## 3 Results and Discussion
### CILTAG and Hydroalcoholic Extract Analysis
#### 3.2.3 The Quantitative Determination of Ascorbic Acid  
In order to ensure the suitability of the method for our determinations, we analyzed the accuracy, precision (repeatability, reproducibility), linearity, and range of the method and the relative specificity of the complex matrix in which ascorbic acid was found. Validation results are presented in Table S2 in the Supplementary Materials File.  
The accuracy and repeatability were verified by injecting one to six standard solutions in triplicate on the same day, and the reproducibility was verified by repeating the determinations on three consecutive days. The specificity was determined using the chromatogram obtained for the analysis of an artificially prepared matrix from standards of ODP-type flavones and polyphenic derivatives (which accompany the ascorbic acid in vegetable-based hydroalcoholic extractive solutions). The linearity, range of concentrations, and stability were determined after statistical processing by the regression of the experimental data obtained from analyzing three sets of standard solutions prepared as described above.  
The calculation of the ascorbic acid concentration was done by the external calibration method using the regression curve obtained when determining the linearity. A total of 20 uL of the diluted sample was injected in duplicate.  
The ascorbic acid in the sample was identified based on the retention time and the UV absorption spectrum of the eluate (ascorbic acid in aqueous solution absorbs UV light at 243 nm). The obtained peaks were pure. The average content of ascorbic acid obtained during storage, from 10 analyses, was 19.66 \(\pm\) 0.85 mg mL\({}^{-1}\).  
The low stability of ascorbic acid in solution is well known and was the reason why the ascorbic acid determination was performed 60 days after the solutions' preparation.  
The prepared extractive solutions were kept for 2 months in parallel at room temperature and in a refrigerator (4-8 \({}^{\circ}\)C). The ascorbic acid content was determined every two weeks by the HPLC method.  
During this time, the ascorbic acid content of the product kept at room temperature decreased by 20%. The obtained average concentrations of ascorbic acid after 60 days were:  
\begin{table}
\begin{tabular}{c c c c} \hline
**Extractive Solutions** & **Identified Compounds** & **Retention Time (t\({}_{\text{R}}\))** & **Maximum Absorbance Wavelength \(\lambda_{\text{max}}\) (nm)** \\ \hline _Medicaginis herba_ & Rosmarinic acid & 30544 & 266, 334 \\  & Rutoside & 19053 & 257, 293, 344 \\ _Trifolini pratense folium_ & Caffeic acid & 10771 & 280 \\  & Rosmarinic acid & 30251 & 254, 350 \\  & Kaempfer & 47787 & 259 \\  & Ferulic acid & 18093 & 216, 234 \\ _Ginkgo bilobae folium_ & Rosmarinic acid & 30877 & 312 \\  & Quercetol & 38845 & 358 \\  & Kaempfer & 48547 & 358 \\ _Myrtilli fruct_s & Chlorogenic acid & 8045 & 218, 242, 325 \\ \hline \end{tabular}
\end{table}
Table 5: Bioactive compounds separated from and identified in the _Medicaginis herba, Trifolini pratense flores, Ginkgo bilobae folium,_ and _Myrtilli fruct_s hydroalcoholic extractive solutions.  
(i) for room temperature, 15.64 \(\pm\) 1.32 mg mL\({}^{-1}\) (79.55%); and (ii) for refrigerated storage, 20.78 \(\pm\) 0.82 mg mL\({}^{-1}\) (105.69%).  
The HPLC method was selected and adapted according to the data in the literature, and the characteristics of the analyzed extractive solutions were suitable for the determinations as can be observed from the validation data.  
By dosing the ascorbic acid by the HPLC method, it was found that the antioxidant preparation \"CILTAG\" contains 20 mg/mL ascorbic acid.