Fingolimod HPLC Assay and Impurity Profiling Methods Following United States Pharmacopeia Monograph Guidelines Using a Purospher STAR C18 Column and UV Detection

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.

Chemical Structures

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

Experimental Conditions

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

Chromatographic Data (System Suitability Solution)

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

Specificity (System Suitability Solution)

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	-

Repeatability (System Suitability Solution)

Peaks	Compound	Area Response (N=3)	Standard Deviation	RSD (%)	RSD (%) (USP43-NF38)
3	Fingolimod	5496.1	2.3	0.04	NMT 0.73%