Development of Gold Standard Ion-Selective Electrode-Based Methods for Fluoride Analysis

Background/Aims: Currently available techniques for fluoride analysis are not standardized. Therefore, this study was designed to develop standardized methods for analyzing fluoride in biological and nonbiological samples used for dental research. Methods: A group of nine laboratories analyzed a set of standardized samples for fluoride concentration using their own methods. The group then reviewed existing analytical techniques for fluoride analysis, identified inconsistencies in the use of these techniques and conducted testing to resolve differences. Based on the results of the testing undertaken to define the best approaches for the analysis, the group developed recommendations for direct and microdiffusion methods using the fluoride ion-selective electrode. Results: Initial results demonstrated that there was no consensus regarding the choice of analytical techniques for different types of samples. Although for several Received: August 27, 2008 Accepted after revision: October 4, 2010 Published online: December 11, 2010 Dr. E. Angeles Martínez-Mier, DDS, MSD, PhD Preventive and Community Dentistry Oral Health Research Institute, Indiana University School of Dentistry 415 Lansing Street, Indianapolis, IN 46202 (USA) Tel. +1 317 274 8822, Fax +1 317 274 5425, E-Mail esmartin @ iupui.edu © 2010 S. Karger AG, Basel Accessible online at: www.karger.com/cre Martínez-Mier et al. Caries Res 2011;45:3–12 4 ic and radioanalytical methods [Venkateswarlu, 1990]. Rapid development in the F analysis field has occurred in the last four decades. However, the development of new techniques has not resulted in F determination becoming simpler or more cost-effective. Some of the newer methods are expensive and complex and can only be used for certain types of samples [Clarkson et al . , 2000]. In spite of significant discoveries made possible by early F analysis techniques, incorrect assumptions were made due to the inherent limitations of those initial methodologies. Good examples of this would be those studies which proposed that there were homeostatic mechanisms maintaining F levels in the body independent of the amount ingested [Singer and Armstrong, 1960] and those that supported the belief that the placenta acted as a partial barrier to the passage of F [Gedalia, 1970]. These erroneous conclusions were reached in part due to the inability of available techniques to measure ionic fluoride instead of total fluoride. At present, the most frequently used techniques for F analysis of samples are gas chromatography [Fresen, 1968], ion chromatography [Michigami et al., 1993; Inoue et al., 1995; Perring and Bourqui, 2002] and the F ion-selective electrode [Frant and Ross, 1966; Gron et al., 1968; Muehlemann, 1969; Iizuka et al., 1970; Clark and Dowdell, 1973; Fagioli et al., 1984; Kissa, 1987; Itai and Tsunoda, 2001; Malde et al., 2001]. The F ion-selective electrode consists of a sensing element bonded into an epoxy body. The F ion-selective electrode produces a potential across a lanthanum fluoride (LaF 3 ) solid ion exchange phase. The measured potential corresponding to the activity of fluoride ions in solution is described by the Nernst equation [Nernst and Schönflies, 1895]. The different approaches currently employed to determine total F may require pretreatment of samples, separation and concentration of F, actual measurement of F ions, calculations of final concentrations per unit of samples, and presentation of the data. Researchers have conducted these necessary steps using many different approaches. Currently available F measurement techniques are not standardized and a universal standard method for F determination has not been established [Clarkson et al., 2000]. Although a variety of techniques are available, none have been accepted for universal use. The current project aimed to review existing analytical techniques for F analysis and identify inconsistencies in their use, between laboratories dedicated to F analysis for dental research, in order to develop a universal gold standard method. Materials and Methods Nine laboratories with an established track record of publications in the area of F analysis for dental research and a history of previous collaborations participated in this effort. In order to use biological samples, Institutional Review Board (IRB) and Institutional Animal Care and Use Committee (IACUC) approvals were obtained at Indiana University (the coordinating site) prior to initiation of the study. The study was undertaken in three phases. In phase 1 comparisons of currently used techniques were conducted. In phase 2, the techniques used by participating laboratories were reviewed and comparative tests conducted to resolve identified differences. In phase 3 the universal gold standard methods were developed and tested in a variety of samples. Phase 1 In phase 1, all laboratories analyzed a standardized set of biological and nonbiological samples, in order to obtain a preliminary measure of agreement. This initial sample set included: standard F solutions (0.0132, 0.02631, 0.0526, 0.2631 and 0.5263  mol F/ml) prepared through the dilution of a commercially available standard fluoride solution (0.1 mol/l NaF, Orion Fisher Scientific Co., Itasca, Ill., USA), beverages (carbonated and noncarbonated, waterand dairy-based), food (homogenized for 1 and 10 min using a tissue homogenizer, single item and pooled meal-based samples), saliva (human, pooled and from individual donors), plasma, and urine (from healthy and systemically compromised donors). Each laboratory analyzed the samples, in duplicate (two aliquots of each sample were analyzed), using the methods they routinely used for F analysis according to sample type. All participating laboratories were asked to provide a description of their own methods. All of the laboratories basically used different modifications of two techniques for F determination: (1) direct analysis using a F ion-selective electrode (Orion No. 96-09 or 94-09; Fisher Scientific Co.) and a pH/ion meter (Orion No. 420A, 720A or EA940) was mainly used for standard solutions; (2) modifications of the hexamethyldisiloxane (HMDS; Sigma Chemical Co., St. Louis, Mo., USA) microdiffusion method of either Taves [1968] or Venkateswarlu [1977] were used for analysis of foods, beverages, urine, saliva, and plasma samples. Direct methods simply involved adding total ionic strength adjustment buffer solutions (TISAB, Orion, Thermo Electron Corp., USA) to the sample for the purpose of adjusting the pH and ionic strength of the standards and samples to the same values. In contrast, the diffusion methods extracted the F from the original sample and transferred it to a trapping solution of small volume so the F concentration in the solution that was finally analyzed was well above the limit of sensitivity of the electrode. Laboratories used this technique when the F in the sample was near or below the limit of sensitivity of the electrode or when the sample was not a liquid. A detailed review of the different standard operating procedures (SOP) used by the collaborating sites in phase 1 demonstrated that a range of different combinations of reagents and techniques were employed, regardless of whether a direct method or diffusion method was used. Phase 2 In phase 2, inconsistencies in the use of direct and diffusion techniques among laboratories were identified. Comparative tests were conducted for both direct and diffusion methods and are