基于液相色谱-质谱联用技术定性定量分析黑豆种皮中可溶型和结合型花青素

Qualitative and Quantitative Analysis of Soluble and Bound Anthocyanins in Black Soybean Seed Coat by High Performance Liquid Chromatography-Mass Spectrometry

以超声波辅助有机溶剂提取法获得黑豆种皮可溶型花青素提取物,再进一步对不含可溶型花青素的黑豆种皮残渣使用酸水解和碱水解以及酸碱/碱酸轮提水解,获得黑豆种皮结合型花青素提取物。采用超高效液相色谱-电喷雾-四极杆飞行时间质谱联用仪分析鉴定各黑豆种皮提取物中所含有的共17种花青素成分,包括11种花青素糖苷类:飞燕草素-3-O-葡萄糖苷、飞燕草素-3-O-半乳糖苷、矮牵牛花素-3-O-葡萄糖苷、矮牵牛花素-3-O-半乳糖苷、天竺葵素-3-O-芸香糖苷、矢车菊素-3-O-葡萄糖苷、矢车菊素-3-O-半乳糖苷、天竺葵素-3-O-己糖苷、芍药花素-3-O-己糖苷、天竺葵素-3-O-(6"-丙二酰葡萄糖苷)和芹菜定-3-O-(6"-丙二酰葡萄糖苷);6种花青素苷元:飞燕草素、矢车菊素、矮牵牛花素、天竺葵素、芹菜定和芍药花素。采用高效液相色谱-电喷雾-三重四极杆质谱联用仪精确定量各类黑豆种皮提取物中的花青素含量,结果表明,酸性结合型花青素提取物中结合型花青素的总含量最高。此外,在黑豆种皮的可溶型花青素提取物中,花青素主要以花青素糖苷类形式存在,苷元含量相对极少;而在结合型花青素提取物中,则主要以花青素苷元为主,糖苷类化合物相对少见。

Ultrasonic-assisted organic solvent extraction was used to obtain soluble anthocyanins from black soybean seed coat, and bound anthocyanins from the extraction residue were obtained by treatment with acid, alkaline, sequential acid/alkali hydrolysis or the reverse sequence. By ultra-high performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry(UPLC-ESI-QTOF-MS), a total of 17 anthocyanin components,including 11 anthocyanin glycosides: delphinidin-3-O-glucoside, delphinidin-3-O-galactoside, petunidin-3-O-glucoside,petunidin-3-O-galactoside, pelargonidin-3-O-rutinoside, cyanidin-3-O-glucoside, cyanidin-3-O-galactoside, pelargonidin-3-O-hexoside, peonidin-3-O-hexoside, pelargonidin-3-O-(6"-malonyl glucoside) and apigeninidin-3-O-(6"-malonyl glucoside);and 6 anthocyanin aglycones: delphinidin, cyanidin, petunidin, pelargonidin, apigeninidin, and peonidin, were identified from these extracts. High performance liquid chromatography coupled with electrospray ionization triple quadrupole mass spectrometry(HPLC-ESI-QQQ-MS) was used to accurately quantify the content of anthocyanins. The total content of bound anthocyanins in the acid extract was the highest. In addition, in the soluble anthocyanin extract, anthocyanins were present mainly in the form of anthocyanin glycoside with lesser amounts of aglycons, whereas in the bound anthocyanin extract,anthocyanin aglycons were the main components but glycosides were rare.