Green Analytical Strategies Based on Natural Deep Eutectic Solvents for the Chemical and Biological Study of Camellia sinensis Leaves
Name: HELENA BEZERRA FERRARI
Publication date: 10/04/2026
Examining board:
Name |
Role |
|---|---|
| CLARICE DIAS BRITTO DO AMARAL | Examinador Externo |
| MARIA TEREZA WEITZEL DIAS CARNEIRO LIMA | Presidente |
| PAULO ROBERTO FILGUEIRAS | Examinador Interno |
Summary: In this study, an ultrasound-assisted extraction (UAE) method using a natural deep eutectic solvent (NADES) was developed for the determination of As, Cd, Cu, Fe, Mn, Pb, and Zn in Camellia sinensis leaf samples. Experimental conditions were optimized, and method settings were evaluated using NIST 1547 and NIST 1573a certified reference materials and Brachiaria brizantha cv. Marandú reference material. While the microwave assisted digestion (MW-AD) reference method uses oxidizing acids combined with hydrogen peroxide, the proposed approach uses solvents derived from molecules such as choline chloride (ChCl) and carboxylic acids (oxalic acid – Ox and malic acid – Ma), as well as xylitol (Xy) and water (Wa). Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES) and Inductively Coupled Plasma Mass Spectrometry (ICP-MS) were used for multi-element determination. The solvents were prepared using ultrasonic heating and made into three solvents: (1) ChCl:Ox:Wa, (2) ChCl:Ma:Wa, and (3) ChCl:Xy:Wa in a 1:1:3 ratio. Fourier Transform Infrared Spectroscopy with Attenuated Total Reflectance (ATR-FTIR) confirmed the formation of solvents through the presence of hydrogen bonds between the components. Furthermore, Thermogravimetric Analysis (TGA) also helped to demonstrate the breaking of these bonds in the mixture formed when subjected to a temperature variation. Parameters such as solvent choice and molar ratio were optimized using univariate methods. Ultrasound time and temperature, and solvent volume were trained in a multivariate optimization. All optimization steps were performed using the reference material Brachiaria brizantha cv. Marandu. The conditions that presented the best analytical response were: solvent ChCl:Ox:Wa, molar ratio 1:1:1, volume of 3.00 mL with ultrasound at 80 °C for 40 min, selected for final comparison with the MW-AD method. The agreement percentages obtained for the reference materials using the developed method ranged from 73% to 90% for Fe and Mn and 80% to 107% for As, Cd, Cu, Pb, and Zn, considered satisfactory compared to the limits established by the Association of Official Analytical Chemists (AOAC). The results of the standard decomposition method served as a reference for evaluating the studies with extraction and eutectic solvent. When comparing the calibration curves between the two methods, greater sensitivity was obtained for the NADES curves. This demonstrates that the method has a greater capacity to respond reliably and measurably to variations in analyte concentration. The limit of detection (LOD) and limit of quantification (LOQ) values for the developed method also showed positive results compared to the reference method, ranging from 0.006 to 2.91 mg kg¹ and 0.019 to 9.70 mg kg¹, respectively. Self organizing maps (SOMs) were applied as an exploratory multivariate tool, allowing the identification of multi-elemental patterns among tea types and highlighting the combined influence of environmental, botanical, and processing-related factors on elemental composition. Cell viability assays were applied to evaluate cytotoxicity in Camellia sinensis samples. The NADES-based method provided results comparable to the reference method and proved to be simple, efficient, safe, and low-cost, in accordance with the principles of Green Analytical Chemistry (GAC) (AGREE: 0.68; AGREEprep: 0.58) as a sustainable alternative for multi-element determination in complex food matrices.
