Fingolimod HPLC Assay & Impurity Profiling (USP)
Introduction
A rapid, accurate, and simple method was implemented for the total chromatographic purity analysis of Fingolimod Hydrochloride by High Performance Liquid Chromatography equipped with a Diode Array Detector. The experimental conditions follow guidelines, with minor allowed modifications, from the USP43-NF38 monograph methods for Fingolimod Hydrochloride Assay and Organic Impurities Profiling. Fingolimod hexyl homolog, Fingolimod heptyl homolog, Fingolimod Hydrochloride, Fingolimod nonyl homolog, Fingolimod decyl homolog, 3-Phenethyl Fingolimod analog, and 2-Phenethyl Fingolimod analog can be resolved with baseline separation utilizing the USP‘s 33-minute gradient method and a Purospher STAR RP-18 HPLC column (150 x 3.0 mm, 3 μm). A 0.1% solution of Phosphoric acid in water and acetonitrile were employed as the mobile phases for the gradient elution. Under applied conditions, system suitability criteria are met, and the method demonstrates good resolution/selectivity, reproducibility, and sensitivity.
Figure 1.Fingolimod HCl
Figure 2.Fingolimod Hexyl Homolog
Figure 3.FingolimodHeptyl Homolog
Figure 4.O-Acetyl Fingolimod
Figure 5.Fingolimod Nonyl Homolog
Figure 6.Fingolimod Decyl Homolog
Figure 7.Fingolimod 3-Phenethyl Analog
Figure 8.Fingolimod 2-Phenethyl Analog
| |||
|---|---|---|---|
| Column | Purospher® STAR RP-18 endcapped (3µm) Hibar RT 150x3.0 mm | Injection Volume | 5 µL |
| Detection | UV @ 215 nm (DAD) | Flow Rate | 0.8 mL/min |
| Mobile Phase | A: 0.1% Phosphoric Acid in H2O B: Acetonitrile | Temperature | Column: 40 °C Autosampler: 10 °C |
| Gradient | See table | Pressure | 135-300 bar |
| Diluent | Mobile Phase A:B (50:50) | ||
| Test Solution | Dissolve 15 mg of Fingolimod Hydrochloride CRS in 25 mL diluent (0.6 mg/mL). | ||
| System Suitability Solution | Dissolve 15.0 mg of USP Fingolimod for System Suitability using 25 mL diluent (0.6 mg/mL). | ||
| Standard Solution | Dilute 1.0 mL of the test solution to 100.0 mL using mobile phase further dilute 1.0 mL of this solution to 10.0 mL using the mobile phase (0.003 mg/mL). | ||
| Time (min) | A (%) | B (%) |
|---|---|---|
| 0 | 80 | 20 |
| 20 | 5 | 95 |
| 23 | 5 | 95 |
| 23.1 | 80 | 20 |
| 33 | 80 | 20 |
| Peaks | Compound | Retention Time (min) | RRT | Resolution | Tailing factor |
|---|---|---|---|---|---|
| 1 | Fingolimod Hexyl Homolog | 9.0 | 0.84 | - | 1.0 |
| 2 | Fingolimod Heptyl Homolog | 10.1 | 0.94 | 11.6 | 1.1 |
| 3 | Fingolimod | 10.7 | 1.00 | 2.1 | 4.4 |
| 4 | O-Acetyl Fingolimod | 11.8 | 1.10 | 3.0 | 0.7 |
| 5 | Fingolimod Nonyl Homolog | 12.3 | 1.15 | 4.2 | 1.2 |
| 6 | Fingolimod Decyl Homolog | 13.5 | 1.26 | 9.8 | 1.4 |
| 7 | 3-Phenethyl Fingolimod Analog | 19.8 | 1.85 | 42.1 | 0.8 |
| 8 | 2-Phenethyl Fingolimod Analog | 20.2 | 1.89 | 2.8 | 1.2 |
| Peaks | Compound | Retention Time (min) | RRT | RRT Requirement (USP43-NF38) | Resolution | Resolution (USP43-NF38) | Tailing | Tailing (USP43-NF38) |
|---|---|---|---|---|---|---|---|---|
| 1 | Fingolimod Hexyl Homolog | 9.0 | 0.84 | 0.82 | - | - | 1.0 | - |
| 2 | Fingolimod Heptyl Homolog | 10.1 | 0.94 | 0.93 | 11.6 | - | 1.1 | - |
| 3 | Fingolimod | 10.7 | 1.00 | 1.00 | 2.1 | - | 4.4 | Not more than (NMT) 5 |
| 4 | O-Acetyl Fingolimod | 11.8 | 1.10 | - | 3.0 | - | 0.7 | - |
| 5 | Fingolimod Nonyl Homolog | 12.3 | 1.15 | 1.13 | 4.2 | 1.2 (between peak 4 and 5) | 1.2 | - |
| 6 | Fingolimod Decyl Homolog | 13.5 | 1.26 | 1.23 | 9.8 | - | 1.4 | - |
| 7 | 3-Phenethyl Fingolimod Analog | 19.8 | 1.85 | 1.97 | 42.1 | - | 0.8 | - |
| 8 | 2-Phenethyl Fingolimod Analog | 20.2 | 1.89 | 2.00 | 2.8 | 0.8 (between peak 7 and 8) | 1.2 | - |
| Peaks | Compound | Area Response (N=3) | Standard Deviation | RSD (%) | RSD (%) (USP43-NF38) |
|---|---|---|---|---|---|
| 3 | Fingolimod | 5496.1 | 2.3 | 0.04 | NMT 0.73% |
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