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A simplified protocol to determine total fluoride concentration in NaF/ silica-based toothpastes

Aim: To determine total fluoride (TF) concentration in Na2FPO3/Ca-based toothpastes, using fluoride ion selective electrode (F-ISE) by the direct technique, it is necessary to use acid (Ac+) to hydrolyze the FPO3 ion and to dissolve insoluble fluoride salts bound to the abrasive. For NaF/silicabased toothpastes, the use of acid is not necessary (Ac-) and a simplified protocol could be followed. Methods: Thus, we evaluated TF concentration in seven brands of NaF/silicabased toothpastes, following the validated conventional Cury’s protocol (Ac+) or a simplification of this protocol (Ac-). Fluoride was analyzed with ISE calibrated with fluoride standard solutions prepared in the same conditions as the samples (Ac+ or Ac-). Results: The mean (±SD; n=21) of TF concentrations (μg F/g) found by Ac+ (971.3±191.2) and Ac(982.4±201.3) protocols were not statistically different (t test, p=0.22). The TF concentrations found agree with those declared by the manufacturers, except for one toothpaste imported from China. Conclusion: The findings suggest that the determination of fluoride in NaF/silica-based toothpastes can be accurately made using a simplified protocol of analysis.


Introduction
Toothpaste is considered the most rational way of fluoride use to control caries and the total fluoride concentration in toothpastes is under legislation control worldwide. Thus, different methods are used to determine fluoride concentration in toothpastes 1 and fluoride ion specific electrode (F-ISE) has been used for a long time 2,3 . The choice of the method for analysis mainly depends on the type of fluoride salt present in the formulation and the abrasive system of the toothpaste.
In toothpastes containing calcium-based abrasives, such as calcium carbonate (CaCO 3 ) or calcium phosphate dihydrate (CaHPO 4 .2H 2 O), sodium monofluorophosphate (Na 2 FPO 3 ) is the source of fluoride salt used due to the chemical incompatibility of sodium fluoride (NaF), stannous fluoride (SnF 2 ) or amine fluoride (AmF) with calcium (Ca ++ ). In formulations Na 2 FPO 3 /Ca-based, part of the total fluoride (TF) is soluble (TSF) as PO 3 F 2ion plus fluoride ion (F -), but part is insoluble (Fins.) bound to the abrasive 4 . NaF, SnF 2 and AmF toothpastes are formulated with hydrated silica (SiO 2 ), in which TF is soluble as F -. Fluoride ion specific electrode (F-ISE) has been successfully used to determine the different forms of fluoride in Na 2 FPO 3 /Ca-based and in NaF/SiO 2 -based toothpastes following the same protocol of analysis 5 .
To determine total fluoride in Na 2 FPO 3 /Ca-based toothpastes with F-ISE, acid is used to hydrolyze FPO 3 2ion and to dissolve insoluble fluoride salts bound to the abrasive 5 . For the determination of TF in Ca-free NaF toothpastes with F-ISE, it is not necessary to use acid because all TF is soluble as Fto be promptly analyzed with the electrode. Although the use of acid is not theoretically necessary and the protocol used since 1980 by Cury et al. (1981) 3 is valid because there is a high correlation (p=0.996) between TF found and that present in 27 NaF/silica-based toothpastes 6 , the protocol could be simplified. Therefore, we evaluated if the step of acid hydrolysis used in the validated protocol of Cury et al. (2010) 5 , could be simplified with accuracy for the determination of TF in NaF/silica-based toothpastes.

Material and Methods
Sampling Seven brands (n=3/brand) of fluoride toothpastes marketed for children were purchased; six in Lima, Peru, and one in Piracicaba, Brazil. Each tube from each brand was purchased in different supermarkets and drugstores and were from different fabrication batches, except for Peppa Pig brand. Table 1 shows information about the toothpastes used. Complying with the purpose of this study, all toothpastes chosen were NaF/silica-based and were analyzed within their expiration time. Toothpaste preparation for fluoride analysis

Statistical analysis
The data of TF found in the 21 samples, analyzed according to the conventional protocol using acid (Ac + ) and those found by the simplified protocol (Ac -), were compared by paired t test at 5%. Figure 3 shows total fluoride (TF) concentration declared by the manufacturer and the mean (SD; n=3) concentrations found by the conventional protocol (Ac + ; acid treatment) and by the simplified one (Ac -; without acid treatment) of the seven toothpastes evaluated. Excluding the unusual finding for Peppa Pig toothpaste, the difference (%) between TF found and that declared for the other six toothpastes was -4.1 and -2.8%, respectively for the conventional and simplified protocol.

Discussion
The protocol of fluoride determination in toothpastes with F-ISE by the direct technique and used since 1980 in Laboratory of Oral Biochemistry from FOP-UNICAMP 3,5,7-20 is considered chemically valid, reliable, and feasible 1 . In addition, it is able to estimate how much of the total fluoride presents in Na 2 FPO 3 /CaCO 3 -based toothpaste is bioavailable to be released in the oral cavity during toothbrushing 21 . However, this protocol could be simplified to determine fluoride in Ca-free toothpaste formulations, which contains ionic fluoride salts, such as NaF.
The findings clearly showed that the simplified protocol used is valid, reliable, and feasible when compared with the results found for the conventional protocol. As shown in figure 4, the mean of total fluoride found in the 21 samples of toothpastes analyzed with the simplified protocol did not statistically differ from the conventional protocol. The results found with the simplified protocol were as reproducible as those found with the conventional protocol, because the variation coefficient (%) of duplicates of analyses (n=21) was 1.3% compared with 1.5% for the conventional.
The simplified protocol is cheaper, consumes less time of work and is easier than the conventional one, because the number of laboratorial steps is reduced from the calibration curve up until the sample determination. Therefore, it can be used instead of the conventional protocol not only for NaF/Ca-free-based toothpastes as well as for SnF 2 or AmF formulations. However, the analyst should check if in fact the commercial toothpaste was not formulated with Na 2 FPO 3 or the abrasive is Ca-free. This warning is important because we have previously found toothpastes containing CaCO 3 as abrasive but according to the manufacturer it was silica 19 .  In all toothpastes, except in Peppa Pig, TF concentration found agreed with the declared by the manufacturer. According to the label of Peppa Pig's toothpaste, it should contain 1450 ppm F but we found 512.0 and 520.7 ppm F, respectively for the conventional and the simplified protocol. This result confirmed our previous fluoride determination in other batches of this same toothpaste 19 , where we found 515.1 ppm F using our conventional protocol 5 .
In addition to the development of this simplified protocol for fluoride determination in toothpastes, the present data confirm the necessity of sanitary vigilance of imported toothpastes from China because if there are approximately 3 times lower TF than the declared in the Peppa Pig brand analyzed, in previous analyses we found the opposite 14 . Also, we have observed in the present study problems with the type of abrasive declared and that found, as it is already described by Chávez et al. (2019) 19 .
Furthermore, the present study confirms that when the use of acid is necessary to hydrolyze PO 3 F 2ion and to dissolve insoluble F bound to abrasive, HCl as used in Cury's protocol (2010) 5 does not produce artifacts in the analysis because the TF concentration found did not statistically differ comparing the results found with the simplified protocol (Figure 3).
In conclusion, the findings suggest that the determination of fluoride in NaF/silica-based toothpastes can be accurately made using a simplified protocol of analysis.