Streamlined Method Validation for Analysis of Formoterol Fumarate and Budesonide in Metered Dose Inhaler with Empower Method Validation Manager Software

This application note presents a validation of UPLC method for formoterol fumarate and budesonide active pharmaceutical ingredients using Empower Method Validation Manager (MVM).

The UPLC method for assay of formoterol fumarate and budesonide drugs was successfully validated for linearity, accuracy, repeatability, intermediate precision, specificity, and robustness. The validation results met the predefined performance requirements, demonstrating that the method is suitable for analysis of the active ingredients in the metered dose inhaler drug product.

Empower 3 MVM Software improved efficiency of the entire validation process and assured adherence to the validation requirements. Statistical evaluation of the data and generation of the validation results were performed within the software, which eliminated the need to export raw data to another software platform for analysis. During the study, Empower MVM tracked every step of the validation process, identifying steps and results that did not meet the defined validation requirements. Utilizing both UV and MS spectral data from an ACQUITY QDa Mass Detector increased confidence and assurance in determination of the chromatographic peak purity.

For additional methods content, please visit methods.waters.com

Benefits

• Improves method validation workflows with Empower Method Validation Manager (MVM) Software 
• Increases conformance of each validation steps with the validation requirements 
• Assures compliance with regulations on data integrity, audit trails, data traceability, and electronic signature sign-off review and approvals

Regulations and quality standards require analytical methods to be validated before implementation in quality control laboratory for routine testing to ensure quality and safety of the drug products. 

The analytical method validation is a complex and demanding process by which laboratory studies are performed to demonstrate that the method generates consistent results with high accuracy and precision for the intended use.^1,2,3 Validation studies must be executed following a protocol with a description of each validation characteristic and predetermined acceptance criteria. This will ensure compliance with the appropriate regulations and guidelines and assure that the method remains appropriate for testing of the drug products. 

In addition, the second stage of the analytical procedure lifecycle approach described in the proposed USP General Chapter <1220>, states that the procedure should be qualified to confirm it generates reproducible values that meet the analytical target profile (ATP) criteria and remain appropriate for testing of the product.⁴ The ATP is a set of performance characteristic requirements based on the intended use of the analytical method and may include criteria for accuracy, precision, specificity, and linearity. These method-specific performance attributes are consistent with traditional validation characteristics. 

In this application note, we present validation of a UPLC method for formoterol fumarate and budesonide active pharmaceutical ingredients in a metered dose inhaler using Empower Method Validation Manager (MVM) software, an option for Empower 3 Chromatography Data Software. The metered dose inhaler containing formoterol fumarate and budesonide is commonly used for treatment of asthma and chronic obstructive pulmonary disease.⁵ The UPLC method was validated for linearity, accuracy, repeatability, intermediate precision, specificity and robustness for analysis of the active ingredients. Employing Empower 3 MVM Software increased efficiency of the entire validation study and assured compliance to the validation requirements. 

Sample description

Standard solutions 

Separate stock solutions were prepared by dissolving budesonide and formoterol fumarate in methanol at 1 mg/mL and 0.1 mg/mL concentration, respectively. The stock solutions were subsequently diluted with diluent (70:30 mobile phase A/acetonitrile) to make a working standard concentration (80/4.5 µg/mL budesonide and formoterol fumarate) and linearity standard solutions described in the study. 

Budesonide and formoterol fumarate metered dose inhaler 

Symbicort inhaler formulation containing 80/4.5 µg of budesonide and formoterol fumarate per actuation was prepared following a procedure described in the literature5 with two exceptions. Samples were diluted with diluent containing a 70:30 mobile phase  A/acetonitrile and filtered through 0.2-µm PTFE syringe filters prior analysis. The final working sample solution contained 80 µg/mL and 4.5 µg/mL of budesonide and formoterol fumarate, respectively. 

UPLC method for formoterol, budesonide, and budesonide related compounds

The UPLC method validated in this study was developed using a software-assisted Quality by Design (QbD) approach.⁶ An example of the UPLC chromatographic separation for formoterol, budesonide, and budesonide-related compounds is shown in Figure 1.

Method validation

The purpose of this study is to demonstrate that the UPLC method is suitable for the determination of the active ingredients, formoterol and budesonide in metered dose inhaler combination dosage form. Therefore, the method was validated following the regulatory recommendations for quantitation of major components of bulk drug substances or active ingredients in finished pharmaceutical products.^2,3 The validation attributes included linearity, accuracy, repeatability, intermediate precision, specificity and robustness. System precision was evaluated for each chromatographic run using five replicate injections of the working standard sample, as recommended in the USP General Chapter <621> on Chromatography.⁷ The system precision criteria included:

• %RSD of retention times: ≤1.0%
• %RSD of peak areas: ≤2.0%
• USP resolution: ≥2.0
• Peak tailing: ≤1.5

Example of system suitability determination (Figure 2) showed that the results were well below the acceptance criteria. A resolution mixture with formoterol and budesonide at the working level spiked with 0.5% of related compounds confirmed that the USP resolution between all the peaks was ≥2.0 (Figure 1). 

Using Empower 3 MVM Software streamlined the entire validation process, from creating a validation protocol to acquiring, analyzing, approving, and reporting validation data. During the study, Empower 3 MVM Software checked the adherence of each validation step against the requirements and flagged any results that did not meet the specifications (Figure 3). Built-in statistical calculations eliminated the need to export data to another software to generate validation results. Validation report templates available within the validation project allowed quick generation of summary reports for each validation test.

1. Linearity

Linearity was demonstrated by analyzing five different concentration levels for both the formoterol and budesonide ranging from 80 to 120% of the working sample concentration. Using Empower MVM Software, we calculated the regression equation and correlation coefficient for plot of average peak area against each drug concentration (Figure 4). Method exhibited an acceptable linearity, both analytes with correlation coefficients (R²) ≥0.999.

