Green TLC Method Simultaneously Tracks Two Arthritis Drugs in Plasma

Rheumatoid arthritis patients routinely take a combination of two very different drugs: methotrexate, the backbone disease-modifying therapy that suppresses the misdirected immune attack on the joints, and etoricoxib, a selective COX-2 inhibitor that dulls the pain and inflammation the disease leaves behind. The pairing works clinically, but it carries a hidden analytical burden. Etoricoxib can slow the kidneys’ clearance of methotrexate, allowing the drug to accumulate in the bloodstream and raising the risk of dose-dependent toxicity. Monitoring both compounds in a single blood sample has therefore been a genuine unmet need, because until now no published method could measure the two drugs together in one analytical run.

A team of pharmaceutical analytical chemists at Beni-Suef University in Egypt has filled that gap with a surprisingly low-tech solution: thin-layer chromatography, the workhorse technique many laboratories associate with undergraduate teaching labs rather than cutting-edge bioanalysis. Writing in the journal Discover Chemistry, Salwa Tohamy, Eglal Abdelaleem, Shaimaa Mohamed and Nessreen Abdelhamid describe a validated TLC-densitometric method that quantifies methotrexate and etoricoxib simultaneously in spiked human plasma, using the antihistamine cyproheptadine as an internal standard to correct for variations in sample handling. The work is notable not only for its analytical performance but also for the unusual rigor with which its environmental credentials were audited.

The chemistry behind the separation is elegant in its simplicity. After precipitating plasma proteins with methanol and centrifuging the mixture, the researchers spotted ten-microliter aliquots onto silica gel plates using an automated CAMAG Linomat 5 applicator. The development chamber was saturated for fifteen minutes with a mobile phase of methanol and isopropanol in a 9.5 to 0.5 ratio, a deliberately benign solvent pair chosen to avoid the toxic acetonitrile and corrosive ammonia that conventional methods often demand. Ultraviolet densitometry at 285 nanometers then read off the separated bands, which migrated to cleanly resolved positions: plasma components stayed near the baseline at a retardation factor of 0.04, cyproheptadine traveled to 0.36, methotrexate to 0.73, and etoricoxib to 0.83.

Getting there required systematic optimization. Early solvent trials, including methanol-ethyl acetate, ethanol-ethyl acetate and acetone-ethyl acetate mixtures, failed to move the highly polar methotrexate off the baseline at all, underscoring how much elution strength methanol contributes. A propanol-methanol blend also left the drug stranded, while a methanol-isopropanol mixture at 7 to 3 produced severe peak tailing. Acidic modifiers such as acetic and formic acid did nothing to fix the tailing; ammonia solution did, but only at volumes the team considered too corrosive for a green method. Pushing the methanol fraction higher solved the problem without additives: the final 9.5 to 0.5 composition eliminated tailing, produced sharp symmetrical peaks and achieved baseline resolution of every component in the mixture.

The method was validated against the principal bioanalytical criteria set by the US Food and Drug Administration, the same standards that govern the assays used in pharmacokinetic studies submitted for drug approval. Calibration curves were linear from 0.2 to 0.9 micrograms per band for methotrexate and from 0.5 to 1.1 micrograms per band for etoricoxib. Quality-control samples prepared at four concentration levels, spanning the lower limit of quantification through low, mid and high quality controls, met the acceptance limits for both intra-day and inter-day accuracy and precision. The drugs also proved stable through repeated freeze-thaw cycles and bench-top handling, and extraction recoveries were satisfactory. System suitability testing confirmed adequate retention, peak symmetry and resolution across every run.

What distinguishes the study from a routine validation paper is its sustainability audit, which the authors conducted with six complementary assessment tools rather than the single greenness metric that is common in the field. The modified Green Analytical Procedure Index, the Analytical Green Star Area, the Environmental Performance and Practicality Index, the Carbon Footprint Reduction Index, the Blue Applicability Grade Index and the Sustainability of Analytical Methods Index each probe a different dimension of a method’s footprint, from solvent toxicity and waste volume to energy demand, carbon emissions and alignment with the United Nations Sustainable Development Goals. Under the EPPI framework, the method scored 76.8 overall, with a practicality score of 75 and an environmental impact score of 78.7. The CaFRI tool returned a score of 72, reflecting minimal waste generation of less than one milliliter per sample and low energy consumption, since TLC requires no pumps to drive the mobile phase.

The SAMI evaluation, the first framework built directly on the seventeen Sustainable Development Goals, awarded the method its highest impact score for contributions to quality education, gender equality, affordable and clean energy, responsible consumption and production, and global partnerships. The method scored positively on six further goals, including good health and well-being, industry innovation, sustainable cities, climate action and the protection of aquatic and terrestrial ecosystems, largely because it replaces chloroform and acetonitrile with biodegradable methanol and isopropanol and consumes a fraction of the energy that high-pressure liquid chromatography demands. Compared directly with reported HPLC, UPLC and LC-MS/MS methods for these drugs, the TLC approach scored best on every sustainability index, while the mass spectrometry methods performed worst, burdened by pumps, vacuum systems, gas generators and heavy instrumentation.

The researchers also used a web-based prediction platform to ask whether the drug combination itself poses a metabolic risk. Roughly eighty percent of drugs that undergo oxidative metabolism are processed by the cytochrome P450 enzyme family, and interactions at these enzymes can push co-administered drugs to unsafe levels or starve them below therapeutic thresholds. Using the Way2Drug platform and the Invariant Accuracy of Prediction approach, the team calculated negative IAP values for all major CYP enzymes evaluated, with the probability of active interaction lower than that of passive interaction for each one. The ORCA severity rating system classified the methotrexate-etoricoxib combination as class 4 negative, supporting a prediction of low CYP-mediated interaction risk. The authors are careful to note the limits of that in-silico result, however: renal clearance and transporter-mediated interactions fall outside the model’s scope and still require experimental and clinical study, which is precisely where the new analytical method is expected to help.

The practical upshot is a method that trades some sensitivity for accessibility. The authors acknowledge that HPLC and mass spectrometry remain more sensitive, and that incurred sample reanalysis would be needed before the TLC assay could support formal therapeutic drug monitoring. But for quality control laboratories, particularly in resource-limited settings, the advantages are compelling: twenty samples can be processed in parallel on a single plate, solvent consumption stays below five milliliters per test, no high-purity reagents or high-pressure instrumentation are required, and maintenance and training demands are minimal. In an era when analytical chemistry is being asked to account for its own environmental bill, the study makes a case that the humble TLC plate, refined with modern validation standards and audited with modern sustainability metrics, still has a serious role to play in pharmaceutical science.

Subject of Research: Development and sustainability evaluation of a TLC-densitometric method for simultaneous determination of methotrexate and etoricoxib in spiked human plasma

Article Title: A novel TLC method for the concurrent bioanalysis of methotrexate and etoricoxib in spiked human plasma with comprehensive sustainability evaluation

Article References: Tohamy, S. I., Abdelaleem, E. A., Mohamed, S. M., & Abdelhamid, N. S. (2026). A novel TLC method for the concurrent bioanalysis of methotrexate and etoricoxib in spiked human plasma with comprehensive sustainability evaluation. Discover Chemistry, 3(1), Article 442. https://doi.org/10.1007/s44371-026-00891-3

Image Credits: AI Generated

DOI: 10.1007/s44371-026-00891-3

Keywords: thin-layer chromatography, methotrexate, etoricoxib, rheumatoid arthritis, spiked human plasma, bioanalytical validation, green analytical chemistry, sustainability metrics, drug-drug interaction prediction, densitometry, cyproheptadine internal standard, pharmaceutical quality control