Determination of Boron Isotope Ratios in Tooth Enamel by Inductively Coupled Plasma Mass Spectrometry ( ICP-MS ) After Matrix Separation by Ion Exchange Chromatography

Boron isotopes in teeth has been a new proxy for dietary reconstructions and its resistance to diagenetic alteration. In this study a method using inductively coupled plasma source mass spectrometry (ICP-MS) for the measurement of boron isotope ratio in human dental enamel has been developed. Human dental enamel were digested with HNO3-H2O2 in a microwave system. Boron in solution was separated from the matrix components using Amberlite IRA-743 resin. The factors that may affect precision and accuracy in isotope ratio determination by ICP-MS, including memory effects, mass bias drift, and concentration effects, were investigated to obtain optimum conditions. Then, the B/B ratios in teeth were measured. The results showed that 2% of HNO3 + 2% of NH3•H2O, selected as the diluent/rinse solution could be effective in the elimination of boron memory effect. There was no concentration effect on boron isotope ratios when the ratio of samples B concentration to standard B concentration (refers to Csample/Cstd) varied from 0.5 to 2. The result of B/B ratios in tooth enamel by sex and age fluctuated over a broad range, ranged from 0.2007 to 0.2574. This method is expected to be used for boron isotope ratio analyses in archeometry, forensic identification, paleoecology, and other disciplines in the future.


Introduction
Human teeth are valuable archives of the life history and behaviour of vertebrates.The bioapatite of the skeletal remains records in its element and isotope composition information about the diet, physiology and mobility as well as climate and environmental conditions.][3] Boron is shown to be an essential element for plants early this century and there is now evidence that it is also necessary for humans.Boron is distributed throughout the human body with the highest concentration in the bones and dental enamel.It is surprising that boron was found in teeth in the range as high as 25-85 ppm.Boron was found to be significantly increased in carious teeth than non-carious teeth despite loss of minerals during cariogenesis. 4Because food provides most of the boron ingested daily by terrestrial mammals (e.g., ca.90% in humans), the boron isotope of bioapatite could potentially be a new paleodietary proxy. 5,6he determination of boron isotope ratios ( 10 B/ 11 B) has been carried out by a variety of methods.These include atomic absorption spectrometry, [7][8][9] thermal ionisation mass spectrometry (TIMS), [10][11][12][13][14] multicollector inductively coupled plasma mass spectrometry (MC-ICP-MS), [15][16][17][18] glow discharge mass spectrometry (GDMS), the secondary ion mass spectrometry (SIMS), 19 laser ablation multicollector inductively coupled plasma mass spectrometry (LA-MC-ICP-MS), [20][21][22] spark source mass spectrometry, 23 and inductively coupled plasma mass spectrometry (ICP-MS), etc. [24][25][26][27][28][29][30][31][32][33][34][35] The objective of this work was to investigate the possibility of using ICP-MS for the determination of boron isotope ratios ( 10 B/ 11 B) in tooth enamel after pre-treatment

Instrumentation
Instrumentation included a ICP-MS (Perkin Elmer-Nexion 300D, PerkinElmer Corporation, USA) with an S10 autosampler, optimized by using a standard 89 Y solution at a concentration of 10 ppb in 2% HNO 3 , the MARS 6 microwave-assisted digestion system (CEM Microwave Technology Ltd operates, USA) and the Milli-Q ultrapure water system (Millipore Corporation, USA).

Reagents and materials
Boron isotopic reference materials: National Institute of Standards and Technology (NIST) boric acid SRM 951 (formerly NBS SRM 951 of the National Bureau of Standards, USA).A certified reference material of bone ash (SRM NIST 1400) and bone meal (SRM NIST 1486) from National Institute of Standards and Technology were also used.Milli-Q H 2 O (18.2 MΩ at 25 °C) from Millipore (Elix-Millpore, USA).HNO 3 was obtained from the Beijing Institute of Chemical Reagent and purified using the SavillexTM DST-100 sub-boiling distillation system (Minnetonka, MN, USA).H 2 O 2 (30 wt.% in H 2 O) was from Sigma-Aldrich Co. LLC.

