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Multi-elemental matrix-matched calcium hydroxyapatite reference materials for laser ablation: Evaluation on teeth by laser-induced breakdown spectroscopy
Spectrochimica Acta Part B 159 (2019) 105650
Contents lists available at ScienceDirect
Spectrochimica Acta Part B journal homepage: www.elsevier.com/locate/sab
Multi-elemental matrix-matched calcium hydroxyapatite reference materials for laser ablation: Evaluation on teeth by laser-induced breakdown spectroscopy☆
T
Mauro Martineza, Courtney Bayneb, Dylan Aiellob, Matthew Julianc, Romain Gaumec,d,e, ⁎ Matthieu Baudeleta,b,e, a
National Center for Forensic Science, University of Central Florida, Orlando, FL 32826, USA Chemistry Department, University of Central Florida, Orlando, FL 32816, USA Materials Science and Engineering Department, National Center for Forensic Science, 12354 Research Parkway Suite 225, Orlando, FL 32826, USA d NanoScience Technology Center, University of Central Florida, Orlando, FL 32816, USA e CREOL – The College of Optics and Photonics, University of Central Florida, Orlando, FL 32816, USA b c
ARTICLE INFO
ABSTRACT
Keywords: LIBS Standards Hydroxyapatite Osseous material
Laser ablation has become the most accepted method for direct sampling of solid materials, as in elemental trace analysis by laser-induced breakdown spectroscopy. However, the scarcity of matrix-matched standards or reference materials (such as NIST reference materials for instance) is an obstacle to quantitative analysis, particularly in the area of hard biological materials analysis, such as bones and teeth. If existing, the corresponding certified standards often consist of powders, then compacted as pellets which density, cohesive strength and dopant incorporation are not always representative of the samples under investigation, thereby casting serious uncertainties on calibration protocols. Here, we report on the synthesis of hydroxyapatite reference ceramic materials that can be doped controllably with various elements of interest. The fabrication process involves spiking during synthesis, prior to a hard sintering of pelletized ceramics. As a result, these materials have good chemical homogeneity and ablation properties that mimic those of naturally calcified biological tissues.
1. Introduction Bones, teeth, and calcified tissues present a notable importance in the fields of forensics, archeology, anthropology and medical diagnostics. These materials can provide information about health, diet, age, sex and migrations [1–6], and help in the post-mortem identification of an individual [7]. Hydroxyapatite (HA), with chemical formula Ca10(PO4)6(OH)2, is the inorganic phase present in most of these calcified materials. Synthetic HA has received much attention in recent years due to its excellent biocompatibility [8,9] and is usually prepared by a wet-chemistry approach. Depending on the precipitation conditions, including pH, ionic strength, temperature and the presence of structuring agents, HA can be obtained with a calcium-to‑phosphorus molar ratio similar to that of natural bone and teeth [10], thus, rendering this material an ideal candidate for clinical applications [9,11]. Elemental analysis is one of the many strategies used to extract valuable information from bones, teeth and calcifications. Different
techniques have been used in the past for this purpose. X-ray fluorescence (XRF) [12,13] is typically used to analyze major and trace elements [14,15], along with micro-XRF [16], although this technique is not sensitive enough to very light elements and the limits of detection (LOD) are between tens and hundreds of μg.g−1. Inductively coupled plasma – mass spectrometry (ICP-MS) is also used for bulk analysis of trace elements [17,18]. This technique achieves the best LOD for a wide range of elements. However, the sample is consumed in the digestion process and any information about elemental distribution is lost. Laser ablation (LA-) ICP-MS has proven advantageous because of its unique solid sampling approach, while maintaining low LODs. By providing elemental imaging capability and avoiding sample destruction, LA-ICPMS has opened new perspectives in elemental distribution imaging and the recovery of elemental incorporation history in bones, teeth, and calcified tissues [3,19–23]. Similarly, laser-induced breakdown spectroscopy (LIBS) [24,25] has also been successfully applied to qualitative and quantitative elemental analysis of calcified materials [26–29].
Selected Paper from the 10th International Conference on Laser-Induced Breakdown Spectroscopy (LIBS 2018) held in Atlanta, GA, USA, October 21–26 2018. Corresponding author at: National Center for Forensic Science, University of Central Florida, Orlando, FL 32826, USA. E-mail address: [email protected] (M. Baudelet).
