Abstract. Laser ablation-inductively coupled plasma-mass spectrometry has been used to generate elementalfingerprints of individual teeth, which have been sectionedto provide a sample which represents the time axis of toothdevelopment. The ablated area is of the order of 100 µmacross, and the ablation process is reproducible, measuredusing phosphorus. Studies on modern teeth reveal that themercury and gold concentrations decrease from the outer(mouth exposed) part of the tooth to the inner newly de-posited material. In contrast, the lead content increasesfrom the outer part to the inner reflecting the exogenousorigin of the mercury and gold, and the endogenous originof the lead from the blood supply. Studies on a 19th cen-tury tooth from Spitzbergen reveal the presence of a rangeof lanthanide elements which occur naturally in the area.Studies of the concentrations of other elements present inteeth permit the comparison of data between teeth via theuse of element/element ratios. The calcium/phosphorusratio is recommended for this purpose.

Introduction

Inductively coupled plasma-atomic emission spectrome-try (ICP-OES) has rapidly been established as a versatiletechnique for multielement quantitation in various matri-ces ([1–3] and references therein). More recently, the useof mass spectrometry has been used to provide rapid multi-element profiling of samples ([4, 5] and references therein).Addition of laser ablation as a sample preparation step hasallowed the direct determination of many elements insolid samples [6–9]. The majority of applications of thetechnique have been in the area of inorganic analysis [10–14]. Whilst it is very convenient to use laser ablation, theprocedures for calibration are necessarily complex. Beingessentially a solid phase sampling technique, the use ofparticulate standard reference materials is inappropriate

because of possible heterogeneity of the sample at thepoint of ablation. Like-wise variation on laser output powerbetween firings, variation of power density at the surface,light scattering and variations in aerosol transport efficiencyout of the cell, have all been cited as possible causes of er-rors in quantitation [15, 16]. To overcome some of thesedifficulties, techniques such as the simultaneous nebulisa-tion of aqueous standards [17] have been employed. Morerecently, matrix matching by fusion [18] and by pelletiza-tion [19] has been developed.

Few applications of laser ablation ICP-MS to biologi-cal matrices have appeared although standard orchardleaves (NIST SRM 1571) and tomato leaves (NIST SRM1573) have been studied [20]. Calibration was against awell established reference material (Bowens Kale). Multi-ple laser shots were used and averages taken and the au-thors considered the results to be reasonably encouraging.However, the application of LA-ICP-MS to the assess-ment of the metal content of biological samples such astissue, blood, hair, teeth and fingernails is not well estab-lished. For such samples, absolute quantitation is not anissue as inter-sample comparison is usually desired. Hence,providing the variables identified previously [15, 16] arestandardised, then the trace element profiles of varioussamples can be compared.

Teeth are a sample of particular interest epidemiologi-cally because the rate of deposition is well known andhence a single tooth contains within it a time axis and henceencapsulates information on exposure to metals which be-come deposited in the tooth.

Mineralised tissue sequester trace elements and theamount present in a particular dental tissue is the cumula-tive function of previous exposures [21]. This is not thecase with other tissues, for example blood, where the in-corporation is transient. Furthermore, the mineralised tis-sue of the teeth, unlike bone, do not remodel, are not sub-ject to turnover and are relatively easy to collect followingroutine extraction or shedding. As a result, teeth are anideal site at which to study exposure to trace elements.

Fresenius J Anal Chem (1996) 354 :254–258

Correspondence to: M. Cooke

Trace element profiling of dental tissues using laser ablation-inductively coupled plasma-mass spectrometry

Alan Cox1, Fergus Keenan1, Michael Cooke1, John Appleton2

1 Environmental Research Centre, School of Science, Sheffield Hallam University, City Campus, Sheffield S1 1WB, UK2 Oral Biology Unit, Clinical Dental Sciences, School of Dentistry, University of Liverpool, Liverpool L69 3BX, UK

Received: 11 April 1995 / Accepted: 27 April 1995

© Springer-Verlag 1996

Fresenius’ Journal of

Teeth develop within fairly well defined time limitsand their tissues grow incrementally. By the selection ofthe appropriate teeth therefore and biopsying a particularpart of the tooth, the exposure over a particular time inter-val can be investigated. Laser ablation ICP-MS is a rapidand sensitive method of sampling and chemically analysingdiscrete areas of tooth substance. This preliminary studydescribes a method in which equivalent areas are sampledin molar teeth from individuals exposed to trace elements asa result of their life styles or from environmental pollution.

