Determination of Main Alkylamides Responsible for Numbing Sensation in Green Sichuan Pepper Through Quantitative Analysis of Multi-Components by a Single Marker with Nonivamide as an Alternative Reference Standard.

The unsaturated long-chain fatty acid amide derivatives in Sichuan pepper are the components responsible for its characteristic numbing sensation. Due to the susceptibility to oxidation of these compounds, it is difficult to maintain a consistent numbing taste in pepper products, and a significant challenge is faced in the quantitative analysis of these components because the reference standards required for such analysis are very expensive, owing to the difficulty in their isolation and purification. More critically, the accuracy of determination results is always influenced by the oxidation reaction during their storage in the air. In this study, High-Performance Liquid Chromatography (HPLC) coupled with the Quantitative Analysis of Multi-components by a Single Marker (QAMS) method was employed to quantitatively analyze four main alkylamides in 28 batches of Sichuan pepper samples. The analysis revealed differences in the compositional profiles of Green Sichuan Pepper from various sources. The results obtained from both the external standard method (ESM) and the QAMS method were consistent, with standardized mean differences (SMDs) below 5.0%. Furthermore, within the QAMS method, two different internal standards, hydroxyl-α-sanshool and Nonivamide, were used. The SMD between the results obtained using these two different internal standards, respectively, was also within 5%. In conclusion, the stable and cost-effective Nonivamide can serve as a viable alternative reference standard for the HPLC-based determination of the major numbing components in Sichuan pepper.