Abstract: Objective: To improve the detection sensitivity and accuracy of steroidal sapogenins (such as tigogenin and sarsasapogenin) in Allium vegetables, this study established an analytical method based on stable isotope labeling derivatization combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Methods: The 3-hydroxy group of steroidal sapogenins was derivatized with D0/D5-pyridine and trifluoromethanesulfonic anhydride (Tf2O) for charge labeling, and internal standard quantification was performed using the peak area ratio between D0 and D5 isotope derivatives. Results: The optimized derivatization reaction was completed within 10 minutes at room temperature, and the derivatives showed excellent stability. The proposed method demonstrated a wide linear range (0.5–200 ng/mL) with a coefficient of determination (R²) > 0.999 4, a limit of detection of 0.2 ng/mL (95% lower than that of the non-derivatized method), and excellent precision (intra-day relative standard deviation (RSD) ≤ 4.02%, and inter-day RSD ≤ 4.36%), and satisfactory spiked recoveries (95.5%–104.2%). This method was successfully applied to the analysis of actual Allium samples, revealing the distribution characteristics of tigogenin and sarsasapogenin in green onions, scallions, onions, and garlic. Conclusion: This strategy enhances the ionization efficiency and compensates for matrix effects through charge labeling. It provides a reliable method for the highly sensitive and accurate quantification of steroidal sapogenins in Allium vegetables and can serve as a reference for the analysis of other natural products with poor ionization efficiency.

Key words: Allium vegetables; steroidal sapogenins; derivatization; liquid chromatography-tandem mass spectrometry; isotope labeling