Lasers Med Sci DOI 10.1007/s10103-016-1905-z
ORIGINAL ARTICLE
Analysis of heterogeneous gallstones using laser-induced breakdown spectroscopy (LIBS) and wavelength dispersive X-ray fluorescence (WD-XRF) Brij Bir S. Jaswal 1 & Vinay Kumar 1 & Jitendra Sharma 1 & Pradeep K. Rai 2 & Mohammed A. Gondal 3 & Bilal Gondal 4 & Vivek K. Singh 1
Received: 22 December 2015 / Accepted: 5 February 2016 # Springer-Verlag London 2016
Abstract Laser-induced breakdown spectroscopy (LIBS) is an emerging analytical technique with numerous advantages such as rapidity, multi-elemental analysis, no specific sample preparation requirements, non-destructiveness, and versatility. It has been proven to be a robust elemental analysis tool attracting interest because of being applied to a wide range of materials including biomaterials. In this paper, we have performed spectroscopic studies on gallstones which are heterogeneous in nature using LIBS and wavelength dispersive X-ray fluorescence (WD-XRF) techniques. It has been observed that the presence and relative concentrations of trace elements in different kind of gallstones (cholesterol and pigment gallstones) can easily be determined using LIBS technique. From the experiments carried out on gallstones for trace elemental mapping and detection, it was found that LIBS is a robust tool for such biomedical applications. The stone samples studied in the present paper were classified using the Fourier transform infrared (FTIR) spectroscopy. WD-XRF spectroscopy has been applied for the qualitative and quantitative analysis of major and trace elements present in the gallstone which was compared with the LIBS data. The results
* Vivek K. Singh [email protected]
1
Department of Physics, Shri Mata Vaishno Devi University, Kakryal, Katra 182320, Jammu and Kashmir, India
2
Department of Urology and Nephrology, Opal Hospital, DLW Road, Varanasi 221010, Uttar Pradesh, India
3
Laser Research Group, Physics Department, King Fahd University of Petroleum and Minerals, Box 5047, Dhahran 31261, Saudi Arabia
4
Department of Gastroenterology, University of Chicago Pritzker School of Medicine, Chicago, IL 60637, USA
obtained in the present paper show interesting prospects for LIBS and WD-XRF to study cholelithiasis better. Keywords LIBS . WD-XRF . FTIR . Cholesterol gallstones . Pigment gallstone . Trace elements
Introduction Laser-induced breakdown spectroscopy (LIBS) is a wellestablished spectro-chemical technique based on spectral analysis of radiation emitted by a micro-plasma induced on the target surface by a laser pulse. The relative simplicity of the LIBS principle makes this technique remarkably alluring for a huge assortment of uses [1–3]. The first analytical use of laser plasma on surfaces and hence the birth of LIBS is dated back to 1962–1963 [4, 5]. Applications of LIBS are growing rapidly and continue to extend its horizon to include a wide variety of materials. The enthusiasm for LIBS is due to various specialized points of interest over other existing analytical techniques. These advantages include small sample requirements, no requirement for vacuum, minimal or no sample preparation, rapid analysis time, multi-elemental capability, and online and stand-off detection [1, 2]. Furthermore, light elements such as carbon (C), hydrogen (H), nitrogen (N), and oxygen (O), which are the primary chemical constituents of organic samples, can also be analyzed using LIBS [6, 7]. LIBS has demonstrated its ability to analyze a large number of biological specimens such as kidney stones, teeth, bone, nail, skin, microorganisms, and viruses [8–18]. In literature, only few applications of LIBS to gallstones are reported [10, 19–24]. LIBS has been used extensively by Singh et al. [10, 19–21] and Pathak et al. [22, 23] to study the
Lasers Med Sci
different types (cholesterol, mixed, and pigmented) of gallbladder stones. Recently, Gondal et al. [24, 25] developed a laser sensor based on LIBS for analysis of gallbladder stones specifically for determination of heavy metals such as chromium (Cr), lead (Pb), cadmium (Cd), nickel (Ni), and mercury (Hg). In the present paper, we have discussed the application of LIBS to different kinds (cholesterol and pigment) of gallstones. Currently, X-Ray fluorescence (XRF), which is a strong competitor technique of LIBS, is being used to analyze the elemental contents in gallstones and urinary stones [14]. The advantages of XRF are (i) it can be used on-site; (ii) no sample preparation is required; (iii) it is a quick technique; and (iv) theoretically almost all components present in the objects can be determined. In comparison to LIBS, it is a non-destructive technique, whereas LIBS is micro-destructive. The processes of recording, analyzing results, and calibration of the system for quantitative XRF analysis are simpler than in LIBS. Handling of the XRF system is much simpler, and its dimensions are generally smaller than those of a LIBS system. LIBS can perform analysis of stratigraphy, unlike XRF. When calibration of the LIBS system is performed, the determination of concentrations of elements using LIBS in materials is more reliable than with the XRF. Concentration limits for elements that can be detected are lower in the case of LIBS than XRF [14]. Comparing detection limits, LIBS tends to have somewhat lower limits than XRF for many elements. In addition, LIBS is the only technique that has a point detection capability, which is necessary for studying stones samples. The joint use of LIBS and XRF allowed for an accurate determination of stones’ elemental composition, which is helpful in elucidating the exact role of major, minor, and trace elements in their pathogenesis and their etiology as well. In the present paper, we have applied WD-XRF spectroscopy to quantify the major and trace elements present in some of the gallstones and the results are compared with the results obtained from LIBS data. Different kinds of gallstone (cholesterol and pigment) studied in the present work have been studied and classified using FTIR spectroscopy. Fig. 1 Snapshot of gallstone samples used in the present study
Materials and methods The gallstone samples were collected from Opal Hospital, Varanasi, India. The digital photographs of the gallstone samples (G1, G2, G3) were shown in Fig. 1. The received stones were dry and stored in sealed pots after being washed in de-ionized water to remove all traces of urine, blood, and other possible contaminants. The as-received stone samples were examined macroscopically before being powdered in a mortar-pestle arrangement. The powder samples were then used subsequently for experimental studies using FTIR, LIBS, and WD-XRF. The FTIR spectra of gallstone samples were recorded in the transmittance mode in the region, 4000−400 cm−1 with the spectral resolution of 1 cm−1 using the Fourier transform infrared spectrophotometer (Perkin Elmer, model RZX). The qualitative measurements of major and trace elements of gallstones have been performed utilizing LIBS technique. The quantitative measurements of the chemical constituents of few gallstones were performed using WD-XRF spectrometer (Model: S8 TIGER, Make: Bruker, Germany). A 4 KWatt Rh anode X-ray tube equipped with proportional flow counter and scintillation counter detectors has been used as energy source. No calibration was required as samples have been analyzed in standard less software BQuant Express^. For WD-XRF spectrometer, the relative standard deviation (RSD) was 5 % and the limit of detection (LOD) was 1 ppm.
