A comparison between two ion selective electrode (ISE) potentiometric

methods is reported for determining the amount of fluoride in hard dental tissue after

placement of fluoride-releasing dental restorations. The two methods are: (1) the

direct method involving linear calibration (LC), and (2) a spiking method involving

multiple standard additions (MA). Results showed that measurements performed by

the LC method underestimate the amount of fluoride released by up to 30%.

Recovery tests demonstrated that the use of MA and blank correction procedures is

useful for an accurate and sensitive ISE determination of fluoride in hard dental

tissues

Cagetti, Maria Grazia

Campus, Guglielmo Giuseppe

Gaspa, Leonardo

Pilo, Maria Itria

Sanna, Gavino

Scanu, Roberta

Spano, Nadia

UnissResearch

Research report
Fluoride 40(2)111–115
April-June 2007
Performance of two potentiometric fluoride determination methods
in hard dental tissue
Campus, Gaspa, Pilo, Scanu, Spano, Cagetti, Sanna
111111PERFORMANCE DIFFERENCES OF TWO POTENTIOMETRIC 
FLUORIDE DETERMINATION METHODS 
IN HARD DENTAL TISSUE
 Guglielmo Campus,a,b,c Leonardo Gaspa,b,d Maria Pilo,e Roberta Scanu,b,e Nadia Spano,e 
Maria Grazia Cagetti,c Gavino Sannab,e,f
Sassari and Milano, Italy
SUMMARY: A comparison between two ion selective electrode (ISE) potentiometric
methods is reported for determining the amount of fluoride in hard dental tissue after
placement of fluoride-releasing dental restorations. The two methods are: (1) the
direct method involving linear calibration (LC), and (2) a spiking method involving
multiple standard additions (MA). Results showed that measurements performed by
the LC method underestimate the amount of fluoride released by up to 30%.
Recovery tests demonstrated that the use of MA and blank correction procedures is
useful for an accurate and sensitive ISE determination of fluoride in hard dental
tissues. 
Keywords: Dental materials; Fluoride ISE potentiometry; Linear calibration; Multiple spiking 
addition.
INTRODUCTION
In dental research, the potentiometric ion selective electrode (ISE) method1-4 is
commonly used for fluoride determinations. The method is fast, simple, and
specific.5 A direct quantification method, using a linear calibration (LC) plot, is
often preferred, but it is unable to detect and remove bias due to matrix effects.6
More powerful methods of quantification, based on spiking of analytical samples
(e.g., the standard addition method),7,8 are available, although they are rarely used
for this purpose.1,9 The aim of this study was to compare ISE determinations of
the amount of fluoride in hard dental tissue after use of fluoride releasing
restorative materials by the direct LC method versus a constant volume multiple
addition (MA) method. 
MATERIALS AND METHODS
Specimen preparation: Twenty-five 3-mm thick root disks from the coronal third
of the roots, measuring approximately 6 mm in width and length were obtained
from extracted bovine incisor teeth. The discs were embedded in a methlyl
methacrylate resin (Leocryl, Leone Italia) and abraded through cementum with
600-grit silicone carbide paper. In the area of the exposed dentin surface near the
root-crown junction, a cavity was prepared, 1.0 mm in depth and 1.5 mm in
diameter, using a diamond burr on a high speed drill (SUPERtorque 655 Kavo,
Germany) under water-spray. Next, the specimens were randomly divided into
five groups and treated respectively with: 1) a 3rd generation self-etching adhesive
(Xeno III, Dentsply, Germany)10; 2) an ionomeric cement (Ketac-Cem
