Sequential extraction of the phosphate and collagen fractions of small bone samples for analysis of multiple isotope systems (δ¹⁸O_PO4, δ¹³C, δ¹⁵N)

Rationale Stable isotope analyses are used on precious archeological and paleontological materials despite their destructive nature, because the information gained by these methods on, for example, feeding habits, migration and health of individuals cannot otherwise be obtained. We approached this issue by devising a new sequential extraction scheme aimed at producing multiple (O, C, N) isotope proxies from small amounts of sample. Methods The new extraction scheme includes dissolution of the bone in dilute HNO(3)followed by separate treatments of the collagenous and phosphate fractions. The collagen fraction is treated further adopting the methods presented in the literature for collagen extraction, modified to accommodate small sample sizes. The phosphate-containing fraction is purified from organic contaminants by H(2)O(2)and the phosphate is precipitated as Ag(3)PO(4)following methods presented in the literature. The use of HF as demineralization agent is also tested. Results A starting amount ofca2 mg produced enough material for meaurement by isotope ratio mass spectrometry of the collagen C and N isotope compositions and bone phosphate O isotope composition. We show that the isotopic data obtained from the sequential extraction scheme are comparable with the isotopic composition measured following conventional methodologies that are usually based on 100-500 mg sample sizes. Conclusions The new sequential extraction scheme combines the preparation for stable isotope analysis of bone mineral and organic phases, thus minimizing the amounts of sample needed and damage caused on a sample piece. The method may allow analysis of skeletal samples previously excluded from isotope analysis due to material limitations.