☆ ⁎
https://doi.org/10.1016/j.sab.2019.105650 Received 19 March 2019; Received in revised form 17 July 2019; Accepted 18 July 2019 Available online 19 July 2019 0584-8547/ © 2019 Elsevier B.V. All rights reserved.
Spectrochimica Acta Part B 159 (2019) 105650
M. Martinez, et al.
Specific examples include the use of LIBS in strontium (Sr) quantification in teeth [30,31], the investigation of carious teeth ex vivo as well as in vivo [32], the qualitative and quantitative analysis of kidney stones [33,34], and the study of paleoclimate in calcified materials [35,36]. However, one of the major challenges in LA-based elemental analysis techniques of calcified materials is the availability of proper calibrated materials. Previous studies report the use of NIST SRM 610 or 612 glass reference materials [19,37,38], although ablation of glass and calcified materials is different and may lead to incorrect quantification results [39], or bone meal (SRM 1486) and bone ash (SRM 1400) [40]. A limitation of the glass standards concerns the available concentration range for calibration. In an effort to obtain adequate calibration materials, chemically similar to naturally-occurring calcified materials, with diverse dopants and over a wide concentration range, Ugarte et al. [39] reported the development of a matrix-matched HA calibration material for LA-ICP-MS. Their approach consisted in co-precipitating HA with analytes of interest and pelletizing these synthesized powders for calibration. Their synthesis showed quantitative analyte co-precipitation as well as low LODs, and they used this reference material for the analysis of fish dorsal spine. This methodology was used for similar applications in the quantification of Mn in teeth and bones by LA-ICPMS [41,42]. However, particle size and pellet compaction can both affect the ablation process yielding variations in the calibration curves [43]. A natural extension to this approach consists in increasing the density of the HA pellets by a sintering process [10,44,45], thereby reducing the difference in sample strength and ablation behavior between the standard and the sample. This work proposes a protocol for the preparation of HA reference materials being spiked during co-precipitation, pelletized and sintered. With this process, we obtained solid standard materials in pellet forms that can be used for elemental quantification analysis of calcified materials using LIBS. Traditional ICP-MS was used to verify and validate the data obtained by LIBS on teeth using these reference materials.
Table 1 Concentration (in μg.g−1) in the doped HA reference materials after calcination and sintering (obtained by ICP-MS, 1σ standard deviation between parentheses).
2. Experimental
tetrahydrate (Ca(NO3)2·4H2O, Acros Organic) and ammonium dihydrogen phosphate (NH4H2PO4, Acros Organic). Aqueous solutions of NH4H2PO4 (0.48 M) and Ca(NO3)2·4H2O (1 M) were prepared in ultrapure water (18 MΩ) and pH-adjusted to a value of 10 by adding ammonium hydroxide (NH4OH, Fisher Scientific). A well-stirred solution of analytes of interest was added at a concentration of 1000 μg.g−1 to the calcium nitrate solution. The concentration of each element in the spiking solutions was adjusted to achieve suitable concentrations on the desired HA standards. The Ca(NO3)2·4H2O solution was added dropwise and under magnetic stirring to the NH4H2PO4 solution. The mixture was then shaken on an orbital shaker and aged for 5 days. The precipitate was subsequently filtered, washed with ultra-pure water, dried in an oven at 80 °C overnight, lightly ground in an alumina mortar, and calcined at 450 °C for three hours. This new hydroxyapatite crystalline powder was pelletized in a die through uniaxial pressing at 2.5 MPa, followed by cold isostatic pressing at 200 MPa. The resulting samples were consolidated by pressure-less sintering process in air at 1200 °C, using heating and cooling rates of 2 °C/min and a soaking time of 2 h. A part of each pellet was dissolved for the determination of the elemental content by ICP-MS and the calculation of the recovery percentage.