Results and discussion FTIR study of stones The FTIR spectra of gallstone samples (G1, G2, and G3) were recorded in the transmittance mode in the spectral region 4000–400 cm−1 with the spectral resolution of 1 cm−1 which is shown in Fig. 2. The FTIR spectra of gallstone samples were compared with the FTIR spectra of standard cholesterol, bilirubin and calcium carbonate (CaCO3).
Lasers Med Sci Fig. 2 FTIR spectra of gallstone samples (G1, G2, G3) used in the present study
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G1 G3
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1082 1033
603 562
1377 1248 1405
2934 2867
3398
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1668 1626 1571
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855
1056
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3289
% Transmitance
FTIR Spectra of Gallstone Samples G1, G2, G3
1000
500
-1 Wavenumber (cm )
The FTIR spectra of the gallstone samples (G1, G2, G3) were recorded in transmittance mode in the spectral region 4000– 400 cm−1 with spectral resolution 1 cm−1. The overlapped FTIR spectra of gallstone samples (G1, G2, G3) is shown in Fig. 2. The type of stone, the principal chemical components of each type and its corresponding IR bands from our study along with literature values [26–29] are given in Table 1. To make the identification of IR bands simpler, the reference spectra of cholesterol, bilirubin, and CaCO3 were also recorded and compared with the FTIR spectra of gallstone specimens. Cholesterol in the gallstone samples identified by the presence of strong characteristic bands between 2800 and 3000 cm−1 (as shown in Fig. 2) due to asymmetric and symmetric stretching vibration of CH2 and CH3 groups [27]. The bands appeared in Table 1
the FTIR spectrum of the gallstones (G1 and G2) are at 2934, 2867, 1466, 1377, and 1056 cm−1 which are the major and strong bands indicating the presence of cholesterol in the gallstone samples. The observed broad and intense band around 3400 cm−1 is due to −OH stretching of cholesterol molecule. The characteristics bands of cholesterol in gallstones sample G3 have also been appeared but the intensities are very low which indicates the presence of cholesterol in gallstone sample G3 in a less amount than that of gallstones G1 and G2. Concluding all above the major constituents of gallstone sample G1 and G2 is cholesterol and hence the samples G1 and G2 are classified as cholesterol type gallstone. In the FTIR spectrum of gallstone G3, the characteristic bands in the region between 1500 and 1700 cm−1 due to
Type and IR bands of principal and other components observed in gallstones with literature values [25–28]
S. no. Gallstones (Appearance) Classification
Principal component Principal IR bands (other components) observed (cm−1)
Literature value [25–28]
1
White color with center yellowish
Cholesterol gallstone Cholesterol
2934, 2867, 1466, 1377, 1056
2925 (CH2 and CH3 asymmetric stretching), 2860 (CH2 and CH3 symmetric stretching), 1460, 1380 (CH2 and CH3 bending), 1050 (C−C stretching)
2
White with center yellowish and brownish Black
Cholesterol gallstone Cholesterol
2934, 2867, 1466, 1377, 1056
Same as above
Pigment gallstone
1668, 1626, 1571
3
Bilirubin Calcium carbonate
Calcium phosphate
1670, 1640 (OC=O stretching), 1575 (C=C stretching) 873, 855, 712 1082, 1082 (O–C stretching), 875 (C−O bending), 856 (CO23 − out-of-plane deformation), 713 (OCO bending) 1033, 603, 562 1036 (stretching vibrations of PO34 group), 603, 562
C II (657.8 nm)
Pb I (600.2 nm)
Cr I (520.8 nm)
Ca I (442.5 nm)
K I (484.9 nm)
Fe I (364.7 nm)
Na II (267.2 nm) Cr II (286.2 nm)
LIBS Signal Intensity (a.u.)
475.4
475.2
475.0
300
400
500
600
700
800
Wavelength (nm)
Fig. 4 LIBS spectra of cholesterol gallstone(G2) in the spectral range 250–850 nm
pigment gallstone (G3) were Ca, Na, Fe, Ti, Cr, F, Si, Mg, C, K, Br, Ag, Ni, and Na. All the elements present in the gallstones were identified by spectral analysis using the National Institute of Standards and Technology (NIST) database [30]. The spectral lines identified as 768.7 nm for Ag; 518.2 and 815.4 nm for Br; 657.8 nm for C; 286.2, 520.8, and 403.9 nm for Cr; 837.5 and 774.5 nm for Cl; 824.8 and 442.8 nm for Ca; 757.3 and 424.6 nm for F; 364.7 and 268.9 nm for Fe; 484.9 and 691.1 nm for K; 634.7 nm for Mg; 822.3 nm for N; 267.2 nm for Na; 809 nm for Ni; 600.2 nm for Pb; 586.8, 580.7, and 634.7 nm for Si; 774.1 nm for Sn; and 428.7, 500.0, and 346.2 nm for Ti.
