Dalfampridine

This article is compiled based on the United States Pharmacopeia (USP) – 2025 Edition
Issued and maintained by the United States Pharmacopeial Convention (USP)
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1 DEFINITION 

Dalfampridine contains NLT 98.0% and NMT 102.0% of dalfampridine (C₅H₆N₂). 

2 IDENTIFICATION

Change to read: 

A. Spectroscopic Identification Tests 〈197〉, Infrared Spectroscopy: 197K or 197A (ERR 1-Oct-2020) 

B. The retention time of the major peak of the Sample solution corresponds to that of the Standard solution, as obtained in the Assay. 

3 ASSAY 

3.1 Procedure 

Solution A: 3.03 g/L of sodium 1-heptanesulfonate, 1.36 g/L of monobasic potassium phosphate, and 1.15 g/L of phosphoric acid in water

Solution B: Acetonitrile 

Mobile phase: See Table 1. 

Table 1 

Diluent: Solution A and Solution B (90:10) 

System suitability stock solution: 0.04 mg/mL of USP Dalfampridine Related Compound A RS prepared as follows. Transfer a suitable quantity of USP Dalfampridine Related Compound A RS to an appropriate volumetric ask and add 20% of the total ask volume of Diluent. Sonicate for about 5 min. Allow to cool to room temperature and dilute with Diluent to volume. Use within 24 h. 

System suitability solution: 0.0002 mg/mL of USP Dalfampridine Related Compound A RS from System suitability stock solution and 0.2 mg/mL of USP Dalfampridine RS in Diluent prepared as follows. Transfer a suitable quantity of USP Dalfampridine RS to an appropriate volumetric ask, add an appropriate volume of System suitability stock solution, and add 25% of the total ask volume of Diluent. Sonicate for NLT 5 min. Allow to cool to room temperature and dilute with Diluent to volume. Pass the resulting solution through a suitable lter, discard NLT the rst 2 mL, and use the ltrate. 

Standard solution: 0.2 mg/mL of USP Dalfampridine RS in Diluent prepared as follows. Transfer a suitable quantity of USP Dalfampridine RS to an appropriate volumetric ask and add 25% of the total ask volume of Diluent. Sonicate for NLT 5 min. Allow to cool to room temperature and dilute with Diluent to volume. Pass the resulting solution through a suitable lter and use the ltrate. 

Sample solution: 0.2 mg/mL of Dalfampridine in Diluent prepared as follows. Transfer a suitable quantity of Dalfampridine to an appropriate volumetric ask and add 25% of the total ask volume of Diluent. Sonicate for NLT 5 min. Allow to cool to room temperature and dilute with Diluent to volume. Pass the resulting solution through a suitable lter and use the ltrate. 

Chromatographic system 

(See Chromatography 〈621〉, System Suitability.) 

Mode: LC 

Detector: UV 275 nm 

Column: 4.6-mm × 10.0-cm; 3.5-µm packing L1 

Column temperature: 30° 

Flow rate: 2 mL/min 

Injection volume: 10 µL 

System suitability 

Samples: System suitability solution and Standard solution 

[Note—See Table 2 for the relative retention times.] 

Suitability requirements 

Resolution: NLT 2.0 between dalfampridine and dalfampridine related compound A, System suitability solution 

Tailing factor: NMT 2.0, Standard solution 

Relative standard deviation: NMT 1.0%, Standard solution 

Analysis 

Samples: Standard solution and Sample solution 

Calculate the percentage of dalfampridine (C₅H₆N₂) in the portion of Dalfampridine taken:  

Result = (r_u/r_s) × (C_s/C_u) × 100 

r_u = peak response from the Sample solution 

r_s = peak response from the Standard solution 

C_s = concentration of USP Dalfampridine RS in the Standard solution (mg/mL) 

C_u = concentration of Dalfampridine in the Sample solution (mg/mL) 

Acceptance criteria: 98.0%–102.0% 

4 IMPURITIES 

4.1 Residue on Ignition 〈281〉 

Ignition temperature: 800° 

Acceptance criteria: NMT 0.3% 

4.2 • Limit of Dalfampridine Related Compound B and Dalfampridine Related Compound C 

[Note—This test should be conducted if dalfampridine related compound B and dalfampridine related compound C are possible from the manufacturing process.] 

Solution A, Solution B, Mobile phase, and Diluent: Prepare as directed in the Assay. 

Standard solution: 2 µg/mL each of USP Dalfampridine Related Compound B RS and USP Dalfampridine Related Compound C RS in Diluent Sensitivity solution: 0.1 µg/mL each of USP Dalfampridine Related Compound B RS and USP Dalfampridine Related Compound C RS from Standard solution in Diluent 

Sample solution: 2000 µg/mL of Dalfampridine in Diluent prepared as follows. Transfer a suitable quantity of Dalfampridine to an appropriate volumetric ask and add 25% of the total ask volume of Diluent. Sonicate for NLT 5 min. Allow to cool to room temperature and dilute with Diluent to volume. 