Sample: nominal concentration S1: 0 μg.g
−1
S2: 216 μg.g−1
S3: 650 μg.g−1
S4: 1083 μg.g−1
Element
After calcination
After sintering
Co Mn V Ni Sr Co Mn V Ni Sr Co Mn V Ni Sr Co Mn V Ni Sr
15.4 (0.2) 9.0 (0.5) Not detected 57 (4) 54.2 (2) 71 (2) 130 (2) 137 (7) 138 (6) 120 (3) 340 (5) 345 (5) 375 (12) 275 (3) 234 (1) 630 (14) 621 (8) 694 (13) 670 (6) 414 (4)
Not detected Not detected Not detected Not detected 7 (3) 64 (4.4) 58 (1) 85 (6) 41 (4) 45 (1) 338 (4) 351 (6) 382 (12) 213 (5) 204 (3) 660 (6) 641 (6) 709 (8) 650 (14) 403 (3)
Table 2 Figures of merit for the HA standard material analyzed by LIBS.
2.1. Instrumentation LIBS data was acquired using a J200 LIBS system (Applied Spectra Inc., Fremont, CA). The instrument consists of a 266 nm Nd:YAG laser (8 ns) with variable energy and ablation spot size, and a 5-channel spectrometer covering a spectral range from 185 to 1040 nm (acquisition delay of 1 μs for 1 ms acquisition duration) in order to obtain a good signal-to-noise ratio. An argon background atmosphere (flow of 1 L/min) is created to avoid interaction with nitrogen and oxygen in the air. HA calibration materials were analyzed with 100 μm spot size, using 10 laser pulses per location with a total of 5 × 5 = 25 locations, every spot separated by 215 μm to cover over an approximate 2 × 2 mm2 area, and an energy of 15 mJ to build the calibration curve. In order to take in account any inhomogeneity on the surface, three 5 × 5 grids were measured per sample. To evaluate the internal homogeneity on the standard material, a transversal cross-section was obtained and imaged by LIBS. The elemental imaging of the teeth samples was performed with the same laser ablation parameters. Data processing was performed using Applied Spectra's Aurora Data Analysis Software package. ICP-MS analysis was performed with a PlasmaQuant Elite (Analytik Jena, Germany).
Analyte
Wavelength
R2
LOD (μg.g−1)
LOQ (μg.g−1)
Co I Mn II V II Ni II Sr II
345.3 nm 257.6 nm 309.3 nm 221.6 nm 407.7 nm
0.994 0.983 0.986 0.996 0.994
49 130 55 42 21
91 219 101 78 38
2.3. Validation samples
2.2. Preparation of hydroxyapatite reference materials
The validation of the reference materials was performed by analyzing two different human teeth obtained from an archaeological collection [46]. Prior to analyses, the teeth were repeatedly washed in an ultrasonic bath for 10 min in distilled water and rinsed in ultrapure water to remove impurities. Then the teeth were mounted in epoxy resin and sliced longitudinally with a diamond saw.
Four HA calibration materials containing cobalt (Co), magnesium (Mg), manganese (Mn), nickel (Ni), vanadium (V) and strontium (Sr) were prepared following the co-precipitation procedure reported by Ugarte et al. [39]. The four calibration samples were labeled S1 through S4 by increasing nominal concentration: 0 (i.e. blank), 216, 650 and 1083 μg g−1. HA was precipitated from pure calcium nitrate 2
Spectrochimica Acta Part B 159 (2019) 105650
M. Martinez, et al.
Fig. 1. (Left) Maps of LIBS signal (normalized to the maximum signal in S4 for each element) for a cross section from the spiked HA pellets for each final reference material samples. Refer to Table 1 (fourth column) for the concentration values. (Right) Calibration curves obtained from the four reference material samples. The red line shown the linear model and the red shaded area is the 95% confidence area for this model. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) 3
Spectrochimica Acta Part B 159 (2019) 105650
M. Martinez, et al.
Fig. 2. (Left)Teeth samples used for validation. The red square is the area mapped by LIBS, the dotted yellow area being sampled for further ICP-MS analysis. (Center) Sr concentration maps obtained by LIBS within the red rectangle. The scale is shown on the top right of the figure from 0 to 300 μg.g−1. (Right) Distribution of the Sr LIBS concentration within the yellow dotted area, in comparison with the corresponding bulk ICP-MS value (red line). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
average value. A normalization step was added to reduce the effect of a potential difference in material density between the standards (considered similar between them) and the samples. The determination coefficient for each linear calibration curve is shown as well, with a lowest value of R2 = 0.983 for Mn and highest of R2 = 0.996 for Ni. The LOD and LOQ were determined from the 95% confidence bands of the linear model [47]. The different reference samples were cross-sectioned with a diamond saw to measure the bulk homogeneity of the element distribution. Fig. 1 (left section) shows the elemental concentration mapping obtained using the LIBS calibration curve through the section of different standard material. These figures demonstrate that the doping process was homogenous inside the standard material. Co and Mn present an outlier region on one edge of their cross-section, certainly because that part was damaged after cutting.
