Comparing the Performance of Demineralization Agents (hcl and EDTA) for Stable Isotope Analysis of Bone Collagen with Implications for Quality Control Criteria and Collagen Yield

Stable isotope analysis of ancient bone collagen is a powerful technique for studying diet, migration, and ecology in archeological contexts. These analyses are, however, limited by collagen preservation and prohibitively low collagen yields. Harsh chemical demineralization is required to isolate the collagen from the mineral component of the bone, which in turn reduces the yield of material available for analysis. Demineralization is typically performed using hydrochloric acid (HCl), which reduces collagen yield via acid hydrolysis of peptide bonds. An alternative to a strong acid (HCl) treatment is the neutral chelating agent ethylenediaminetetraacetic acid (EDTA). To our knowledge, it has never been empirically tested whether EDTA treatment reduces collagen loss relative to HCl in samples that are known to be poorly preserved (i.e., low yields and/or collagen extracts failing other collagen quality control [QC] criteria). We tested the effect of the demineralization agent on collagen yield, stable isotope, and elemental composition of poorly preserved ancient bone samples. Collagen yield was significantly higher in EDTA-treated samples; however, this did not translate into a greater number of samples passing relevant quality control criteria. Stable isotope compositions (SIC) were also not significantly different between treatments. The atomic C:N ratio of samples treated with EDTA was significantly lower than equivalent samples treated with HCl, which is likely a product of increased deamidation of asparagine and glutamine residues when collagen is demineralized with HCl relative to EDTA. We conclude that although EDTA treatment may reduce collagen loss relative to HCl treatment, this does not necessarily increase the probability of producing reliable-stable isotope data from bone samples yielding low amounts of collagen. Based on our isotopic data, we suggest the following provisional collagen QC criteria for EDTA-demineralized samples: collagen yield > 2%, wt% C > 4%, wt% N > 2%, and atomic C:N ratio between 2.80 and 3.25.