Evaluation of free and total fluoride concentration in mouthwashes via measurement with ion-selective electrode

doi:10.21203/rs.2.11615/v1

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Evaluation of free and total fluoride concentration in mouthwashes via measurement with ion-selective electrode

https://doi.org/10.21203/rs.2.11615/v1

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Background

Mouthwashes are among the currently used oral hygiene products aimed at preventing and reducing dental caries. This is accomplished by a remineralization process, due to the presence of fluoride ions; however, mouthwashes should also contain an appropriate amount of unbound or free fluorides to provide bioavailability. The aim of this study was to compare active fluoride concentration and total fluoride concentration in mouthwashes.

Methods

Fluorine-containing mouthwashes from various companies and manufacturers (Colgate Total Plax Classic Mint®, Colgate Total Plax Gentle Mint®, Colgate Total Plax Fresh Mint®, Oral B Advantage®, Reach Fresh Mint®, Foramen®, Lacalut Sensitive®, Sensodyne®, Vesna F®, and Lacalut Fresh®) were selected as study objects. Fluoride measurements were carried out using the method of potentiometry.

Results

Free fluoride:total fluoride ratio was more than 80% for six samples and less than 63% for three samples. Two samples had more than 70% and less than 80% of unbound fluoride, respectively. Rinse containing sodium monofluorophosphate (Na2PO3F) had more than 50% of free fluoride, while the rinses containing amine fluoride (AmF) had 94%. The difference in activity can be explained by binding of fluoride ions by components contained in mouthwashes, such as coloring agents, polymeric compounds, etc. The lowest activity was observed for aluminum fluoride (AlF3) rinse, due to possible generation of strong fluoride complexes. This implies that fluoride ions will not be uptaken by tooth tissue and may even be washed away from it, compromising the efficacy of mouthwashes.

Conclusions

There is an urgent need to develop a method for evaluation of free fluorides in mouthwashes for proper updating of national and international guidelines.

Dentistry

fluorides

mouthwashes

potentiometry

ion-selective electrode

According to the World Health Organization, dental caries is a disease prevalent among 60%–90% of school-age children and almost 100% of adults worldwide [1]. There are numerous reports regarding the influence of comprehensive prevention programs on the prevalence of caries, including topical fluoridation provided by the application of fluoride-containing toothpastes, mouthwashes, gels, foams, and varnishes [2-8]. In addition to the fluorides, other inorganic elements are essential to mineralize hard tooth tissues and increase their resistance to caries. Toothpastes and mouthwashes containing sodium phosphate, calcium glycerophosphate, and zinc oxide exhibit a pronounced anticaries action [9-11]. Mouthwashes are additional products for oral hygiene. Most of the currently available mouthwashes can be divided into four groups, depending on their therapeutic action: (1) mouthwashes reducing oral malodor; (2) mouthwashes decreasing dental plaque formation due to antibacterial action; (3) mouthwashes containing various concentrations of fluorine compounds and able to affect the mineralization of dental hard tissue; and (4) mouthwashes preventing or reducing gingivitis [12-17].Various fluorine and phosphorus compounds are included as active components in the mouthwashes of the second and the third groups. Basic organic and inorganic fluorine compounds, contained in products for prophylaxis of oral cavity diseases, are sodium fluoride (NaF), sodium monofluorophosphate (Na₂PO₃F), amine fluoride (AmF), aluminum fluoride (AlF₃), and stannous fluoride (SnF₂) [18,19]. The active mineral supplements could be phosphate salts, for instance, nano-sized sodium hexametaphosphate and sodium trimetaphosphate, which are capable of changing the solubility of enamel after adsorption on its surface [20,21].Mouthwashes containing fluorine compounds are divided into groups, depending on fluoride ion concentration: 0,05% sodium fluoride solutions can be used daily; and 0,2% sodium fluoride solutions are recommended for weekly or fortnightly application [22]. Rošin-Grget et al. reviewed various theories of the cariostatic action of fluorine. According to one of these theories, fluoride ions penetrate into the lattice of hydroxyapatite, Ca₁₀(PO₄)₆(OH)₂, resulting in the formation of fluorohydroxyapatite, Ca₁₀(PO₄)₆(OH)F, which is more resistant to acids [23].According to another theory, during the exposure of the tooth surface to NaF, the reaction with hydroxyapatite results in the formation of poorly soluble crystals of calcium fluoride [23]:Ca₁₀(PO₄)₆(OH)₂+ 20NaF 10CaF₂ + 6Na₃PO₄ + 2NaOH