Table 3 Comparison between ICP-MS and LIBS on the two validation samples. Sample
Tooth 1 Tooth 2
Ni (μg.g−1)
Sr (μg.g−1)
ICP-MS
LIBS
ICP-MS
LIBS
36 ± 2 Not detected
Not detected Not detected
105 ± 5 115 ± 6
105 ± 25 139 ± 40
3. Results and discussion 3.1. Elemental recovery During the synthesis of the doped HA phase, dopant losses (including poor co-precipitation, evaporation loss during calcination and sintering) and contamination may occur. Recovery measurements at the different steps of the synthesis process can help correct and improve the procedure in the future. Table 1 shows the ICP-MS concentration results for three different steps in the synthesis of the standard materials. As one could expect, the optimal conditions for HA precipitation are not conducive to a complete incorporation of the analytes selected for this study. Furthermore, after calcination, only about 50 to 60% of the expected analyte concentration was recovered, while the blanks showed some contamination. After sintering, very negligible loss was observed for the larger concentrations (samples S3 and S4), whereas losses were larger in the lower concentration pellets (S1 and S2). For instance, Ni decreased by 70% from 138 to 41 μg.g−1 in pellet S2. These losses allowed the blank pellets to have Co, Mn and Ni in concentration below the detection limit for the ICP-MS.
3.3. Validation by LIBS elemental imaging and ICP-MS analysis Two teeth samples were analyzed under the same LIBS conditions as the calibration materials in order to validate the use of the reference materials for quantitative analysis. An elemental image was obtained using the calibration curves established above. Fig. 2 shows the elemental concentration maps for Sr using LIBS. The red rectangle represent the section of the teeth that was imaged by LIBS. The yellow dotted area shows the location that was sampled by a grinding tool after LIBS analysis for counter-analysis by ICP-MS (respectively 10 and 17 mm3 for the two samples). The ICP-MS results are shown in Table 3. Only Ni and Sr showed concentrations above LOQ for ICP-MS. The corresponding LIBS measurements within the same location are displayed as a histogram summarizing the Sr concentration distribution in each region of interest (Fig. 2, right section) and compared to the ICPMS result (vertical line). The first two samples show an average concentration for the area close to the ICP-MS result. Table 3 shows the average LIBS concentrations and their standard deviation for the data extracted from the yellow dotted area, and the corresponding ICP-MS values. These Sr concentration results are consistent with values expected in human teeth, as Hare et al. [48] reported concentrations around 120 μg g−1 for modern samples.
3.2. Calibration curve and homogeneity on HA standard material Calibration curves were built with the LIBS signal from each of the 25 points of a 2 × 2 mm2 grid on three different locations on the pellet surface (in order to take in account any inhomogeneity on the surface) for a total of 75 points per sample. From each spectrum, the signal from the singly ionized species of Co, Mn, Ni, Sr and V emission lines (as well as Ca for normalization – Ca I emission line at 442.6 nm or Ca II emission line at 220.86 nm for the corresponding ionic stage of the analyte) was integrated. The list of emission lines used for the data extraction are shown in Table 2. The normalized signal for the 75 points was averaged to build the calibration curve versus the concentration obtained by ICP-MS for every standard material (Fig. 1, right section). The error bars represent the 1σ standard deviation on each side of the
4. Conclusion Targeting the lack of matrix-matched standards for the LIBS analysis of calcified or osseous materials, this paper introduces the synthesis of ablation-matched calibrated hydroxyapatite ceramic materials made by co-precipitation of five analytes. The incorporation of these analytes 4
Spectrochimica Acta Part B 159 (2019) 105650
M. Martinez, et al.
proved to be homogeneous throughout the bulk and could be used as improved solid reference material in the analysis of bones, teeth or calcifications in laser-ablation-based analytical techniques. We demonstrated the good agreement between bulk ICP-MS analysis and spatially resolved LIBS studies on two teeth samples. While more work is necessary to improve on the incorporation of more elements into this reference matrix, this high-density hydroxyapatite opens the way to new certified materials and the quantitative use of LIBS for multiple fields of research such as biological archeology, forensic anthropology, and cancer research with calcified tissues.