Radiopaque, 3M ESPE AG, Germany) plus a thin layer of bonding agent (Clerafil
aDental Institute, University of Sassari, Italy; bNanoworld Institute, Nanoscience Laboratories,
University of Sassari, Italy; cSchool of Dentistry, University of Milan, “S.Paolo Hospital” WHO
Collaborating Centre of Milan for Epidemiology and Community Dentistry, Milano, Italy;
dBiomedical Science Department, University of Sassari, Italy;  eChemistry Department,
University of Sassari, Italy;  fFor correspondence: Prof. Gavino Sanna, Chemistry Department,
Sassari University, Via Vienna, 2, I-07100 Sassari, Italy. E-mail: sanna@uniss.it
Research report
Fluoride 40(2)111–115
April-June 2007
Performance of two potentiometric fluoride determination methods
in hard dental tissue
Campus, Gaspa, Pilo, Scanu, Spano, Cagetti, Sanna
112112SE bond); 3) a light-cured bonding system consisting of a self-etching primer and
a bonding agent (Clearfil SE bond, Kuraray, Europe GmbH, Germany); 4) an
ionomeric cement (Ketac-Cem Radiopaque, 3M ESPE AG, Germany); 5) a control
group with no restorative material on dentinal surface. The specimen were then
submitted to a demineralizing-remineralization procedure under four 24-hr cycles:
1 hr in a demineralizing solution11 and 23 hr in a remineralizing solution.12
Analytical procedure: A) SAMPLE MINERALIZATION AND PRE-TREATMENT: Six grams of
the dentinal sample powder, obtained from each specimen by abrasion with 600-
grit silicone carbide paper, was transferred to a 50-mL volumetric flask and
dissolved in 40 mL of 37% HCl solution and then diluted with water to the mark.
A 5.0-mL aliquot of the above solution was transferred to a 25-mL volumetric
flask, diluted 1:1 with water, neutralized with a 6 M NaOH solution to pH 4.5, and
made up to the mark with water. B) LC FLUORIDE EVALUATION: Six solutions (each
containing 2.39 mol/L of HCl neutralized with NaOH) were prepared containing
10.0, 5.0, 1.0, 0.5 and 0.1 mg F–/L, respectively. The last one was kept as the
analytical blank. In a beaker, 25.0 mL of each solution was mixed 1:1 with TISAB
solution, and the potential was measured beginning with the blank solution. Each
sample was determined in quintuplicate. C) MA FLUORIDE DETERMINATION: In a beaker,
25.0 mL of each sample was mixed 1:1 with TISAB solution and the potential was
measured, beginning with the most diluted one. Later, three consecutive additions
of fluoride standard solution were made, collecting the potential value after each
addition. Table 1 reports the concentration and the volume of each addition. The
Gran’s-like linearization procedure7,13 provided the analytical concentration. Each
sample was determined in quintuplicate. D) BLANK EVALUATION: To increase the
accuracy and precision in the measurement of very low fluoride concentrations
(up to few µg/L), the Villa procedure15 was always used. 
Equipment: A Model 96.09 F– ISE, a Model 900200 Ag/AgCl reference
electrode, and a Model 290 mV digital meter, all by Orion, were used.
Temperature was 20±0.5ºC. Appropriate fixed volume Eppendorf Research Series
2100 pipettes were used. Everywhere possible, glassware was replaced with
polyethylene labware.
Table 1. Operating parameters of MA method 
Material 
Concentration of standard spiking  
fluoride solution (mg F–/L) 
Standard volume  
of each addition (µL) 
1 (Xeno) 50        100 
2 (CVI+ClearS-bond) 100        100 
3 (ClearS-bond) 50          50 
4 (Cement VI) 500          50 
5 (Dentine, control) 50          50 
Blank 50          50 
 
