The State Standard of Russian Federation Р 51577-2000 specifies only the total amount of fluoride ions in liquid oral hygiene products. We failed to find articles dedicated to the determination of free fluoride concentration in mouthwashes; however, several authorities have been focusing on the studies aimed at free fluoride analysis in toothpaste since 2005 [25-27]. The participants of European Organization for Caries Research Workshop, which was held to discuss the issues related to the methodology for determination of potentially available fluoride in toothpastes, have emphasized the necessity of developing methods measuring fluoride bioavailability . The development of techniques providing the assessment of free fluoride in mouthwashes is also required. Mouthwashes selected for fluoride measurements do not represent all the variety of brands commercially available in Russia, however even the small sample size indicates great discrepancies between the obtained data on fluoride content.
The experiment showed the results to be almost identical for the Colgate Total Plax (Gentle Mint) ® and Colgate Total Plax (Fresh Mint) ® solutions, with both methods.
Significant differences in concentrations determined by the method of standard addition and by the modified method of quantitative assessment were observed for such solutions as Oral B®, Reach®, and Foramen®. The fact that an increase in total fluoride content was observed with the modified method may be explained by a change in the ionic strength of the solution during the measurement; or by partial binding of fluoride ions by certain components of the mouthwashes; or possibly by polymeric compounds such as poloxamers and polyphenylene oxides. Analogous observations were made for the Sensodyne® solution, which does include polymeric compounds. This assumption is supported by the fact that in standard solutions prepared with Albavit solution containing polymeric compounds, the determined concentration was systematically higher than the theoretical concentration; whereas systematically lower results were obtained with model solutions in the procedure of fluoride ion determination using TISAB solution.
In those preparations lacking polymeric compounds, results of fluoride ion determination were identical at the constant ionic strength and modified method within the error of experiment. To confirm these assumptions, we determined concentration and activity of the test solutions following 10-fold dilution. The behavior of investigated values of activity and concentration also differs in both groups of preparations: while concentration for the preparations not containing polymeric compounds showed on average a 10-fold change following dilution, the 10-fold dilution of preparations containing polymeric compounds resulted in an 18-to-20-fold change of these values.
These trends are not observed for mouthwash containing AmF (Lacalut Sensitive®) as a source of active fluorine, even though it contains polymeric compounds (polyethylene glycol) in its formulation; and the mode of change of both activity and concentration following dilution corresponds to that of mouthwashes that do not contain polymeric compounds. It is noteworthy that after each measurement of AmF mouthwash the electrode continued retaining the potential when being immersed in distilled water and even required additional washing or mechanical cleaning of membrane surface. Therefore, one could presume that high free fluoride to total fluoride ratio (94%) could be due to adsorption of AmF on the surface of the electrode membrane.
The determinable concentrations are almost identical for the Colgate Total Plax® mouthwashes, which only vary by type of coloring agent. However, for the Colgate Total Plax (Classic Mint®) mouthwash, the decrease in the activity of fluoride ions can be explained by the difference in the type of colorant added at approximately the same concentration of total fluoride. Therefore, it is reasonable to assume that the coloring agent added to Colgate Total Plax (Classic Mint®) mouthwash could bind free fluorides.
The analysis of the results, obtained for different series of the same mouthwash, revealed differences in the content of active fluorine for the Oral B® rinses and almost identical values for the Reach Fresh mint® solutions. The comparative analysis of test solutions containing NaF or AmF as a source of fluoride ions showed that there is no significant difference between the measured values of active fluorine and those calculated theoretically, in both procedures. Thus, both organic and inorganic sources of fluoride have similar potential against tooth decay, which can be confirmed by clinical data .
A particularly low activity of fluoride ions in comparison with the declared one was observed for the Lacalut Fresh® solution, in which AlF3 served as the source of fluoride ions. The results of determination of fluoride concentration by standard method showed an abnormal increase in the activity of fluoride ions following the initial dilution with TISAB and the standard behavior following the secondary dilution. When a standard of NaF was added to the solution, a sharp reduction of the potential was observed instead of an increase. Thus, the standard procedure for this preparation turned out to be inapplicable due to a possible interaction between the components of buffer solution TISAB and the components of the test solution. Therefore, to determine the total concentration of fluoride ions, the modified method was selected. To understand the practically 700-fold fluoride ion activity decrease compared to declared values, additional studies were performed, in particular the pH determination of test solutions. The results showed that Lacalut Sensitive® and Lacalut Fresh® solutions have the lowest pH values (4,2 and 4,4, respectively). In the experiment, the pH values of these solutions were adjusted to a value of 7,00, with the electromotive force for the Lacalut Sensitive® solution changing insignificantly (within 2%), whereas for Lacalut Fresh® the change was over 16%. To elucidate the reasons for the observed behavior, we added the precise aliquot of NaF standard solution to the test sample. The addition of the known amount of NaF led to a 6,5-fold increase in the activity of fluoride ions, whereas their theoretically calculated activity should have increased 100-fold. This allows one to conclude that the obtained result is the consequence of fluoride ion binding by the solution components into a non-ionized species of the fluorine compounds (complexes or poorly dissociating dimers H2F2) . To evaluate the binding capacity of fluoride ions, the experiment was carried out according to the modified method by doubling the concentration of the standard. In this case, the known concentration of NaF was added to the Lacalut Fresh® solution containing the standard until the activity doubled. The result of the experiment showed that the addition of 75% of the required amount of NaF was followed by the doubling of fluoride ion activity, with the further addition of a minimal amount of fluoride ions, leading to a dramatic increase in the activity (300-fold!). This provides quantitative proof of depletion of fluoride ion binding capacity (i.e., the component binding fluoride ions has completely reacted). Interestingly, the pH value of the solution dramatically changed: from 4,2 to 6,5 (the pH value of the standard solution of NaF is 7,7). The results of the experiment may be attributable to possible generation of strong complexes due to fluoride ion binding by the components of the test solution, particularly by AlF3 salts (Kinst [AlF6]3- = 2,1⋅10-21) , rather than to formation of non-ionized fluorine compounds due to acidic pH value (e.g., the pH shift of the Lacalut Sensitive® solution resulted in insignificant changes). Thus, a study of fluoride ion activity in the Lacalut Fresh® solution suggests that fluoride ions will not be uptaken by tooth tissue and may even be washed away from it; thus, this supplement will have no effect when applied according to the directions for use of the preparation (5–7 drops/100 ml water).
The present study has some limitations since it does not reproduce in vivo conditions, i.e. dilution by saliva and influence of saliva components on the release of fluoride ions. It is known that phosphatase, one of saliva components, hydrolyzes Na2PO3F, releasing fluoride ions [25, 32]. Thus, it may be necessary to add components reproducing artificial saliva during sample preparation in order to avoid underestimated results when measuring Na2PO3F mouthwashes. The method of potentiometry itself does not account for the influence of mouthwash components on the work of the electrode.