Isotope databases (“isoscapes”) of human tissues play an important role in global forensic investigations. It is thus essential to have a standardised sample preparation and measurement protocol for stable isotope analysis of human tissues. In this context, accurate δ 18 O analysis of nitrogen-containing organic samples is limited by analytical challenges, especially the interference of NO + ions in the mass spectrometer. Towards this, we report a comprehensive sample preparation protocol to clean the hair tissue samples and demonstrated the use of a mixer mill for homogenous cryogenic grinding. We have also outlined a complete workflow for the measurement of δ 18 O values in human tissues via Flash 2000 HT EA-IRMS, including instrument configuration and set-up, as well as data acquisition and processing parameters. Two data acquisition strategies- N 2 dilution and N 2 removal methods- are presented, along with method validation using both international and in-house laboratory reference materials. The average precision and accuracy for N 2 dilution method were 0.3‰ and for N 2 removal method were 0.4‰. While the primary focus of the study is human hair, the described protocol is applicable to any nitrogen-containing organic sample, such as feathers, hooves, bone collagen, nails, horns, and claws. Additionally, we have discussed alternative strategies to address the limitations of the method. Most importantly, the mentioned protocol will allow under-resourced laboratories to measure accurate and precise δ 18 O values using existing high-temperature conversion (HTC) elemental analysers without incurring additional instrumentation costs. • Comprehensive guide for δ 18 O analysis of organic compounds • Cryogenic grinding of human tissues using a mixer mill • Two methods for mitigating m/z 30 interference for δ 18 O analysis • High-throughput δ 18 O analysis protocol without costly hardware modifications
Agrawal et al. (2026) studied this question.