Methods for Isolating, Identifying, and Quantifying Anthocyanin Metabolites in Clinical Samples
Methods for Isolating, Identifying, and Quantifying Anthocyanin Metabolites in Clinical Samples
复制标题
DOI：
10.1021/ac500565a
复制
发表时间：
2014-10-21
影响因子：
7.4
通讯作者：
Kay, Colin D.
中科院分区：
文献类型：
--
作者：
de Ferrars, Rachel M.;Czank, Charles;Kay, Colin D.
直到最近，花青素苷的代谢命运相对未知，主要是由于它们在生理pH下的不稳定性以及缺乏用于从生物样品中分离和鉴定其代谢物的公开方法。本工作的目的是建立用于提取和定量存在于尿液、血清和粪便样品中的花青素代谢物的方法。获得35种商业分析物和10种合成分析物，包括已知和预测的花青素苷的人类和微生物代谢物，作为参考标准。针对有机改性剂、离子改性剂、移动的相梯度、流速、柱类型、MS源和化合物依赖性参数优化HPLC和MS/MS条件。考察了吸附剂、溶剂、酸、防腐剂、洗脱剂和蒸发等因素对固相萃取效率的影响。HPLC-MS/MS方法验证证明线性（R（2），0.997 +/- 0.002）和灵敏度可接受（检测限（LOD）：尿液，100 +/- 375 nM;血清，104 +/- 358 nM;粪便138 +/- 344 nM），最终SPE方法提供的尿液回收率为88.3 +/- 17.8%，血清为86.5 +/- 11.1%，粪便为80.6 +/- 20.9%。最终的方法应用于来自花青素干预研究的临床样品，其中45种建模代谢物中的36种在尿液，血浆或粪便样品中检测到。所描述的方法为鉴定和定量广泛系列的花青素苷代谢物提供了合适的通用性，用于探索吸收、分布、代谢和消除的未来临床研究。
The metabolic fate of anthocyanins until recently was relatively unknown, primarily as a result of their instability at physiological pH and a lack of published methods for isolating and identifying their metabolites from biological samples. The aim of the present work was to establish methods for the extraction and quantification of anthocyanin metabolites present in urine, serum, and fecal samples. 35 commercial and 10 synthetic analytes, including both known and predicted human and microbial metabolites of anthocyanins, were obtained as reference standards. HPLC and MS/MS conditions were optimized for organic modifier, ionic modifier, mobile phase gradient, flow rate, column type, MS source, and compound dependent parameters. The impact of sorbent, solvent, acid, preservative, elution, and evaporation on solid phase extraction (SPE) efficiency was also explored. The HPLC-MS/MS method validation demonstrated acceptable linearity (R(2), 0.997 +/- 0.002) and sensitivity (limits of detection (LODs): urine, 100 +/- 375 nM; serum, 104 +/- 358 nM; feces 138 +/- 344 nM), and the final SPE methods provided recoveries of 88.3 +/- 17.8% for urine, 86.5 +/- 11.1% for serum, and 80.6 +/- 20.9% for feces. The final methods were applied to clinical samples derived from an anthocyanin intervention study, where 36 of the 45 modeled metabolites were detected within urine, plasma, or fecal samples. The described methods provide suitable versatility for the identification and quantification of an extensive series of anthocyanin metabolites for use in future clinical studies exploring absorption, distribution, metabolism, and elimination.