Wu Huaxin,Huang Ting,Zhang Wei,Yao Huan,Li Xueyao,Su Ping,Yang Yi
In the biomedical and chemical fields, purity assessment of compounds is a critical step in ensuring product quality and safety. In this study, a method for purity quantification was proposed as two-signal suppression–internal standard correction–high-performance liquid chromatography–quantitative nuclear magnetic resonance (TSS-ISC-HPLC-qNMR). The two-signal suppression effectively suppresses the interference of solvent signals in the NMR spectra, and the internal standard correction eliminates the influence of many variables during sample preparation and analysis. The purity result (99.89%) of the quantification of certified reference material of acesulfame (AF) by this method matched with the certified value (99.88%), which verified the accuracy of the method. The purity result (with standard deviation) of 94.42% ± 0.10% for the quantification of the low-purity drug digitoxin by the method matched with the purity result of 94.42% ± 0.11% by qNMR with deconvolution (as a validated method), which proved that the method could be used for the quantification of low-purity compounds. The methodology achieved a substantial reduction in total analysis time, from 28 to 4 h (considering only the routine analysis time after optimization), in contrast to the ISC-HPLC-qNMR approach. Additionally, it effectively decreased bias to undetectable with a standard deviation of 0.10%, while the bias of the TSS-HPLC-qNMR method was 0.93%. The present method ensures the accuracy of the quantitative results while demonstrating a low economic burden and significant time efficiency, which shows great potential for application in the accurate quantitative analysis of low-purity organic compounds. Graphical
Digitoxin is a cardiac glycoside used in the therapy of heart failure and arrhythmias, and has potential as an anti-cancer and anti-influenza treatment. However, digitoxin has the disadvantage of a narrow therapeutic window meaning that it is easy to cause toxicity. Consequently, there is an urgent need to accurately monitor the plasma concentration of patients. In this study, an anti-digitoxin monoclonal antibody (mAb) 5D2 was developed and purified, and found to have outstanding specificity, sensitivity, and affinity. The half inhibitory concentrations (IC 50 ) and the limit of detection (LOD) values of mAb 5D2 in human plasma determined by ic-ELISA were 0.205 ng/mL and 0.010 ng/mL, while the cross-reactivity with other analogs was negligible. Based on mAb 5D2, a colloidal gold-based immunochromatographic assay was developed for the purpose of quickly monitoring the concentration of digitoxin in human plasma. The detection results could be obtained within 10 min, and were in agreement with those of liquid chromatography-tandem mass spectrometry (LC-MS/MS). The strip could be evaluated semi-quantitatively by the naked eye, with a visual LOD (vLOD) of 0.5 ng/mL and a cut-off value of 10 ng/mL. Meanwhile, the linear range was between 0.319 ng/mL and 2.406 ng/mL, which was obtained quantitatively by the strip reader. Therefore, the immunochromatographic strip could offer a new tool for rapid, accurate, and portable detection of digitoxin.
Background and Purpose Cardiac glycosides (CGs), traditionally prescribed for heart failure and arrhythmias, show anticancer potential. However, their mechanisms for preferential inhibition of tumour tissue and constituent malignant cells are not fully elucidated. This study aims to elucidate the therapeutic benefits of CGs in targeting specific tumours and dissect their multi-targeting mechanisms that confer their cytotoxicity against malignant cells. Experimental Approach We designed an integrated workflow to identify therapeutic CGs with high toxicity to certain cancers, investigating their multi-target effects, assessing their toxicity to malignant cells and analysing the prognostic relevance of CGs' target genes. The computational findings were confirmed through gene knockdown, cell viability assays, reactive oxygen species (ROS) measurements and so forth. Key Results CGs modulate multiple genes crucial for ion homeostasis, oxidative stress and apoptosis, with a particularly strong inhibitory effects on uveal melanoma (UVM). Notably, digitoxin suppresses UVM cell proliferation and induces ROS levels by simultaneously targeting STAT3 and KLF5. Single-cell transcriptomic analysis revealed that malignant cells are likely more vulnerable to CGs due to their higher expression of CG target genes compared with surrounding cells in the UVM microenvironment. Conclusions and Implications Given UVM's limited options, our study highlights the potential of digitoxin as a promising novel therapeutic agent for this aggressive and rare ocular cancer. Our comprehensive approach is effective in identifying the potent, cancer-specific therapeutic agents from herbal plants.
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