Liquid Chromatography coupled with Mass Spectrometry as an Analytical Strategy to Assess the Occurrence of Potentially Toxic Cyanogenic Glycosides in Edible Microgreens

Microgreens are increasingly promoted as sustainable, nutrient-dense foods, yet their content of potentially harmful specialized metabolites remains poorly explored. Here, we developed and applied a reversed-phase liquid chromatography–electrospray ionization high-resolution mass spectrometry (RPLC-ESI-HRMS) method for the detection of cyanogenic glycosides (CNGs) in edible microgreens. Method optimization, performed using dhurrin and lotaustralin as model standards, showed that positive ion detection of sodium adducts provided the most informative and selective HRMS/MS response, with diagnostic fragmentation patterns suitable for CNG recognition in complex matrices. Quantitative validation for lotaustralin showed excellent linearity (R2 = 0.998), low detection/quantification limits (LOD 0.16 mg/L; LOQ 0.53 mg/L), good extraction recovery, and a negligible matrix effect. Application of the method revealed a clear species-dependent profile. No detectable CNGs were found in broccoli raab and kale microgreens, supporting their safety as ready-to-eat products in this respect. In contrast, flax microgreens contained four CNGs: linamarin, lotaustralin, linustatin, and neolinustatin. Monoglycosylated species predominated, with lotaustralin quantified at 5.5 ± 0.6 mg/g dry weight and linamarin estimated at even higher levels. Diglycosylated CNGs were present at much lower concentrations and displayed multiple chromatographic peaks, consistent with the occurrence of structurally related isomeric forms. These quantitative results are specific to the flax microgreen samples analyzed here, obtained by pooling the lyophilized material obtained from several plants; thus, they do not account for biological variability among individual plants. Based on the measured CNG levels, flax microgreens showed a non-negligible cyanogenic potential. Assuming 1, 10 and 25% conversion to hydrogen cyanide, the estimated release would be, respectively, about 3, 33 and 81 mg HCN/kg of fresh flax microgreens, values lower than the current EU limit (150 mg HCN/kg of edible product) for flaxseed intended for direct consumption but comparable to values reported for other foods. These findings highlight the need to complement the nutritional evaluation of novel microgreens with targeted toxicological screening.