Greener, Smarter HPLC: Scientists Rebuild Antifungal Drug Testing With Quality by Design and Sustainability Metrics

A team of pharmaceutical scientists in India has unveiled a new analytical method for measuring the antifungal drug itraconazole that is not only highly accurate but also explicitly scored for its environmental footprint. The study, published in Discover Green Chemistry, combines the statistical rigor of Quality by Design (QbD) with a battery of greenness and whiteness assessment tools, offering a template for how routine pharmaceutical quality control could become more sustainable without sacrificing reliability. The work arrives at a moment when laboratories worldwide are under growing pressure to reduce the toxic solvent waste that chromatography inevitably generates.

Itraconazole is a potent broad-spectrum antifungal used against organisms including Candida, Aspergillus, Cryptococcus, and Blastomyces, and it appears in both oral and topical formulations, particularly for skin infections. Because the molecule is highly lipophilic and poorly soluble, considerable formulation effort has gone into improving its bioavailability, which makes reliable measurement of the drug in finished products essential. Stability is a particular concern: exposure to heat, light, acid, alkali, or oxidants can degrade itraconazole and undermine both efficacy and safety over a product’s shelf life. A validated, stability-indicating analytical method is therefore a regulatory necessity, not a luxury.

The researchers chose reverse-phase high-performance liquid chromatography (RP-HPLC) as their platform. Compared with ultraviolet spectrophotometry, high-performance thin-layer chromatography, or ultra-performance liquid chromatography, RP-HPLC offers a favorable balance of sensitivity, selectivity, reproducibility, and cost for routine regulatory-grade analysis. Earlier methods for itraconazole, the authors note, typically optimized chromatographic conditions by trial and error and rarely accounted for stress-induced degradation products, especially in topical gel formulations, where robust quality control has lagged behind work on tablets and nanoparticles.

The study’s central innovation is its systematic development framework. Following ICH Q8, Q9, and Q10 guidelines, the team began by defining an Analytical Target Profile and three critical analytical attributes: theoretical plate count, peak tailing, and retention time. An Ishikawa fishbone diagram mapped the risk factors, and a twelve-run Plackett–Burman screening design tested five variables at two levels each: flow rate, mobile phase composition, buffer pH, column temperature, and injection volume. Flow rate and mobile phase composition emerged as the dominant drivers of resolution and symmetry, while pH and temperature played moderate roles, narrowing the field for the next stage.

Those surviving factors were then fed into a three-factor, three-level Box–Behnken design, with center-point replicates to confirm reproducibility across the design space. Multiple linear regression in Design Expert software produced polynomial models whose validity was checked through coefficients of determination, PRESS statistics, and lack-of-fit analysis. The models proved impressively predictive: the retention time model achieved an R-squared of 0.9758 with an F-value of 95.22, and the theoretical plate model reached an R-squared of 0.9964 with an F-value of 427.84. Three-dimensional response surface plots visualized how acetonitrile content and flow rate interacted to shape peak quality, and numerical optimization with a desirability function converged on a final method using a 90:10 acetonitrile-to-triethylamine buffer ratio at pH 3.0, a 1.0 mL/min flow rate, and 25 degrees Celsius, delivering a tailing factor of 1.07, a retention time of 5.95 minutes, and roughly 5250 theoretical plates with a desirability score of 1.0.

Validation under ICH Q2(R1) guidelines produced strong numbers across the board. Linearity held from 1 to 25 micrograms per milliliter with a correlation coefficient of 0.999. Recovery studies spiked at 80, 100, and 120 percent levels returned values between 96.8 and 99.3 percent, while intra-day and inter-day precision at three concentration levels kept relative standard deviations below 2 percent. The limits of detection and quantification were 2.12 and 6.43 micrograms per milliliter, respectively, and deliberate perturbations of solvent composition, flow, and temperature shifted results by less than 1.5 percent, confirming robustness. Applied to a marketed itraconazole gel, the method assayed the drug at 100.27 percent of label claim with no interference from excipients.

