Sustainable high-throughput microwell spectrophotometric methods for avapritinib quality control via charge-transfer complexation

www.nature.com/scientificreports OPEN Sustainable high-throughput microwell spectrophotometric methods for avapritinib quality control via charge-transfer complexation Awadh M. Ali1,5, Mohammed S. Alsalhi1,5, Weam M. Othman2, Antonio Frontera3, Waleed Alahmad4 & Ibrahim A. Darwish1 Avapritinib (AVA) is a breakthrough targeted therapy as the first potent inhibitor approved for gastrointestinal stromal tumors (GIST) with PDGFRA D842V mutations. It lacks convenient analytical methods for routine quality control. This study addresses this gap by developing and validating two novel, green, high-throughput microwell spectrophotometric methods (MW-SPMs) for AVA quantification in pharmaceutical tablets—representing the first employment of charge-transfer complexation for AVA analysis. The methods exploit rapid charge-transfer complex (CTC) formation between AVA (electron donor) and two π-acceptors: 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) and chloranilic acid (CLA). The reactions, conducted in 96-well plates using only 200 µL total volume, yielded stable colored complexes with absorption maxima at 460 nm and 520 nm for the reactions with DDQ and CLA, respectively. Following ICH-compliant optimization and validation, both methods demonstrate excellent linearity (3.13–100 µg/well for DDQ; 6.25–100 µg/well for CLA), precision (RSD ≤ 1.8%), and accuracy (recoveries: 98.0–101.7%). Job’s method confirmed 1:1 stoichiometry, while density functional theory (DFT) uniquely revealed the molecular basis for the stronger binding affinity of DDQ over CLA through dominant π–π stacking and hydrogen-bonding interactions, demonstrating how computational analysis can guide reagent selection in analytical method development. A comprehensive multi-metric sustainability assessment—utilizing ten tools including AES, AGREE, BAGI, RGB, and RAPI—confirmed the methods’ excellent greenness (e.g., AES score = 90), superior practicality, and robust analytical performance, achieving a high White Index of 94.2%. With a substantially higher throughput of ~ 500 samples/hour and minimal solvent consumption (200µL/well), the proposed MW-SPMs offers a rapid, sustainable, and cost-effective alternative to conventional chromatographic and spectrofluorimetric techniques for routine pharmaceutical analysis, aligning with multiple United Nations Sustainable Development Goals. Keywords Avapritinib, Charge-transfer complex, Microwell spectrophotometry, Green analytical chemistry, High-throughput analysis, Sustainability assessment, White analytical chemistry Avapritinib (AVA) is a novel, potent, and highly selective type I tyrosine kinase inhibitor that targets mutant PDGFRA and KIT, particularly PDGFRA D842V and KIT D816V mutants that drive gastrointestinal stromal tumors (GIST) and systemic mastocytosis1,2. The chemical structure of AVA is given in Fig. 1, and it is chemically named as: (1S)-1-(4-fluorophenyl)-1-(2-{4-[6-(1-methyl-1H-pyrazol-4-yl)pyrrolo[1-f]triazin-4-yl]piperazin1-yl}pyrimidin-5-yl)ethanamine. It is approved for the treatment of adults with unresectable or metastatic GIST harboring PDGFRA exon 18 mutations and for advanced forms of aggressive systemic mastocytosis, 1Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O. Box 2457, 11451 Riyadh, Saudi Arabia. 2Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy, Misr University for Science and Technology, 6Th October City, Egypt. 3Department de Química, Universitat de Les Illes Balears, Crta. de Valldemossa Km 7.5, 07122 Palma, Spain. 4Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand. 5Awadh M. Ali and Mohammed S. Alsalhi contributed equally to this work. email: ; Scientific Reports | (2026) 16:15874 | https://doi.org/10.1038/s41598-026-51872-6 1 Fig. 1. The chemical structures of Avapritinib (AVA), 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ) and 2,5-chloro-3,6-dihydroxy-1,4-benzoquinone (chloranilic acid: CLA). representing a paradigm shift and reflecting a pivotal role in the management of these otherwise refractory malignancies1,2. AVA possesses high lipophilicity (log P ~ 3.5) and poor aqueous solubility, characteristics that necessitate reliable analytical methods for quality control of its solid dosage forms1,2. AVA is marketed under the trade name of AYVAKIT™ tablets for oral use, manufactured by Blueprint Medicines Corporation (Cambridge, MA, USA)3. Despite its clinical success, AVA’s efficacy is hindered by a narrow therapeutic index, necessitating careful dose management to balance its potent anticancer effects against the risk of significant adverse effects3. The narrow therapeutic index and chronic therapy of AVA necessitate rigorous, reliable, and sustainable quality control (QC) methods for its dosage forms. Existing analytical methods for AVA include chromatographic techniques such as HPLC-UV4–10and UPLC-MS/MS11–13. Although these methods offer high sensitivity and selectivity, they require sophisticated instrumentation, long analysis times, and multistep sample preparation, which are laborious for routine QC laboratories. Spectrofluorimetric methods using derivatization14–16 or micelle enhancement17have also been reported, but they involve time-consuming, temperature-controlled reaction steps and introduce variables that limit robustness and reproducibility. These methods consume considerable volumes of organic solvents and generate significant chemical waste, conflicting with Green Analytical Chemistry (GAC) principles18,19. While acetonitrile is not a green solvent by strict standards, the miniaturized method (200 µL /well) drastically reduces solvent consumption and waste, which is the primary contributor to its overall greenness. Several charge-transfer based spectrophotometric methods have been reported for related pharmaceutical compounds, demonstrating the utility of π-acceptor reagents for analytical applications20–22. Therefore, a simpler, faster, and inherently parallel alternative is needed. Driven by GAC concepts, recent trends prioritize methods that minimize solvent consumption, reduce hazardous waste, and lower energy requirements while maintaining analytical performance suitable for QC21,22. Additionally, high-throughput strategies that allow the simultaneous or parallel processing of large numbers of samples are increasingly adopted to improve productivity and reduce per-sample cost in pharmaceutical QC laboratories23,24. Microwell-based methods using 96-well plates have emerged as attractive platforms that naturally integrate green and high-throughput features25,26and have been successfully applied for drug quantification in dosage forms27–29. Microwell spectrophotometric methods (MW-SPMs) employ microplates and absorbance readers to measure multiple samples in parallel using micro-volumes of sample solutions and reagents, leading to reduced solvent consumption and minimal waste generation. The batch-w (...truncated)