It should be noted that the equilibrium shift in both chemical processes is determined by the activity of fluoride ions in the oral fluid, which in turn depends on the concentration of free fluoride ions in the applied prophylactic and therapeutic preparations. Various ingredients of mouthwashes, such as colourants, flavouring agents, sweeteners, preservatives, surfactants, etc., can chemically bind fluoride ions. Such bound fluoride is not effective in preventing and reducing dental caries. Thus, the control and the determination of «active» fluoride can determine the quality and, hence, the therapeutic efficacy of mouthwashes.

Fluorine-containing mouthwashes currently used in dental practice can be divided into three types: (1) therapeutic mouthwashes that can be purchased over-the-counter or prescribed only under the supervision of a doctor; (2) cosmetic mouthwashes mainly aimed at freshening breath; and (3) mouthwashes that are a combination of these two types [24].

Since the first type consists of therapeutic agents, the content of biologically active compounds is controlled by the corresponding normative and technical documentation and by appropriate methods of quantitative analysis of total fluoride.

The quality of the second type of preparations is in many cases determined only by the technical specifications in manufacturing and may not provide for the quantitative determination of the active substances in the final product. The aim of this study was to compare active-fluoride and total-fluoride concentrations in mouthwashes.

Fluorine-containing mouthwashes from various companies and manufacturers (Colgate Total Plax Classic Mint^®, Colgate Total Plax Gentle Mint^®, Colgate Total Plax Fresh Mint^®, Oral B Advantage^®, Reach Fresh Mint^®, Foramen^®, Lacalut Sensitive^®, Sensodyne^®, Vesna F^®, and Lacalut Fresh^®) were selected as study objects (Table 1).The method of direct potentiometry was used to determine fluoride ion activity using fluoride ion–selective electrode based on lanthanum fluoride. Standard silver chloride electrode was used as a reference electrode. Electromotive force of a galvanic cell was measured by standard laboratory ion meter И160М.To obtain calibration curves, standard solutions of NaF (5⋅10^-1-5⋅10^-5 M) were prepared using deionized water, as well as Albavit solution, which does not contain fluoride ions and is similar in composition to the test mouthwashes. Calibration curve data is given in Table 2 and Table 3 and in Figure 1.

To determine total fluoride concentration in mouthwashes, the method of standard addition was applied, using total ionic strength adjustment buffer (TISAB), which sets constant ionic strength during measurement and which also precomplexes interfering ions (e.g., Al³⁺). Since the results of quantitative determination of fluoride ions on standard mixtures prepared with aqueous solution and Albavit solution were slightly differentiated (i.e., the results obtained in the first case were systematically lower compared to those obtained theoretically, whereas in the second case the results were found to be higher), we attempted to modify the procedure by using the method of double increase in concentration without preliminary dilution with TISAB solution in order to determine possible binding of fluoride ions by components contained in the mouthwashes.

The accuracy of measurement of fluoride ion concentrations in standard solutions of NaF is shown in Table 4. Similar measurements for NaF solution prepared with Albavit solution are represented in Table 5.

Results of fluoride analysis are shown in Table 6. Most of the analyzed mouthwashes contained NaF as a source of fluoride ions; other types of fluoride agents were Na₂PO₃F, AmF, and AlF_3.The free fluoride:total fluoride ratio was more than 80% for six samples and less than 63% for three samples. Two samples had more than 70% and less than 80% of unbound fluoride, respectively. The fluoride ions have shown the best activity in the NaF-containing mouthwashes, regardless of their formulation. Mouthwash containing Na₂PO₃F had more than 50% of free fluoride, while AmF-containing mouthwashes had 94% of free fluoride. The lowest activity was observed for AlF₃ mouthwash.

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 mouthwashes containing AmF as a source of active fluorine, even though they contain polymeric compounds (polyethylene glycol) in their formulation; and the mode of change of both activity and concentration following dilution corresponds to that of mouthwashes that do not contain polymeric compounds.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)^® preparation, 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. The analysis of the results of the determination of different series of the same preparation revealed differences in the content of active fluorine for the Oral B^® preparations and almost identical results for the Reach^® 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 [25].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, poorly dissociating dimers H₂F₂, etc.) [26]. 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 the 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 (K_inst[AlF₆]^3- = 2,1⋅10^-21) [27], 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).

