The analytical challenge begins with the drug’s unusual chemistry. Cefpodoxime proxetil is sold as a racemic mixture of R- and S-epimers arising from an asymmetric carbon in the proxetil side chain, which means any credible assay must resolve two chromatographic peaks, designated CFP-A and CFP-B, rather than one. Earlier methods relied on solid-phase extraction, liquid chromatography coupled to mass spectrometry, or tandem MS platforms that are expensive and often unavailable in academic or resource-limited laboratories. The new work, published in Discover Chemistry, deliberately takes the opposite approach: a conventional HPLC system with ultraviolet detection, a simple liquid-liquid extraction, and a workflow that a modest laboratory can reproduce without specialized instrumentation.

Getting the chemistry to cooperate required careful optimization. The team screened five mobile phase compositions and found that acetonitrile-water mixtures produced fronting peaks, while methanol-buffer systems at neutral pH caused tailing or splitting because the molecule’s carboxylic acid group, with a pKa of roughly 3.2, was partially ionized. The winning formulation, a 20 millimolar phosphate buffer at pH 3.0 mixed with acetonitrile and methanol in a 40:10:50 ratio, suppressed that ionization and delivered symmetric peaks: CFP-A eluting at 7.4 minutes with an asymmetry of 1.22 and over 8,000 theoretical plates, CFP-B at 8.3 minutes, and a resolution of 2.1 between the two epimers. Baseline separation of everything, including the internal standard, was achieved within fifteen minutes.

Sample preparation was optimized with equal rigor. Four extraction solvents were tested, and tert-butyl methyl ether emerged as the clear winner, recovering 62.24 percent of CFP-A and 58.25 percent of CFP-B from spiked plasma, consistent with the drug’s moderate lipophilicity. A 2³ full factorial design explored centrifugation time, plasma volume, and vortex time, and a descriptive Pareto analysis showed vortex time to be the dominant factor. Seven candidate internal standards were evaluated before eplerenone was selected; it extracted efficiently at 88.39 percent recovery, eluted cleanly at 10.8 minutes, and shared just enough structural similarity with the analyte to normalize sample-to-sample variability without risking metabolic cross-reactivity.

Validation followed the US Food and Drug Administration’s 2018 bioanalytical guidance, and the numbers are impressive for a UV-based method. The assay was linear from 200 to 6,400 nanograms per milliliter using weighted 1/X² regression, a model chosen after unweighted fitting showed the concentration-dependent heteroscedasticity typical of bioanalytical data, with correlation coefficients exceeding 0.999. Inter-day accuracy ranged from minus 4.05 to plus 3.15 percent relative error across quality control levels, and precision stayed below 1.8 percent relative standard deviation, far inside the ±15 percent regulatory window. Blank plasma from six independent donors showed interference of less than 6 percent of the lower limit of quantification response, and the analyte proved stable on the bench, through three freeze-thaw cycles, over thirty days at minus 20 degrees Celsius, and for four hours in the autosampler.

Beyond the assay itself, the researchers synthesized cefpodoxime acid by refluxing the prodrug with dilute sodium hydroxide, then confirmed the product’s identity through melting point, thin-layer chromatography, infrared spectroscopy, and proton NMR. The spectral evidence told a clean chemical story: the ester carbonyl stretch at 1762 wavenumbers that dominates the prodrug’s infrared spectrum vanished in the metabolite, replaced by a broad hydroxyl stretch and a carboxylic acid carbonyl, while the NMR spectrum lost the isopropyl signals of the proxetil group entirely. The recrystallized product reached 98.7 percent chromatographic purity, though the modest yield limited how much full co-validation of the metabolite as a quantified analyte the team could complete, a limitation they acknowledge openly.

The kinetic experiments delivered perhaps the most practically useful finding. In 0.1 normal sodium hydroxide at 37 degrees Celsius, cefpodoxime proxetil hydrolyzed with clean first-order kinetics, a rate constant of 0.049 per minute, and a half-life of just 14.0 minutes, with the metabolite peak accounting for 96.5 percent of theoretical yield by ninety minutes. The alkaline conditions were chosen as a forced stress, not to mimic physiology, but the message for any laboratory handling these samples is stark: under basic conditions the prodrug does not survive long. Maintaining appropriate pH during plasma collection and preparation is essential to prevent ex vivo conversion from silently corrupting pharmacokinetic data.

Biological and computational checks rounded out the picture. In broth microdilution assays, the synthesized metabolite inhibited Escherichia coli ATCC 25922 at the same minimum inhibitory concentration as the parent drug, 125 nanograms per milliliter, and came within a single two-fold dilution step against Bacillus subtilis ATCC 6633, a difference the authors correctly attribute to the inherent granularity of the method. TOPKAT in silico toxicity predictions, all performed within the software’s validated optimum prediction space, flagged neither compound as mutagenic, developmentally toxic, or carcinogenic, with only mild ocular irritancy predicted for both. The authors are careful to frame these as preliminary computational screens rather than substitutes for experimental toxicology.

