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

The review’s authors, Ravi Patel of ThermoFisher Scientific, Dipen Purohit of Navinta LLC, and Krupalkumar Morker of Frontage Laboratories, compiled analytical methods published between 2010 and 2025 for the simultaneous estimation of the two antihypertensive agents in pharmaceutical formulations and biological matrices. Their motivation stems from a growing tension in pharmaceutical quality control: the same laboratories that validate drug safety generate streams of toxic solvent waste, consume large amounts of energy, and expose analysts to hazardous chemicals. As sustainability expectations rise across the industry, the environmental footprint of the analytical methods themselves has come under scrutiny alongside the performance metrics that regulators traditionally demand.

The two drugs at the center of the assessment are clinically complementary. Amlodipine besylate is a long-acting dihydropyridine calcium channel blocker that relaxes blood vessels by inhibiting calcium ion influx into vascular smooth muscle and cardiac cells, reducing peripheral resistance and the heart’s oxygen demand. Indapamide, a thiazide-like diuretic, works differently: it blocks sodium reabsorption in the kidney’s distal convoluted tubule, increasing sodium and water excretion and lowering plasma volume, while also exerting vasodilatory effects independent of its diuretic action. Combined in a single pill, the two agents achieve better blood pressure control at lower doses than either drug alone, minimizing side effects and improving patient compliance. Hypertension affects approximately 1.28 billion adults worldwide, and according to the World Health Organization nearly 46 percent of those affected are unaware of their condition, making reliable, affordable quality control for these medicines a genuine global health matter.

To quantify environmental performance, the reviewers applied two complementary scoring systems. The first, the Analytical GREEnness metric, or AGREE, was developed at Gdańsk University of Technology and evaluates a method against the twelve principles of green analytical chemistry. It produces a radial, clock-like diagram in which each segment is color-coded from red, indicating poor greenness, to green, indicating strong performance, and yields a cumulative score from 0 to 1. The second tool, the Analytical Eco-Scale, starts from an ideal score of 100 points and deducts penalty points for hazardous reagents, excessive solvent quantities, high energy consumption, waste generation, and safety risks. Methods scoring 75 or above are classified as excellent green analysis, those between 50 and 75 as acceptable, and anything below 50 as ecologically unacceptable.

The results were strikingly consistent. Spectrophotometric methods, which measure how much ultraviolet or visible light a dissolved drug absorbs, achieved AGREE scores between 0.62 and 0.66 and Eco-Scale scores as high as 97. A first derivative ratio spectrophotometry method topped the ranking with an Eco-Scale score of 97, while absorbance ratio, area under the curve, ultraviolet absorbance correction, and Vierordt’s simultaneous equations methods all clustered around AGREE scores of 0.62 to 0.66 with Eco-Scale values of 91. The reasons are straightforward: these techniques require minimal solvent, typically just methanol, need no buffer solutions, involve little or no sample preparation, consume very little energy, and generate almost no waste. For routine quality control of tablets in resource-conscious settings, the review suggests they remain hard to beat.

High-performance thin-layer chromatography, or HPTLC, produced a more nuanced picture that hinged entirely on solvent choice. A stability-indicating HPTLC method using ethanol, ethyl acetate, and triethylamine achieved the single highest AGREE score of the entire comparison, 0.70, along with an Eco-Scale score of 91, demonstrating that planar chromatography can be genuinely green when built on renewable, low-toxicity solvents. By contrast, a conventional HPTLC method relying on dichloromethane, a hazardous chlorinated solvent, scored only 0.50 on AGREE with an Eco-Scale value of 83. The lesson, the authors argue, is that the same instrumental platform can occupy opposite ends of the sustainability spectrum depending on the chemistry chosen to run it.

Conventional reversed-phase high-performance liquid chromatography, the workhorse of pharmaceutical analysis, fared less well. AGREE scores for RP-HPLC methods ranged from 0.47 to 0.61, with Eco-Scale values between 80 and 87. The environmental burden comes from the technique’s fundamental operating model: continuous pumping of mobile phase through a column at flow rates typically between 0.6 and 1.5 milliliters per minute, using organic solvents such as acetonitrile and methanol mixed with phosphate, acetate, or citrate buffers. Every analysis flushes liters of solvent mixture into waste containers over time, and the pumps and detectors draw continuous power. Methods employing phosphate buffers and triethylamine scored at the lower end of the chromatographic range, while stability-indicating variants with optimized solvent compositions achieved slightly better values.

Yet the review also identified a promising path forward within chromatography itself. An RP-HPLC method developed under Analytical Quality by Design, or AQbD, principles, using a mobile phase of methanol and 0.1 percent orthophosphoric acid at pH 4.5, reached an AGREE score of 0.61 and an Eco-Scale score of 87, among the best chromatographic results. AQbD is a systematic framework that identifies the critical method parameters affecting performance and deliberately optimizes them, and the review shows it can be steered toward environmental goals as well as analytical ones. Similarly, an eco-friendly RP-HPLC method for amlodipine impurity profiling that substituted ethanol for more problematic solvents satisfied both greenness metrics while maintaining full separation of the drug’s known impurities, and an optimized UPLC method for amlodipine recorded a low environmental impact value on the HPLC-EAT scale alongside excellent reproducibility.

