## 5 Materials and Methods  
In vitro studies were conducted on 45 human teeth (premolars and molars) that were free from caries, enamel defects and demineralization, removed for orthodontic reasons after obtaining the prior consent of the patients. Each tooth was cut along the long axis with the buccal and palatal surface intact. Until the start of the study, the specimens were stored in thymol solution. Each specimen, after washing and drying, was coated with acid-resistant varnish while leaving exposed enamel (4 mm/4 mm) on the smooth buccal and palatal surface (Figure 4a,b). Three fluoride varnishes were used for the purposes of the study: one first-generation varnish (Duraphat) and two second-generation varnishes (MI Varnish and Enamel Varnish). Table 2 provides information on the type and composition of the applied agents. The test material was divided into nine groups of 10 specimens each, depending on the type of varnish applied and the pH of the environment (Table 3). The prepared specimens were weighed before and after the application in order to accurately determine the amount of the preparation applied, using a Radwag laboratory balances with a readability of 3 decimal places. The specimens were then placed in sealed plastic containers and immersed in 5 mL of artificial saliva at pH = 4.0 for groups DV/A, MV/A and EV/A; pH = 5.0 for groups DV/B, MV/B and EV/B; and pH = 7.0 for groups DV/C, MV/C and EV/C. The composition of artificial saliva was as follows: NaCl (0.4 g), KCl (4.0 g), urea (1.0 g), Na\({}_{2}\)S-9H\({}_{2}\)O (0.005 g), NaH\({}_{2}\)PO\({}_{4}\):2H\({}_{2}\)O and CaCl\({}_{2}\):2H\({}_{2}\)O (0.795 g). pH = 7.0 was achieved with 1 M NaOH, whereas pH = 4.0 and 5.0 were achieved with the use of 1 M HCL. During the measurements, the specimens were stored at 37 \({}^{\circ}\)C. The \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Fluoride Varnish** & **Code** & **Manufactures** & **Ingredients** \\ \hline Colgate® Duraphat & DV & Colgate Oral Care, Sydney, NSW, Australia & 30–60\% colophonium, 10–30\% ethanol, 5\% sodium fluoride, other ingredients \\ \hline MI VarnishTM & MV & GC, Tokyo, Japan & 30–50\% polyvinylacetate, 10–30\% hydrogenated rosin, 20–30\% ethanol, 5\% sodium fluoride, 1–5\% CPP-ACP, 1–5\% silicon dioxide \\ \hline Embrace VarnishTM & EV & Pulpdetent, Watertown, MA, USA & 5\% nitrium fluoride with CXP (xylitol coated calcium and phosphate) \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Type and composition of studied varnishes. Figure 4: (a) Varnish–coated enamel specimen. (b) Diagram of prepared sample. \begin{table}
\begin{tabular}{c c c} \hline \hline
**Fluoride Varnish** & **Group** & **pH of Artificial Saliva** \\ \hline \multirow{3}{*}{Duraphat} & DV/A & 4.0 \\  & DV/B & 5.0 \\  & DV/C & 7.0 \\ \hline \multirow{3}{*}{MI Varnish} & MV/A & 4.0 \\  & MV/B & 5.0 \\  & MV/C & 7.0 \\ \hline \multirow{3}{*}{Embrace Varnish} & EV/B & 4.0 \\  & EV/B & 5.0 \\ \cline{1-1}  & EV/C & 7.0 \\ \hline \hline \end{tabular}
\end{table} 
Table 3: Experimental groups. determination of fluoride levels in the artificial saliva environment was performed three times each after 1, 2, 24, 48 and 168 h, and followed by calculation of the average value. In any case, after the measurement period, 5 mL of eluate was used for the determination; then the sample was dried and placed into a fresh artificial saliva solution. The ORION 9609 Model ion selective electrode combined with the CPI-551 Elmetron (Thermo Fisher Scientific Co., Waltham, MA, USA) microcomputer were used for the determination of fluoride ions. The system was calibrated before each subsequent determination using TISAB. Measurements of the released fluoride ion concentration were expressed in ppm, later converted into \(\upmu\)mol/L and in relation to the quantity of applied varnish converted into \(\upmu\)g/mg.