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Article

Cuscohygrine and Hygrine as Biomarkers for Coca Leaf Chewing: Analytical Challenges in GC-MS Detection and Implications for the Differentiation of Cocaine Use in Forensic Toxicology

by
Nélida C. Rubio
1,
Iván Alvarez-Freire
1,
Pamela Cabarcos-Fernández
1,*,
María J. Tabernero-Duque
1,
Inés Sánchez-Sellero
1,
Antonio Moreda-Piñeiro
1,
Pilar Bermejo-Barrera
2 and
Ana M. Bermejo-Barrera
2
1
Forensic Toxicology Service, Forensic Sciences Institute, Faculty of Medicine, University of Santiago de Compostela, Rúa San Francisco, s/n, 15782 Santiago de Compostela, Spain
2
Trace Element, Spectroscopy and Speciation Group (GETEE), Institute of Materials iMATUS, Department of Analytical Chemistry, Nutrition and Bromatology, Faculty of Chemistry, University of Santiago de Compostela, Avenida das Ciencias, s/n, 15782 Santiago de Compostela, Spain
*
Author to whom correspondence should be addressed.
Separations 2025, 12(8), 201; https://doi.org/10.3390/separations12080201
Submission received: 30 June 2025 / Revised: 23 July 2025 / Accepted: 25 July 2025 / Published: 30 July 2025
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Abstract

Cuscohygrine (CUS) and hygrine (HYG) are pyrrolidine alkaloids proposed as biomarkers of coca leaf consumption, a culturally accepted practice in some Latin American countries. Differentiating legal coca use from illicit cocaine consumption holds forensic importance. While LC-MS/MS is preferred, GC-MS remains widely used in Latin American toxicology labs due to accessibility. This study critically evaluates the analytical limitations of GC-MS for detecting CUS and HYG in biological matrices. Key parameters—injector temperature (180–290 °C), injection mode (split/splitless), solvent, liner condition, and matrix—were systematically studied. GC-MS showed significant limitations: low-abundance, non-specific fragments (m/z 42, 84, 98, 140) failed to meet the identification criteria in SIM mode. Thermal degradation of CUS to HYG and CUS-d6 to HYG-d3 was observed, especially with splitless injection and aged liners. Matrix effects produced signal enhancement ranging from +29% to +316%, meaning that analyte responses in biological samples were significantly higher than in neat standards, likely due to reduced degradation or adsorption. Although deuterated internal standards (CUS-d6) partially corrected signal variability and matrix enhancement, these corrections were not sufficient to overcome the fundamental limitations of GC-MS, including poor ion specificity and compound instability. These findings support the need for LC-MS/MS-based approaches for reliable alkaloid detection and question the suitability of GC-MS for CUS analysis in forensic toxicology contexts.
Keywords: cuscohygrine; hygrine; coca leaf; gas chromatography- mass spectrometry; forensic toxicology; cocaine cuscohygrine; hygrine; coca leaf; gas chromatography- mass spectrometry; forensic toxicology; cocaine
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MDPI and ACS Style

Rubio, N.C.; Alvarez-Freire, I.; Cabarcos-Fernández, P.; Tabernero-Duque, M.J.; Sánchez-Sellero, I.; Moreda-Piñeiro, A.; Bermejo-Barrera, P.; Bermejo-Barrera, A.M. Cuscohygrine and Hygrine as Biomarkers for Coca Leaf Chewing: Analytical Challenges in GC-MS Detection and Implications for the Differentiation of Cocaine Use in Forensic Toxicology. Separations 2025, 12, 201. https://doi.org/10.3390/separations12080201

AMA Style

Rubio NC, Alvarez-Freire I, Cabarcos-Fernández P, Tabernero-Duque MJ, Sánchez-Sellero I, Moreda-Piñeiro A, Bermejo-Barrera P, Bermejo-Barrera AM. Cuscohygrine and Hygrine as Biomarkers for Coca Leaf Chewing: Analytical Challenges in GC-MS Detection and Implications for the Differentiation of Cocaine Use in Forensic Toxicology. Separations. 2025; 12(8):201. https://doi.org/10.3390/separations12080201

Chicago/Turabian Style

Rubio, Nélida C., Iván Alvarez-Freire, Pamela Cabarcos-Fernández, María J. Tabernero-Duque, Inés Sánchez-Sellero, Antonio Moreda-Piñeiro, Pilar Bermejo-Barrera, and Ana M. Bermejo-Barrera. 2025. "Cuscohygrine and Hygrine as Biomarkers for Coca Leaf Chewing: Analytical Challenges in GC-MS Detection and Implications for the Differentiation of Cocaine Use in Forensic Toxicology" Separations 12, no. 8: 201. https://doi.org/10.3390/separations12080201

APA Style

Rubio, N. C., Alvarez-Freire, I., Cabarcos-Fernández, P., Tabernero-Duque, M. J., Sánchez-Sellero, I., Moreda-Piñeiro, A., Bermejo-Barrera, P., & Bermejo-Barrera, A. M. (2025). Cuscohygrine and Hygrine as Biomarkers for Coca Leaf Chewing: Analytical Challenges in GC-MS Detection and Implications for the Differentiation of Cocaine Use in Forensic Toxicology. Separations, 12(8), 201. https://doi.org/10.3390/separations12080201

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