Analysis of 3‐nitropropionic acid in Fabaceae plants by HPLC‐MS/MS.

Introduction: 3‐Nitropropionic acid (3‐NPA) is a toxic compound that can accumulate in esterified form in the Fabaceae family. In the Lotae tribe, many species have been identified as 3‐NPA producers (e.g., Securigera varia), while some of the genetically close Lotae plants were formerly reported as 3‐NPA‐free (e.g., Lotus corniculatus and Anthyllis vulneraria). These plants are used as forage and have a tradition in ethnomedicine, also, the extracts of A. vulneraria are used in cosmetics. Objectives: Our aim was to investigate the 3‐NPA content of these selected Fabaceae species and to develop a validated quantitative method to evaluate 3‐NPA concentrations in extracts of different herbal parts and cosmetic products. Materials and Methods: A UHPLC‐ESI‐Orbitrap‐MS/MS method was applied for detection and identification of 3‐NPA derivatives in the form of glucose esters. For the quantitative analysis, an optimized sample processing method was developed. The free 3‐NPA content was determined using HPLC‐ESI‐MS/MS. Results: 3‐NPA esters could be detected in all three species, but their quantity showed a high variation. S. varia contained 0.5–1.0 g/100 g of 3‐NPA, while in L. corniculatus samples only trace quantities were detectable, below the LOQ (25 ng/ml). Most of the A. vulneraria samples showed similarly low concentrations, but one sample had 3‐NPA levels comparable to S. varia. 3‐NPA could not be detected in the tested cosmetics containing A. vulneraria extracts. Conclusions: Using highly sensitive analytical methods, new 3‐NPA‐containing species were identified. The developed validated quantitative method is suitable for the determination of 3‐NPA concentrations in herbal samples. The nitropropionic acid (NPA) content of Securigera varia (L.), Lotus corniculatus L., and Anthyllis vulneraria L. was analyzed. The presence of glucose esters of nitropropionic acid were confirmed by UHPLC‐ESI‐Orbitrap‐MS/MS. An optimized sample preparation protocol was developed along with a validated HPLC‐ESI‐MS/MS method for quantitative purposes. NPA content of S. varia samples were determined, and the substance was detected in Lotus corniculatus and A. vulneraria, indicating that these species are able to produce the toxin in low quantities. [ABSTRACT FROM AUTHOR]