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5
Contents lists available at ScienceDirect
Spectrochimica Acta Part B journal homepage: www.elsevier.com/locate/sab
Multi-elemental matrix-matched calcium hydroxyapatite reference materials for laser ablation: Evaluation on teeth by laser-induced breakdown spectroscopy☆
T
Mauro Martineza, Courtney Bayneb, Dylan Aiellob, Matthew Julianc, Romain Gaumec,d,e, ⁎ Matthieu Baudeleta,b,e, a
National Center for Forensic Science, University of Central Florida, Orlando, FL 32826, USA Chemistry Department, University of Central Florida, Orlando, FL 32816, USA Materials Science and Engineering Department, National Center for Forensic Science, 12354 Research Parkway Suite 225, Orlando, FL 32826, USA d NanoScience Technology Center, University of Central Florida, Orlando, FL 32816, USA e CREOL – The College of Optics and Photonics, University of Central Florida, Orlando, FL 32816, USA b c
ARTICLE INFO
ABSTRACT
Keywords: LIBS Standards Hydroxyapatite Osseous material
Laser ablation has become the most accepted method for direct sampling of solid materials, as in elemental trace analysis by laser-induced breakdown spectroscopy. However, the scarcity of matrix-matched standards or reference materials (such as NIST reference materials for instance) is an obstacle to quantitative analysis, particularly in the area of hard biological materials analysis, such as bones and teeth. If existing, the corresponding certified standards often consist of powders, then compacted as pellets which density, cohesive strength and dopant incorporation are not always representative of the samples under investigation, thereby casting serious uncertainties on calibration protocols. Here, we report on the synthesis of hydroxyapatite reference ceramic materials that can be doped controllably with various elements of interest. The fabrication process involves spiking during synthesis, prior to a hard sintering of pelletized ceramics. As a result, these materials have good chemical homogeneity and ablation properties that mimic those of naturally calcified biological tissues.
1. Introduction Bones, teeth, and calcified tissues present a notable importance in the fields of forensics, archeology, anthropology and medical diagnostics. These materials can provide information about health, diet, age, sex and migrations [1–6], and help in the post-mortem identification of an individual [7]. Hydroxyapatite (HA), with chemical formula Ca10(PO4)6(OH)2, is the inorganic phase present in most of these calcified materials. Synthetic HA has received much attention in recent years due to its excellent biocompatibility [8,9] and is usually prepared by a wet-chemistry approach. Depending on the precipitation conditions, including pH, ionic strength, temperature and the presence of structuring agents, HA can be obtained with a calcium-to‑phosphorus molar ratio similar to that of natural bone and teeth [10], thus, rendering this material an ideal candidate for clinical applications [9,11]. Elemental analysis is one of the many strategies used to extract valuable information from bones, teeth and calcifications. Different
techniques have been used in the past for this purpose. X-ray fluorescence (XRF) [12,13] is typically used to analyze major and trace elements [14,15], along with micro-XRF [16], although this technique is not sensitive enough to very light elements and the limits of detection (LOD) are between tens and hundreds of μg.g−1. Inductively coupled plasma – mass spectrometry (ICP-MS) is also used for bulk analysis of trace elements [17,18]. This technique achieves the best LOD for a wide range of elements. However, the sample is consumed in the digestion process and any information about elemental distribution is lost. Laser ablation (LA-) ICP-MS has proven advantageous because of its unique solid sampling approach, while maintaining low LODs. By providing elemental imaging capability and avoiding sample destruction, LA-ICPMS has opened new perspectives in elemental distribution imaging and the recovery of elemental incorporation history in bones, teeth, and calcified tissues [3,19–23]. Similarly, laser-induced breakdown spectroscopy (LIBS) [24,25] has also been successfully applied to qualitative and quantitative elemental analysis of calcified materials [26–29].
Selected Paper from the 10th International Conference on Laser-Induced Breakdown Spectroscopy (LIBS 2018) held in Atlanta, GA, USA, October 21–26 2018. Corresponding author at: National Center for Forensic Science, University of Central Florida, Orlando, FL 32826, USA. E-mail address: [email protected] (M. Baudelet).
