Retention-Guided LC-MS/MS Enables Discovery and Structural Assignment of Trace Steviol Glycosides from in Vitro Glycosylation Reactions

IF 6.7 1区农林科学 Q1 AGRICULTURE, MULTIDISCIPLINARY

Journal of Agricultural and Food Chemistry Pub Date : 2026-10-17 DOI:10.1021/acs.jafc.6c04961

Takehiro Watanabe,Kohki Fujikawa,Kazunari Iwaki,Tadayoshi Hirai,Jun Murata

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引用次数: 0

Abstract

Abstract Stevia rebaudiana produces diverse steviol glycosides (SVGs), but many positional isomers remain difficult to distinguish by MS/MS alone because of similar fragmentation patterns and low abundance. Here, we established a retention-guided LC–MS/MS workflow for discovery and structural characterization of trace SVGs generated by in vitro glycosylation using crude enzyme extracts from Stevia leaves. Reversed-phase retention trends associated with sugar identity and linkage patterns enabled chromatographic discrimination of isomeric SVGs, including rebaudiosides D and I, which exhibit indistinguishable MS/MS and MS3 spectra. Enzymatic reactions using UDP–13C6-glucose and UDP-xylose generated rare sugar-chain elongated derivatives, including rebaudioside FX1 and the previously unreported compound Reb FX0 carrying Glcβ(1–2)Glcβ1– at C-13 and Xylβ(1–2)Glcβ1– at C-19. Structural assignments were confirmed by comparison with semisynthetic standards. This integrated analytical–biochemical approach provides a practical platform for discovery-oriented characterization of trace SVGs and supports compositional analysis of stevia-derived ingredients and bioconversion products relevant to food applications.

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保留导向的lc -MS/MS能够从体外糖基化反应中发现痕量甜菊醇糖苷并进行结构分配

甜菊糖（Stevia rebaudiana）产生多种甜菊醇糖苷（SVGs），但由于许多位置异构体的断裂模式相似且丰度较低，因此难以通过MS/MS单独区分。在这里，我们建立了一个保留引导的LC-MS /MS工作流程，用于发现和结构表征甜菊叶粗酶提取物体外糖基化产生的痕量SVGs。与糖的特性和连锁模式相关的反相保留趋势使得包括雷鲍迪苷D和I在内的同分异构体SVGs具有不可区分的MS/MS和MS3光谱。利用udp - 13c6 -葡萄糖和udp -木糖的酶促反应生成了罕见的糖链延长衍生物，包括雷鲍迪苷FX1和以前未报道的化合物Reb FX0，在C-13和C-19上携带Glcβ(1-2)Glcβ1 -和Xylβ(1-2)Glcβ1 -。通过与半合成标准的比较，确定了结构分配。这种综合的分析-生化方法为痕量svg的发现导向表征提供了一个实用的平台，并支持甜菊糖衍生成分和与食品应用相关的生物转化产品的成分分析。

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来源期刊

Journal of Agricultural and Food Chemistry 农林科学-农业综合

CiteScore

9.90

自引率

8.20%

发文量

1375

审稿时长

2.3 months

期刊介绍： The Journal of Agricultural and Food Chemistry publishes high-quality, cutting edge original research representing complete studies and research advances dealing with the chemistry and biochemistry of agriculture and food. The Journal also encourages papers with chemistry and/or biochemistry as a major component combined with biological/sensory/nutritional/toxicological evaluation related to agriculture and/or food.