Stable carbon isotope and accurate mass studies for source determination and identification of cochineal insects and lake pigments

Differentiation between the main types of cochineal dyestuffs, used as a principal red colourant throughout history, remains challenging. Nonetheless, accurate cochineal source identification would provide corroborating evidence to historical accounts, unveiling ancient practices and historical trade routes. Herein, stable carbon isotope analysis is proposed, for the first time, as a tool for source investigation of colourants on cultural heritage objects. Through bulk analysis, the isotopic signatures of Mexican, Armenian, and Polish cochineal insects were confirmed to reflect those of their host plants (CAM, C 4 , and C 3 , respectively). Compound-specific isotope analysis (CSIA), widely used for provenancing archaeological lipids, was then utilised to investigate the insect lipid extracts, revealing that Mexican, Armenian, and Polish cochineals have distinct fatty acid profiles and isotopic signatures. A new protocol, based on direct inlet pyrolysis-gas chromatography-combustion-isotope ratio mass spectrometry (DIP-GC-C-IRMS), was developed to target a known biomarker of the main colourant carminic acid (CA), thereby obtaining the isotopic signature of the colourant itself. Although Armenian cochineal harvested from C 3 plants has not yet been investigated, results so far reveal distinct CA δ 13 C values (δ 13 C CA ) for Armenian and Mexican cochineal lakes and paint replicas, suggesting that this method may be able to differentiate between the three main cochineal sources. The presence of the known marker in similar red insect-derived lakes (kermes and lac) was also investigated. In addition to providing the first example of CSIA of lake pigments, this is the first study that utilises DIP-GC-QTOF-MS/MS as a complementary technique. By offering additional structural information for CA derivatives, a structure and mechanism of formation for a newly identified biomarker is proposed.