At the opposite extreme sat the most technologically advanced approach. UPLC–MS/MS and LC–MS/MS methods, which couple ultra-performance liquid chromatography with tandem mass spectrometry, recorded some of the lowest scores in the assessment, with an AGREE value of 0.47 and an Eco-Scale score of 68. These hyphenated techniques are unmatched for sensitivity and specificity, capable of quantifying amlodipine, indapamide, and other antihypertensives in human serum for therapeutic drug monitoring and bioequivalence studies. But that power carries a cost: high-purity solvents, buffer salts, energy-intensive instrumentation, and complex solvent systems all weigh heavily against them on greenness metrics. The review does not suggest abandoning them, since bioanalytical work in plasma and serum has few alternatives, but it does highlight that their routine use where simpler methods suffice carries a substantial and often unexamined environmental price.

The broader message of the review extends well beyond these two drugs. Current ICH validation guidelines focus on precision, accuracy, and robustness but say little about sustainability, and the authors argue that green chemistry principles should be integrated from the earliest stages of method development rather than assessed as an afterthought. Their recommendations include replacing hazardous solvents with greener alternatives such as ethanol and ethyl acetate, minimizing solvent volumes, adopting AQbD frameworks to optimize flow rates and compositions, and pursuing miniaturization and automation. Spectrophotometric and AQbD-assisted HPTLC approaches, they conclude, offer the best combination of green credentials and analytical reliability for routine pharmaceutical quality control. As regulators and the public increasingly expect the pharmaceutical industry to align with sustainable development goals, the environmental scorecard of the laboratory bench, this review suggests, deserves the same rigor as the assay results it produces.

Subject of Research: Greenness assessment of analytical methods for quantifying amlodipine besylate and indapamide

Article Title: Comparative greenness assessment of analytical methods for the estimation of amlodipine besylate and indapamide using green analytical chemistry principles

Article References: Comparative greenness assessment of analytical methods for the estimation of amlodipine besylate and indapamide using green analytical chemistry principles. (n.d.). https://doi.org/10.1007/s44509-026-00013-3

Image Credits: AI Generated

DOI: 10.1007/s44509-026-00013-3

Keywords: green analytical chemistry, amlodipine besylate, indapamide, AGREE metric, Analytical Eco-Scale, spectrophotometry, RP-HPLC, HPTLC, UPLC-MS/MS, Analytical Quality by Design, pharmaceutical analysis, sustainability

The study, published in the journal Discover Electrochemistry, was led by Sandeep Kurundawade and Sharanappa T. Nandibewoor of KLE Technological University in Hubballi, together with colleagues at Atria Institute of Technology in Bengaluru and J.S.S. Banashankari Arts, Commerce and S.K. Gubbi Science College in Dharwad. Their choice of modifier was polyaniline, one of the oldest and best understood conducting polymers. Polyaniline is prized in materials science for its unique p-type doping chemistry, its reversible redox transitions between the leucoemeraldine, emeraldine and pernigraniline states, and its protonic conductivity, all of which allow it to alter the electronic behavior of an electrode surface. Compared with its polymeric cousins polypyrrole and polythiophene, polyaniline also offers superior environmental resilience, an essential property for a sensor intended for repeated laboratory use.

Synthesis of the polymer followed a classical route: oxidative polymerization of aniline in 1.5 molar hydrochloric acid, initiated by the gradual, drop-wise addition of ammonium persulphate while the reaction mixture was stirred. The slow addition is not decorative; it prevents over-oxidation and promotes uniform chain growth. The reaction mixture’s shift to the characteristic dark green color of polyaniline’s emeraldine salt signaled success. The researchers then characterized the product with scanning electron microscopy, energy dispersive X-ray spectroscopy, and X-ray diffraction. Electron microscopy revealed a cohesive network of uniformly packed spherical agglomerates, while the diffraction pattern showed a broad peak near 2θ equal to 25 degrees, a signature of the polymer’s π-π stacked aromatic framework and its mildly crystalline, emeraldine-phase structure.

The heart of the sensor is a glassy carbon electrode, a workhorse of electroanalytical chemistry with a geometric area of just 0.09 square centimeters. A 1 milligram sample of polyaniline was dispersed in dimethylformamide by sonication and drop-cast onto the polished electrode, with the deposited volume optimized at 4 microliters after testing across a 2 to 8 microliter range. To measure how much the coating enlarged the electrochemically active surface, the team employed a ferricyanide redox probe and the Randles-Sevcik equation. The bare electrode’s active area was 0.04 square centimeters; with polyaniline it grew to 0.156 square centimeters, a 4.4-fold increase that translates into a far denser network of electroactive sites and correspondingly faster charge transfer.