☆ ⁎
https://doi.org/10.1016/j.sab.2019.105650 Received 19 March 2019; Received in revised form 17 July 2019; Accepted 18 July 2019 Available online 19 July 2019 0584-8547/ © 2019 Elsevier B.V. All rights reserved.
Spectrochimica Acta Part B 159 (2019) 105650
M. Martinez, et al.
Specific examples include the use of LIBS in strontium (Sr) quantification in teeth [30,31], the investigation of carious teeth ex vivo as well as in vivo [32], the qualitative and quantitative analysis of kidney stones [33,34], and the study of paleoclimate in calcified materials [35,36]. However, one of the major challenges in LA-based elemental analysis techniques of calcified materials is the availability of proper calibrated materials. Previous studies report the use of NIST SRM 610 or 612 glass reference materials [19,37,38], although ablation of glass and calcified materials is different and may lead to incorrect quantification results [39], or bone meal (SRM 1486) and bone ash (SRM 1400) [40]. A limitation of the glass standards concerns the available concentration range for calibration. In an effort to obtain adequate calibration materials, chemically similar to naturally-occurring calcified materials, with diverse dopants and over a wide concentration range, Ugarte et al. [39] reported the development of a matrix-matched HA calibration material for LA-ICP-MS. Their approach consisted in co-precipitating HA with analytes of interest and pelletizing these synthesized powders for calibration. Their synthesis showed quantitative analyte co-precipitation as well as low LODs, and they used this reference material for the analysis of fish dorsal spine. This methodology was used for similar applications in the quantification of Mn in teeth and bones by LA-ICPMS [41,42]. However, particle size and pellet compaction can both affect the ablation process yielding variations in the calibration curves [43]. A natural extension to this approach consists in increasing the density of the HA pellets by a sintering process [10,44,45], thereby reducing the difference in sample strength and ablation behavior between the standard and the sample. This work proposes a protocol for the preparation of HA reference materials being spiked during co-precipitation, pelletized and sintered. With this process, we obtained solid standard materials in pellet forms that can be used for elemental quantification analysis of calcified materials using LIBS. Traditional ICP-MS was used to verify and validate the data obtained by LIBS on teeth using these reference materials.
Table 1 Concentration (in μg.g−1) in the doped HA reference materials after calcination and sintering (obtained by ICP-MS, 1σ standard deviation between parentheses).
2. Experimental
tetrahydrate (Ca(NO3)2·4H2O, Acros Organic) and ammonium dihydrogen phosphate (NH4H2PO4, Acros Organic). Aqueous solutions of NH4H2PO4 (0.48 M) and Ca(NO3)2·4H2O (1 M) were prepared in ultrapure water (18 MΩ) and pH-adjusted to a value of 10 by adding ammonium hydroxide (NH4OH, Fisher Scientific). A well-stirred solution of analytes of interest was added at a concentration of 1000 μg.g−1 to the calcium nitrate solution. The concentration of each element in the spiking solutions was adjusted to achieve suitable concentrations on the desired HA standards. The Ca(NO3)2·4H2O solution was added dropwise and under magnetic stirring to the NH4H2PO4 solution. The mixture was then shaken on an orbital shaker and aged for 5 days. The precipitate was subsequently filtered, washed with ultra-pure water, dried in an oven at 80 °C overnight, lightly ground in an alumina mortar, and calcined at 450 °C for three hours. This new hydroxyapatite crystalline powder was pelletized in a die through uniaxial pressing at 2.5 MPa, followed by cold isostatic pressing at 200 MPa. The resulting samples were consolidated by pressure-less sintering process in air at 1200 °C, using heating and cooling rates of 2 °C/min and a soaking time of 2 h. A part of each pellet was dissolved for the determination of the elemental content by ICP-MS and the calculation of the recovery percentage.