Collection and treatment of permanent teeth
The permanent teeth were collected from 7 to 79 years old male and female humans who have lived in Shaanxi (NW China) for many years. 36The healthy teeth were extracted from sample providers who do not smoke or drink.The teeth were healthy permanent teeth extracted due to impacted wisdom tooth deformity, orthodontic treatment, or other reasons.Extracted teeth with complete crowns, no caries or mottled enamel, complete enamel development, and no obvious wear or defects on the morsal surface were selected.The selected carious teeth exhibited clear cavities.The teeth were soaked in acetone solution for 24h.[39][40][41] The microwave digestion conditions used in this study for teeth digestion were adapted to those previously used for biological samples following the method of Li et al. 36 with some modification.Teeth samples were weighed, ground to powder using mortar and pestle, and weighed again before the digestion (depending on the tooth type and size, they ranged from 0.0090 to 0.7350 g).In this study, powdered teeth samples were pre-dissolved in 3 mL of 50% HNO 3 solution and 2 mL of 30% H 2 O 2 solution using microwave digestion tank (The MARS 6 microwave-assisted digestion system, CEM Microwave Technology Ltd operates, USA).The tank was then gently shaken.Approximately 3 mL Milli-Q water was added dropwise, and the samples were digested according to procedure in Table 1.
For the certified reference material (NIST SRM 1400 and NIST SRM 1486), an amount of ca.0.0500 g of sample was digested in microwave as described above.

Separation and enrichment of B with an ion-exchange process
The mineral phases of tooth enamel are mostly hydroxyapatite crystals of various structures and composition with incorporated trace elements.Major elements found in enamel are Ca, P, Na, Mg, and Cl.Their mean concentrations are well known, and their approximate concentrations are 37% Ca; 18% P; 0.4% Mg; 0.7% Na, and 0.28% Cl.Because of unavoidable matrix interference during the measurement of the 10 B and 11 B isotopes with ICP-MS, even after microwave digestion of the samples, it was necessary to separate B from all the other elements and matrix components still present.An ion exchange procedure using Amberlite IRA 743 was adapted and optimized for this purpose. 18,33The detailed methods of the experiment are listed in Table 2.  instrument conditions.The ICP-MS operating parameters are given in Table 3.
Determination of the B isotope ratio, 11 B/ 10 B, by means of ICP-MS is complicated by a large mass discrimination effect (because of the relatively large mass difference between the two B isotopes) and the drift in the mass discrimination during measurement, which may lead to a concomitant drift in the measured isotope ratio.Therefore, to take into account the variations in mass bias with time, we employed the correction method using the NIST SRM 951 as standard sample. 31The isotopic ratio of boron ( 10 B/ 11 B) meas was calculated by equations (1) and ( 2): ( ) Here, the signal intensities of 10 B and 11 B in the sample solution is represented as 10 B samp and 11 B samp , the certified standard value of the 10 B/ 11 B ratio for NIST SRM 951 is represented as ( 10 B/ 11 B) cert , and the measured value of the isotopic ratio of boron for each sample as ( 10 B/ 11 B) meas .The signal intensities of 10 B and 11 B in the blank solution and NIST SRM 951 solution averaged over n measurements are represented in the Table 4.The measurements were repeated 10 times for each sample and the average was set as the measured value.

Boron memory effects
The high sensitivity of ICP-MS makes this technique suitable, reliable and rapid for boron determinations.However, measuring boron at ultratrace levels by ICP-MS often present a significant memory effect.Over the past decades, most researchers have used several methods to eliminate boron memory effect.One was direct injection nebulization (d-DIHEN), 29 and another was using different diluents/rinse solution, including water, nitric acid, Triton X-100, ammonia and mannitol in water, in nitric acid and in ammonia. 26These attempts has been achieved satisfactory results.
Several reagents, including water and 2% of HNO 3 , 2% of HNO 3 + 2% NH 3 •H 2 O and 2% of HNO 3 + 2% mannitol were tested and evaluated according to the memory effect, the analytical precision and the background.For each reagent, an equivalent blank and a 100 ng mL -1 standard solution were analyzed.The ion-time response for 11 B + was continuously monitored.First, the signal was collected with the reagent blank for about 5 min, then the B solution with the same reagent was introduced for 5 min, and finally the reagent blank was introduced again as the flush solution for about 10 min.Table 5 and Figure 1 gives the B ion-time response (counts s -1 ) for the selected reagents.Figure 1 gives the B ion-time response (counts s -1 ) for the selected reagents.The results show that 2% HNO 3 + 2% NH 3 •H 2 O and 2% HNO 3 + 2% mannitol exhibit similar and significant memory effects.Then 2% HNO 3 + 2% NH 3 •H 2 O was selected for sample analysis in this text.