480
Ni II (809.6 nm)
495
Ag I (768.7 nm)
510
F II (424.6 nm)
525
Si II (580.7 nm) Si I (623.7 nm) Mg II (634.7 nm) C II (657.8 nm) K I (691.1 nm)
Cr I (403.9 nm)
LIBS spectra of black pigment gallstone (G3)
Ti II (346.2 nm)
Sn II (774.1 nm)
C II (657.8 nm)
K I (691.1 nm)
Si II (586.8 nm)
Ti I (428.7 nm)
K I (484.9 nm) Ti I (500.0 nm) Br II (518.2 nm)
476
Fe I (364.7 nm)
477
LIBS spectra of cholesterol gallstone (G1)
Na II (267.2 nm) Cr II (286.2 nm)
LIBS Signal Intensity (a.u.)
478
Br I (815.4 nm) Ca II (824.8 nm) Cl II (837.5 nm)
The LIBS spectra of cholesterol gallstones (G1, G2) and pigment gallstone (G3) were recorded in the spectral range 250– 850 nm which are shown in Figs. 3, 4, and 5. The elements detected in (i) cholesterol gallstone (G1) were Na, Cr, Fe, Ti, K, Br, Si, C, K, Sn, Ca, and Cl (ii) cholesterol gallstone (G2) were Na, Cr, Fe, Ca, K, Cr, Pb, C, F, N, and Cl; and (iii)
475.6
Fe I (268.9 nm)
LIBS of cholesterol and pigment gallstones (G1, G2, G3)
LIBS spectra of cholesterol gallstone (G2)
LIBS Signal Intensity (a.u.)
stretching vibration of C=C, CO, and C−N at 1571, 1626, and 1668 arising from bilirubin and bilirubinate salts [27]. The broad and strong band nearly at 3398 cm−1 is due to the −OH stretching of cholesterol and NH stretching vibration of pyrrole of bilirubin present in the gallstone sample. In addition to these, some bands have also been appeared particularly at 1248, 699 cm−1. The absorption intensities of these bands in gallstones sample G3 are more pronounced than those of gallstones G1 and G2 indicating bilirubin as the major constituents of gallstone sample G3. Thus, the gallstone sample G3 can be classified as pigment gallstones. In addition to the presence of the characteristic bands of bilirubin, some more bands with measurable intensities have been appeared particularly at 712, 855, and 873 cm−1 which are the characteristics bands of CaCO3 [27]. The strong bands at 1033 cm−1 and the double bonds at 603 and 562 cm−1 come from phosphate [28]. Therefore, complete analyses of FTIR spectra of gallstones revealed that cholesterol was the major component detected with the minute presence of bilirubin in cholesterol type gallstones (G1, G2) and bilirubin was the major component detected in pigmented gallstone (G3). Calcium carbonate and phosphates are also detected in pigmented gallstones. Thus, in conclusion, the stone samples G1 and G2 were classified as cholesterol gallstone and the sample G3 was classified as pigment gallstone.
F I (757.3 nm) Cl I (774.5 nm) N I (822.3 nm ) Cl I (837.5 nm)
Lasers Med Sci
475 300
400
500
600
700
800
Wavelength (nm) Fig. 3 LIBS spectra of cholesterol gallstone (G1) in the spectral range 250–850 nm
300
400
500
600
700
800
Wavelength (nm) Fig. 5 LIBS spectra of black pigment stone in the spectral range 250– 850 nm
Lasers Med Sci
To verify our LIBS data, we have analyzed pigment gallstone qualitatively and quantitatively using WD-XRF spectroscopy. The typical WD-XRF spectrum of pigment gallstone is shown in Fig. 6. The spectrum was analyzed qualitatively which clearly shows the presence of elements particularly C, O, Ca, P, S, Na, Cl, Br, Ru, Fe, Mg, K, Cu, and Si. The LIBS data shows the presence of elements particularly C, Ca, K, Mg, Si, and Br in pigment gallstone which was further confirmed by WD-XRF spectroscopy. Few more elements such as S, Cl, Ru, and Cu were detected using WD-XRF spectroscopy. The elements quantified in pigment gallstone using WDXRF were Na, Cl, S, Ca, P, Br, Ru, and Fe and their concentrations are 0.06, 0.03, 0.02, 0.02, and 0.01 % and 22, 19, and 15 ppm, respectively.
Conclusion Formation of gallstones takes place over a long period of time causing heterogeneous distribution of various elements within the same stone. We have analyzed heterogeneous gallstones utilizing LIBS and WD-XRF techniques. Using LIBS, we
KCps
C KA1
Cu KA1
Fe KA1
Ca KA1
P KA1 S KA1
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2 3
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Na KA1 Mg KA1
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The LIBS signal intensity of the mineral elements detected in pigment stone is greater than that of cholesterol stones and hence reflects its corresponding concentration of elements in stone samples. It was observed that pigment gallstone were rich in minerals and heavy metals. The common elements detected in all samples were C, Ca, Na, Cr, Fe, and K. The presence of C in all types of gallstones is due to the presence of cholesterol and bilirubin in stone samples. The elements Na and Cl are detected in cholesterol gallstones (G1, G2) which may be due to the presence of NaCl in the bile of the patients. Cr is the heavy and toxic metal which is detected in all kinds of gallstones. Recently, Jaswal et al. [31] studied the center and surface parts of cholesterol gallstone (white in color) using time-of-flight secondary ion mass spectrometry (TOFSIMS) and reported the presence of heavy elements particularly, Pb and Cr, which in turn authenticates our results. Elements Br, Ti, and Si are detected only in cholesterol (G1) and pigment stones (G3). The heavy elements particularly Ag and Ni are the only minerals which are detected only in pigment gallstones (G3). Pb and Sn were detected only in cholesterol gallstones G1 and G2, respectively. Fluorine (F) was absent in cholesterol gallstone (G1).