Sample: nominal concentration S1: 0 μg.g
−1
S2: 216 μg.g−1
S3: 650 μg.g−1
S4: 1083 μg.g−1
Element
After calcination
After sintering
Co Mn V Ni Sr Co Mn V Ni Sr Co Mn V Ni Sr Co Mn V Ni Sr
15.4 (0.2) 9.0 (0.5) Not detected 57 (4) 54.2 (2) 71 (2) 130 (2) 137 (7) 138 (6) 120 (3) 340 (5) 345 (5) 375 (12) 275 (3) 234 (1) 630 (14) 621 (8) 694 (13) 670 (6) 414 (4)
Not detected Not detected Not detected Not detected 7 (3) 64 (4.4) 58 (1) 85 (6) 41 (4) 45 (1) 338 (4) 351 (6) 382 (12) 213 (5) 204 (3) 660 (6) 641 (6) 709 (8) 650 (14) 403 (3)
Table 2 Figures of merit for the HA standard material analyzed by LIBS.
2.1. Instrumentation LIBS data was acquired using a J200 LIBS system (Applied Spectra Inc., Fremont, CA). The instrument consists of a 266 nm Nd:YAG laser (8 ns) with variable energy and ablation spot size, and a 5-channel spectrometer covering a spectral range from 185 to 1040 nm (acquisition delay of 1 μs for 1 ms acquisition duration) in order to obtain a good signal-to-noise ratio. An argon background atmosphere (flow of 1 L/min) is created to avoid interaction with nitrogen and oxygen in the air. HA calibration materials were analyzed with 100 μm spot size, using 10 laser pulses per location with a total of 5 × 5 = 25 locations, every spot separated by 215 μm to cover over an approximate 2 × 2 mm2 area, and an energy of 15 mJ to build the calibration curve. In order to take in account any inhomogeneity on the surface, three 5 × 5 grids were measured per sample. To evaluate the internal homogeneity on the standard material, a transversal cross-section was obtained and imaged by LIBS. The elemental imaging of the teeth samples was performed with the same laser ablation parameters. Data processing was performed using Applied Spectra's Aurora Data Analysis Software package. ICP-MS analysis was performed with a PlasmaQuant Elite (Analytik Jena, Germany).
Analyte
Wavelength
R2
LOD (μg.g−1)
LOQ (μg.g−1)
Co I Mn II V II Ni II Sr II
345.3 nm 257.6 nm 309.3 nm 221.6 nm 407.7 nm
0.994 0.983 0.986 0.996 0.994
49 130 55 42 21
91 219 101 78 38
2.3. Validation samples
2.2. Preparation of hydroxyapatite reference materials
The validation of the reference materials was performed by analyzing two different human teeth obtained from an archaeological collection [46]. Prior to analyses, the teeth were repeatedly washed in an ultrasonic bath for 10 min in distilled water and rinsed in ultrapure water to remove impurities. Then the teeth were mounted in epoxy resin and sliced longitudinally with a diamond saw.
Four HA calibration materials containing cobalt (Co), magnesium (Mg), manganese (Mn), nickel (Ni), vanadium (V) and strontium (Sr) were prepared following the co-precipitation procedure reported by Ugarte et al. [39]. The four calibration samples were labeled S1 through S4 by increasing nominal concentration: 0 (i.e. blank), 216, 650 and 1083 μg g−1. HA was precipitated from pure calcium nitrate 2
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Fig. 1. (Left) Maps of LIBS signal (normalized to the maximum signal in S4 for each element) for a cross section from the spiked HA pellets for each final reference material samples. Refer to Table 1 (fourth column) for the concentration values. (Right) Calibration curves obtained from the four reference material samples. The red line shown the linear model and the red shaded area is the 95% confidence area for this model. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) 3
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Fig. 2. (Left)Teeth samples used for validation. The red square is the area mapped by LIBS, the dotted yellow area being sampled for further ICP-MS analysis. (Center) Sr concentration maps obtained by LIBS within the red rectangle. The scale is shown on the top right of the figure from 0 to 300 μg.g−1. (Right) Distribution of the Sr LIBS concentration within the yellow dotted area, in comparison with the corresponding bulk ICP-MS value (red line). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
average value. A normalization step was added to reduce the effect of a potential difference in material density between the standards (considered similar between them) and the samples. The determination coefficient for each linear calibration curve is shown as well, with a lowest value of R2 = 0.983 for Mn and highest of R2 = 0.996 for Ni. The LOD and LOQ were determined from the 95% confidence bands of the linear model [47]. The different reference samples were cross-sectioned with a diamond saw to measure the bulk homogeneity of the element distribution. Fig. 1 (left section) shows the elemental concentration mapping obtained using the LIBS calibration curve through the section of different standard material. These figures demonstrate that the doping process was homogenous inside the standard material. Co and Mn present an outlier region on one edge of their cross-section, certainly because that part was damaged after cutting.
