The clinical backdrop gives the study its urgency. Community-acquired bacterial pneumonia remains a leading cause of illness and death worldwide, particularly among elderly patients, the immunocompromised, and those with chronic respiratory or cardiovascular disease. As antimicrobial resistance erodes the effectiveness of conventional antibacterial therapies, new agents with activity against resistant organisms have become a pressing need. Nafithromycin, a semisynthetic ketolide derived from the macrolide class, answers that call by inhibiting bacterial protein synthesis through binding to the 50S ribosomal subunit. Unlike older macrolides, it shows enhanced ribosomal affinity and retains activity against macrolide-resistant pathogens, with broad-spectrum coverage of Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, and atypical respiratory organisms. Favorable pharmacokinetics, including prolonged penetration and residence in pulmonary tissue, add to its clinical appeal.

Yet a new drug is only as trustworthy as the analytical methods that verify its content, and the literature on Nafithromycin has been thin. Published work has concentrated on LC-MS/MS bioanalysis for pharmacokinetic studies and on chromatographic impurity profiling, including a stability-indicating method for related impurities reported by Ahirrao and colleagues. While powerful, such techniques demand sophisticated instrumentation, costly solvents, extensive sample preparation, and highly trained personnel, which limits their routine use in many quality control laboratories. The Pune team set out to close that gap with methods that any reasonably equipped laboratory could run, while also meeting the freshly revised ICH Q2(R2) guideline, the International Council for Harmonisation’s science- and risk-based framework for analytical procedure validation.

The spectrophotometric approach is elegantly simple. Nafithromycin exhibits maximum absorbance at 275 nanometers in a mixed solvent of methanol and 10 millimolar ammonium acetate buffer at pH 9.0, blended 70:30 by volume. Direct absorbance measurement at that wavelength, using a Shimadzu UV-Visible spectrophotometer with 1 centimeter quartz cells, produced a well-defined spectrum with stable baseline behavior. The chromatographic method used a Waters Alliance e2695 system with a photodiode array detector, separating the drug on a Phenomenex Kinetex C18 column of 150 by 4.6 millimeters with 5 micrometer particles. The same methanol-buffer mobile phase was delivered isocratically at 1.0 milliliter per minute, with detection at 275 nanometers, a 20 microliter injection volume, and a total run time of just 10 minutes.

Buffer selection proved decisive. Preliminary trials with unbuffered solvent systems produced broader peaks and poorer reproducibility, because Nafithromycin carries ionizable functional groups whose charge state varies with pH. The 10 millimolar ammonium acetate buffer at pH 9.0 held the drug in a consistent ionization state, sharpening peak symmetry and improving chromatographic performance. Under the optimized conditions, Nafithromycin eluted in roughly 2.085 minutes as a sharp, symmetrical peak, a retention time short enough to keep throughput high in a busy quality control laboratory. System suitability testing with six replicate injections confirmed that peak area variability stayed below 2.0 percent relative standard deviation, theoretical plate counts exceeded 2000, and tailing factors remained under 2.0.

Validation followed the full ICH Q2(R2) menu of performance characteristics. Both methods showed linear responses across a concentration range of 2 to 12 micrograms per milliliter, with correlation coefficients above 0.999; the UV method yielded a regression equation of y = 0.0101x minus 0.0003 with R-squared of 0.9989, while the HPLC method produced y = 89471x plus 2600 with an almost perfect R-squared of 0.9999. Precision was exemplary: repeatability gave a relative standard deviation of 0.83 percent for the UV method and a striking 0.09 percent for HPLC, and intermediate precision values, assessed across days, instruments, and concentration levels, ranged from 0.25 to 1.60 percent for UV and just 0.08 to 0.17 percent for HPLC, all comfortably below the 2.0 percent acceptance threshold.

Trueness, assessed by standard addition at 80, 100, and 120 percent levels, produced mean recoveries of 99.90 to 100.00 percent for the UV method and 99.92 to 99.95 percent for HPLC, confirming that tablet excipients did not interfere with the determination. Sensitivity differed sharply between the techniques: limits of detection and quantitation were 0.46 and 1.39 micrograms per milliliter for UV, but only 0.05 and 0.166 micrograms per milliliter for HPLC, a roughly tenfold sensitivity advantage for the chromatographic approach. Robustness testing, which deliberately perturbed wavelength, solvent grade, and sonication time for UV, and flow rate, mobile phase composition, and wavelength for HPLC, left results essentially unchanged, with relative standard deviations between 1.19 and 1.36 percent for UV and 0.19 to 0.31 percent for HPLC. Prepared solutions remained stable for 24 hours, with peak area variation under 0.5 percent.

