Fluticasone Propionate Assay by HPLC-UV following USP Monograph

Eddy Tan, R&D Scientist

R&D APAC Lab (Singapore)

Fluticasone propionate

Abstract

The Purospher^® STAR RP-18e Hibar^® HPLC column is used for the assay of fluticasone propionate following the USP monograph. This is a reverse phase isocratic separation of fluticasone propionate from fluticasone propionate related compound D.

• Introduction
• Experimental
• Results and Discussion
• Conclusion

Introduction

Fluticasone propionate is a glucocorticoid and is available as nasal sprays to treat sneezing, itchy or runny nose, or other symptoms caused by hay fever. It is also available as creams and ointments for topical applications to treat itchy skin.

The Purospher^® STAR RP-18e Hibar^® HPLC column, 250 x 4.6 mm, 5 µm, is used to demonstrate the system suitability criteria for the assay of fluticasone propionate following the USP monograph.

Experimental

Mobile Phase Preparation

• For buffer, weigh 1.15 g of monobasic ammonium phosphate in a 1 L volumetric flask and adjust with phosphoric acid to pH of 3.5 ± 0.05.
• For mobile phase, make up methanol, acetonitrile and buffer as 50:15:35 parts respectively.

System Suitability Solution, 0.05 mg/mL USP Fluticasone Propionate Resolution Mixture RS

• Weigh ~10 mg of USP Fluticasone Propionate Resolution Mixture RS into a 100 mL volumetric flask.
• Add ~90 mL of mobile phase and sonicate for 15 min.
• Top-up to mark with mobile phase and mix well. This is the System Suitability stock standard (~0.1 mg/mL).
• Dilute to a concentration of 0.05 mg/mL with mobile phase.

Fluticasone Propionate Solutions for Assay and Linearity Test

• Weigh ~10 mg of fluticasone propionate CRM into a 100 mL volumetric flask.
•  Add ~90 mL of mobile phase to it and sonicate for 15 min.
• Top-up to mark with mobile phase and mix well. This is the fluticasone propionate stock standard (~0.1 mg/mL).
• Dilute to 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07 mg/mL with mobile phase in 5 mL volumetric flasks.

Fluticasone propionate solution for assay and relative standard deviation (RSD): 0.04 mg/mL.

Fluticasone propionate solutions for linearity, LOD and LOQ: 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07 mg/mL.

HPLC Parameters
Column:	Purospher® STAR RP-18e 5 µm Hibar® RT 250-4.6 mm (1.51456)
Detection:	UV, 239 nm
Mobile phase:	Methanol:acetonitrile:buffer (50:15:35), isocratic
Flow rate:	1.5 mL/min
Pressure drop:	300 bar (or ~4350 psi)
Column temp.:	40 oC
Detector:	UV, 239 nm
Injection:	20 µL
Samples:	System Suitability Solution: 0.05 mg/mL USP Fluticasone Propionate Resolution Mixture RS in mobile phase (MP) Fluticasone Propionate CRM solutions for assay and linearity test: 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, and 0.07 mg/mL fluticasone propionate in MP

Results and Discussion

Figure 1. 0.05 mg/mL USP Fluticasone propionate resolution mixture RS.

The relative retention times for fluticasone propionate and fluticasone propionate related compound D are 1.00 and 1.10 respectively.

Table 1. Resolution between fluticasone propionate and related compound D for 0.05 mg/mL USP fluticasone propionate resolution mixture RS

Injection number	Resolution (USP)
Injection 1	2.90
Injection 2	2.90
Injection 3	2.90
Injection 4	2.89
Injection 5	2.90
Average	2.90
Standard Deviation	0.004
RSD	0.15%

Acceptance Criteria for System Suitability

Resolution: NLT 1.5 between fluticasone propionate and fluticasone propionate related compound D.

Figure 2. Fluticasone propionate 0.04 mg/mL standard solution.

Table 2. Reproducibility of repeated injections of 0.04 mg/mL fluticasone propionate CRM solution

Injection number	Peak Area
Injection 1	14.4618
Injection 2	14.4392
Injection 3	14.4089
Injection 4	14.4391
Injection 5	14.4354
Average	14.4369
Standard Deviation	0.019
RSD	0.13%

Acceptance Criteria for Standard Solution
Relative standard deviation (RSD): NMT 2%

Table 3. Linearity, LOD and LOQ for fluticasone propionate

Concentration (mg/mL)	Mean Area (mAU*min)
0.0100	3.581
0.0200	7.067
0.0300	10.773
0.0400	14.483
0.0500	17.877
0.0601	21.472
0.0701	25.231
STEYX	0.094
Slope	359.88
LOD (mg/mL)	0.00086
LOQ (mg/mL)	0.0026
R2	0.9999

Figure 3. Linearity plot for fluticasone propionate solutions 0.01 to 0.07 mg/mL.

Conclusion

The Purospher^® STAR RP-18e Hibar^® RT column is able to comply with the system suitability criteria indicated in the USP monograph for the assay of fluticasone propionate. The resolution between fluticasone propionate and fluticasone propionate related compound D is greater than 1.5. The RSD of the standard solution is not more than 2%. Both criteria are within the assay specifications as defined in the monograph.

The column is robust with good reproducibility for repeated analyses (data not shown here). In addition, the column offers good linearity from 0.01 to 0.07 mg/mL fluticasone propionate with R² of 0.9999. The Limit of Detection (LOD) for fluticasone propionate is 0.86 µg/mL and the Limit of Quantification (LOQ) is 2.6 µg/mL. 

References

1. USP Monographs, Fluticasone Propionate. USP-NF. Rockville, MD: United States Pharmacopeia. (2022). https://doi.org/10.31003/uspnf_m34220_05_01