Effects of demineralization protocols on hydrogen and oxygen isotope ratios in fossil bone collagen

The effect of three commonly used preparation protocols on bone collagen δ 2 H and δ 18 O values was investigated, using six generally well-preserved faunal bones (cattle, bison, horse) ranging in age from the Holocene to Late Pleistocene. The treatments tested were ethylene diamine tetraacetic acid demineralization without gelatinization and hydrochloric acid demineralization with and without gelatinization. There are significant shifts in δ 18 O values among preparation methods. No systematic shifts in bone collagen δ 2 H values were noted among the three treatments, and most offsets were low (< 2–3 ‰) and smaller than typical measurement uncertainties. A few sample/treatment combinations yielded slightly higher inter-treatment δ 2 H differences (∼ 5–7 ‰). Lower mass fraction of hydrogen in HCl-demineralized samples suggests (greater) molecular alterations to the collagen structure, with possibilities including deamidation and/or peptide bond hydrolysis. The use of 2 H-enriched water ( Δ 2 H + 258 ‰) in the demineralization and gelatinization solutions systematically elevated resultant collagen δ 2 H values; however, given the expected water isotopic variation among labs this should be an insignificant source of inter-lab variation. The δ 2 H values appear to be fairly robust in these samples. Further work across a greater range of samples from different burial environments and diagenetic states is recommended.