New Green Analytical Methods Put India’s First Homegrown Ketolide Antibiotic to the Test

Nafithromycin, the novel ketolide antibiotic developed by Wockhardt Ltd. and approved in India in 2024 for community-acquired bacterial pneumonia, has taken a significant step toward robust pharmaceutical quality control. In a study published in Discover Chemistry, researchers at S.S.P. Shikshan Sanstha’s Siddhi College of Pharmacy in Pune report the first combined development, validation, and environmental assessment of two complementary analytical techniques for quantifying the drug in tablet form. The work, led by Vaishnavi Arjun Singh with Hitanshi Darji and PravinKumar Sable, delivers both a simple ultraviolet spectrophotometric method and a reverse-phase high-performance liquid chromatographic method, each validated under the newest international regulatory framework and screened for ecological sustainability using two widely recognized greenness evaluation tools.

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