
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.

Acid degradation
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.

Alkali degradation
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.

Peroxide degradation
The standard solution was subjected to oxidative degradation by refluxing with 5 mL of 30% v/v hydrogen peroxide (H 2 O 2 ) 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.

Method validation
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.

Stability-indicating tests
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 (R T ) 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.

Method development and optimization of chromatographic conditions
To achieve satisfactory separation of dalfampridine, different buffer at various pH conditions and solvents of different proportions were tested as binary and tertiary eluents. However, the buffer was adjusted at pH 3.0 with dilute OPA and ACN to achieve good satisfactory results at a flow rate of 0.5 mL/min measured at a detection of 262 nm. The optimized chromatogram and optimized conditions are mentioned in Fig. 2 and Table 1, respectively. System suitability results The system suitability parameters such as retention time, tailing factor, and theoretical plates for optimized standard mixture chromatogram are tabulated in Table 2.

Method validation Linearity
The linearity sample preparation was carried out 4 to 15 ppm (ST-1). The calibration curve (Fig. 3) was plotted with a concentration of standard solutions against mean peak areas.

Range
It is derived from linearity. The range was evaluated by measuring different concentrations of standard solutions to dalfampridine, i.e., 25 to 75 ppm (50 to150%) as shown in ST-2.

Specificity
The standard and sample solution peaks were found at R T 4.5 min within system suitability test acceptance criteria as presented in SF-5. As per degradation studies, the interference with degradants was nil, and the purity angle for the sample solution is less than the purity threshold which indicates the peak is pure. Hence, the results of specificity of the developed method were found to be in acceptance criteria. The results are presented in ST-3.

Accuracy (% Recovery)
The recovery studies show the accuracy in the proposed method; the known amount of pure drug concentrations was spiked in placebo at three different levels, i.e., 50%, 100%, and 150%, and the study was carried out in triplicates for each level. Accuracy was calculated in terms of the percentage of recovery. The results are tabulated in Table 3.

Precision
System precision, method precision, and intermediate precision are the three levels for evaluation. Each level was investigated by six replicate injections of concentration 50 ppm (100%) of dalfampridine. The results of precision were expressed in terms of % assay and RSD and are tabulated in ST-4.

Robustness
The robustness was believed to be unaffected when small, deliberate changes like flow change, buffer pH change, wavelength change, and column temperature were performed at 100% test (50 ppm) concentration.   The R T for the control sample was found at 4.9 min. Then, R T was shifted when the flow rate was changed and found at 6.2 min and 4.1 min at a flow rate of 0.4 and 0.6 mL/min, respectively. The results are summarized in Table 4.

Ruggedness
The ruggedness was carried out by injecting 6 replicate injections of dalfampridine sample solution of conc. 50 ppm, i.e., 100% strength. The analysis was carried out by using two different analysts; hence, the analysis was carried out twice on a system. The results after the test were found within limits, and %RSD was found to be less than 2. The results of ruggedness are tabulated in Table 5.

Limit of detection and limit of quantitation
It was calculated by applying the formula given for LOD and LOQ.
The values for LOD and LOQ after calculation were found to be 0.711 μg/mL and 2.154 μg/mL, respectively.

Discussion
Few methods like UV [12][13][14] spectroscopy and RP-HPLC [15][16][17][18][19] were reported for the estimation of dalfampridine. In the present method, dalfampridine was eluted at 4.5 min with a run time of 10 min. The present method was developed using 0.1% buffer at pH 3.0 ± 0.05 adjusted with diluted OPA and ACN in the ratio of 60:40% v/v. The method was developed with the minimum or reduced amount of organic solvents as the mobile phase which results in a more sensitive and cost-effective method. The analytical methods like stability-indicating RP-HPLC are responsible for idiosyncrasy between active pharmaceutical ingredients from any degradation products  formed under given conditions. These are the methods where the effect of stressors like pH, temperature, and other conditions helps in understanding the stability of the drug during storage conditions and analysis; the results are summarized in Table 6.

Analysis of marketed sample
The method is applicable for the analysis of dalfampridine in marketed tablet dosage forms of 10 mg. The percentage assay results were found to be 98.3% and the amount found was up to 9.83 mg. The results are summarized in Table 7.

Conclusion
An accurate, precise, specific, stability-indicating, isocratic RP-HPLC method was developed for the estimation of dalfampridine in bulk and tablet dosage form. The compound was evaluated by forced degradation pertaining to several stress conditions, where the developed method separates the compound and degradants successfully and estimated the active contents. The method was successfully validated according to USFDA guidelines for all the parameters which were found within acceptance criteria. The developed method may be useful for routine analysis of dalfampridine tablets or for assay of dalfampridine tablets from stability batches.