0,2
0,4
0,6
1
2
3
4
5
6
7
8
9
10
20
KeV
Fig. 6 A typical WD-XRF spectra of black pigment stone
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54
56
Lasers Med Sci
have successfully analyzed a large number of elements such as Na, Cr, Fe, Ti, K, Br, Si, C, K, Sn, Ca, Cl, Pb, F, N, F, Mg, Br, Ag, and Ni in gallstone samples. Qualitative and quantitative analysis of pigment gallstone have been done using LIBS and WD-XRF techniques. Using WD-XRF, the elements detected successfully were C, O, Ca, P, S, Na, Cl, Br, Ru, Fe, Mg, K, Cu, and Si; however, the LIBS data shows the presence of elements particularly C, Ca, K, Mg, Si, and Br in pigment gallstone which was confirmed by WD-XRF data. Using WD-XRF, the quantified elements in pigment gallstone were Na, Cl, S, Ca, P, Br, Ru, and Fe. In LIBS, for the quantitative analysis of biological samples such as gallstones certified reference materials (CRM) are required to draw the calibration curves and to get the CRM for heterogeneous gallstones is extremely difficult and also LIBS is very sensitive to matrix effect. However, no calibration is required in WD-XRF as samples have been analyzed in standard less software BQuant Express^. Thus, the joint use of LIBS and WD-XRF allow accurate determination of stones’ elemental composition, which is helpful in elucidating the exact role of trace elements in their pathogenesis and their etiology as well. FTIR has been used for the successful characterization of cholesterol and pigment gallstones. In addition to cholesterol and bilirubin, calcium carbonate and calcium phosphate are also noticed in black pigment gallstone. Our results revealed that LIBS and WD-XRF are the promising techniques which are capable to analyze stone samples present inside the human body. The results obtained in the present paper show interesting prospects for LIBS and WDXRF to study cholelithiasis better. Acknowledgments We are thankful to Central Instrumentation Laboratory (CIL) and Sophisticated Analytical Instrumentation Facility (SAIF), Punjab University, Chandigarh for providing FT-IR and WDXRF experimental facilities, respectively. The authors are also thankful to Professor S. B. Rai and Prof. S.N. Thakur from the Department of Physics, Banaras Hindu University, Varanasi for their valuable suggestions and discussions during the course of the present work. Prof. Gondal is thankful to KFUPM for supporting this work through project RG 1421.
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Cremers DA, Radziemski LJ (2006) Handbook of laser-induced breakdown spectroscopy. Wiley & Sons, Chichester 2. Miziolek A, Palleschi V, Schecter I (2006) Laser-induced breakdown spectroscopy (LIBS) fundamentals and applications. Cambridge University Press, New York 3. Singh JP, Thakur SN (2007) Laser-induced breakdown spectroscopy. Amsterdam 4. Brech F, Cross L (1962) Optical microemission stimulated by a ruby maser. Appl Spectrosc 16:59 5. Debras-Guedon J, Liodec N (1963) Comptes Rendus Hebdomadaires des Seances de I’. Acad Sci 257:3336–3339 6. Baudelet M, Boueri M, Yu J, Mao SS, Piseltelli V, Mao XL, Russo RE (2007) Time-resolved ultraviolet laser-induced breakdown spectroscopy for organic material analysis. Spectrochim Acta B 62:1329–1334
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Li J, Lu J, Lin Z, Gong S, Xie C, Chang L, Yang L, Li P (2009) Effects of experimental parameters on elemental analysis of coal by laserinduced breakdown spectroscopy. Opt Laser Technol 41(8):907–913 Fang X, Ahmad SR, Mayo M, Iqbal S (2005) Elemental analysis of urinary calculi by laser induced plasma spectroscopy. Lasers Med Sci 20:132–137 Singh VK, Rai AK, Rai PK, Jindal PK (2009) Cross-sectional study of kidney stones by laser-induced breakdown spectroscopy. Lasers Med Sci 24:749–759 Singh VK, Singh V, Rai AK, Thakur SN, Rai PK, Singh JP (2008) Quantitative analysis of gallstones using laser-induced breakdown spectroscopy. Appl Opt 47(31):G38–G47 Singh VK, Kumar V, Sharma J (2015) Importance of laser-induced breakdown spectroscopy for hard tissues (bone, teeth) and other calcified tissue materials. Laser Med Sci 30:1763–1778 Singh VK, Rai AK (2011) Prospects for laser-induced breakdown spectroscopy for biomedical applications: a review. Laser Med Sci 26:673–687 Singh VK, Kumar V, Sharma J, Khajuria Y (2014) Importance of laser induced breakdown spectroscopy for biomedical applications: a comprehensive review. Mater Focus 3:169–182 Jaswal BBS, Singh VK (2015) Analytical assessments of gallstones and urinary stones: a comprehensive review of the development from laser to LIBS. Appl Spectrosc Rev 50(6):473–498 Rehse SJ, Salimnia H, Miziolek AW (2012) Laser-induced breakdown spectroscopy (LIBS): an overview of recent progress and future potential for biomedical applications. J Med Eng Technol 36:77–89 Anzano J, Lasheras RJ (2009) Strategies for the identification of urinary calculus by laser induced breakdown spectroscopy. Talanta 79:352–360 Oztoprak BG, Gonzalez J, Yoo J, Gulecen T, Mutlu N, Russo RE, Gundogdu O, Demir A (2012) Analysis and classification of heterogeneous kidney stones using laser induced breakdown spectroscopy (LIBS). Appl Spectrosc 66:1353–1361 Khalil AAI, Gondal MA, Shemis M, Khan IS (2015) Detection of carcinogenic metals in kidney stones using ultraviolet laser-induced breakdown spectroscopy. Appl Opt 54(8):2123–2131 Singh VK, Rai V, Rai AK (2009) Variational study of the constituents of cholesterol stones by laser-induced breakdown spectroscopy. Laser Med Sci 24:27–33 Singh VK, Rai AK (2012) Spatial distribution of minerals across the mixed gallstones using LIBS. Asian J Spectrosc. 