Mouthwashes are oral hygiene products aimed at enhancing the remineralization process and reducing demineralization due to the presence of fluoride ions; however, mouthwashes should also contain an appropriate amount of free fluorides to provide bioavailability. The mouthwashes analyzed in this study have various free fluoride:total fluoride ratios, which could lead to a decrease in the caries-preventive effect. Thus, there is an urgent need to develop a method for evaluation of free fluorides in mouthwashes for proper updating of national and international guidelines.

Ethics approval and consent to participate

Not applicable.

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Not applicable.

Availability of data and material

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Funding

There was no funding support available for this research.

Authors' contributions

VR was the supervisor of the project. All authors contributed to writing the manuscript, read and approved the final version of the manuscript.

Acknowledgements

Not applicable.

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Table 1. Composition of fluoride-containing mouthwashes.

Label	Composition
	Active ingredients		Et	Glyc	Sorb	Other ingredients
	fluorides	phosphates	Et	Glyc	Sorb	Other ingredients
Colgate Total Plax Classic mint^®	NaF	disodium hydrogen phosphate	+	+	+	Sodium lauryl sulfate, sodium methyl cocoyl taurate, menthol, saccharin, aqua, flavourings, colourants
Colgate Total Plax Gentle mint^®	NaF	disodium hydrogen phosphate	+	+	+	Sodium lauryl sulfate, sodium methyl cocoyl taurate, menthol, saccharin, aqua, flavourings, colourants
Colgate Total Plax Fresh mint^®	NaF	disodium hydrogen phosphate	+	+	+	Sodium lauryl sulfate, sodium methyl cocoyl taurate, menthol, saccharin, aqua, flavouring agents, colourants
Oral B Advantage^®	NaF	–	+	+	–	Methylparaben, propylparaben, poloxamer 407, cetylpyridine chloride, sodium saccharin, aqua, flavouring agents, colourants
Reach Fresh mint^®	NaF	disodium hydrogen phosphate, sodium dihydrogen phosphate	+	+	+	copolymer РОЕ, sodium benzoate, polyphenylene oxide, POE (20) sorbitan monooleate, perfume, cetylpyridinium chloride, aqua, colourants
Foramen^®	NaF	–	–	+	+	Potassium nitrate, PEG-40, allantoin, flavour additive, sodium methylparaben, sodium propylparaben, lemonacid, sodium benzoate, cetylpyridine chloride, aqua, sodium saccharinate
Lacalut sensitive^®	AmF	–	–	+	–	Cocamidopropyl betaine, chlorhexidine bigluconate, aqua, aluminum lactate, propylene glycol, PEG-40, flavouring agent
Sensodyne^®	NaF	disodium hydrogen phosphate, sodium dihydrogen phosphate	–	+	+	Sodium benzoate, poloxamer 338, potassium chloride, PEG-60, aqua, cetylpyridine chloride, sodium saccharinate, sodium chloride, methylparaben, flavoring agent
Vesna F^®	Na₂PO₃F	_	_	_	_	Aqua, xylitol, calcium glycerophosphate, polyoxyethylene sorbitan monooleate, sodium benzoate, citric acid, food flavour, colourants
Lacalut Fresh^®	AlF₃	_	_	_	_	Aqua, isopropyl alcohol, PEG-40 hydrogenated castor oil, flavors, peppermint extract, aluminum lactate, chlorhexidine bigluconate, sodium saccharinate, allantoin, bisabolol

* Et – ethanol, Glyc – glycerol, Sorb – sorbitol

Table 2. Calibration of ISE in NaF standard aqueous solutions.

