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