Alkaloid variation trend during the processing of nine-processed tangerine peel based on UPLC-MS/MS

【Objective】 To analyze the variation patterns of alkaloid compounds during the processing of nine-processed tangerine peel， which could provide theoretical basis for optimizing its processing procedures and quality control.【Method】 Citrus peels from four key processing stages of nine-processed tangerine peel， raw material （CP1）， soa-king （CP2）， pickling （CP3）， and aging （CP4）， were selected as experimental samples. Ultra-performance liquid chromatography coupled with tandem mass spectrometry （UPLC-MS/MS） was used in combination with principal component analysis （PCA）， cluster analysis， and orthogonal partial least squares discriminant analysis （OPLS-DA） to investigate variation trend in alkaloid metabolites at the four key processing stages. Differential metabolites were identified through significance testing， fold change analysis， K-means cluster， and KEGG pathway annotation.【Result】 A total of 33 alkaloids and their derivatives were identified across the four key processing stages of nine-processed tangerine peel. These included 10 indole alkaloids， 3 quinoline alkaloids， each 2 of organic amines， pyridines， acridones， and phenethylamines， each 1 of quaternary ammonium compound， piperidine， pyrrole， and purine， and 8 alkaloid derivatives. Synephrine showed the highest relative content， followed by stachydrine. Based on the criteria of Fold Change ≥ 1.5 or Fold Change ≤ 0.67， together with variable importance in projection （VIP） ≥ 1， 30 significantly differential metabolites were screened， accounting for 90.9% of the total alkaloids. The comparisons of CP1 vs CP2， CP2 vs CP3， CP3 vs CP4， and CP1 vs CP4 contained 22， 17， 10， and 23 significantly differential metabolites respectively. During the overall proces-sing of nine-processed tangerine peel， the relative contents of alkaloids showed a general decreasing trend， with indole alkaloids exhibiting the most pronounced decline， and the major changes mainly occurring during the soaking stage. The up-regulated differential metabolites included DL-2-aminoadipic acid， nicotine glucuronide， acetylcholine， and betaine， whereas the down-regulated differential metabolites were mainly indole alkaloids， such as 4-aminoindole， N-acetyl-5-hydroxytryptamine， and methoxyindoleacetic acid. The main KEGG metabolic pathways annotated for the differential metabolites included tryptophan metabolism， biosynthesis of secondary metabolites， ABC transporters， as well as glycine， serine， and threonine metabolism.【Conclusion】 During the processing of nine-processed tangerine peel， soaking is the key stage for the reduction and transformation of alkaloids contents. In practical production， soaking parameters， such as soaking time and temperature， can be optimized to balance the retention of major active components with the removal of bitter substances. Meanwhile， the contents of major active components， such as synephrine， and the transformation of indole compounds should be monitored.