133–137 Singh VK, Pathak AK, Rai PK, Rai PK, Jindal PK (2009) LIBS: an efficient technique to study stone formation in human body. Bull Laser Spectrosc Soc India 18:50–72 Pathak AK, Kumar R, Singh VK, Agrawal R, Rai S, Rai AK (2012) Assessment of LIBS for spectrochemical analysis: a review. Appl Spectrosc Rev 47:14–40 Pathak AK, Singh VK (2011) Study of different concentric rings inside gallstones with LIBS. Laser Med Sci 26:531–537 Gondal MA, Shemis MA, Gondal B, Khalil AAI (2016) Gallbladder stones analysis using pulsed UV laser induced breakdown spectroscopy. J Med Bioeng. 5(2). Gondal MA, Shemis MA, Khalil AAI, Nasr MM, Gondal B (2016) Laser Produced Plasma Diagnosis of Carcinogenic Heavy Metals in Gallstones. J Anal At Spectrom 31:506–514 Zhou XS, Shen GR, Wu JG, Li WH, Xu YZ, Weng SF, Soloway RD, Fu XB, Tian W, Xu Z, Shen T, Xu GX, Wentrup-Byrne E (1997) A spectroscopic study of pigment gallstones in China. Biospectroscopy 3:371–380 Kleiner O, Ramesh J, Huleihel M, Cohen B, Kantarovich K, Levi C, Polyak B, Marks RS, Mordehai J, Cohen Z, Mordechai S (2002) A comparative study of gallstones from children and adults using FTIR spectroscopy and fluorescence microscopy. BMC Gastroenterol. 2.
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Liu G, Xing D, Yang H, Wu J (2002) Vibrational spectroscopic study of human pigment gallstones and their insoluble materials. J Mol Struct 616:187–191 Gupta U, Singh VK, Kumar V, Khajuria Y (2015) Experimental and theoretical spectroscopic studies of calcium carbonate (CaCO3). Mater Focus 4:164–169
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NIST Atomic Spectra Database, http://physics.nist.gov/ PhysRefData/ASD/lines_form.html Jaswal BBS, Kumar V, Swart HC, Sharma J, Rai PK, Singh VK (2015) Multi-spectroscopic analysis of cholesterol gallstone using TOF-SIMS, FTIR and UV–vis spectroscopy. Appl Phys B 121:49– 56
ORIGINAL ARTICLE
Analysis of heterogeneous gallstones using laser-induced breakdown spectroscopy (LIBS) and wavelength dispersive X-ray fluorescence (WD-XRF) Brij Bir S. Jaswal 1 & Vinay Kumar 1 & Jitendra Sharma 1 & Pradeep K. Rai 2 & Mohammed A. Gondal 3 & Bilal Gondal 4 & Vivek K. Singh 1
Received: 22 December 2015 / Accepted: 5 February 2016 # Springer-Verlag London 2016
Abstract Laser-induced breakdown spectroscopy (LIBS) is an emerging analytical technique with numerous advantages such as rapidity, multi-elemental analysis, no specific sample preparation requirements, non-destructiveness, and versatility. It has been proven to be a robust elemental analysis tool attracting interest because of being applied to a wide range of materials including biomaterials. In this paper, we have performed spectroscopic studies on gallstones which are heterogeneous in nature using LIBS and wavelength dispersive X-ray fluorescence (WD-XRF) techniques. It has been observed that the presence and relative concentrations of trace elements in different kind of gallstones (cholesterol and pigment gallstones) can easily be determined using LIBS technique. From the experiments carried out on gallstones for trace elemental mapping and detection, it was found that LIBS is a robust tool for such biomedical applications. The stone samples studied in the present paper were classified using the Fourier transform infrared (FTIR) spectroscopy. WD-XRF spectroscopy has been applied for the qualitative and quantitative analysis of major and trace elements present in the gallstone which was compared with the LIBS data. The results
* Vivek K. Singh [email protected]
1
Department of Physics, Shri Mata Vaishno Devi University, Kakryal, Katra 182320, Jammu and Kashmir, India
2
Department of Urology and Nephrology, Opal Hospital, DLW Road, Varanasi 221010, Uttar Pradesh, India
3
Laser Research Group, Physics Department, King Fahd University of Petroleum and Minerals, Box 5047, Dhahran 31261, Saudi Arabia
4
Department of Gastroenterology, University of Chicago Pritzker School of Medicine, Chicago, IL 60637, USA
obtained in the present paper show interesting prospects for LIBS and WD-XRF to study cholelithiasis better. Keywords LIBS . WD-XRF . FTIR . Cholesterol gallstones . Pigment gallstone . Trace elements
