An inter‐laboratory comparative study into sample preparation for both reproducible and repeatable forensic ²H isotope analysis of human hair by continuous flow isotope ratio mass spectrometry

Abstract

Stable isotope analysis of organic materials for their hydrogen ((2)H), carbon ((13)C), nitrogen ((15)N) or oxygen ((18)O) isotopic composition using continuous flow isotope ratio mass spectrometry (CF-IRMS) is an increasingly used tool in forensic chemical analysis. (2)H isotopic analysis can present a huge challenge, especially when dealing with exhibits comprising exchangeable hydrogen such as human scalp hair. However, to yield forensic data that are fit for purpose, analysis of the (2)H isotopic composition of the same homogeneous human hair sample by any laboratory worldwide must yield the same isotopic composition within analytical uncertainty. This paper presents longitudinal (2)H isotope data for four human hair samples of different provenance, measured by three different laboratories whose sample preparation was based on a two-stage H exchange equilibration method. Although each laboratory employed varying means to comply with the generic features of the sample preparation protocol such as the (2)H isotopic composition of exchange waters or drying down of samples prior to analysis, within each laboratory the Principle of Identical Treatment (P.I.T.) was applied for each individual experiment. Despite the variation in materials and procedures employed by the three laboratories, repeatable and reproducible 'true' (2)H isotope values (δ(2)H(hair,true)) were determined by each laboratory for each of the four stock samples of human scalp hair. The between-laboratory differences for obtained δ(2)H(hair,true) values ranged from 0.1 to 2.5 ‰. With an overall 95% confidence interval of ±2.8 ‰, these differences were not significantly different, which suggests that the general method of two-stage exchange equilibration carried out at ambient temperature is suitable for accurately and reproducibly determining 'true' δ(2)H-values for hair and other proteins provided that certain key conditions are met.

Critique: Measuring Hydrogen Stable Isotope Abundance of Proteins to Infer Origins of Wildlife, Food and People

“…Tap water [71] VSMOW Human hair (values determined using IUPAC guidelines) [52] Human hair [71] 34 ‰ Measuring hydrogen stable isotope abundance to infer origins of wildlife, food & people | PersPective www.future-science.com Ambient humidity at the point of origin is -65 ‰ too, so its total d 2 H value as measured at this location would also be -65 ‰. Published data put the typical exchange fraction of finely chopped hair at ambient at 0.1 [47,48,54] ues vary greatly if participating laboratories did not determine or correct for the molar exchange fraction of these materials as part of the ILC [75]. Not surprisingly therefore, ILC samples of horse hair and paper that had been allowed to equilibrate with ambient humidity in the participating laboratories, yielded Huber estimates of the mean values (H15) of d 2 H total associated with H15 SDs of ±11.1 and 7.4 ‰, respectively [76].…”

“…While the first two problems are easily overcome with drying and careful sample handling, the latter problem has attracted a wide variety of disparate approaches that range from dealing with this challenge [47,[52][53][54][55][56] to not addressing the problem [57,58]. One might argue that for studies purely concerned with looking at differences in H isotopic composition between and among samples, knowledge of the molar exchange fraction and 'true' d 2 H-values of nonexchangeable hydrogen might not be an issue, so long as all samples have been treated in an identical fashion, and having been stored, exposed to the same humidity under identical conditions (temperature, relative humidity and stable isotopic composition of humidity), thus …”

“…To Subsequent to equilibration and prior to H isotope ana lysis, samples must be dried down either in an evacuated desiccator containing noncaking phosphorous pentoxide as a desiccant or in a container connected to a continuously operating vacuum line (vacuum drying oven or freeze dryer arrangement) for at least 7 days to remove residual moisture traces from the samples [47,54,55]. To avoid back-exchange of equilibrated samples with ambient humidity, samples have to be loaded quickly (e.g., in <30 min) on to a Zero-Blank autosampler that can be sealed (i.e., isolated from ambient) and, once sealed, can be purged with a dry helium so samples are kept in a dry, inert, atmosphere while waiting their turn to be analyzed [47,54,55,77]. Following this protocol permits strictest adherence to the PIT and will therefore avoid any ambiguity that would otherwise arise or could otherwise be construed.…”

Expanding the Isotopic Toolbox: Applications of Hydrogen and Oxygen Stable Isotope Ratios to Food Web Studies

“…Regardless of the method used, we emphasize that the methods must be reliable and reproducible, and reported with sufficient detail in publications. The interchange of materials among laboratories can aid in assuring repeatability in δ 2 H and δ 18 O measurements (e.g., Meier-Augenstein et al, 2011), given that it is unrealistic to expect that all laboratories are equipped in exactly the same way.…”

Forensic Mass Spectrometry

“…In forensic applications, isotope ratio analysis provides information about the natural or synthetic origins of scheduled drugs (110)(111)(112)(113)(114)(115)(116)(117)(118)(119)(120), organic and inorganic explosives (76,121,122), petroleum hydrocarbons (such as in ignitable liquids) (65,66,(123)(124)(125)(126), and various other forensic materials (such as fibers, matches, and paint chips) (127)(128)(129)(130)(131). The ability to analyze hard and soft human tissue has also provided helpful investigative leads about the geographic origins of humans, especially in John Doe and Jane Doe cases where fingerprints, DNA, and dental records are not found in databases (132)(133)(134)(135)(136)(137)(138)(139). As global isotope landscapes and databases become more readily available (140)(141)(142)(143)(144)(145)(146), the determination of human geographic provenance and human behaviors or biometrics (147) should become more common.…”