Experimental

1 Tooth preparation. Individual teeth were prepared by sectioninghorizontally using a diamond saw to avoid metal contamination ofthe cut surface. The surface thus exposed revealed dentine de-posited across the lifetime of the tooth ranging from new dentineadjacent to the pulp to the new dentine at the cement-dentine junc-tion. Tooth sections were mounted on slides for insertion into theablation chamber.

2 Instrumentation. A VG Instruments’ PQII + ICP mass spectro-meter and a VG Laserlab were used for this work. The VG Laser-Lab system utilises a 500 mJ Nd: YAG laser operating at 1064 nm.In the present work all measurements were made in the Q-switchedmode, other laser instrument parameters are given in Table 1.

In operation, radiation from the laser was focussed onto thesurface of the prepared tooth sample inside the ablation chamber.The chamber was mounted on a computer controlled stepper mo-tor driven X-Y translation stage. This enabled the ablation site ofthe sample to be precisely controlled. A charge coupled device(CCD) camera was mounted above the chamber to permit theviewing of the sample via a video monitor.

The ablated material was removed from the chamber by a con-tinuous stream of argon gas and transferred directly to the ICP

source. Operating conditions for the Plasma Quad instrument aregiven in Table 2.

Results and discussion

Individual tooth samples were prepared for analysis bysectioning horizontally using a diamond saw. This proce-dure yielded a smooth, contamination free, dentine surfacerepresenting a time axis from recent (near pulp) to oldest(near cementum) across the tooth. The analytical tech-nique employed combines the use of laser ablation to pro-duce a suitable aliquot of sample for analysis with an in-ductively coupled plasma source which renders the toothmaterial down to its individual elements. Ionisation oc-curs in the plasma and the ionic species enter the massspectrometer which separates them by atomic mass andcharge, i.e. the mass/charge ratio. As there are less than100 naturally occurring elements with the heaviest (ura-nium) having a mass of 238 amu, high resolution is notneeded in order to profile the elemental distributionwithin the ablated, atomised sample. The result is a massspectrum of all the elements present in the sample.

The laser used for this preliminary study was a Nd :YAG laser with an output power of 500 mJ which ablatedan area of approximately 100 µm in diameter to a depth ofsome 30 µm. The ablated material is in the form of a mi-crofine dispersion rather than a true vapour. After ablationthe sample is entrained in an inert gas stream which trans-fers it into the plasma source. The use of an argon plasmasource rather than the flame or graphite furnace systemsof conventional atomic absorption spectrometers virtuallyeliminates the matrix interference problems often encoun-tered with these two techniques.

The types of information may be obtained by using themass spectrometer as a detector. Qualitative informationas to the identity of the elements present across the massrange studied (100–240 amu) provides a fingerprint pat-tern which characterises the sample. Semi-quantitative in-formation can also be derived from the data by measuringthe area response for each mass and then relating it to the

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Table 1. Typical laser ablation parameters for the analysis of teeth

Laser type Nd: YAGLaser mode Q-switchedLaser input energy (V) 720Shot repetition rate (Hz) 100Sampling scheme Single spot

Table 2. Typical ICP-MS operating conditions for laser ablation ofteeth

Forward power/kw 1.35

Gas flow ratescoolant (l min–1) 14auxiliary (l min–1) 0.5carrier (l min–1) 0.91

Scan conditionsdwell time (µs) 160channels (/amu) 20scan range m/z 6 –240

Skip regions m/z 11 – 2232 – 4179.5– 80.5

Acquisition time(s) 60 Fig.1. Repetitive sampling of a single tooth for reproducibility ofthe ablation procedure determined by measurement of phosphorus

response for a major element such as phosphorus (mass =31) which is mono-isotopic and common to all samples.An alternative would be to normalise to an element/ele-ment ratio. We have found that the calcium/phosphorusratio in teeth is approximately 1.51, (RSD 3.62%), for allteeth studied which correlates with established values.The proposal to use target element/major element ratios asa basis for inter-sample comparison is dependent upon therepeatability of the ablation procedure. To evaluate this, asingle tooth was sampled six times and the phosphorus re-sponse measured. The mean was 27769, the range wasfrom 25319 to 30947 of the mean and the RSD was 8.2%.