Introduction Laser-induced breakdown spectroscopy (LIBS) is a wellestablished spectro-chemical technique based on spectral analysis of radiation emitted by a micro-plasma induced on the target surface by a laser pulse. The relative simplicity of the LIBS principle makes this technique remarkably alluring for a huge assortment of uses [1–3]. The first analytical use of laser plasma on surfaces and hence the birth of LIBS is dated back to 1962–1963 [4, 5]. Applications of LIBS are growing rapidly and continue to extend its horizon to include a wide variety of materials. The enthusiasm for LIBS is due to various specialized points of interest over other existing analytical techniques. These advantages include small sample requirements, no requirement for vacuum, minimal or no sample preparation, rapid analysis time, multi-elemental capability, and online and stand-off detection [1, 2]. Furthermore, light elements such as carbon (C), hydrogen (H), nitrogen (N), and oxygen (O), which are the primary chemical constituents of organic samples, can also be analyzed using LIBS [6, 7]. LIBS has demonstrated its ability to analyze a large number of biological specimens such as kidney stones, teeth, bone, nail, skin, microorganisms, and viruses [8–18]. In literature, only few applications of LIBS to gallstones are reported [10, 19–24]. LIBS has been used extensively by Singh et al. [10, 19–21] and Pathak et al. [22, 23] to study the
Lasers Med Sci
different types (cholesterol, mixed, and pigmented) of gallbladder stones. Recently, Gondal et al. [24, 25] developed a laser sensor based on LIBS for analysis of gallbladder stones specifically for determination of heavy metals such as chromium (Cr), lead (Pb), cadmium (Cd), nickel (Ni), and mercury (Hg). In the present paper, we have discussed the application of LIBS to different kinds (cholesterol and pigment) of gallstones. Currently, X-Ray fluorescence (XRF), which is a strong competitor technique of LIBS, is being used to analyze the elemental contents in gallstones and urinary stones [14]. The advantages of XRF are (i) it can be used on-site; (ii) no sample preparation is required; (iii) it is a quick technique; and (iv) theoretically almost all components present in the objects can be determined. In comparison to LIBS, it is a non-destructive technique, whereas LIBS is micro-destructive. The processes of recording, analyzing results, and calibration of the system for quantitative XRF analysis are simpler than in LIBS. Handling of the XRF system is much simpler, and its dimensions are generally smaller than those of a LIBS system. LIBS can perform analysis of stratigraphy, unlike XRF. When calibration of the LIBS system is performed, the determination of concentrations of elements using LIBS in materials is more reliable than with the XRF. Concentration limits for elements that can be detected are lower in the case of LIBS than XRF [14]. Comparing detection limits, LIBS tends to have somewhat lower limits than XRF for many elements. In addition, LIBS is the only technique that has a point detection capability, which is necessary for studying stones samples. The joint use of LIBS and XRF allowed for an accurate determination of stones’ elemental composition, which is helpful in elucidating the exact role of major, minor, and trace elements in their pathogenesis and their etiology as well. In the present paper, we have applied WD-XRF spectroscopy to quantify the major and trace elements present in some of the gallstones and the results are compared with the results obtained from LIBS data. Different kinds of gallstone (cholesterol and pigment) studied in the present work have been studied and classified using FTIR spectroscopy. Fig. 1 Snapshot of gallstone samples used in the present study
Materials and methods The gallstone samples were collected from Opal Hospital, Varanasi, India. The digital photographs of the gallstone samples (G1, G2, G3) were shown in Fig. 1. The received stones were dry and stored in sealed pots after being washed in de-ionized water to remove all traces of urine, blood, and other possible contaminants. The as-received stone samples were examined macroscopically before being powdered in a mortar-pestle arrangement. The powder samples were then used subsequently for experimental studies using FTIR, LIBS, and WD-XRF. The FTIR spectra of gallstone samples were recorded in the transmittance mode in the region, 4000−400 cm−1 with the spectral resolution of 1 cm−1 using the Fourier transform infrared spectrophotometer (Perkin Elmer, model RZX). The qualitative measurements of major and trace elements of gallstones have been performed utilizing LIBS technique. The quantitative measurements of the chemical constituents of few gallstones were performed using WD-XRF spectrometer (Model: S8 TIGER, Make: Bruker, Germany). A 4 KWatt Rh anode X-ray tube equipped with proportional flow counter and scintillation counter detectors has been used as energy source. No calibration was required as samples have been analyzed in standard less software BQuant Express^. For WD-XRF spectrometer, the relative standard deviation (RSD) was 5 % and the limit of detection (LOD) was 1 ppm.
Results and discussion FTIR study of stones The FTIR spectra of gallstone samples (G1, G2, and G3) were recorded in the transmittance mode in the spectral region 4000–400 cm−1 with the spectral resolution of 1 cm−1 which is shown in Fig. 2. The FTIR spectra of gallstone samples were compared with the FTIR spectra of standard cholesterol, bilirubin and calcium carbonate (CaCO3).
