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Abstract：
Spinach (Spinacia oleracea) is one of the most famous vegetables worldwide, rich in essential metabolites for various health benefits. It is a valuable plant source that has the potential to be a nutraceutical. This study aimed to evaluate the single characteristic marker compound to establish the validation of HPLC-DAD methods applied to the development of a nutraceutical using spinach samples. Six metabolites (1-6) were identified from the spinach samples such as freeze-dried spinach (FDS) and spinach extract concentrate (SEC) by LC-Q-TOF/MS analysis. Among the six metabolites, 3\',4\',5-trihydroxy-3-methoxy-6,7-methylenedioxyflavone 4\'-glucuronide (TMG) was selected as a marker compound due to its highest abundance and high selectivity. The specificity, accuracy, linearity, precision, repeatability, limit of detection (LOD), and limit of quantification (LOQ) of TMG in the spinach samples (FDS and SEC) were validated according to AOAC international guideline. The specificity was confirmed by monitoring the well separation of the marker compound from other compounds of spinach samples in the base peak intensity (BPI) and ultraviolet (UV) chromatogram. The calibration curve of TMG (15.625~500 μg/mL) had reasonable linearity (R2 = 0.999) considered with LOD and LOQ values, respectively. Recovery rate of TMG was 93-101% for FDS and 90-95% for SEC. The precision was less than 3 and 6% in the intraday and interday. As a result, the HPLC-DAD validation method of TMG in the spinach samples (FDS and SEC) was first established with AOAC and KFDA regulations for approving functional ingredients in functional foods.
摘要：
菠菜（Spinaciaoleracea）是世界上最著名的蔬菜之一，富含各种健康益处的必需代谢产物。它是一种有潜力成为营养食品的有价值的植物来源。本研究旨在评估单一特征标记化合物，以建立HPLC-DAD方法的验证，该方法适用于使用菠菜样品开发营养食品。通过LC-Q-TOF/MS分析从菠菜样品如冻干菠菜(FDS)和菠菜提取物浓缩物(SEC)中鉴定出六种代谢物(1-6)。在六种代谢物中，3\',4\',5-三羟基-3-甲氧基-6,7-亚甲基二氧黄酮4'-葡糖苷酸（TMG）因其最高的丰度和高选择性而被选择为标记化合物。特异性，准确度,线性度精度,重复性,检测限（LOD），根据AOAC国际指南验证了菠菜样品（FDS和SEC）中TMG的定量限（LOQ）。通过在基峰强度(BPI)和紫外(UV)色谱图中监测标记化合物与菠菜样品的其他化合物的良好分离来确认特异性。TMG的校准曲线(15.625~500μg/mL)与LOD和LOQ值具有合理的线性关系(R2=0.999),分别。TMG的回收率对于FDS为93-101%,对于SEC为90-95%。日内和日间的精度分别小于3％和6％。因此,菠菜样品中TMG的HPLC-DAD验证方法（FDS和SEC）首先是根据AOAC和KFDA法规建立的，用于批准功能性食品中的功能性成分。