As no suitable standard reference materials are cur-rently available for teeth, and in view of the requirementfor qualitative data and for inter-sample comparisonrather than absolute quantitation, we suggest that the prin-ciple of element/element ratioing be adopted.

Figure 1 shows a scanning electron micrograph of thetooth sample used to generate the phosphorus data. Theablated areas lie along the inner i.e. newest dentine next tothe pulp. They are not circular as expected, presumablybecause the incident laser beam has not impinged on thesurface of the sample at right angles. The major dimen-

sion of the crater is of the order of 100 µm. There is a halolike effect around the crater which could indicate thatsome of the ablated material falls back onto the surface ofthe tooth rather than be transferred to the plasma source.Visually however, the process is reproducible and thiscorrelates with observed data for phosphorus.

Figure 2 shows an electron micrograph of a single ab-lation crater. The non-circular shape is apparent and thereis evidence of structural damage at the bottom of the im-pact crater. Other ablation sites show similar morphology.Towards the edge of the picture the fall-out of ablated ma-terial can be seen as lighter spots. The blackish spots ad-jacent to the crater are dentinal tubules made distinctiveby vapour outgassing at the time of ablation.

Using conventional ICP-optical emission spectrome-try, the major elements in a selection of teeth were mea-sured. Various element/element ratios were then calcu-lated. The results are given in Table 3. Samples 1 and 2, 4and 5, 6 and 7 are duplicate pairs. From these ratios sev-eral observations can be drawn. Firstly, the calcium/phos-phorus and calcium/magnesium ratios are relatively con-stant across all samples (Ca/P: mean 1.51, RSD = 3.62%;Ca/Mg: mean 31.2, RSD = 6.3%). In contrast the calcium/sodium and phosphorus/sodium ratios appear to show in-ter-sample variability but intra-sample constancy. Comparethe results for teeth 1, 3, 5 with those for 8, 9 and 10. Asthe calcium/phosphorus ratio appears approximately con-stant it would appear that the sodium concentration is aninter-sample variable. A similar, although less pronounced,pattern is observed for strontium. These preliminary re-sults appear to suggest that element/sodium ratios mightbe a more responsive parameter than the element/phos-phorus ratio for the comparison of relative levels of heavyelements in teeth samples. We are currently analysing amuch larger group of teeth to evaluate the most reliableelemental ratios for inter-sample comparison.

For simple fingerprinting of metal profiles however, therepeatability of the ablation process coupled with a stan-dardisation of response scales for the data provides a suit-able basis for the primary screening studies reported here.

Examples of the type of information obtained fromteeth are shown in Fig. 3. The sample in the lower trace isan early nineteenth century human tooth from the Spitz-

256

Fig. 2. A single ablation site showing the non-circular ablation crater

Table 3. Element/element ratios in teeth measured by ICP-OES. Tooth sample number

Element ratio 1 2 3 4 5 6 7 8 9 10 Mean RSD

Ca/P 1.54 1.55 1.46 1.50 1.58 1.55 1.52 1.55 1.45 1.41 1.51 3.62Ca/Mg 29.9 31.7 31.6 31.6 29.2 31.1 30.6 36.3 30.2 29.8 31.2 6.30Ca/Na 38.8 38.8 35.7 37.9 38.5 44.3 45.6 45.0 45.5 47.0 41.7 9.88Ca/K 685 890 801 809 1010 464 881 4721 4346 3045 791a 22.2Ca/Sr 1860 1710 2354 2020 2022 1750 1640 1621 1380 1334 1770 17.5Na/Sr 48.0 43.7 65.9 53.3 48.6 39.5 35.6 34.6 30.5 28.3 42.8 26.9P/Na 25.1 25.0 24.4 25.3 26.3 28.6 30.0 29.0 31.3 33.4 27.8 11.1P/Sr 1202 1102 1602 1349 1280 1129 1069 1046 950 947 1170 17.2P × 103 143 137 130 131 126 130 133 135 136 141 134 3.93Ca × 103 221 212 191 196 199 201 202 209 197 199 203 4.36