Lasers Med Sci Fig. 2 FTIR spectra of gallstone samples (G1, G2, G3) used in the present study
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603 562
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FTIR Spectra of Gallstone Samples G1, G2, G3
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-1 Wavenumber (cm )
The FTIR spectra of the gallstone samples (G1, G2, G3) were recorded in transmittance mode in the spectral region 4000– 400 cm−1 with spectral resolution 1 cm−1. The overlapped FTIR spectra of gallstone samples (G1, G2, G3) is shown in Fig. 2. The type of stone, the principal chemical components of each type and its corresponding IR bands from our study along with literature values [26–29] are given in Table 1. To make the identification of IR bands simpler, the reference spectra of cholesterol, bilirubin, and CaCO3 were also recorded and compared with the FTIR spectra of gallstone specimens. Cholesterol in the gallstone samples identified by the presence of strong characteristic bands between 2800 and 3000 cm−1 (as shown in Fig. 2) due to asymmetric and symmetric stretching vibration of CH2 and CH3 groups [27]. The bands appeared in Table 1
the FTIR spectrum of the gallstones (G1 and G2) are at 2934, 2867, 1466, 1377, and 1056 cm−1 which are the major and strong bands indicating the presence of cholesterol in the gallstone samples. The observed broad and intense band around 3400 cm−1 is due to −OH stretching of cholesterol molecule. The characteristics bands of cholesterol in gallstones sample G3 have also been appeared but the intensities are very low which indicates the presence of cholesterol in gallstone sample G3 in a less amount than that of gallstones G1 and G2. Concluding all above the major constituents of gallstone sample G1 and G2 is cholesterol and hence the samples G1 and G2 are classified as cholesterol type gallstone. In the FTIR spectrum of gallstone G3, the characteristic bands in the region between 1500 and 1700 cm−1 due to
Type and IR bands of principal and other components observed in gallstones with literature values [25–28]
S. no. Gallstones (Appearance) Classification
Principal component Principal IR bands (other components) observed (cm−1)
Literature value [25–28]
1
White color with center yellowish
Cholesterol gallstone Cholesterol
2934, 2867, 1466, 1377, 1056
2925 (CH2 and CH3 asymmetric stretching), 2860 (CH2 and CH3 symmetric stretching), 1460, 1380 (CH2 and CH3 bending), 1050 (C−C stretching)
2
White with center yellowish and brownish Black
Cholesterol gallstone Cholesterol
2934, 2867, 1466, 1377, 1056
Same as above
Pigment gallstone
1668, 1626, 1571
3
Bilirubin Calcium carbonate
Calcium phosphate
1670, 1640 (OC=O stretching), 1575 (C=C stretching) 873, 855, 712 1082, 1082 (O–C stretching), 875 (C−O bending), 856 (CO23 − out-of-plane deformation), 713 (OCO bending) 1033, 603, 562 1036 (stretching vibrations of PO34 group), 603, 562
C II (657.8 nm)
Pb I (600.2 nm)
Cr I (520.8 nm)
Ca I (442.5 nm)
K I (484.9 nm)
Fe I (364.7 nm)
Na II (267.2 nm) Cr II (286.2 nm)
LIBS Signal Intensity (a.u.)
475.4
475.2
475.0
300
400
500
600
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800
Wavelength (nm)
Fig. 4 LIBS spectra of cholesterol gallstone(G2) in the spectral range 250–850 nm
pigment gallstone (G3) were Ca, Na, Fe, Ti, Cr, F, Si, Mg, C, K, Br, Ag, Ni, and Na. All the elements present in the gallstones were identified by spectral analysis using the National Institute of Standards and Technology (NIST) database [30]. The spectral lines identified as 768.7 nm for Ag; 518.2 and 815.4 nm for Br; 657.8 nm for C; 286.2, 520.8, and 403.9 nm for Cr; 837.5 and 774.5 nm for Cl; 824.8 and 442.8 nm for Ca; 757.3 and 424.6 nm for F; 364.7 and 268.9 nm for Fe; 484.9 and 691.1 nm for K; 634.7 nm for Mg; 822.3 nm for N; 267.2 nm for Na; 809 nm for Ni; 600.2 nm for Pb; 586.8, 580.7, and 634.7 nm for Si; 774.1 nm for Sn; and 428.7, 500.0, and 346.2 nm for Ti.
480
Ni II (809.6 nm)
495
Ag I (768.7 nm)
510
F II (424.6 nm)
525
Si II (580.7 nm) Si I (623.7 nm) Mg II (634.7 nm) C II (657.8 nm) K I (691.1 nm)
Cr I (403.9 nm)
LIBS spectra of black pigment gallstone (G3)
Ti II (346.2 nm)
Sn II (774.1 nm)
C II (657.8 nm)
K I (691.1 nm)
Si II (586.8 nm)
Ti I (428.7 nm)
K I (484.9 nm) Ti I (500.0 nm) Br II (518.2 nm)
476
Fe I (364.7 nm)
477
LIBS spectra of cholesterol gallstone (G1)
Na II (267.2 nm) Cr II (286.2 nm)
LIBS Signal Intensity (a.u.)
478
Br I (815.4 nm) Ca II (824.8 nm) Cl II (837.5 nm)
The LIBS spectra of cholesterol gallstones (G1, G2) and pigment gallstone (G3) were recorded in the spectral range 250– 850 nm which are shown in Figs. 3, 4, and 5. The elements detected in (i) cholesterol gallstone (G1) were Na, Cr, Fe, Ti, K, Br, Si, C, K, Sn, Ca, and Cl (ii) cholesterol gallstone (G2) were Na, Cr, Fe, Ca, K, Cr, Pb, C, F, N, and Cl; and (iii)
475.6
Fe I (268.9 nm)
LIBS of cholesterol and pigment gallstones (G1, G2, G3)
LIBS spectra of cholesterol gallstone (G2)
LIBS Signal Intensity (a.u.)
stretching vibration of C=C, CO, and C−N at 1571, 1626, and 1668 arising from bilirubin and bilirubinate salts [27]. The broad and strong band nearly at 3398 cm−1 is due to the −OH stretching of cholesterol and NH stretching vibration of pyrrole of bilirubin present in the gallstone sample. In addition to these, some bands have also been appeared particularly at 1248, 699 cm−1. The absorption intensities of these bands in gallstones sample G3 are more pronounced than those of gallstones G1 and G2 indicating bilirubin as the major constituents of gallstone sample G3. Thus, the gallstone sample G3 can be classified as pigment gallstones. In addition to the presence of the characteristic bands of bilirubin, some more bands with measurable intensities have been appeared particularly at 712, 855, and 873 cm−1 which are the characteristics bands of CaCO3 [27]. The strong bands at 1033 cm−1 and the double bonds at 603 and 562 cm−1 come from phosphate [28]. Therefore, complete analyses of FTIR spectra of gallstones revealed that cholesterol was the major component detected with the minute presence of bilirubin in cholesterol type gallstones (G1, G2) and bilirubin was the major component detected in pigmented gallstone (G3). Calcium carbonate and phosphates are also detected in pigmented gallstones. Thus, in conclusion, the stone samples G1 and G2 were classified as cholesterol gallstone and the sample G3 was classified as pigment gallstone.