To prove the method was genuinely stability-indicating, the team subjected both bulk drug and gel to forced degradation under ICH Q1A(R2) conditions. Acidic hydrolysis with 0.1 N hydrochloric acid degraded about 15 percent of bulk itraconazole versus 12 percent in the gel; alkaline stress caused 12 and 10 percent degradation; oxidative stress with 3 percent hydrogen peroxide proved harshest at 18 and 15 percent; and thermal and photolytic stresses caused only 3 to 5 percent loss. In every case the chromatographic method cleanly separated the parent drug from its degradants, and in every case the gel matrix offered modest protection, likely through buffering excipients or physical shielding from ultraviolet light.

What distinguishes this work from a competent validation exercise is its metrological and environmental accounting. Following ISO/IEC 17025, the researchers estimated measurement uncertainty from peak area repeatability, weighing of standards and samples, and reference standard purity, arriving at a combined standard uncertainty of 1.45 percent and an expanded uncertainty of plus or minus 2.9 percent at 95 percent confidence for a 20 microgram per milliliter sample. On the sustainability side, the method scored 0.71 on the AGREE metric and 0.72 on AGREEprep, 72.5 on both the RAPI and White Analytical Chemistry indices, 85 on ComplexGAPI, and 50 out of 100 on each RGBfast dimension. The scores describe a method that is analytically excellent and moderately green: strengths included small sample amounts, minimal sample treatment, no derivatization, and a short run time, while penalties stemmed from hazardous, non-renewable solvents, namely methanol, acetonitrile, and triethylamine, and roughly 106 milliliters of liquid waste per analysis.

The quantitative solvent accounting is nonetheless encouraging. The optimized method consumes about 6 milliliters of mobile phase per six-minute run, far below the 15 to 25 milliliters typical of conventional HPLC, and energy demand was estimated at under 0.05 kilowatt-hours per analysis. Because QbD-driven experimental design replaced trial-and-error optimization, the team estimates that repeated experiments and solvent wastage fell by 40 to 50 percent. The authors also mapped the method against the United Nations Sustainable Development Goals, finding positive contributions to SDG 3 on health and SDG 9 on industry and innovation, with partial alignment on clean water, clean energy, and responsible consumption, but clear gaps on climate action due to the solvent burden.

The study’s broader message is methodological: sustainability can be quantified and built into analytical development from the start rather than audited after the fact. By coupling QbD’s design spaces and desirability functions with AGREE, ComplexGAPI, and White Analytical Chemistry, the researchers demonstrate a workflow in which robustness, uncertainty, and environmental impact are optimized together. They point toward further gains through micro- or ultra-HPLC columns, greener solvent substitutions such as ethanol or propylene carbonate, and waste recycling, changes that prior literature suggests could push AGREE scores above 0.8. For an industry whose laboratories collectively churn through enormous volumes of acetonitrile every day, that combination of statistical discipline and ecological accountability may prove as influential as the method itself.

Subject of Research: Development of a green chemistry and Quality by Design-based RP-HPLC method for itraconazole analysis in pharmaceutical formulations

Article Title: Green chemistry integrated quality by design based RP-HPLC method for analysis of Itraconazole in pharmaceutical formulations

Article References: Arghode, R., Trivedi, S. S., Bondre, S., Hussain, U., & Gupta, K. (2026). Green chemistry integrated quality by design based RP-HPLC method for analysis of Itraconazole in pharmaceutical formulations. Discover Green Chemistry, 1(1), Article 8. https://doi.org/10.1007/s44509-026-00010-6

Image Credits: AI Generated

DOI: 10.1007/s44509-026-00010-6

Keywords: itraconazole, RP-HPLC, Quality by Design, green analytical chemistry, White Analytical Chemistry, AGREE, ComplexGAPI, Box-Behnken design, stress degradation, method validation, pharmaceutical analysis, sustainability