Methods for the Analysis of Flunisolide Nasal Spray

To jump-start the standard development process and have earlier stakeholder engagement, USP is piloting a new approach for developing and sharing information with our stakeholders. Through a collaboration between USP’s Small Molecules Department and Global Analytical Development Laboratory, methods are being developed and validated for drug substances and drug products for which no monographs are currently available. The Emerging Standards are intended to improve USP’s official standards elaboration process by increasing transparency and allowing for broader stakeholder participation by publishing on the USP website prior to formal notice and comment through publication in the Pharmacopeial Forum.  

Flunisolide Nasal Spray has been evaluated and shown to be a suitable candidate for development under this new program. The methods, in this document, are being published to help analyze Flunisolide Nasal Spray. Additional method development and validation information is provided to justify the use of method parameters. 

Certain commercial software, instruments, or material may be identified in this document to specify adequately the experimental procedure. Such identification does not imply approval, endorsement, or certification by USP of a particular brand or product, nor does it imply that the software, instrument, or material is necessarily the best available for the purpose or that any other brand or product was judged to be unsatisfactory or inadequate.

This document is not a USP compendial standard and is intended to serve as a resource for informational purposes only. It does not reflect USP or USP’s Expert Body opinions of future revisions to the official text of the USP-NF. Parties relying on the information in this document bear independent responsibility for awareness of, and compliance with, any applicable federal, state, or local laws and requirements.

Flunisolide is an inhaled corticosteroid with anti-inflammatory actions and used in the treatment of asthma. It is often prescribed as treatment for allergic rhinitis and its principal mechanism of action involves activation of glucocorticoid receptors. Flunisolide is a glucocorticoid receptor agonist. It is administered either as an oral metered-dose inhaler for the treatment of asthma or as a nasal spray for treating allergic rhinitis. Corticosteroids are naturally occurring hormones that prevent or suppress inflammation and immune responses. When given as an intranasal spray, flunisolide reduces watery nasal discharge (rhinorrhea), nasal congestion, postnasal drip, sneezing, and itching at the back of the throat that are common allergic symptoms¹.

This document summarizes method validation, as well as robustness and forced degradation study results. It also describes the final procedures that may be suitable for the identification, and the strength and purity determination of flunisolide in the presence of various impurities and excipients in Flunisolide Nasal Spray. Summary of validation data and representative chromatographic results are included.

3.1 Flunisolide and Impurities Standards

USP Reference Standards for flunisolide, desonide, flurandrenolide were used. Flunisolide 17 beta acid (related compound A), flunisolide 11-keto (related compound B), flunisolide 21-aldehyde (related compound C), 9 alfa bromo flunisolide (related compound D), dihydroxycorticosterone acetate, epoxyhydrocortisone acetate and bromohydrocortisone acetate were procured from another supplier. Chemical structures of flunisolide and related impurities are shown in Figures 1 and 2.

 

Figure 1: Flunisolide

 

Figure 2a. Desonide

Figure 2b. Flurandrenolide

Figure 2c. Flunisolide 17-beta acid (Related Compound A)

Figure 2d. Flunisolide 11-keto (Related Compound B)

Figure 2e. Flunisolide 21-aldehyde (Related Compound C)

Figure 2f. 9-alpha bromo flunisolide (Related Compound D)

Figure 2g. Dihydroxycorticosterone acetate

Figure 2h. Epoxy hydrocortisone acetate

Figure 2i. Bromo hydrocortisone acetate

Figure 2: Flunisolide Impurities

3.2 Samples

Flunisolide Nasal Spray from one commercial source was used to evaluate methods described in this document.

3.3 Reagents

Potassium dihydrogen phosphate (ACS grade) was obtained from Merck and acetonitrile (HPLC grade) was from Qualigens. Water was obtained from the Sartorius water purification system.

Forced degradation study was performed by exposing flunisolide API to acid, base, oxidation, heat, humidity, and light conditions. The stressed samples and a control sample (unstressed) were analyzed and compared.  The chromatograms were processed at 245 nm to detect the degradation impurities of flunisolide. The PDA data from 200-400 nm showed homogeneity of UV spectrum for the flunisolide peak indicating the peak is free from coelution. The obtained results for each of the forced degradation condition are presented in Table 1.