Research report
Fluoride 40(2)111–115
April-June 2007
Performance of two potentiometric fluoride determination methods
in hard dental tissue
Campus, Gaspa, Pilo, Scanu, Spano, Cagetti, Sanna
113113Reagents: Analytical grade reagents were used throughout. NaF used for
standard was 99.99%. Water was ultra pure grade. The TISAB solution was
purchased from Thermo-Orion (code 94-09-11).
Statistical analysis: The data and the robustness of the analytical methods used
were statistically analyzed using Student’s t-test (p<0.05). 
RESULTS
Table 2 reports the total fluoride concentration by means of an LC plot. The
equation of the calibration line obtained is y = (–59.1±0.2)x + (159.2±0.3), with a
correlation coefficient R= 0.9999. Table 2 shows the analytical data obtained using
the MA method.  
Analytical values for samples 2, 4, and 5 are quite different from those obtained
using the LC plot (Student’s t-test). An analytical interference is evidenced by the
substantial differences of their slope values, respect to the blank one. The matrix
interference in each situation was evaluated by a normalization of the slopes of the
different experimental MA lines. A dimensionless parameter, matrix index (MI),
has hence been defined and evaluated (Table 2):  
where asample and ablank are the slopes of both linear regressions considered,
Csample and Cblank are the concentrations of the relevant fluoride spiking solutions
(Table 1). Values of MI close to 1 mean that data relative to the matrix of the
sample are affected by the same bias as the analytical blank, i.e. a situation of “no
interference.” On the other hand, MI lower (or higher) than 1 implies an analyte
underestimation (or overestimation) by matrix interference. Group 1 and 3
samples gave a matrix index close to 1 (the blank value), whereas in groups 2 and
4 a significant matrix effect was observed (0.78–0.71 MI). Samples from group 5
(dentin, control group) showed an MI value of 0.77. 
                       
                              MI 
  
   =  
 asampleCblank
 ablankCsample
Table 2. Fluoride amounts in the bulk material evaluated by means of LC and MA methods  
Group Samplea LC method 
C F–±SDb (mg/kg) 
MA method 
C F–±SD (mg/kg) 
Matrix index 
1 9.380±0.394 9.853±0.453 0.98 
2 17.81±0.69 21.27±0.53 0.78 
3 2.632±0.126 2.667±0.139 1.03 
4 45.33±0.50 53.72±0.64 0.71 
5 2.094±0.107 2.938±0.147 0.77 
Blank -  1 
 aEach sample has been measured in quintuplicate. bSD = estimated standard deviation.  
Research report
Fluoride 40(2)111–115
April-June 2007
Performance of two potentiometric fluoride determination methods
in hard dental tissue
Campus, Gaspa, Pilo, Scanu, Spano, Cagetti, Sanna
114114DISCUSSION
Nowadays, dentinal root caries is an increasing problem15 and to prevent them,
it is essential to consider their cause and how to protect against them. Fluoride is
considered an important tool16-17 in arresting the processes of secondary caries;
otherwise, in relation to water fluoridation, debate is still open,18-19 and a
precautionary approach to the use of fluoride would consider all the available
evidence on efficacy, safety, and alternatives. 
The need to obtain high reproducibility and reliability in the measurement of
fluoride in solutions or in dental tissues is therefore essential.1 Comparison of data
sets in Table 2 shows significant differences. Linear calibration always tends to
underestimate the fluoride amount. The MI value reveals that groups 2, 4, and 5
(i.e., the “inorganic-based” materials) evidence a strong matrix interference that, if
not corrected, leads to substantial under-evaluation of the amount of fluoride
released (up to ca. 30% for sample 4 ionomeric cement). It is interesting to note
that, in both methods the dentinal samples (i.e., the control group), show MI
values not too far from the value observed for ionomeric cements. In the same
conditions polymer-based materials (samples 1 and 3) show low or negligible
matrix interference.
Recovery tests, performed in triplicate with samples 3 and 4 (i.e., a pure organic
and an inorganic-based material, respectively) show good accuracy: the recoveries
were between 96 and 102% (sample 3) and between 98 and 101% (sample 4).
These results show that the proposed method is bias-free. Another advantage to
combine the MA ISE potentiometric determination of fluorides with the blank
evaluation/subtraction is represented by a significant lowering and
“customization” of the limit of detection. In this way, the limit of detection is
related to the nature of the sample, and not to a data set of standard solutions, as
usually happens in the LC method. 
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Research report
Fluoride 40(2)111–115
April-June 2007
Performance of two potentiometric fluoride determination methods
in hard dental tissue
Campus, Gaspa, Pilo, Scanu, Spano, Cagetti, Sanna
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Performance of two potentiometric fluoride determination methods in hard dental tissue

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