A Validated Trigonelline-Based Method for the Standardization and Quality Control of Trigonella foenum-graecum L

Fenugreek, or Trigonella foenum-graecum L, is an edible and medicinal plant of the Fabaceae family. Fenugreek seeds are rich in proteins, lipids, and essential nutrients, and they also contain various phytochemicals, including flavonoids, steroidal saponins, coumarin, and alkaloids such as trigonelline. Trigonelline (TG) is a bioactive plant alkaloid initially extracted from fenugreek seeds. A substantial portion of fenugreek's health benefits may rely on the presence of TG. This study addresses the critical need for a fast, green, and economical method that overcomes inefficiencies, high solvent usage, and sensitivity limitations in the quantification of TG.

Fenugreek seeds from various origins were extracted using three green solvents: acetone (ACt), ethanol (EtOH), and water (H ₂O). The UPLC-MS/MS method was developed and validated using a green mobile phase of H ₂O: EtOH, and an r ²-value of 0.999 in the linearity range of 0.1-500 ppb was adopted. The method was validated with an accuracy of 98.6% for trace analysis of TG using a small amount (10 mg) of fenugreek samples from five different origins.

The average extract yield was 5.36 mg/100 mg with a standard deviation (SD) of 6.3, with the highest extract yield observed in H ₂O. The ESI (+ve) of the UPLC-MS/MS resulted in the fragmentation pattern ( m/z) 138→94.10→92.05→78.20. The TG quantification revealed an average TG concentration of 181.4 ppb (SD = 176.4), with the highest amount of TG in H ₂O extract (mean = 392.7, SD = 132.4 ppb), followed by EtOH (mean = 91.9, SD = 83.3 ppb) and ACt (mean = 59.5, SD = 30.9 ppb). The TG amount observed in the validation step substantiated the efficiency and reproducibility of the developed method.

The method may be used as an effective tool for a green, rapid, economical, and eco-friendly extraction and quantification of TG in diverse matrices of pharmaceutical, cosmeceutical, herbal, and food products.