In this study, we compared the elemental and isotopic composition of modern and ancient bone samples pre-treated using different demineralization agents with acidic and neutral pH. The purpose of our research was to examine if demineralization using a mineral acid such as hydrochloric acid (HCl) significantly alters the δ15N and δ13C values of bone collagen. Evidence from the elemental and amino acid composition of the samples were incorporated alongside isotopic compositions to provide a holistic view of the effect of demineralization agents on the composition of bone collagen. The stable isotope compositions of collagen extracts were also compared against equivalent whole bone samples to assess whether whole bone has a stable isotope composition that is comparable to collagen demineralized with a neutral agent. Our results demonstrate that bone demineralization using either ethylenediaminetetraacetic acid (EDTA) or HCl yields collagen extracts with stable isotope compositions that are not significantly different, indicating that mineral acid does not alter δ15N and δ13C values of bone collagen. The results comparing whole bone and extracted collagen stable isotope compositions indicate that whole bone cannot be used as an effective replacement for bone collagen due to the significantly different stable isotope compositions between these sample materials. In ecological and archaeological studies performing stable isotope analysis on bone, sample pre-treatment to isolate collagen is a necessity to obtain the most reliable and reproducible isotopic measurements.

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.

Stable carbon (δ 13 C) and nitrogen (δ 15 N) isotope compositions of bone and dentine collagen extracted from museum specimens have been widely used to study the paleoecology of past populations. Due to possible systematic differences in stable isotope values between bone and dentine, dentine values need to be transformed into bone-collagen equivalent using a correction factor to allow comparisons between the two collagen sources. Here, we provide correction factors to transform dentine δ 13 C and δ 15 N values into bone-collagen equivalent for two toothed whales: narwhal and beluga.We sampled bone and tooth dentine from the skulls of 11 narwhals and 26 belugas. In narwhals, dentine was sampled from tusk and embedded tooth; in beluga, dentine was sampled from tooth. δ 13 C and δ 15 N were measured and intraindividual bone and dentine isotopic compositions were used to calculate correction factors for each species. We detected differences in δ 13 C and δ 15 N. In narwhals, we found (i) lower average δ 13 C and δ 15 N in bone compared with dentine; (ii) no difference in dentine δ 13 C between tusk and embedded tooth. For belugas, we also detected lower δ 13 C and δ 15 N in bone compared with tooth dentine. The correction factors provided by the study enable the combined analysis of stable isotope data from bone and dentine in these species.