Effects of B concentration on the measured B isotope ratios
The term concentration effects used here refers to the phenomenon that mass fractionation during plasma source mass spectrometry varies with changes in concentration of sample solutions compared to the standard solution under a given set of working conditions.This phenomenon may be regarded as a special case of matrix effects.So, it was important to maintain the similar concentration of B in sample and standard solution.
To investigate the feasibility of concentration effect, a series of measurements have been carried out using NIST SRM 951 B solutions with B concentrations varying from 0.05 to 1.5 ppm (refers to C sample ) vs. the same NIST SRM 951 B solution at a fixed concentration of 0.5 ppm (refers to C std ).The measured B isotope ratios of the "sample" varing with C sample /C std was plotted in Figure 2.These variations cannot be explained by molecular interferences, but must result from instrumental mass fractionation.This implies that the instrumental fractionation of B isotopes varies according to the B concentration introduced into the mass spectrometer, at least for the given set of working conditions.It can be seen from Figure 2, when C sample /C std varied from 0.5 to 2, and there was no effect on boron isotope ratios.

Reproducibility of the measurement
To test the analytical precision and accuracy of the methods above, the boron isotope ratios of NIST SRM 951 were measured in 220 days.The results are illustrated in Figure 3.The reproducibility of all the measurements shown in Figure 3 fell below 0.2% RSD, indicating the long-term reproducibility of measurement.

Determination of 10 B/ 11 B ratios in tooth enamel
Using the above described experimental procedures, the B isotopes in the enamel of the extracted teeth were chemically separated and measured.The result of 10 B/ 11 B ratios in tooth enamel by sex and age were shown in Table 6 and Figure 4.In order to answer these two questions, the 10 B/ 11 B of teeth has been compared with other stable isotope proxies used in ecology, analyzed on the same specimens, to determine the influence of diet (δ 13 C), 42,43 water sources (δ 18 O), 42,43 trophic levels (δ 15 N, δ 44 / 42 Ca), [44][45][46] as well as with geological and vegetation maps ( 87 Sr/ 86 Sr), 36,47,48 should be investigated the influence of the local bedrock and plant matter contributions, respectively.The Sr isotope composition (i.e., 87 Sr/ 86 Sr ratio) of the healthy teeth and carious teeth at the same region in Shaanxi (NW of China) was measured by Li et al. 36 Their results demonstrate that 87 Sr/ 86 Sr does not appear to be affected by the caries formation, age or sex.The 87 Sr/ 86 Sr ratio in the enamel of the healthy and carious teeth of individuals of varying ages and genders ranged between 0.710935 and 0.711037, which falls into the range of the 87 Sr/ 86 Sr found in the local, naturally occurring water, soils and rocks.

Conclusions
We present here a method to measure 10 B/ 11 B ratios in tooth enamel by ICP-MS after pre-treatment by ion exchange separation.The 10 B/ 11 B ratio in the enamel of the healthy teeth and carious teeth fluctuated over a broad range, ranged from 0.2007 to 0.2574.It is anticipated that this technique offers the potential for using B isotope ratios to trace the geochemical cycling of B in scientific archaeology, forensic identification, paleoecology, and other disciplines.The data in Figure 4 indicate that the 10 B/ 11 B ratio in the enamel of the healthy teeth and carious teeth by sex and age fluctuated over a broad range, ranged from 0.2007 to 0.2574.Boron has two naturally occurring isotopes, 10 B (19.9%) and 11 B (80.1%).A relatively large mass difference (10%) between the two isotopes and high volatility results in significant boron isotopic variation from -70‰ to +75‰ in natural materials; thus, boron isotopes have numerous applications in geochemistry, isotope hydrology, oceanography, environmental sciences, cosmology, and nuclear technology. 13The 10 B/ 11 B ratio in the enamel of the teeth could potentially be a new

11 B
/ 10 B isotope ratio measurements 11 B/ 10 B isotope ratio was determined by inductively coupled plasma mass spectrometry (ICP-MS, Perkin Elmer-Nexion 300D, PerkinElmer Corporation, USA) with an S10 autosampler.The ICP-MS instrument operating parameters were established by automatically optimizing the

Figure 1 .Figure 2 .
Figure 1.Comparison of background values with various washing solutions.

Figure 4 .
Figure 4. 10 B/ 11 B isotope ratios in the tooth samples (each group has one sample).
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Table 1 .
Microwave digestion programs for teeth

Table 3 .
Experimental conditions used for the ICP-MS measurements

Table 2 .
Columns for B separations using Amberlite IRA 743 resin

Table 4 .
The measurement procedure for boron isotope ratios

Table 5 .
Relationship between boron ion strength and changes with the cleaning time

Table 6 .
The 10 B/ 11 B ratios in tooth enamel by sex and age