When the methods were applied to marketed MIQNAF tablets, each labeled at 400 milligrams, the UV assay returned 99.42 plus or minus 0.42 percent of the label claim and HPLC returned 99.71 plus or minus 0.14 percent, both within pharmacopeial acceptance criteria. A one-way analysis of variance comparing the two sets of assay results yielded a calculated F-value of 2.58 against a critical value of 4.96 at 95 percent confidence, meaning no statistically significant difference between the methods. That statistical equivalence matters practically: laboratories without HPLC infrastructure can rely on the spectrophotometric method, while those needing higher sensitivity or chromatographic selectivity can adopt the HPLC procedure with confidence that both deliver the same answer.

The greenness assessment distinguishes this study from most method-validation reports. Using the Green Analytical Procedure Index, a pictogram-based tool scoring sample preparation, reagents, energy use, occupational hazards, and waste, both methods showed predominantly green and yellow zones, with limited red zones tied mainly to methanol consumption and solvent waste in the chromatographic procedure. The Analytical GREEnness Metric Approach, which evaluates compliance with the twelve principles of green analytical chemistry on a scale approaching 1.0, awarded scores of 0.72 for the UV method and 0.75 for the HPLC method. These figures indicate good environmental compliance for routine work, though the authors note that solvent usage remains the main ecological footprint to manage.

The study’s authors are careful to scope their claims: the HPLC method is intended for routine assay rather than impurity profiling, and the work represents, to their knowledge, the first comparative development, ICH Q2(R2)-based validation, statistical comparison, and greenness assessment of UV and RP-HPLC methods for Nafithromycin. As India’s first indigenous ketolide moves through clinical use against drug-resistant respiratory pathogens, the availability of validated, economical, and environmentally responsible quality control methods ensures that every batch of the drug can be verified quickly and reliably. In an era when analytical chemistry is increasingly judged not only by accuracy but by sustainability, this dual-method platform offers a template for how new antibiotics can be brought under rigorous, green quality control from the start of their market life.

Subject of Research: Development and validation of green UV spectrophotometric and RP-HPLC methods for quantitative determination of the antibiotic Nafithromycin

Article Title: Development, validation, and greenness assessment of UV spectrophotometric and RP-HPLC methods for the quantitative determination of Nafithromycin

Article References: Singh, V. A., Darji, H., & Sable, P. (2026). Development, validation, and greenness assessment of UV spectrophotometric and RP-HPLC methods for the quantitative determination of Nafithromycin. Discover Chemistry, 3(1), Article 466. https://doi.org/10.1007/s44371-026-00920-1

Image Credits: AI Generated

DOI: 10.1007/s44371-026-00920-1

Keywords: Nafithromycin, ketolide antibiotic, RP-HPLC, UV spectrophotometry, ICH Q2(R2), method validation, green analytical chemistry, GAPI, AGREE, community-acquired bacterial pneumonia, pharmaceutical quality control, ANOVA

The stakes behind this seemingly technical achievement are considerable. Impurity control sits at the heart of pharmaceutical safety: trace contaminants that slip past inadequate analytical methods can compromise therapeutic efficacy or, in the worst cases, pose direct risks to patients. Sertraline, chemically known as (1S,4S)-4-(3,4-dichlorophenyl)-N-methyl-1,2,3,4-tetrahydronaphthalen-1-amine hydrochloride, is a selective serotonin reuptake inhibitor that works by blocking serotonin reabsorption at presynaptic nerve terminals, enhancing neurotransmission in the central nervous system. During its synthetic preparation, structurally related impurities can arise, and regulators such as the International Council for Harmonisation require comprehensive impurity profiling and stability evaluation to guarantee product quality throughout a medicine’s shelf life.

Existing methods for sertraline impurity testing, the authors note, have been limited by empirical optimization, long run times, high solvent consumption, inadequate impurity resolution, and limited robustness. The drug substance is official in both the United States Pharmacopoeia and the European Pharmacopoeia, yet the pharmacopeial approaches rely on gas chromatography for specified organic impurities. The research team set out to close this gap by combining two modern paradigms: Analytical Quality by Design, often abbreviated AQbD, which applies systematic, risk-based experimentation to analytical method development, and Green Analytical Chemistry, which seeks to minimize the environmental footprint of laboratory work.

The journey to the final method was one of iterative refinement. Early attempts using an isocratic elution of potassium dihydrogen phosphate and acetonitrile on a conventional column failed entirely, with all impurity peaks eluting at the same retention time. Switching to methanol improved matters only marginally, producing broad peaks, while gradient programs and a phenyl column achieved separation but with unsatisfactory peak shapes caused by isomeric compounds. The breakthrough came with a pentafluorophenyl-modified C18 column, the ACE 5 C18 PFP, whose special surface chemistry offers multiple retention mechanisms, including hydrophobic, pi-pi, dipole-dipole, and hydrogen-bonding interactions, providing enhanced selectivity for structurally similar aromatic impurities that defeat ordinary C18 columns.

