Detection of saffron (Crocus sativus L.) adulteration using 1H NMR spectroscopy

Abstract

Crocus sativus L. (saffron) is the most valuable spice worldwide, yet its limited production and high market value make it highly susceptible to adulteration with plant substitutes and synthetic dyes. In this study, ¹H Nuclear Magnetic Resonance (NMR) spectroscopy was applied as a rapid, non-destructive technique for saffron authentication and detection of eight plant adulterants and one synthetic dye in a single experiment. Methanolic and hexane extracts were prepared to capture a broad polarity range of metabolites from authentic saffron, known adulterants, and commercial brands. Spectral comparison targeted key saffron markers, including picrocrocin, crocin, and crocetin. Most brand samples closely matched the authentic reference, with minor variations that can attributed to seasonal and geographical factors, whereas the ZK sample lacked diagnostic saffron peaks and instead displayed resonances consistent with Sudan IV and Arnica montana, confirming adulteration. Quantitative NMR (qNMR) was further employed for picrocrocin determination, and the method was validated in terms of selectivity, specificity, linearity (R² > 0.998), precision (intra- and inter-day RSD < 5.5 %), robustness (consistent results across varied scan numbers and relaxation delays). The method demonstrated a limit of detection (LOD) of 0.443 µg/mL and a limit of quantification (LOQ) of 1.342 µg/mL for picrocrocin, indicating high sensitivity and suitability for reliable quantification. Overall, these findings establish ¹H NMR spectroscopy as a robust qualitative analytical platform for both authentication and detection of adulteration of saffron. Its integration into routine quality control and regulatory frameworks could strengthen consumer protection and ensure the integrity of high-value herbal and food products.