2. Accuracy

Accuracy of the method was demonstrated by assessing recoveries of formoterol and budesonide drugs spiked to the Symbicort inhaler sample solutions at 80%, 100%, and 120% levels in triplicates. The average % recovery for the accuracy samples (Figure 5) ranged  from 97.8 to 101.5% with %RSD ≤1.42%, which met the acceptance criteria of 95–105% and %RSD ≤5.0%. 

3. Repeatability

To demonstrate repeatability, six separate preparations of Symbicort inhaler sample spiked with formoterol and budesonide at 100% level were examined for recovery. For analyst A, the average % recovery and %RSD of % recoveries for budesonide and formoterol met  the specification of 100 ± 5.0% and %RSD ≤2.0%, respectively (Figure 6). 

4. Intermediate precision

For intermediate precision, the second analyst prepared and analyzed six separate preparations of the Symbicort inhaler sample spiked at 100%. The results generated by analyst A and B met the criteria for recovery and %RSD (Figure 6). 

5. Specificity

Specificity demonstrates that the analytes can be separated and accurately measured in the presence of other components that may be expected to be present, such as impurities, degradation products, and matrix components. In addition, the spectral peak purity test is recommended to demonstrate that the chromatographic peak represents a single component or is spectrally homogenous. 

The spectral purity of each active ingredient was demonstrated by spiking Symbicort inhaler sample formulation with related compounds. Both UV and MS spectral data were used to demonstrate that each active ingredient is not coeluting with other components in the sample (Figure 7). Peak table showed that the purity angle was below the threshold angle, confirming that each active ingredient is spectrally homogenous or representing one component (Figure 7A). The Mass Analysis Window from the Empower 3 Software with the purity spectrum showed spectral data across each peak – which is at the leading, apex, and trailing regions of the peak (Figure 7B). The top and bottom plots represent UV and MS spectrum, respectively. The mass spectrum showed presence of one mass (m/z) across each peak, specific for each active ingredient, which confirmed presence of a single component. Overall, utilizing both UV and MS spectra we demonstrated that our analytes are not subject to interference with any components of the formulation and/or the related compounds.

6. Robustness

For robustness, we studied the effect of changes in column temperature (33 ± 2.0 °C) and flow rate (0.35 ± 0.35 mL/min) on the USP resolution between peaks, with a goal of achieving a minimum USP resolution of ≥2.0. A full factorial experimental design in the Empower 3 MVM Software allowed us to investigate the interaction of the two factors and generated an experimental study design with combination of different chromatographic conditions (Figure 8). The robustness study showed that the USP resolution remains ≥2.0 across all conditions (Figure 9). 

Continued method performance verification

After implementation in the QC laboratory for routine use, method performance should be continuously monitored and verified to assure that it remains fit-for-purpose and meets the ATP goals throughout the method’s lifecycle. Routine monitoring may include trend analysis of the system suitability data, tracking analytical results, out-of-specification, or other parameters as appropriate. Trending using control charts of the Empower 3 Software provides graphical representation of the method performance characteristics, which allows quick identification of any out-of-trend data or deviations from the performance goals.⁸ As for example, monitoring the USP resolution between critical pair is important to assure acceptable separation is achieved for accurate quantitation (Figure 10).

The UPLC method for assay of formoterol fumarate and budesonide drugs was successfully validated for linearity, accuracy, repeatability, intermediate precision, specificity, and robustness. The validation results met the predefined performance requirements, demonstrating that the method is suitable for analysis of the active ingredients in the metered dose inhaler drug product. 

Empower 3 MVM Software improved efficiency of the entire validation process and assured adherence to the validation requirements. Statistical evaluation of the data and generation of the validation results were performed within the software, which eliminated the need to export raw data to another software platform for analysis. During the study, Empower MVM tracked every step of the validation process, identifying steps and results that did not meet the defined validation requirements. Utilizing both UV and MS spectral data from an ACQUITY QDa Mass Detector increased confidence and assurance in determination of the chromatographic peak purity. 

Finally, control charts of the Empower 3 Software enable quick trend analysis of the method performance characteristics, which helps to verify that the method continuously generates fit-for-purpose results. 

1. U.S. Department of Health and Human Service, Food and Drug Administration, Center for Drug Evaluation and Research (CDER). Analytical Procedures and Methods Validation for Drugs and Biologics, Guidance for Industry. July 2015.
2. USP General Chapter, <1225>, Validation of Compendial Procedures, The United States Pharmacopeia Convention, official May 1, 2013.
3. ICH Q2B, Validation of Analytical Procedures, International Conference on Harmonization, November 1996.
4. Proposed New USP General Chapter: The Analytical Procedure Lifecycle <1220>, The United States Pharmacopeia Convention. Pharmacopeia Forum, 43(1), Jan–Feb, 2017.
5. Salem YA, Shaldam MA, El-Sherbiny DT, El-Wasseef DR, El-Ashry SM. Simultaneous Determination of Formoterol Fumarate and Budesonide Epimers in Metered Dose Inhaler using Ion-Pair Chromatography. Journal of Chromatographic Science. 2017, 55, 1013–1020. 
6. Alkhateeb FL, Rainville P. Applying a Software-Assisted Analytical Quality-by-Design Approach for the Analysis of Formoterol, Budesonide, Related Compounds. Waters Application Note. 720006654EN. 2019.
7. USP General Chapter, <621>, Chromatography, USP36-NF31, The United States Pharmacopeia Convention, official December 2013. 
8. Maziarz M, Rainville P, Naughton S. Continued Performance Verification of Analytical Procedures Using Control Charts of Empower Chromatography Data Software. Waters White Paper. 720006581EN. 2019.