What elevates the study above a routine method validation is its sustainability accounting. Using the AGREE metric, which scores adherence to the twelve principles of green analytical chemistry on a scale from 0 to 1, the method earned 0.65, reflecting a simple workflow held back mainly by the organic solvent consumed in extraction. The White Analytical Chemistry framework, which balances analytical performance, environmental impact, and practical efficiency, returned component scores of 75, 44, and 63 respectively, for an overall score of 61. No previously published cefpodoxime method has reported standardized greenness metrics, making this one of the first cefpodoxime bioanalytical platforms to be graded on environmental terms.

For pharmacokinetic, bioavailability, and bioequivalence studies, the practical implications are considerable. A validated, economical UV-based assay that resolves both epimers and can chromatographically monitor the active metabolite on the same platform gives generic manufacturers and academic laboratories a realistic alternative to mass spectrometry. The authors position the current work as a foundation: once larger quantities of characterized cefpodoxime acid become available, the same chromatographic system could be extended to a fully validated simultaneous parent-metabolite assay. In an era when antibiotic development desperately needs reliable bioanalytical tools, a method that is fast, cheap, regulatorily compliant, and honestly graded for its environmental cost is a quiet but genuine advance.

Subject of Research: Development and validation of an RP-HPLC bioanalytical method for quantifying cefpodoxime proxetil epimers and its active metabolite cefpodoxime acid in human plasma

Article Title: Development and validation of an RP-HPLC method for cefpodoxime proxetil epimers in human plasma with metabolite characterization, hydrolysis kinetics, and sustainability assessment

Article References: Chhajed, S. S., Chikhale, H. U., Sonawane, S. S., & Panja, A. (2026). Development and validation of an RP-HPLC method for cefpodoxime proxetil epimers in human plasma with metabolite characterization, hydrolysis kinetics, and sustainability assessment. Discover Chemistry, 3(1), Article 572. https://doi.org/10.1007/s44371-026-01013-9

Image Credits: AI Generated

DOI: 10.1007/s44371-026-01013-9

Keywords: cefpodoxime proxetil, cefpodoxime acid, RP-HPLC, bioanalytical validation, human plasma, hydrolysis kinetics, prodrug, cephalosporin antibiotics, green analytical chemistry, AGREE metric, White Analytical Chemistry, TOPKAT toxicity prediction

The clinical pairing is a familiar one in oncology wards. Cytarabine, chemically known as cytosine arabinoside or Ara-C, is a pyrimidine nucleoside analogue that leukemia cells take up and phosphorylate into an active triphosphate form. Once converted, it is incorporated into DNA and blocks DNA polymerase, halting the proliferation of the rapidly dividing cells that drive acute myeloid leukemia, the most common acute leukemia in adults. But cytarabine is also classified as an emetogenic agent, meaning it frequently triggers nausea and vomiting, particularly at high doses or in combination regimens. That is where granisetron comes in. As a selective antagonist of the 5-hydroxytryptamine type-3 receptor, it blocks serotonin signaling in both the gastrointestinal tract and the central nervous system, preventing and treating chemotherapy-induced nausea and vomiting in adult and pediatric patients alike.

Because the two drugs travel together in a patient’s bloodstream, measuring them simultaneously in a biological matrix is clinically valuable for therapeutic drug monitoring, pharmacokinetic studies, and bioanalytical applications. Yet, according to the research team led by Salwa I. Tohamy, Eglal A. Abdelaleem, Adel Lashien, Mahmoud M. Amin, and Nessreen S. Abdelhamid, no reverse-phase HPLC method had previously been reported for the simultaneous determination of cytarabine and granisetron in spiked human plasma. Existing options had drawbacks: many published HPLC methods handled only a single analyte or demanded complex sample preparation and long analysis times, while liquid chromatography-tandem mass spectrometry, though exquisitely sensitive, requires expensive instrumentation that is not routinely available in every laboratory.

The new method relies on a Thermo Scientific Dionex Ultimate 3000 HPLC system equipped with a reverse-phase C18 column measuring 25 centimeters long with a 4.6 millimeter internal diameter and 5 micrometer particles. Separation was achieved using gradient elution with a mobile phase composed of water containing 0.1 percent triethylamine, adjusted to pH 6 with orthophosphoric acid, mixed with methanol. Detection was performed at 290 nanometers using a diode array detector. The gradient program completes within 15 minutes, followed by a 3-minute re-equilibration step, giving a total run time of 18 minutes per sample. Getting there required considerable optimization. The team screened ethanol, acetonitrile, and phosphate buffer combinations, and tested isocratic methanol-buffer mixtures at several ratios, but none resolved the two drugs adequately. Gradient systems based on methanol and acetonitrile caused granisetron peak tailing and overlap with the internal standard. The breakthrough came with the addition of 0.1 percent triethylamine, which sharply reduced tailing and improved separation.