Impedance measurements reinforced that picture. Nyquist plots, fitted to a Randles equivalent circuit, yielded charge transfer resistance values of 17.03 ohms for the bare electrode and 11.57 ohms for the modified one, while the calculated heterogeneous charge transfer rate constants rose from 1.56 × 10⁻⁵ to 2.3 × 10⁻⁵ centimeters per second and exchange currents climbed from 1.508 × 10⁻³ to 2.219 × 10⁻³ amperes. In plain terms, the polymer film does not merely add surface area; it actively lubricates the movement of electrons between the drug molecules in solution and the electrode beneath. An immersion-time study further showed that the analytical signal peaked after 40 seconds of accumulation, beyond which the response declined, likely because the available surface had saturated or an inhibitory molecular layer had formed.

With the hardware characterized, the team turned to the electrochemistry of oxybutynin itself. Cyclic voltammetry in phosphate buffer at pH 7.4, scanned between 0.5 and 1.5 volts, produced a single anodic peak with no counterpart on the reverse scan, marking the oxidation as irreversible. On the modified electrode the peak current reached 76.7 microamperes against 42.6 microamperes on bare glassy carbon, and the peak potential shifted slightly downward from 1.191 to 1.185 volts, evidence that polyaniline lowers the overpotential and eases the oxidation. Varying the pH from 3.0 to 9.2 revealed a maximum response at 7.4, close to physiological pH, and a peak potential that tracked pH with a slope of 64 millivolts per pH unit, near the Nernstian ideal of 59, indicating equal numbers of electrons and protons in the reaction.

Scan rate studies completed the mechanistic portrait. The peak current scaled linearly with the square root of the scan rate, and a log-log analysis produced a slope of 0.3285, close to the theoretical value of 0.5 for diffusion control. Applying the Laviron equation to the relationship between peak potential and scan rate gave a transfer-coefficient-electron product of 1.14, which resolved to approximately two electrons when the standard transfer coefficient of 0.5 was assumed. Combined with the pH result, the oxidation of oxybutynin at the modified electrode is therefore a diffusion-controlled, irreversible process involving two electrons and two protons, proceeding through successive electron-transfer and deprotonation steps toward an oxidized imine-type intermediate and ultimately an amine-based product.

The analytical performance is where the sensor earns its headline. Using square-wave voltammetry, a technique prized for its ability to reject background currents, the team recorded a linear response across 0.04 to 10 micromolar oxybutynin, with a detection limit of 9.16 nanomolar and a limit of quantification of 30.5 nanomolar, calculated by the standard equations based on the regression intercept’s standard deviation. That sensitivity compares favorably with previously reported oxybutynin sensors, which the authors note have tended to be either expensive, laborious to construct, or both. Selectivity testing with 100-fold excesses of citric acid, gum acacia, D-glucose, dopamine, L-ascorbic acid, sucrose, and common inorganic salts produced peak potential shifts all below the 2 percent tolerance limit, confirming the sensor’s robustness in messy real-world matrices.

The practical demonstrations may prove the most consequential part of the work. Pulverized commercial 5 milligram tablets, dissolved and diluted into the linear range, yielded recoveries in excellent agreement with the label claim, opening the door to routine pharmaceutical quality control. Human urine diluted 100-fold and spiked with known drug concentrations, along with tap water, Krishna River water from Belagavi in Karnataka, and RO-purified water, were all analyzed with acceptable recoveries, extending the sensor’s reach into pharmacokinetic monitoring and environmental surveillance of pharmaceutical contamination. Stability testing showed consistent within-day signals and retention of a high percentage of the initial response after 15 days of storage in a sealed container. Taken together, the results suggest that a coating of a cheap, easily synthesized conducting polymer can transform a routine glassy carbon electrode into a precision instrument, one well suited to clinical laboratories, quality control departments, and environmental monitoring programs that have historically been priced out of high-sensitivity drug analysis.

Subject of Research: Ultrasensitive electrochemical detection of the drug oxybutynin hydrochloride using a polyaniline-modified glassy carbon electrode sensor

Article Title: Ultrasensitive electrochemical detection of oxybutynin hydrochloride using polyaniline modified sensor

Article References: Kurundawade, S., Megalamani, M. B., Naik, K., Yaraguppi, D. A., & Nandibewoor, S. T. (2026). Ultrasensitive electrochemical detection of oxybutynin hydrochloride using polyaniline modified sensor. Discover Electrochemistry, 3(1), Article 73. https://doi.org/10.1007/s44373-026-00161-y

Image Credits: AI Generated

DOI: 10.1007/s44373-026-00161-y

Keywords: oxybutynin hydrochloride, polyaniline, electrochemical sensor, glassy carbon electrode, square-wave voltammetry, cyclic voltammetry, overactive bladder, pharmaceutical analysis, detection limit, conducting polymer, electrocatalysis, pharmacokinetics