Nevirapine holds a special place in HIV medicine. It was the first non-nucleoside reverse transcriptase inhibitor, or NNRTI, approved by the United States Food and Drug Administration for the treatment of HIV-1 infection, and it remains a mainstay of combination therapy in many parts of the world. But the drug comes with baggage. Its toxicity profile includes serious dermatological reactions, Stevens-Johnson Syndrome and significant hepatotoxicity, and most nevirapine-based regimens require twice-daily dosing. That dosing burden, in part because drug levels cannot always be maintained sufficiently high, has contributed to the emergence of multi-drug resistant strains of HIV. To address these therapeutic shortcomings, researchers have proposed novel delivery systems, including niosomes, tiny surfactant-based vesicles stabilised with dicetyl phosphate and cholesterol, which the same research group previously developed using Span 20 or Span 80 surfactants.

Measuring a drug accurately across such diverse matrices, from crystalline bulk powder to lipid vesicles, demands a robust analytical method. Existing techniques for nevirapine include gradient elution, ion-pair reversed-phase HPLC, conventional isocratic HPLC, gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry. Most were designed for plasma or dosage form analysis and suffer from long run times that make them poorly suited to routine quality control laboratories, where large numbers of samples must be processed quickly. Worse, many were developed by the one-factor-at-a-time approach, in which a single variable is adjusted while everything else is held constant. This strategy requires a large set of experiments and, critically, cannot reveal how factors interact with one another, potentially compromising the optimisation process.

The AQbD philosophy turns method development on its head. Rather than stumbling toward a workable separation, the analyst first identifies the critical analytical attributes that define success and the method parameters that influence them, then uses science- and risk-based understanding to build a defensible design space within which the method reliably performs. The cornerstone of this approach is design of experiments, and the researchers chose one of its most powerful tools: the Central Composite Design. This second-order design allows five levels of each input factor to be evaluated with far fewer experiments than a three-level full factorial design would demand, while still capturing curvature and interaction effects in the response surface.

Three input parameters were judged significant in preliminary studies: mobile phase flow rate, the fraction of organic solvent, and column temperature. The responses monitored were the retention time of nevirapine and the resolution factor between nevirapine and carbamazepine, the internal standard chosen after candidates including efavirenz and tenofovir disoproxil fumarate were considered. Twenty experiments, run in randomised order and in triplicate to minimise bias, mapped the design space. Analysis of variance revealed that flow rate and organic solvent content dominated both responses, with F-values reaching 545 and 823 for retention time and 2,578 for the solvent effect on resolution, while column temperature was statistically negligible across the range explored. Quadratic terms confirmed measurable curvature, meaning the relationships were not strictly linear, and a modest interaction between flow rate and solvent composition was detected.

The chemistry behind these effects is intuitive. Increasing the organic solvent content weakens the hydrophobic interaction between nevirapine and the C18 stationary phase, shortening retention, while a higher flow rate simply sweeps the analyte through the column faster. Resolution, by contrast, decreased as either solvent fraction or flow rate rose, because faster elution leaves less time for the analyte and internal standard to separate. Numerical optimisation using a desirability function in Design-Expert software converged on a set-point of 1.0 mL/min flow, 57.5:42.5 acetonitrile-to-water, and 25 degrees Celsius. Experimental verification in five replicates delivered a nevirapine retention time of 4.10 minutes, carbamazepine at 6.7 minutes, and a resolution factor of 13, with prediction errors of under four percent against the model’s forecasts.

Validation followed ICH Q2 (R1) guidelines and covered the full battery of required parameters. The calibration curve was linear from 0.5 to 200 micrograms per millilitre with a correlation coefficient of 0.9994, an unusually wide range that spans both low-dose and high-concentration applications. Repeatability and intermediate precision both produced relative standard deviations well below the five percent acceptance criterion, with some concentrations showing variability of less than 0.05 percent. Accuracy experiments at low, medium and high levels recovered between 99.1 and 101.5 percent of the theoretical concentration, with bias under two percent. The limit of quantitation was 0.5 micrograms per millilitre, confirmed experimentally with a precision of 0.65 percent, and the limit of detection was set at 0.033 micrograms per millilitre by convention.

Specificity and stability-indicating capability were then put to the test. Chromatograms of blank mobile phase, standards and tablet extracts showed no interference at the analyte retention time, confirming that excipients in both commercial Aspen nevirapine 200 mg tablets and in-house 400 mg sustained-release tablets did not disturb the measurement. Forced degradation studies subjected nevirapine to eight hours of reflux at 90 degrees Celsius in 30 percent hydrogen peroxide, 0.1 M hydrochloric acid, 0.1 M sodium hydroxide, neutral water and intense light at 500 watts per square metre. Recovery fell to 84.7 percent under oxidative stress, 90.7 percent under acidic conditions and 87.3 percent under alkaline stress, while the drug remained essentially stable under neutral and photolytic conditions. Crucially, no degradation peaks appeared at the analyte retention time in any experiment, demonstrating that the method can distinguish intact nevirapine from its breakdown products.

Real-world application sealed the case. The commercial tablets assayed at 98.35 percent of label claim and the in-house sustained-release tablets at 99.13 percent, both comfortably within pharmacopoeial expectations. When applied to niosomes, the method quantified both free drug in the supernatant and entrapped drug released by destroying the vesicle pellet with n-propanol, yielding entrapment efficiencies of 96.8 percent for optimised Span 20 systems and 98.0 percent for Span 80 systems, with values of 92.4 to 94.1 percent even in cholesterol-containing formulations. Compared with published methods, most of which rely on laborious phosphate or acetate buffers that shorten column life, generate waste and complicate preparation, the buffer-free isocratic system offers clear advantages in cost, sustainability and throughput. The authors argue that the AQbD framework, by explicitly defining a design space in which the method tolerates small fluctuations in flow, temperature and solvent ratio, produces analytical procedures that are not merely validated once but robust by construction, a lesson with universal application for pharmaceutical laboratories worldwide.

Subject of Research: Development and validation of a stability-indicating RP-HPLC method using Analytical Quality by Design for quantifying nevirapine in bulk, tablet and niosome formulations

Article Title: The use of QbD for the development and validation of a stability-indicating RP-HPLC method for the quantitation of nevirapine in bulk, tablet and niosome formulations

Article References: Witika, B. A., & Walker, R. B. (2026). The use of QbD for the development and validation of a stability-indicating RP-HPLC method for the quantitation of nevirapine in bulk, tablet and niosome formulations. Frontiers in Analytical Science, 6, Article 1735125. https://doi.org/10.3389/frans.2026.1735125

Image Credits: AI Generated

DOI: 10.3389/frans.2026.1735125

Keywords: nevirapine, Analytical Quality by Design, RP-HPLC, Central Composite Design, response surface methodology, stability-indicating method, niosomes, HIV, pharmaceutical analysis, ICH Q2 validation, forced degradation, carbamazepine internal standard