Table 3 Comparison between ICP-MS and LIBS on the two validation samples. Sample
Tooth 1 Tooth 2
Ni (μg.g−1)
Sr (μg.g−1)
ICP-MS
LIBS
ICP-MS
LIBS
36 ± 2 Not detected
Not detected Not detected
105 ± 5 115 ± 6
105 ± 25 139 ± 40
3. Results and discussion 3.1. Elemental recovery During the synthesis of the doped HA phase, dopant losses (including poor co-precipitation, evaporation loss during calcination and sintering) and contamination may occur. Recovery measurements at the different steps of the synthesis process can help correct and improve the procedure in the future. Table 1 shows the ICP-MS concentration results for three different steps in the synthesis of the standard materials. As one could expect, the optimal conditions for HA precipitation are not conducive to a complete incorporation of the analytes selected for this study. Furthermore, after calcination, only about 50 to 60% of the expected analyte concentration was recovered, while the blanks showed some contamination. After sintering, very negligible loss was observed for the larger concentrations (samples S3 and S4), whereas losses were larger in the lower concentration pellets (S1 and S2). For instance, Ni decreased by 70% from 138 to 41 μg.g−1 in pellet S2. These losses allowed the blank pellets to have Co, Mn and Ni in concentration below the detection limit for the ICP-MS.
3.3. Validation by LIBS elemental imaging and ICP-MS analysis Two teeth samples were analyzed under the same LIBS conditions as the calibration materials in order to validate the use of the reference materials for quantitative analysis. An elemental image was obtained using the calibration curves established above. Fig. 2 shows the elemental concentration maps for Sr using LIBS. The red rectangle represent the section of the teeth that was imaged by LIBS. The yellow dotted area shows the location that was sampled by a grinding tool after LIBS analysis for counter-analysis by ICP-MS (respectively 10 and 17 mm3 for the two samples). The ICP-MS results are shown in Table 3. Only Ni and Sr showed concentrations above LOQ for ICP-MS. The corresponding LIBS measurements within the same location are displayed as a histogram summarizing the Sr concentration distribution in each region of interest (Fig. 2, right section) and compared to the ICPMS result (vertical line). The first two samples show an average concentration for the area close to the ICP-MS result. Table 3 shows the average LIBS concentrations and their standard deviation for the data extracted from the yellow dotted area, and the corresponding ICP-MS values. These Sr concentration results are consistent with values expected in human teeth, as Hare et al. [48] reported concentrations around 120 μg g−1 for modern samples.
3.2. Calibration curve and homogeneity on HA standard material Calibration curves were built with the LIBS signal from each of the 25 points of a 2 × 2 mm2 grid on three different locations on the pellet surface (in order to take in account any inhomogeneity on the surface) for a total of 75 points per sample. From each spectrum, the signal from the singly ionized species of Co, Mn, Ni, Sr and V emission lines (as well as Ca for normalization – Ca I emission line at 442.6 nm or Ca II emission line at 220.86 nm for the corresponding ionic stage of the analyte) was integrated. The list of emission lines used for the data extraction are shown in Table 2. The normalized signal for the 75 points was averaged to build the calibration curve versus the concentration obtained by ICP-MS for every standard material (Fig. 1, right section). The error bars represent the 1σ standard deviation on each side of the
4. Conclusion Targeting the lack of matrix-matched standards for the LIBS analysis of calcified or osseous materials, this paper introduces the synthesis of ablation-matched calibrated hydroxyapatite ceramic materials made by co-precipitation of five analytes. The incorporation of these analytes 4
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proved to be homogeneous throughout the bulk and could be used as improved solid reference material in the analysis of bones, teeth or calcifications in laser-ablation-based analytical techniques. We demonstrated the good agreement between bulk ICP-MS analysis and spatially resolved LIBS studies on two teeth samples. While more work is necessary to improve on the incorporation of more elements into this reference matrix, this high-density hydroxyapatite opens the way to new certified materials and the quantitative use of LIBS for multiple fields of research such as biological archeology, forensic anthropology, and cancer research with calcified tissues.
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