1http://dx.doi.org/10.20396/bjos.v19i0.8661689 Volume 19 2020 e201689 Original Article 1 Academic Department of Dentistry for Children and Adolescents, Faculty of Stomatology, Universidad Peruana Cayetano Heredia, Lima, Peru. 2 Department of Biosciences, Piracicaba Dental School, University of Campinas, Piracicaba, SP, Brazil. * Corresponding author: Jaime Aparecido Cury Department of Biosciences, Piracicaba Dental School, University of Campinas Av Limeira, 901, Piracicaba, SP, Brazil, 13414-903 Email: jcury@unicamp.br Received: October 18, 2020 Accepted: November 23, 2020 A simplified protocol to determine total fluoride concentration in NaF/ silica-based toothpastes Jenniffer Quiroz-Torres1 , Cinthia Pereira Machado Tabchoury2 , Carlos Liñán-Durán1 , Antonio Pedro Ricomini-Filho2 , Ailin Cabrera-Matta1 , Jaime Aparecido Cury2,* 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 2- ion and to dissolve insoluble fluoride salts bound to the abrasive. For NaF/silica- based 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/silica- based 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. Key words: Fluorides. Toothpastes. Silicic acid. Dentifrices. Ion-selective electrodes. http://dx.doi.org/10.20396/bjos.v19i0.8661689 mailto:jcury@unicamp.br https://orcid.org/0000-0001-7957-8662 https://orcid.org/0000-0002-7660-5685 https://orcid.org/0000-0003-2669-842X https://orcid.org/0000-0002-6593-6040 https://orcid.org/0000-0002-4493-7036 https://orcid.org/0000-0003-1046-5605 2 Quiroz-Torres et al. 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 concentra- tion in toothpastes1 and fluoride ion specific electrode (F-ISE) has been used for a long time2,3. 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 carbon- ate (CaCO3) or calcium phosphate dihydrate (CaHPO4.2H2O), sodium monofluo- rophosphate (Na2FPO3) is the source of fluoride salt used due to the chemical incompatibility of sodium fluoride (NaF), stannous fluoride (SnF2) or amine flu- oride (AmF) with calcium (Ca++). In formulations Na2FPO3/Ca-based, part of the total fluoride (TF) is soluble (TSF) as PO3F 2- ion plus fluoride ion (F-), but part is insoluble (Fins.) bound to the abrasive4. NaF, SnF2 and AmF toothpastes are for- mulated with hydrated silica (SiO2), in which TF is soluble as F -. Fluoride ion spe- cific electrode (F-ISE) has been successfully used to determine the different forms of fluoride in Na2FPO3/Ca-based and in NaF/SiO2-based toothpastes following the same protocol of analysis5. To determine total fluoride in Na2FPO3/Ca-based toothpastes with F-ISE, acid is used to hydrolyze FPO3 2- ion and to dissolve insoluble fluoride salts bound to the abrasive5. 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 F- to 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 toothpastes6, 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 infor- mation 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. 3 Quiroz-Torres et al. Table 1. Information on the toothpastes analyzed Brand Code Country of Purchase Abrasive agent Fluoridated agent Declared [F] (ppm F) Expiration date Colgate 6+ A1 Peru Silica NaF 1100 01/2020 Colgate 6+ A2 Peru Silica NaF 1100 02/2020 Colgate 6+ A3 Peru Silica NaF 1100 10/2019 Aqua Fresh My Big B1 Peru Silica NaF 1150 02/2019 Aqua Fresh My Big B2 Peru Silica NaF 1150 09/2018 Aqua Fresh My Big B3 Peru Silica NaF 1150 06/2018 Vitis Jr C1 Peru Silica NaF 1000 05/2019 Vitis Jr C2 Peru Silica NaF 1000 12/2019 Vitis Jr C3 Peru Silica NaF 1000 04/2019 Colgate Kids D1 Peru Silica NaF 1100 02/2020 Colgate Kids D2 Peru Silica NaF 1100 02/2020 Colgate Kids D3 Peru Silica NaF 1100 02/2020 Dento Jr E1 Peru Silica NaF 1100 04/2020 Dento Jr E2 Peru Silica NaF 1100 10/2019 Dento Jr E3 Peru Silica NaF 1100 - Peppa Pig F1 Peru Silica NaF 1450 06/2021 Peppa Pig F2 Peru Silica NaF 1450 - Peppa Pig F3 Peru Silica NaF 1450 - Tandy G1 Brazil Silica NaF 1100 02/2020 Tandy G2 Brazil Silica NaF 1100 02/2020 Tandy G3 Brazil Silica NaF 1100 02/2020 Toothpaste preparation for fluoride analysis Toothpastes samples were prepared (Figure 1) for analysis of total fluoride (TF) with F-ISE by the protocol described by Cury et al. (2010)5. An amount of 90 to 110 mg of toothpaste was weighed (± 0.01 mg) and vortexed in 10.0 mL of purified water. Dupli- cate volumes of 0.25 and 1.0 mL of the suspension were transferred to tubes codified Ac+ and Ac-, respectively conventional (Figure 1A) and simplified protocols (Figure 1B). To the tubes Ac+, 0.25 mL of 2.0 M HCl was added and after 1 h at 45 oC the extracts were buffered with 0.50 mL of 1.0 M NaOH plus 1.0 mL of TISAB II. To the tubes Ac-, just 1.0 mL of TISAB II was added. 