# Modern Techniques for Flavonoid Extraction--To Optimize or Not to Optimize?
## 3 Sample Preparation and Hydrolysis  
Generally, flavonoids are isolated from plant tissues, from the food matrix, or from liquid samples (biological fluids or beverages). For some liquid samples, it is sufficient to centrifuge and filter them before analysis, but for some matrices the whole process is much more complex. Flavonoids can be isolated from fresh plant material, but in this case  
\begin{table}
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the samples should be kept at low temperature and the extraction should start soon after harvest to prevent enzymatic or chemical degradation and to modify the particles of the plant material to contribute to the release of bioactive compounds loosely bound to the cell wall polymers [41]. Many different methods can be employed in sample preparation (Figure 2) but nowadays, plant material is most commonly dried by freeze-drying, convection drying, or microwave vacuum drying (Table 3). As is evident from Table 3, most studies reported that freeze-drying is the preferred method for pretreatment of samples to obtain most of the flavonoids. \begin{table}
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**Sources** & **Drying Methods** & **Results** \\ \hline green tea (_Camellia sinensis_) & sun, shade, oven 60 \({}^{\circ}\)C, 80 \({}^{\circ}\)C & the highest total flavonoid \\ leaves [42] & and 100 \({}^{\circ}\)C, microwave and freeze-drying & content was obtained in oven \\ \hline  & freeze-drying and & freeze-drying provides \\  & air-drying [43] & extracts with higher amounts \\  & & of flavonoids \\ \cline{2-3}  & freeze-drying and & freeze-drying is more suitable \\  & air-drying [44] & flavoves; while air-drying was \\ \hline  & shade one day then & the highest flavonoids yield \\ _Dryopteris erythrosarcoma_ & oven-dried at 75 \({}^{\circ}\)C for 48 h, & was from first drying the plant \\ leaves [45] & dried in the sun, then & material in the shade and then \\  & oven-dried at 75 \({}^{\circ}\)C for 48 h, & completing the drying process \\  & oven-dried at 75 \({}^{\circ}\)C for 48 h & in an oven at 75 \({}^{\circ}\)C \\ \hline  & air-drying at ambient & \\  & temperature, oven-drying at & \\  & 40 \({}^{\circ}\)C and 80 \({}^{\circ}\)C, & freeze-drying–prefreezing \\  & with liquid N\({}_{2}\), & freeze-drying of \\ birth leaves [46] & freeze-drying–prefreezing at & leaves frozen at \(-18 \,{}^{\circ}\)C is \\  & \(-18 \,{}^{\circ}\)C, freeze-drying without & preferred as a drying method \\  & prefreezing, storing frozen for & \\  & 12 days without drying, and & immediate extraction of \\  & fresh samples & \\ \hline \hline \end{tabular}
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Table 3: Literature reports on the influence of drying methods on flavonoid content. Figure 2: Main steps in sample preparation before extraction. After drying, the samples are usually ground to a fine powder. This can be done manually with a mortar and pestle or by grinding over a long period of time. For example, [PERSON] et al. [54] studied the effect of particle size on the extraction of flavonoids from grape by-products and reported that the highest amount was obtained with a grinding time of 6.8 min and a solid-liquid ratio of 3 mL/g. The solid-liquid ratio is also an important parameter and depends on the plant tissue and the content of specific flavonoids we want to detect. Sometimes, the use of an excessive amount of the samples can lead to the extraction of other compounds present in the samples, which can lead to a \"matrix effect\" during the analysis, caused by the change in ionization efficiency of the target analyses in the presence of co-eluting compounds in the same matrix. In this case, the flavonoids of interest are difficult to detect. On the other hand, the flavonoids of interest are below the detection limit if we use too small a sample amount. Therefore, this parameter also needs to be optimized.