	Concentration of NaF, mol/l									Average
	0.5	0.1	0.05	0.01	0.005	0.001	0.0005	0.0001	0.00005	Average
рF	0.533	1.119	1.362	2.046	2.320	3.017	3.310	4.000	4.301
EMF	573.0	532.0	524.2	470.0	466.9	409.0	405.5	353.0	338.0
	573.7	531.5	524.7	471.0	467.6	407.6	406.6	350.0	344.4
	572.8	532.3	522.0	471.0	469.2	406.7	408.0	347.6	340.0
		534.4		478.0		410.0		340.0
		536.0		478.0		407.0		345.5
		539.5		473.8		404.0		348.1
X̅	573.2	534.3	523.6	473.6	467.9	407.4	406.7	347.4	340.8
S	0.47	3.03	1.44	3.61	1.18	2.08	1.25	4.40	3.27	2.18
D X	0.69	3.18	2.14	3.79	1.75	2.18	1.86	4.62	4.86	2.53
E(%)	0.12	0.60	0.41	0.80	0.37	0.53	0.46	1.33	1.43	0.58

Table 3. Calibration of ISE in NaF solutions prepared with Albavit solution.

0.553

0.803

1.119

1.362

1.660

2.046

2.320

2.620

3.017

3.310

EMF

(aq.)

594

573

534

520

474

467

420

406

EMF

(Alba)

556

505

442

Table 4. The accuracy of measurement of F^- in NaF standard aqueous solutions.

NaF / TISAB	EMF	+ 10 ml TISAB	V (NaF) added, ml	DЕ	с (F^–)	%	X̅	S	X	E, %
5 / 5	508.2	490.9	9.2	17.3	0.096	96	95.8	1.92	2.21	2.3
3 / 7	495.6	478.5	4.0	17.1	0.095	95
7 / 3	518.4	498.7	19.2	19.7	0.094	94
3 / 7	497.0	478.8	4.2	18.2	0.099	99
5 / 5	505.0	488.0	9.1	17.0	0.095	95

Table 5. The accuracy of measurement of F^- in NaF standard solutions prepared using Albavit solution (4 ml «Alba» + 1 ml NaF).

c(NaF), mol/l	EMF	+ 5 ml TISAB	+ 10 ml TISAB	V (NaF) added, ml	с (F^–) theoretical	с (F^–) actual	c̄ (F^–)
0.5	532.2	505.3	486.9	1.0	0.100	0.104	0.105±0.002
0.5	536.7	509.7	490.8	1.0	0.100	0.106
0.5	534.2	507.2	489.8	1.0	0.100	0.105
0.1	501.3	469.5	450.8	1.0	0.020	0.022	0.022±0.0005

Table 6. The determination of fluoride-ions concentration and activity by the method of standard addition and modified method.

Label	pH	Dilution	F^-, mol/l×10^-3
			Method of standard addition		Modified method
			c (F^–)	a (F^–)	c (F^–)	a (F^–)
Colgate Total Plax Classic mint	7.15	–	9.77	5.52	9.92	5.43
Colgate Total Plax Classic mint		1¸10	0.75	0.89	1.11	1.06
Colgate Total Plax Gentle mint	5.70	–	9.14	8.05	8.91	8.71
Colgate Total Plax Gentle mint		1¸10	1.22	0.75	1.19	1.20
Colgate Total Plax Fresh mint	5.95	–	9.87	9.81	9.39	8.92
Colgate Total Plax Fresh mint		1¸10	0.63	0.47	0.70	0.43
Oral B Advantage	5.70	–	17.23	15.81	25.91	15.52
Oral B Advantage		1¸10	1.42	1.21	1.2	0.98
Oral B Advantage*	5.75	–	16.70	12.30	16.81	6.08
Reach Fresh mint	5.80	–	15.86	14.56	24.09	13.71
Reach Fresh mint		1¸10	1.20	0.71	1.40	1.02
Reach Fresh mint*	5.75	–	18.15	16.10	26.90	14.20
Foramen	5.80	–	14.62	9.11	20.05	9.05
Foramen		1¸10	0.75	0.94	1.20	1.07
Lacalut sensitive	4.40	–	5.48	5.13	8.60	4.20
Lacalut sensitive		1¸10	0.758	0.77	0.15	0.86
Sensodyne	5.70	–	16.25	12.53	23.59	11.12
Sensodyne		1¸10	2.01	1.27	–	–
Vesna F	6.05	–	4.49	2.76	5.20	2.71
Lacalut Fresh	4.20	–	–	0.093	1.30	0.095
Lacalut Fresh		1¸10	–	0.012	0.14	0.012

* preparation of another batch/manufacturer was used

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Evaluation of free and total fluoride concentration in mouthwashes via measurement with ion-selective electrode

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