4 Quiroz-Torres et al. Two calibration curves were made (Figure 2). For samples prepared according to the conventional protocol (Ac+; Figure 1A), standards ranging from 0.25 to 4.0  µg F/mL prepared in 0.25 M HCl, 0.25 M NaOH and TISAB II 50% (v/v) were used. For samples evaluated by the simplified protocol (Ac-; Figure 1B), fluoride standards ranging from 4.0 to 32.0 µg F/mL prepared in TISAB II 50% (v/v) were used. All fluoride standards were prepared from NaF 99.99% (Sigma-Aldrich, St Louis, MO, USA). The accuracy of the analysis was checked with a standard fluoride solution (Orion 940907, Thermo Scientific, Boston, MA, USA) and the average coefficient of variation from triplicates was 1.0 and 2.4%, respectively for the conventional and the simplified protocol. For the analysis, a F-ISE (Orion 96-06; Orion Research Inc., Boston, MA, USA) coupled to an ion analyzer (Orion Star A214; Orion Research Inc., Boston, MA, USA) was used. Fluoride concentration in the samples was determined from linear regression of the logarithm of fluoride concentrations of the standards with the respective mV values (r2=0.999 for both calibration curves), using Excel spreadsheet (Microsoft®). The results were expressed in ppm F (µg F/g; mg F/kg). Calibration curve (A) mV 40.0 50.0 60.0 70.0 80.0 90.0 100.0 110.0 120.0 lo g µg F /m L y = -0.0171x + 1.4244 R2 = 1 0.8000 0.6000 0.4000 0.2000 0.0000 -0.2000 -0.4000 -0.6000 -0.8000 Calibration curve (B) mV -10.0 0.0 10.0 20.0 30.0 40.0 50.0 lo g µg F /m L y = -0.0174x + 1.4022 R2 = 0.9999 1.5000 1.3000 1.1000 0.7000 0.9000 0.5000 Figure 2. Calibration curves (n=3) and data of the correlation between the logarithm of fluoride concentrations in standards and the relative mV generated for the conventional (A) and simplified (B) protocols of analysis. Toothpaste (90-110 mg) 1.0 mL Suspension + 1.0 mL TISAB II Ac- Calibration with fluoride standards ranging from 4.0 to 32.0 µg F/mL, prepared in TISAB II 50% (v/v) Calibration with fluoride standards ranging from 0.25 to 4.0 µg F/mL, prepared in 0.25 M HCl, 0.25 M NaOH and TISAB II 50% (v/v) 0.5 mL 1 M NaOH + 1.0 mL TISAB II 0.25 mL Suspension + 0.25 mL 2 M HCL 1h 45°C ISE Analysis Ac+ (B)(A) Suspension 10 mL H2O ISE Analysis Figure 1. Toothpaste sample preparation for total fluoride determination by the conventional (A) and the simplified protocol (B) 5 Quiroz-Torres et al. Figure 4 shows means (SD; n=21) of fluoride concentration found in the tooth- paste tubes by the conventional protocol and by the simplified one. The difference between the protocols was not statistically significant (p=0.22). The variation coefficient in percentage was 19.8 and 20.5%, respectively by the conventional and simplified protocol. 0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 pp m F (µ g F/ g) Toothpastes Brand Colgate 6+ Aqua Fresh My Big Vitis Jr Colgate Kids Dento Jr Peppa Pig Tandy TF Declared TF Found Conventional TF Found Simplified Figure 3. Total fluoride (TF) declared (ppm F) by the manufacturer and concentrations found using the conventional (Ac+) and the simplified (Ac-) protocol (Mean;SD;n=3) Statistical analysis The data of TF found in the 21 samples, analyzed according to the conventional pro- tocol using acid (Ac+) and those found by the simplified protocol (Ac-), were compared by paired t test at 5%. Results 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 tooth- paste, 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. 6 Quiroz-Torres et al. 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-UNICAMP3,5,7-20 is considered chemically valid, reliable, and feasible1. In addition, it is able to estimate how much of the total fluoride presents in Na2FPO3/CaCO3-based toothpaste is bio- available to be released in the oral cavity during toothbrushing21. However, this proto- col 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 fea- sible 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 cali- bration 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 SnF2 or AmF formulations. However, the analyst should check if in fact the commer- cial toothpaste was not formulated with Na2FPO3 or the abrasive is Ca-free. This warn- ing is important because we have previously found toothpastes containing CaCO3 as abrasive but according to the manufacturer it was silica19. 0 100 200 300 400 500 600 700 800 900 1000 Conventional Protocol Simplified Protocol pp m F ; µ g F/ g Sample Treatment Figure 4. Fluoride concentration (ppm F) found in the toothpastes by the conventional (Ac+) and the simplified (Ac-) protocol; (Mean;SD;n=7;p=0.22) 7 Quiroz-Torres et al. 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 toothpaste19, where we found 515.1 ppm F using our conventional protocol5. In addition to the development of this simplified protocol for fluoride determina- tion 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 opposite14. 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 PO3F 2- ion 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 con- centration found did not statistically differ comparing the results found with the sim- plified protocol (Figure 3). 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