In summary, desonide and flunisolide 11 keto are formed in all the above degradation conditions. Flunisolide 17 beta acid is another known degradant present in stressed samples under base, hydrogen peroxide and light exposures. One major degradation impurity was formed at 0.45 RRT (about 3.84%) under base degradation conditions. All known and unknown impurity peaks are separated from each other and no coelution was observed with the main peak.

Identification of flunisolide in Flunisolide Nasal Spray was evaluated using the HPLC conditions from the Assay section with PDA spectral match and chromatographic retention time match. See the Assay section for method conditions and solution preparations.

5.1 PDA Spectral Match

The validation parameters and results are summarized in Table 2, and representative UV spectra of flunisolide from the Standard solution and Sample solution are shown in Figures 3-4.

5.2 Retention Time Match

The validation parameters and results are summarized in Table 3, and representative chromatograms of the Standard solution and Sample solution are shown in Figures 5 and 6, respectively. 
 

The validated assay procedure for Flunisolide Nasal Spray, using the criteria described in USP General Chapter <1225> Validation of Compendial Procedures² and found to be specific, linear, accurate, precise for the samples evaluated.

6.1 Instruments and Method

The analysis of Flunisolide Nasal Spray was done using Waters Alliance 2695 and Agilent 1260 Infinity series equipped with a photo diode array (PDA) detector. Luna phenyl hexyl, 4.6-mm x 15.0-cm, 3 µm (L11) column from Phenomenex was used. The analysis was performed at 30 ˚C column temperature, with a flow rate of 1.0 mL/min and 50 µL as the injection volume. Autosampler temperature was kept at 5 ˚C. The PDA detector was set at 200-400 nm wavelength and the detection wavelength was at 245 nm with run time of 45 minutes. The separation was achieved by a gradient program as mentioned below.

6.2 Solutions

Mobile Phase A (20 mM potassium dihydrogen phosphate): Weighed and transferred about 10.88 g of potassium dihydrogen phosphate into 4000 mL of water.

Mobile Phase B: Acetonitrile

Diluent: Mixed 800 mL of water and 200 mL of acetonitrile (Diluent is used as a blank).

Flurandrenolide stock solution-1 (250 µg/mL of flurandrenolide): Weighed and transferred about 1.25 mg of flurandrenolide into 5 mL volumetric flask, added 1 mL of acetonitrile, sonicated to dissolve, and diluted to volume with water.

Flurandrenolide stock solution-2 (2.5 µg/mL of flurandrenolide): Transferred 0.5 mL of the Flurandrenolide stock solution-1 into 50 mL volumetric flask and diluted to volume with Diluent.

System suitability solution (125 µg/mL of flunisolide and 1.25 µg/mL of flurandrenolide): Weighed and transferred about 1.25 mg of flunisolide into a 10 mL volumetric flask, added 1.0 mL of acetonitrile, sonicated to dissolve and added 5.0 mL of the Flurandrenolide stock solution-2 and diluted to volume with water.

Standard stock solution (250 µg/mL of flunisolide): Weighed and transferred about 12.5 mg of flunisolide standard into a 50 mL volumetric flask, added 10 mL of acetonitrile, sonicated to dissolve, and diluted to volume with water.

Standard solution (40 µg/mL of flunisolide): Transferred 4.0 mL of the Standard stock solution into 25 mL volumetric flask and diluted to volume with Diluent.

Sample solution (40 µg/mL of flunisolide): Nominally 40 µg/mL of flunisolide in Diluent prepared as follows. Prepared a composite by mixing NLT 2 containers of non-pressurized liquid drug dosage form of Nasal Spray by removing the metered-dose cap and pouring the Nasal Spray solution out of the commercialized containers into the suitable container. Transferred 4.0 mL of the composite solution (each mL is equivalent to 0.25 mg of flunisolide) into 25 mL volumetric flask and diluted to volume with Diluent.

