A team of researchers at Gujarat Technological University in Gandhinagar, India, has now delivered what they describe as the first analytical method for desidustat that is simultaneously green, statistically optimized, and capable of tracking the drug’s degradation. Writing in the journal Discover Green Chemistry, the group led by Parmar Bharvi and Kashyap Thummar combined two frameworks that have been reshaping pharmaceutical analysis: Analytical Quality by Design, often abbreviated AQbD, and the principles of Green Analytical Chemistry. Their chosen tool was high-performance thin-layer chromatography, or HPTLC, a planar chromatographic technique that has long been prized for its low solvent consumption, high sample throughput, and modest operating costs, but which has traditionally been developed through trial and error rather than systematic design.

The scientific logic behind the study begins with risk. In an AQbD workflow, the analyst first defines an Analytical Target Profile, essentially a specification sheet for what the method must achieve, and then identifies every factor that could jeopardize those goals. The team used failure mode and effects analysis, a technique borrowed from engineering, to score potential failure causes by severity, likelihood, and detectability, producing risk priority numbers that ranked each variable. Mobile phase volume, chamber saturation time, and solvent front distance emerged as the critical method parameters, the high-risk factors demanding experimental optimization. Moderate-risk items such as solvent and sample purity were flagged for stringent quality checks, while low-risk factors like sample preparation and detection wavelength were simply placed under routine control. This layered strategy, the authors note, is what separates a designed method from a lucky one.

To optimize the three critical parameters, the researchers turned to a Box-Behnken Design, a response surface methodology that explores quadratic effects and interactions among variables with remarkable economy. Where a traditional approach might demand dozens of experiments, the design required only seventeen runs, evaluated at low, medium, and high levels of each factor. Analysis of variance confirmed that quadratic polynomial models described both the retention factor and the peak area with high coefficients of determination, non-significant lack-of-fit tests, and strong predicted values, meaning the models could genuinely forecast chromatographic behavior rather than merely describe past experiments. Interaction terms proved largely insignificant, simplifying the final model without sacrificing predictive power. The exercise revealed a clear trade-off: longer solvent front distances produced larger peak areas but also broadened the bands and pushed the retention factor upward, eroding specificity.

The optimized conditions settled on a mobile phase of toluene, ethyl acetate, methanol, and formic acid in the ratio 10.5 to 3 to 1.5 to 0.45, a 20-minute chamber saturation, and a development distance of 60 millimeters. Under these conditions, desidustat produced a sharp, well-resolved band at a retention factor of 0.45 plus or minus 0.02, scanned densitometrically at 230 nanometers. The choice of a compact development distance, the authors explain, deliberately favored band quality over raw signal, a decision that reflects the design-space thinking at the core of AQbD: instead of chasing a single best response, the method balances resolution, sensitivity, and robustness simultaneously.

Validation followed the international ICH Q2(R2) guideline, and the numbers were impressive. The method showed excellent linearity across 600 to 1600 nanograms per band, with a correlation coefficient of 0.997. Accuracy, assessed by standard addition at three spiking levels, ranged from roughly 98.84 to 101.87 percent recovery, while precision, measured both within a single day and across days, stayed comfortably below the 2 percent relative standard deviation threshold. Sensitivity limits were established at 220.8 nanograms per band for detection and 517.9 nanograms per band for quantification, calculated by the instrument’s built-in signal-to-noise algorithm. Deliberate small perturbations in wavelength, saturation time, and development distance left the results essentially untouched, confirming robustness, and tablet excipients produced no interfering peaks.

Perhaps the most clinically revealing portion of the work is the forced degradation study, conducted under the spirit of ICH Q1A(R2). The researchers subjected desidustat to hydrolytic, oxidative, thermal, and photolytic stress. The drug proved most vulnerable to acidic hydrolysis, degrading by about 16 percent and yielding a distinct degradation product at a retention factor of 0.78, cleanly separated from the parent peak at 0.45. Under alkaline, oxidative, thermal, and sunlight exposure, degradation remained limited to 3 to 9 percent with no additional bands appearing, indicating the molecule is comparatively stable under those conditions. Because the method resolves the degradant without co-elution and passes peak purity checks, it qualifies as stability-indicating, meaning quality control laboratories can use it to verify not just how much drug is present, but whether the product has held up during storage. Structural identification of the degradation product by mass spectrometry was beyond the scope of the study, the authors acknowledge, but the chromatographic evidence of selective separation stands on its own.

The green credentials of the method were then put to a quantitative test using two widely respected assessment tools. The AGREE metric, which scores analytical procedures against the twelve principles of Green Analytical Chemistry on a scale from 0 to 1, awarded the method 0.87, comfortably within the excellent greenness category above 0.75. The ComplexGAPI tool, which renders the environmental impact of every step from sample preparation to final measurement as a color-coded pentagram, showed predominantly green and yellow zones with minimal red, the single red segment attributable to the toluene in the mobile phase. Even so, the overall environmental burden stays low because each plate requires only 10 to 12 milliliters of mobile phase while analyzing many samples at once, a fraction of the solvent consumed by conventional high-performance liquid chromatography, which typically depends on acetonitrile-rich systems and generates far more waste.

The comparison with earlier desidustat methods underscores why the work matters. Ultraviolet spectrophotometry offers simplicity but limited stability information; reversed-phase HPLC handles dissolution and assay work but at higher cost and solvent expense; liquid chromatography with tandem mass spectrometry delivers trace-level sensitivity for pharmacokinetics and doping control but is overkill and resource-intensive for routine tablet testing. The new HPTLC method occupies a pragmatic middle ground: sensitive enough for quality control, fast enough for high-throughput laboratories, cheap enough for routine deployment, and now formally documented as environmentally sustainable. The AQbD framework also eases method transfer between laboratories, a key regulatory requirement, because the statistically verified design space defines exactly how much the parameters can flex without compromising performance.

Beyond desidustat itself, the study offers a template. The authors argue that the same integration of risk assessment, designed experiments, and greenness evaluation can be extended to other pharmaceutical compounds, and they point toward future applications in impurity profiling and bioanalysis. Funded in part by a seed grant from Gujarat Technological University and published open access, the research arrives at a moment when the pharmaceutical industry is under growing pressure to shrink the environmental footprint of its laboratories. A method that quantifies a kidney-anemia drug with sub-microgram precision while using milliliters rather than liters of solvent may not make headlines the way a new molecule does, but it represents the quieter, cumulative kind of innovation that makes modern medicine both reliable and sustainable.

Subject of Research: Development of a green, stability-indicating HPTLC method using Analytical Quality by Design for quantifying desidustat in pharmaceutical formulations

Article Title: Integration of analytical quality by design and green chemistry principles in the development of an HPTLC method for Desidustat estimation

Article References: Bharvi, P., Thummar, B., Dudhatra, B., Joshi, H., Maheriya, B., Vadalia, J., & Thummar, K. (2026). Integration of analytical quality by design and green chemistry principles in the development of an HPTLC method for Desidustat estimation. Discover Green Chemistry, 1(1), Article 7. https://doi.org/10.1007/s44509-026-00007-1

Image Credits: AI Generated

DOI: 10.1007/s44509-026-00007-1

Keywords: desidustat, HPTLC, Analytical Quality by Design, green analytical chemistry, Box-Behnken design, stability-indicating method, chronic kidney disease anemia, ICH Q2(R2), AGREE metric, ComplexGAPI, pharmaceutical quality control, forced degradation

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