Scientists Validate Quality-by-Design HPLC Method for Brivaracetam and Identify Its Key Degradant

Brivaracetam, an antiepileptic drug sold under the brand name Briviact, now has a newly developed and validated analytical method designed to quantify it in bulk drug substance and to identify its principal degradation product. The method, built on a reverse-phase high-performance liquid chromatography (RP-HPLC) platform and shaped by an Analytical Quality by Design (AQbD) strategy, was reported in the Future Journal of Pharmaceutical Sciences by researchers led by Sandip Kumar Dey, Koustav Dutta, Sumanta Mondal, Sagar Suman Panda and Prasenjit Mondal. The work addresses a persistent gap in pharmaceutical analysis: many published methods for the drug lack a defined design space, robustness data, or the identification of major degradants.

The team’s central finding is the characterization of brivaracetam’s major degradation product, formed under acidic stress, as (S)-2-((R)-2-oxo-4-propylpyrrolidin-1-yl)butanoic acid. The compound has a molecular weight of 213.28 g/mol and the molecular formula C11H19NO3, and it eluted at a retention time of 23.6 minutes, well separated from the brivaracetam peak at roughly 16.8 minutes. Its structure was established using a combination of chromatographic and spectroscopic techniques, including liquid chromatography-mass spectrometry (LC-MS), infrared spectroscopy, proton nuclear magnetic resonance spectroscopy and carbon-13 NMR, and the researchers used the identified structure to outline a plausible degradation pathway.

Brivaracetam itself is a racetam derivative and a molecular analogue of levetiracetam, used alongside other antiepileptic medications to manage partial-onset seizures with or without secondary generalization. It acts by binding to synaptic vesicle glycoprotein 2A (SV2A) with an affinity approximately 20 times higher than levetiracetam. The drug received approval from the European Commission on January 14, 2016, and from the U.S. Food and Drug Administration on May 12, 2016. Because impurities can affect the safety and quality of drug substances, controlling organic impurities is mandatory under ICH Q3A(R2) and Q3B(R2) guidelines, which set qualification thresholds based on maximum clinical dose; for brivaracetam, whose highest daily dose is 200 mg taken as 100 mg twice daily, those thresholds are 0.15% for drug substance and 0.20% for drug product.

The analytical separation was performed on a Waters Alliance HPLC system equipped with an X-Terra RP18 column measuring 250 by 4.6 mm with 5 µm particles. A gradient elution technique combined mobile phase A, consisting of 0.1% w/v perchloric acid in water, with mobile phase B, a water-acetonitrile mixture at a 10:90 v/v ratio, flowing at 1.2 mL per minute. Detection took place at 210 nm, the wavelength of maximum absorption determined by scanning a reference standard solution with a photodiode array detector. The injection volume was 20 µL, the column temperature was held at 30 °C, the autosampler at 25 °C, and the total run time was 35 minutes. The researchers selected perchloric acid over formic or phosphoric acid for its ion-exchange capacity and acid strength, which helps neutralize the positive charge of basic compounds and enhance retention, while avoiding the salt deposition and column obstruction risks associated with phosphate buffers.

What distinguishes the study is its systematic application of Analytical Quality by Design, a science- and risk-based approach that builds quality into an analytical method from the outset rather than relying on end-product testing alone. Using Design Expert software version 13, the team applied a two-level fractional factorial design with resolution V, four center points and no replicates, generating 20 experimental runs. Five independent factors were examined: detection wavelength, flow rate, column oven temperature, the volume of perchloric acid in mobile phase A, and changes in the minor component of mobile phase B. The responses tracked were retention time, theoretical plates, peak asymmetry, the relative standard deviation of replicate reference standard injections, and percent assay.

The statistical analysis revealed which parameters genuinely mattered. Flow rate significantly impacted retention time, tailing factor and theoretical plates, with p-values below 0.05, while column temperature and minor component changes in mobile phase B significantly affected retention time. All other interactions proved statistically insignificant. The center point of the design space fell within acceptable ranges, indicating a robust region where deliberate variations in method parameters do not meaningfully affect the assay result. According to the authors, none of the investigated parameters had a practically relevant impact on assay performance, which reduces the risk of failure during downstream validation testing.

Because impurity reference standards were unavailable, the team relied on forced degradation, or stress testing, to generate analytical samples. Test solutions of brivaracetam were exposed to hydrochloric acid, sodium hydroxide, hydrogen peroxide, heat at 50 °C, humidity of roughly 75 to 80 percent relative humidity, and photolytic light exposure of about 1.2 million lux hours with near-ultraviolet energy of at least 200 watt-hours per square meter. Acid conditions produced the strongest effect, degrading 10 percent of the drug, while base and peroxide stress yielded assay values of 99.1 percent and 100.0 percent with degradation of 1 percent and 0 percent respectively. Under heat, photolytic and humidity conditions, no considerable degradation was observed. In every case, degradant peaks were well separated from the brivaracetam peak, with purity angles below the threshold set by Empower 3 software, confirming the method’s stability-indicating nature.

Validation followed International Council for Harmonization Q2(R2) guidelines and produced consistently strong numbers. The method showed linearity across 50 to 150 percent of the nominal test concentration, spanning 100 to 300 ppm, with a correlation coefficient of 0.9999. Accuracy, assessed through recovery studies at 50, 100 and 150 percent levels, ranged from 99.7 to 100.4 percent. Repeatability and intermediate precision, expressed as percent relative standard deviation of assay on an anhydrous basis, came in at 0.2 percent and 0.3 percent respectively. Reference standard and test solutions remained stable for at least 48 hours at room temperature, and all system suitability parameters met their pre-established acceptance criteria throughout validation.

The environmental footprint of the method was also evaluated using the Analytical Greenness (AGREE) model, which considers health effects, environmental impact, waste generation, energy consumption and safety. The method scored 0.62 on the greenness scale, which the authors characterize as moderate, indicating reasonable environmental performance with room for improvement.

The authors position the work as a corrective to limitations they identified in prior literature. Earlier studies on brivaracetam analysis either omitted forced degradation studies, failed to define a design space, did not demonstrate stability-indicating capability, or identified degradants without developing and validating a quantification method. By combining a defined design space, a characterized major degradant, full ICH-compliant validation and a risk-based development framework in a single method, the study offers quality-control laboratories a tool intended for routine assay and stability analysis of brivaracetam in bulk drug substance. The researchers note that the AQbD framework may also allow modifications within the established method operable design region without full revalidation, potentially reducing time and cost while supporting consistent, reliable performance over the method’s lifecycle.

Subject of Research: Development and validation of a stability-indicating RP-HPLC method for brivaracetam in bulk drug substance using Analytical Quality by Design, with identification of its major degradation product

Article Title: Analytical method development, validation for the estimation of brivaracetam in bulk drug substance by RP-HPLC and identification of its major degradation product: a quality by design approach

Article References: Dey, S. K., Dutta, K., Mondal, S., Panda, S. S., & Mondal, P. (2026). Analytical method development, validation for the estimation of brivaracetam in bulk drug substance by RP-HPLC and identification of its major degradation product: a quality by design approach. Future Journal of Pharmaceutical Sciences, 12(1), Article 81. https://doi.org/10.1186/s43094-026-01010-w

Image Credits: AI Generated

DOI: 10.1186/s43094-026-01010-w

Keywords: brivaracetam, RP-HPLC, Analytical Quality by Design, stability-indicating method, forced degradation, degradation product identification, ICH Q2(R2) validation, pharmaceutical analysis, antiepileptic drug, LC-MS, NMR spectroscopy, green analytical chemistry