The optimized method uses a gradient of two mobile phases built around a 20 millimolar ammonium phosphate buffer adjusted to pH 4.2, mixed with methanol and acetonitrile in carefully tuned ratios. Mobile phase A combines buffer, methanol, and acetonitrile at 80:10:10, while mobile phase B blends methanol, acetonitrile, and buffer at 50:20:30. Separation proceeds on the 250 by 4.6 millimeter column at 40 degrees Celsius with a flow rate of 1.0 milliliter per minute and ultraviolet detection at 254 nanometers. Under these conditions, the six impurities elute at distinct retention times ranging from roughly 4 minutes for impurity-E to nearly 42 minutes for impurity-F, with sertraline itself appearing at about 22 minutes, all fully resolved from blank and placebo signals.

Crucially, the team did not simply tune parameters by trial and error. They identified column temperature and flow rate as critical method parameters and subjected them to a three-level, two-factor Design of Experiments, running thirteen HPLC experiments and modeling the results with response surface methodology. Statistical analysis confirmed that neither variable significantly affected the relative retention times of the key impurities, establishing a design space within which the method remains reliable. This systematic approach, validated against ICH Q2(R2) guidelines, demonstrated excellent linearity across concentration ranges of approximately 0.2 to 5.0 micrograms per milliliter with correlation coefficients exceeding 0.999, precision with relative standard deviations below 2 percent, and recoveries between 99.0 and 103.0 percent.

The method’s stability-indicating power was tested through forced degradation studies that subjected sertraline capsules to acidic, alkaline, oxidative, hydrolytic, thermal, photolytic, and humidity stress. Samples were treated with hydrochloric acid or sodium hydroxide at 60 degrees Celsius, exposed to 10 percent hydrogen peroxide, irradiated with more than 1.2 million lux hours of light per ICH Q1B, heated to 100 degrees Celsius for three days, or held at 90 percent relative humidity. Sertraline proved remarkably stable, with only limited degradation observed under all conditions. Where degradation products did form, they were cleanly resolved from the analyte peak, and photodiode array peak purity analysis confirmed the sertraline peak remained spectrally homogeneous, with satisfactory mass balance values throughout.

Perhaps the most distinctive feature of the study is its environmental assessment. Using two established greenness metrics, the AGREE analytical tool and the Green Analytical Procedure Index, the researchers scored their method at 0.79 and 84 respectively, indicating high environmental compatibility. The method earns green ratings for low sample consumption, minimal preparation steps, automation, avoidance of derivatization, waste control, and energy efficiency. In an era when laboratories worldwide are under pressure to reduce their reliance on hazardous solvents, the deliberate balancing of acetonitrile consumption against chromatographic performance represents a meaningful step toward sustainable pharmaceutical quality control.

The researchers report that sample and mobile phase solutions remained stable for up to 72 hours at room temperature, further supporting the method’s suitability for routine work. With its combination of complete impurity resolution, regulatory-compliant validation, statistically proven robustness, and favorable environmental profile, the AQbD-driven method offers pharmaceutical manufacturers a practical tool for both routine quality control and formal stability testing of sertraline capsules. As analytical laboratories increasingly embrace Quality by Design thinking and green chemistry principles, this study offers a template for how the two movements can converge, transforming the humble chromatogram into evidence of both patient safety and environmental responsibility.

Subject of Research: Development of a stability-indicating RP-HPLC method with Analytical Quality by Design and greenness assessment for quantifying impurities in sertraline hydrochloride dosage forms

Article Title: A stability-indicating RP-HPLC method for quantification of impurities in sertraline hydrochloride finished dosage forms using quality by design with environmental assessment

Article References: Bypaneni, V., Pinnenti, M. K., Temmanaboyina, G., Sambu, P., & Nagisetti, B. M. K. (2026). A stability-indicating RP-HPLC method for quantification of impurities in sertraline hydrochloride finished dosage forms using quality by design with environmental assessment. Discover Chemistry, 3(1), Article 569. https://doi.org/10.1007/s44371-026-01010-y

Image Credits: AI Generated

DOI: 10.1007/s44371-026-01010-y

Keywords: sertraline hydrochloride, RP-HPLC, Analytical Quality by Design, impurity profiling, forced degradation, green analytical chemistry, AGREE, GAPI, pharmaceutical quality control, stability-indicating method, chromatography, antidepressants

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