## 4 Electron Transfer-Based Assays
### Dpph
#### 4.3.2 Comparison of Methodological Approaches Used in Previous Studies of Plant Materials  
In the DPPH assay, several inconsistencies exist, ranging from the preparation of the stock DPPH solution to the calculation of measurement units. The concentrations of both the stock and working DPPH solutions vary across studies (Table 3). Such variations can compromise linearity, especially when the concentration of the dissolved substance is too high. Ideally, the concentration of the working solution should range between 50 and 100 uM to achieve an absorbance of 0.8 \(\pm\) 0.02 [71]. However, some studies either do not report the concentration of the stock DPPH solution or use concentrations significantly higher than 100 uM [75]. These discrepancies in DPPH concentrations make it difficult to compare results across different studies. The absorbance measured in this assay depends on the initial DPPH concentration, which directly affects the final results.  
The most significant inconsistency in the DPPH method concerns the way results are reported. Results are most often expressed as the percentage of neutralized radicals, i.e., the percentage activity (I %) of the tested antioxidant [68; 76; 77]. It is expressed by the formula  
\[\text{I }\%=(\text{A}_{0}-\text{A}_{\text{s}})/\text{A}_{0}\times 100\]  
\(\text{A}_{0}\)--absorbance of control  
\(\text{A}_{\text{s}}\)--absorbance of sample  
Figure 2: The reaction involved in the DPPH assay.  
A key disadvantage of this unit is that it does not account for the initial concentration of the DPPH solution, which directly affects the final absorbance measurement [71]. Moreover, the percentage activity provides no information about the actual amount or concentration of antioxidants in the test sample. Additionally, variations in the volumes of extracts used for analysis are often not accounted for when calculating results in this unit. Therefore, if the concentration of the test DPPH solution is unknown and the volume of extract varies, comparing results across studies may lead to erroneous conclusions [26; 71]. An alternative unit is milligrams of Trolox equivalents per 100 g of sample (mg TE/100 g) [69]. However, this approach also fails to consider the initial concentration of the DPPH solution during result conversion. Some studies have reported results using both units--percentage activity and mg TE/100 g--which allows for comparison of discrepancies between them [68; 69]. In addition, the results presented in these two units were not always directly proportional.  
Due to the instability of the DPPH radical, the test solution should be prepared freshly (ex tempero) and used as soon as possible. When not in use, it should be stored in the dark at refrigeration temperature. Using the same test solution is crucial to ensure consistent absorbance values in both the calibration curve and sample measurements. Only under such conditions can the results be reliably referenced to the standard curve [71].  
These discrepancies and limitations affect both the comparability and interpretation of DPPH assay results. Moreover, many researchers optimize various methodological factors, which further complicates comparison of the obtained results. Nonetheless, such discrepancies and inconsistencies are common to all spectrophotometric methods used to assess antioxidant potential.  
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