Sample preparation is deliberately simple. One milliliter of human plasma is mixed with caffeine, which serves as the internal standard, and diluted with HPLC-grade methanol. The mixture is vortexed for five minutes to precipitate proteins, centrifuged at 4000 rpm for ten minutes, and the clear supernatant is filtered through a 0.22 micrometer syringe filter before a 10 microliter aliquot is injected. Caffeine was chosen after the researchers tested paracetamol, ketoprofen, dapoxetine, and furosemide as alternatives; it offered the right retention time, sharp peak shape, and baseline separation from both drugs, and it is not endogenously present in plasma at interfering levels under the analytical conditions used.

Validation followed the U.S. Food and Drug Administration’s 2018 Bioanalytical Method Validation Guidance, the International Council for Harmonisation M10 guideline from 2022, and relevant principles of ICH Q2(R2). The method proved linear from 0.1 to 20 micrograms per milliliter for cytarabine and 0.3 to 20 micrograms per milliliter for granisetron, in both pure solutions and spiked plasma, with correlation coefficients of 0.9999 across all calibration curves. Accuracy and precision were assessed at five quality control levels, from the lower limit of quantification through the upper limit, and all results fell within the regulatory acceptance limits of plus or minus 15 percent for quality control samples and plus or minus 20 percent at the lower limit. Selectivity testing across six different blank plasma samples showed no interfering peaks at the retention times of either drug or the internal standard.

The researchers also probed how the plasma matrix itself might distort results. Matrix effect values ranged from 92.84 to 102.31 percent for cytarabine and 88.89 to 101.91 percent for granisetron, indicating no significant ion suppression or enhancement and a consistent analytical response across quality control levels. Stability studies confirmed that both drugs survive realistic laboratory handling: samples held at room temperature for six hours, kept in the autosampler for 24 hours, or subjected to three freeze-thaw cycles at minus 20 degrees Celsius all recovered within acceptable limits, with coefficients of variation below 2 percent. Robustness testing, which introduced small deliberate variations in methanol composition, flow rate, and detection wavelength, showed no meaningful impact on performance.

What distinguishes this study from many analytical papers is its self-critical environmental accounting. The team applied three complementary assessment tools. The AGREE metric, which evaluates the twelve principles of green analytical chemistry including energy consumption and waste generation, returned a score of 0.68, above the 0.6 threshold considered acceptable for green analytical methods. The Green Analytical Procedure Index, or GAPI, produced a pictogram with three red, nine yellow, and three green sections, with the predominance of yellow pointing to room for improvement in chemical use efficiency and waste generation. The multicolor assessment framework, which quantifies whiteness in the sense of white analytical chemistry, yielded an overall whiteness score of 68.7 percent, with the reliability component scoring highest at 82.5 percent and the blueness component at 77.5 percent.

Compared with recently reported chromatographic methods for these two drugs, the new approach consumes less solvent, requires less sample preparation, and runs faster, while extending applicability to biological matrices rather than being limited to pharmaceutical dosage forms. A statistical comparison using one-way ANOVA found no significant differences in accuracy or precision relative to the published methods, meaning the greener design did not come at the cost of analytical quality. The authors suggest the method is well suited for routine bioanalytical work and therapeutic drug monitoring in clinical settings, where laboratories may lack mass spectrometry infrastructure but still need reliable, sensitive measurements of these two co-administered drugs.

Beyond its immediate application, the study reflects a broader shift in analytical chemistry, where the environmental footprint of a method is now evaluated as rigorously as its sensitivity and precision. Solvent-heavy chromatography has long been an accepted cost of doing business in pharmaceutical analysis, but frameworks like AGREE, GAPI, and the multicolor assessment give laboratories a standardized vocabulary for demanding better. For leukemia patients, the practical payoff is a faster, cheaper, and cleaner way to ensure that the delicate balance between a life-saving chemotherapy drug and its anti-nausea companion is tracked accurately in the bloodstream, using equipment that most hospital and university laboratories already possess.

Subject of Research: Green gradient RP-HPLC determination of cytarabine and granisetron in spiked human plasma with greenness and whiteness assessment

Article Title: Green gradient RP HPLC for determination of cytarabine and granisetron in spiked human plasma with greenness and whiteness assessment

Article References: Tohamy, S. I., Abdelaleem, E. A., Lashien, A., Amin, M. M., & Abdelhamid, N. S. (2026). Green gradient RP HPLC for determination of cytarabine and granisetron in spiked human plasma with greenness and whiteness assessment. Discover Chemistry, 3(1), Article 559. https://doi.org/10.1007/s44371-026-00983-0

Image Credits: AI Generated

DOI: 10.1007/s44371-026-00983-0

Keywords: cytarabine, granisetron, RP-HPLC, human plasma, acute myeloid leukemia, chemotherapy-induced nausea and vomiting, green analytical chemistry, AGREE metric, GAPI, white analytical chemistry, therapeutic drug monitoring, bioanalytical method validation

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