F I (757.3 nm) Cl I (774.5 nm) N I (822.3 nm ) Cl I (837.5 nm)
Lasers Med Sci
475 300
400
500
600
700
800
Wavelength (nm) Fig. 3 LIBS spectra of cholesterol gallstone (G1) in the spectral range 250–850 nm
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500
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Wavelength (nm) Fig. 5 LIBS spectra of black pigment stone in the spectral range 250– 850 nm
Lasers Med Sci
To verify our LIBS data, we have analyzed pigment gallstone qualitatively and quantitatively using WD-XRF spectroscopy. The typical WD-XRF spectrum of pigment gallstone is shown in Fig. 6. The spectrum was analyzed qualitatively which clearly shows the presence of elements particularly C, O, Ca, P, S, Na, Cl, Br, Ru, Fe, Mg, K, Cu, and Si. The LIBS data shows the presence of elements particularly C, Ca, K, Mg, Si, and Br in pigment gallstone which was further confirmed by WD-XRF spectroscopy. Few more elements such as S, Cl, Ru, and Cu were detected using WD-XRF spectroscopy. The elements quantified in pigment gallstone using WDXRF were Na, Cl, S, Ca, P, Br, Ru, and Fe and their concentrations are 0.06, 0.03, 0.02, 0.02, and 0.01 % and 22, 19, and 15 ppm, respectively.
Conclusion Formation of gallstones takes place over a long period of time causing heterogeneous distribution of various elements within the same stone. We have analyzed heterogeneous gallstones utilizing LIBS and WD-XRF techniques. Using LIBS, we
KCps
C KA1
Cu KA1
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The LIBS signal intensity of the mineral elements detected in pigment stone is greater than that of cholesterol stones and hence reflects its corresponding concentration of elements in stone samples. It was observed that pigment gallstone were rich in minerals and heavy metals. The common elements detected in all samples were C, Ca, Na, Cr, Fe, and K. The presence of C in all types of gallstones is due to the presence of cholesterol and bilirubin in stone samples. The elements Na and Cl are detected in cholesterol gallstones (G1, G2) which may be due to the presence of NaCl in the bile of the patients. Cr is the heavy and toxic metal which is detected in all kinds of gallstones. Recently, Jaswal et al. [31] studied the center and surface parts of cholesterol gallstone (white in color) using time-of-flight secondary ion mass spectrometry (TOFSIMS) and reported the presence of heavy elements particularly, Pb and Cr, which in turn authenticates our results. Elements Br, Ti, and Si are detected only in cholesterol (G1) and pigment stones (G3). The heavy elements particularly Ag and Ni are the only minerals which are detected only in pigment gallstones (G3). Pb and Sn were detected only in cholesterol gallstones G1 and G2, respectively. Fluorine (F) was absent in cholesterol gallstone (G1).
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0,4
0,6
1
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KeV
Fig. 6 A typical WD-XRF spectra of black pigment stone
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have successfully analyzed a large number of elements such as Na, Cr, Fe, Ti, K, Br, Si, C, K, Sn, Ca, Cl, Pb, F, N, F, Mg, Br, Ag, and Ni in gallstone samples. Qualitative and quantitative analysis of pigment gallstone have been done using LIBS and WD-XRF techniques. Using WD-XRF, the elements detected successfully were C, O, Ca, P, S, Na, Cl, Br, Ru, Fe, Mg, K, Cu, and Si; however, the LIBS data shows the presence of elements particularly C, Ca, K, Mg, Si, and Br in pigment gallstone which was confirmed by WD-XRF data. Using WD-XRF, the quantified elements in pigment gallstone were Na, Cl, S, Ca, P, Br, Ru, and Fe. In LIBS, for the quantitative analysis of biological samples such as gallstones certified reference materials (CRM) are required to draw the calibration curves and to get the CRM for heterogeneous gallstones is extremely difficult and also LIBS is very sensitive to matrix effect. However, no calibration is required in WD-XRF as samples have been analyzed in standard less software BQuant Express^. Thus, the joint use of LIBS and WD-XRF allow accurate determination of stones’ elemental composition, which is helpful in elucidating the exact role of trace elements in their pathogenesis and their etiology as well. FTIR has been used for the successful characterization of cholesterol and pigment gallstones. In addition to cholesterol and bilirubin, calcium carbonate and calcium phosphate are also noticed in black pigment gallstone. Our results revealed that LIBS and WD-XRF are the promising techniques which are capable to analyze stone samples present inside the human body. The results obtained in the present paper show interesting prospects for LIBS and WDXRF to study cholelithiasis better. Acknowledgments We are thankful to Central Instrumentation Laboratory (CIL) and Sophisticated Analytical Instrumentation Facility (SAIF), Punjab University, Chandigarh for providing FT-IR and WDXRF experimental facilities, respectively. The authors are also thankful to Professor S. B. Rai and Prof. S.N. Thakur from the Department of Physics, Banaras Hindu University, Varanasi for their valuable suggestions and discussions during the course of the present work. Prof. Gondal is thankful to KFUPM for supporting this work through project RG 1421.
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