A A B C C D D E F G

a Omitting samples 8, 9, 10

257

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bergen region (latitude 78°N, longitude 15°E). The sam-ple in the upper trace is a modern tooth (again human)from Krakow in Poland. To obtain this data each toothwas sampled in the dentine region approximately midwayacross the time axis. Clearly, there are major differencesin elemental composition between these two samples. Con-sidering first the mass range 100–130 amu. The Spitzber-gen tooth contains significant amounts of tin (masses =112–120) which is absent in the modern tooth. Also, theantimony concentration is much higher than for the mod-ern tooth. Silver is present in both, as is antimony. How-ever, when the mass range 160–190 is considered, the dif-ferences become extreme. For the modern tooth (Fig.3,upper trace) there are no elements present in detectableconcentration. The older tooth (lower trace) shows the pres-ence of a range of elements including holmium, thulium,lutetium, hafnium and tantalum together with tungsten. Inthe range 190–240 the modern tooth shows only lead andmercury. The older tooth shows a lower concentration ofmercury, a much higher abundance of lead (masses 204,206, 207, 208) and also the presence of bismuth. Thoriumand uranium are also present as is to be expected in viewof the masses observed in the region 164–186.

It has been established that exposure to an element ei-ther via ingestion or inhalation leads to incorporation intocalciferous material such as teeth and bone [22]. Lead is acommon element environmentally and exposure can arisenaturally through the presence of lead ores, through expo-sure to lead containing alloys, e.g. pewter (which alsocontains tin) or from the use of lead additives in petrol.The older tooth contains both tin and lead whereas themodern tooth contains only lead. In contrast, the newertooth contains more mercury than does the older, whichsuggests a modern source such as dental amalgam.

Additional studies of ablation sites situated along thetime axis (inner to outer) of a modern tooth show interest-ing variations in the amount of lead, gold and mercuryfound at each site. For lead the concentration generallydecreases from the inner i.e. newest material to the outeri.e. older material. For mercury and gold the reverse gra-dient is observed. It may be argued therefore that the leadfound in teeth is endogenous in nature and arrives therevia the bloodstream. In contrast, the mercury and gold arepresent in the outer layers as contaminants from buccalexposure to these elements following dental treatment, oris deposited preferentially in the early years of growth.This is presently considered unlikely.

The origin of the range of lanthanide elements is lessobvious. Lananide elements occur naturally in severalparts of the world including the northern part of Scan-danavia and Russia, and Spitzbergen lies to the north ofthis region. The most likely explanation is that this oldertooth was from a person who was exposed to the environ-ment in this area possibly through a nomadic existence.The tin, lead and, possibly, antimony content could arisefrom the use of utensils made of pewter. Notwithstandingthe route for incorporation it is clear from these prelimi-nary results that laser ablation-ICP-MS can be used toprovide elemental fingerprints from teeth which illustratethe incorporation of these elements into the tooth duringits lifetime of development.

Conclusion

Laser ablation ICP-MS provides a rapid yet simple meansof obtaining both qualitative and semi-quantitative infor-mation about the metals deposited in teeth. Such informa-tion has value in providing information about exposure tothese metals over the lifetime of the tooth. Hence it pro-vides supporting evidence in epidemiological studies andprovides a means of retrospectively investigating previousexposure for correlation with current health status. Evi-dence is produced which suggests that metals may be in-corporated into the tooth structure either endogenouslyfrom the bloodstream or exogenously from buccal expo-sure. Absolute quantitation is difficult to achieve becauseof the nature of the sample and the lack of suitable stan-dard reference materials. However, by use of element/ele-ment ratios inter-sample comparison can be made. Thenature of the sample ensures that the information con-tained in the sample is encapsulated and hence free fromcontamination until the surface is exposed by cutting witha diamond saw. Even then the cut surface may be ablatedclean and the freshly exposed underlying material sam-pled. Hence this type of sample has great potential as asource of retrospective information in support of medicalinvestigations.

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