Amino acid d 13 C analysis of hair proteins and bone collagen using liquid chromatography/isotope ratio mass spectrometry: paleodietary implications from intra-individual comparisons z Maanasa Raghavan 1 * ,y , James S. O. McCullagh 2 , Niels Lynnerup 3 and Robert E. M. Hedges 1 1 Research Laboratory for Archaeology and the History of Art, Dyson Perrins Building, South Parks Road, Oxford OX1 3QY, UK 2 Chemistry Research Laboratory, Mansfield Road, Oxford OX1 3TA, UK 3 Laboratory of Biological Anthropology, Department of Forensic Medicine, University of Copenhagen, DK 2200 Copenhagen, Denmark Received 31 July 2009; Revised 25 November 2009; Accepted 26 November 2009 We report a novel method for the chromatographic separation and measurement of stable carbon isotope ratios (d 13 C) of individual amino acids in hair proteins and bone collagen using the LC- IsoLink system, which interfaces liquid chromatography (LC) with isotope ratio mass spectrometry (IRMS). This paper provides baseline separation of 15 and 13 of the 18 amino acids in bone collagen and hair proteins, respectively. We also describe an approach to analysing small hair samples for compound-specific analysis of segmental hair sections. The LC/IRMS method is applied in a historical context by the d 13 C analysis of hair proteins and bone collagen recovered from six individuals from Uummannaq in Greenland. The analysis of hair and bone amino acids from the same individual, compared for the first time in this study, is of importance in palaeodietary reconstruction. If hair proteins can be used as a proxy for bone collagen at the amino acid level, this validates compound-specific isotope studies using hair as a model for palaeodietary reconstruction. Our results suggest that a small offset observed in the bulk d 13 C values of the hair and bone samples may be attributed to two factors: (i) amino acid compositional differences between hair and bone proteins, and (ii) differential turnover rates of the tissues and the amino acid pools contributing to their synthesis. This application proposes that hair may be a useful complementary or alternative source of compound-specific paleodietary information. Copyright # 2010 John Wiley & Sons, Ltd. Reconstruction of paleodiet has been a focal point in archaeological science primarily because it has the potential to shed light on several archaeologically pertinent issues relating to health and nutrition, subsistence patterns, environmental conditions, economic and social status among others. 1–5 Food, in archaeological contexts, is a rare commodity because by its very nature it tends to be consumed or, if not, is susceptible to degradation. Conven- tional means of inferring past diets have included residue analyses and zooarchaeology, which offer direct evidence of food items, and, indirectly, consumption patterns by study- ing tissues from consumers. Hair, with regard to isotopic paleodietary reconstruction, presents several advantages over other ubiquitously ana- lysed tissues such as bones and teeth. First, sampling is non- invasive with minimal visible destruction to archaeological samples and, importantly, it is also straightforward to sample modern individuals who play an important role in helping understand the relationship between isotopic patterns and diet. 6–8 Second, since hair is metabolically inert post-keratinisation, and grows at an average rate of 1 centimeter per month, chronological studies of diets can be undertaken that address issues relating to changing diets and seasonality. 4,8–12 In the context of paleodiet, the initial tenet ’you are what you eat’ proposed that dietary macromolecules (proteins, carbohydrates and lipids) are incorporated into consumer tissues without any significant fractionation. 13 However, much work has been done since on the differential isotopic compositions of carbon and nitrogen in diet and consumers, and numerous studies have shown that food components are routed as heterogeneous pools to different body tissues. 14–17 For instance, bone collagen d 13 C is enriched over dietary protein by 4–5% while hair d 13 C is enriched by 1–2%, and both tissues have d 15 N enrichment values of 2–3%. 13,15,18–21 However, relatively few studies have quantified the inherent RAPID COMMUNICATIONS IN MASS SPECTROMETRY Rapid Commun. Mass Spectrom. 2010; 24: 541–548 Published online in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/rcm.4398 *Correspondence to: M. Raghavan, Center for GeoGenetics, Natural History Museum of Denmark, University of Copenha- gen, Universitetsparken 15, DK 2100 Copenhagen, Denmark. E-mail: mraghavan@snm.ku.dk y Present address: Center for GeoGenetics, Natural History Museum of Denmark, University of Copenhagen, Universitet- sparken 15, DK 2100 Copenhagen, Denmark. z Presented at SIMSUG 2009, held 14–15 January 2009 at the University of Glasgow. Copyright # 2010 John Wiley & Sons, Ltd.