Phosphate oxygen isotope analysis on microsamples of bioapatite: removal of organic contamination and minimization of sample size Felicitas B. Wiedemann-Bidlack 1 * ,y , Albert S. Colman 2z and Marilyn L. Fogel 2 1 Department of Anthropology, The George Washington University, 2110 G St NW, Washington DC 20009, USA 2 Geophysical Laboratory, Carnegie Institution of Washington, 5251 Broad Branch Road, Washington DC 20015, USA Received 8 January 2008; Revised 20 March 2008; Accepted 22 March 2008 Modern and fossil teeth record seasonal information on climate, diet, and migration through stable isotope compositions in enamel and dentine. Climatic signals such as seasonal variation in meteoric water isotopic composition can be recovered through a microscale histology-based sampling and isotopic analysis of enamel phosphate oxygen. The phosphate moiety in bioapatite is particularly resistant to post mortem diagenesis. In order to determine the phosphate oxygen isotope composition of enamel, phosphate must be chemically purified from other oxygen sources in the enamel lattice and matrix, mainly hydroxyl and carbonate ions, and trace quantities of organics. We present a wet chemical technique for purifying phosphate from microsampled enamel and dentine. This technique uses a sodium hypochlorite oxidation step to remove interferences from residual organic constituents of the enamel and/or dentine scaffold, isolates phosphate as relatively large and easily manipulated Ag 3 PO 4 crystals by using a strongly buffered, moderate-temperature microprecipitation, and preserves the oxygen isotope composition of the initial tooth phosphate. The reproducibility of phosphate oxygen isotope compositions thus determined (measured as d 18 O, V-SMOW scale) is typically 0.2–0.3% (1 s.d.) on samples as small as 300 mg of enamel or dentine, a considerable improvement over available techniques for analyses of bioapatite phosphate oxygen. Copyright # 2008 John Wiley & Sons, Ltd. Many studies have employed d 18 O analyses of vertebrate bone and teeth to investigate animal diet, habitat, environ- mental conditions, or climate change. 1–5 These studies use the correlation between the d 18 O of meteoric water, which depends on geography and climate, 6,7 and the empirical correlations between the local d 18 O of drinking water/diet and the d 18 O incorporated into mineralized tissues such as bone and teeth during their formation. 8 The d 18 O recorded in the mineral phase is species-specific as it depends on the d18O of body water sources and the fractionations associated with water loss mechanisms. 9–11 The mineral phase of bone is the same as that of dentine and mammalian tooth enamel, namely, carbonated hydroxyapatite: Ca 5 (PO 4 ) 3 (OH) with carbonate substituting for the phosphate or hydroxyl group. 12,13 However, bone, dentine, and enamel are different in crystallite size and packing, and the relative amounts of mineral versus organic material. 14 Tooth enamel has become a preferred sample material for paleo-environmental studies because the preservation of a biogenic isotope signal in a fossil specimen is often better in enamel than in the more porous dentine and bone. 15 Enamel consists of densely packed and relatively large apatite crystals, resulting in a high mineral content (96 wt %). 16 These attributes make tooth enamel more stable against post mortem diagenetic alteration than dentine and bone which are only 70 wt % and 60 wt % mineralized, respectively. 15,17 Currently, the oxygen isotope composition of biological apatite is measured using a variety of techniques. Some target the oxygen derived from the carbonate group (CO 2 3 ), 18–21 others focus on the phosphate (PO 3 4 ) oxygen, 3,22 and some measure all oxygen-containing groups of the sample, i.e., carbonate, phosphate and hydroxyl groups. 10,23,24 The sample pretreatment and processing are less complicated for the oxygen isotope analysis of carbonate 25,26 than that for phosphate. 22,27–30 Although carbonate analysis offers infor- mation on two elements carrying dietary information (i.e. carbon and oxygen), d 18 O CO3 appears to be more prone to diagenetic alteration than phosphate d 18 O(d 18 Op) because the P–O bond is energetically more stable than the C–O bond (for review, see Kohn and Cerling 31 ). There persists, however, a need for an effective method for the pretreatment of microscale samples to purify phosphate from organic- containing bioaptite. Restrictions in sample size are of increasing importance as the retrieval of serial samples from small incremental growth RAPID COMMUNICATIONS IN MASS SPECTROMETRY Rapid Commun. Mass Spectrom. 2008; 22: 1807–1816 Published online in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/rcm.3553 *Correspondence to: F. B. Wiedemann-Bidlack, Department of Biomineralization, The Forsyth Institute, Boston MA 02115, USA. E-mail: fbidlack@forsyth.org y Present address: Department of Biomineralization, The Forsyth Institute, Boston MA 02115, USA. z Present address: Department of the Geophysical Sciences, University of Chicago, Chicago, IL 60637, USA. Copyright # 2008 John Wiley & Sons, Ltd.