6.3 Validation Parameters and Results

The optimized chromatographic conditions were checked for robustness and validated by evaluating specificity, linearity, precision, and accuracy as described in USP General Chapter <1225> Validation of Compendial Procedure². The system suitability requirements and results are summarized in Table 4. The validation parameters, solutions, and results for Flunisolide Nasal Spray are summarized in Table 5. Representative chromatograms of the Standard solution and Sample solution are shown in Figures 5 and 6, respectively.

The developed HPLC method was validated for the OI procedure, using the criteria described in USP General Chapter <1225> Validation of Compendial Procedure² and found to be specific, accurate, precise, robust, linear, and free from interference for the samples evaluated.

Bromo hydrocortisone is a process impurity which is not controlled in the drug product. The same method could be used for analysis of bromo hydrocortisone, if needed (RRT about 1.3).  However, due to the instability of the impurity in the solution, the standards and samples should be freshly prepared and analyzed within 1 hour of preparation.

7.1 Instruments and method

The analysis of Flunisolide Nasal Spray was performed using the same instruments and method as described in the Assay section.

7.2 Solutions

Prepare Mobile Phase A, Mobile Phase-B, Diluent and System suitability solution as per the Assay procedure.

Preparation of Individual stock solutions: Individual stock solutions consisting of 250 µg/mL flunisolide 17 beta acid, dihydroxycorticosterone acetate, flunisolide 21-aldehyde, desonide, flunisolide, flurandrenolide, flunisolide 11-keto, epoxyhydrocortisone acetate and 9 alfa bromo flunisolide were individually prepared by dissolving appropriate amounts of each standard in Diluent.

Preparation of Sensitivity solution: A solution consisting of 0.0625 µg/mL each of flunisolide 17 beta acid, dihydroxycorticosterone acetate, flunisolide 21-aldehyde, desonide, flunisolide, flurandrenolide, flunisolide 11-keto, epoxyhydrocortisone acetate and 9 alfa bromo flunisolide was prepared by combining appropriate volumes of Individual stock solutions in Diluent.

Preparation of Standard solution: A solution consisting of 0.125 µg/mL each of flunisolide 
17 beta acid, dihydroxycorticosterone acetate, flunisolide, flurandrenolide, flunisolide 11-keto, epoxyhydrocortisone acetate, 9 alfa bromo flunisolide and 0.5 µg/mL for desonide and flunisolide 21 aldehyde was prepared by combining appropriate volumes of Individual stock solutions in Diluent.

Preparation of Sample solution: Nominally 125 µg/mL of flunisolide in Diluent prepared as follows. Prepared a composite by mixing NLT 2 containers of non-pressurized liquid drug dosage form of Nasal Spray by removing the metered-dose cap and pouring the Nasal Spray solution out of the commercialized containers into the suitable container. Transferred 2.5 mL of the composite solution (each mL is equivalent to 0.25 mg of flunisolide) into 5 mL volumetric flask, added 1 mL of acetonitrile and diluted to volume with water.

Preparation of Robustness solution: A solution consisting of 125 µg/mL of flunisolide and 1.25 µg/mL of each of flunisolide 17 beta acid, dihydroxycorticosterone acetate, flurandrenolide, flunisolide 11-keto, epoxyhydrocortisone acetate, 9 alfa bromo flunisolide and 0.5 µg/mL for desonide and flunisolide 21 aldehyde was prepared by combining appropriate volumes of Individual stock solutions in Diluent.

7.3    Validation Parameters and Results

The system suitability parameters and results are summarized in Table 6. The validation parameters and results are summarized in Table 7. Representative chromatograms of Blank, System suitability solution, Sensitivity solution, Spiked sample solution, and Sample solution are presented in Figures 7-11. Linearity was established for flunisolide, and related impurities and relative response factors presented in Table 8, whereas accuracy and repeatability were established for flunisolide related impurities.

The Sensitivity solution was considered as limit of quantitation (LOQ) for each analyte at 0.0625 µg/mL with respect to the sample concentration of 125 µg/mL.