Chromatographic system: Proceed as directed in the Assay, except use an Injection volume of 30 µL. 

System suitability 

Samples: Standard solution and Sensitivity solution 

[Note—See Table 2 for the relative retention times.] 

Suitability requirements 

Relative standard deviation: NMT 10% each for dalfampridine related compound B and dalfampridine related compound C, Standard solution 

Signal-to-noise ratio: NLT 10 each for dalfampridine related compound B and dalfampridine related compound C, Sensitivity solution

Analysis 

Samples: Standard solution and Sample solution 

Calculate the percentage of dalfampridine related compound B and dalfampridine related compound C in the portion of Dalfampridine taken: 

Result = (r_u/r_s) × (C_s/C_u) × 100 

r_u = peak response of dalfampridine related compound B or dalfampridine related compound C from the Sample solution U 

r_s = peak response of dalfampridine related compound B or dalfampridine related compound C from the Standard solution S 

C_s = concentration of USP Dalfampridine Related Compound B RS or USP Dalfampridine Related Compound C RS in the Standard solution (µg/mL) 

C_u = concentration of Dalfampridine in the Sample solution (µg/mL) 

Acceptance criteria: NMT 0.0075% each for dalfampridine related compound B and dalfampridine related compound C

4.3 Organic Impurities 

Solution A, Solution B, Mobile phase, Diluent, and System suitability solution: Prepare as directed in the Assay. Standard solution: 2 µg/mL of USP Dalfampridine RS in Diluent 

Sensitivity solution: 0.1 µg/mL of USP Dalfampridine RS from Standard solution in Diluent 

Sample solution: 200 µg/mL of Dalfampridine in Diluent prepared as follows. Transfer a suitable quantity of Dalfampridine to an appropriate volumetric ask and add 25% of the total ask volume of Diluent. Sonicate for NLT 5 min. Allow to cool to room temperature and dilute with Diluent to volume. Pass the resulting solution through a suitable lter and use the ltrate. 

Chromatographic system: Proceed as directed in the Assay, except use a Detector wavelength of 265 nm for isonicotinamide. System suitability 

Samples: System suitability solution, Standard solution, and Sensitivity solution 

[Note—See Table 2 for the relative retention times.] 

Suitability requirements 

Resolution: NLT 2.0 between dalfampridine and dalfampridine related compound A, System suitability solution Tailing factor: NMT 2.0 for dalfampridine, System suitability solution 

Relative standard deviation: NMT 5.0% for dalfampridine, Standard solution 

Signal-to-noise ratio: NLT 10, Sensitivity solution 

Analysis 

Samples: Standard solution and Sample solution 

Calculate the percentage of isonicotinamide in the portion of Dalfampridine taken: 

Result = (r_u/r_s) × (C_s/C_u) × (1/F) × 100 

r_u = peak response of isonicotinamide at 265 nm from the Sample solution 

r_s = peak response of dalfampridine at 275 nm from the Standard solution 

C_s = concentration of USP Dalfampridine RS in the Standard solution (µg/mL) 

C_u = concentration of Dalfampridine in the Sample solution (µg/mL) 

F = relative response factor (see Table 2) 

Calculate the percentage of dalfampridine related compound A and any other unspecied impurity in the portion of Dalfampridine taken: 

Result = (r_u/r_s) × (C_s/C_u) × (1/F) × 100 

r_u = peak response of dalfampridine related compound A or any unspecied impurity at 275 nm from the Sample solution U 

r_s = peak response of dalfampridine at 275 nm from the Standard solution 

C_s = concentration of USP Dalfampridine RS in the Standard solution (µg/mL) 

C_u = concentration of Dalfampridine in the Sample solution (µg/mL) 

F = relative response factor (see Table 2) 

Acceptance criteria: See Table 2. Disregard peaks less than 0.05%. 

Table 2 

a This impurity is quantied using the Limit of Dalfampridine Related Compound B and Dalfampridine Related Compound C test.

b The sum of all impurities from the test for Organic Impurities and the Limit of Dalfampridine Related Compound B and Dalfampridine Related Compound C test. 

5 SPECIFIC TESTS 

Water Determination 〈921〉, Method I, Method Ia: NMT 0.3% 

6 ADDITIONAL REQUIREMENTS 

Packaging and Storage: Preserve in tight containers. 

USP Reference Standards 〈11〉 

USP Dalfampridine RS 

USP Dalfampridine Related Compound A RS 

4-Aminopyridine 1-oxide. 

C₅H₆N₂O      110.11

USP Dalfampridine Related Compound B RS 

3,5-Dibromopyridin-4-amine. 

C₅H₄Br₂N₂      251.91 

USP Dalfampridine Related Compound C RS 

1,3-Di(pyridin-4-yl)urea. 

C₁₁H₁₀N₄O          214.23