建立一种基于美国官方分析化学师协会(Association of Official Analytical Chemists, AOAC)方法检测黑果枸杞及其制品中花青素含量的改进pH示差法。考察了黑果枸杞及其制品中花青素的最佳提取和检测条件,通过液相色谱-三重四级杆串联质谱法鉴别出黑果枸杞中花青素的具体化学结构,并计算出混合花青素的平均摩尔质量。通过分光光度法测得混合花青素的平均摩尔消光系数,对改进后的pH示差法进行方法学验证和花青素的含量测定。结果显示,最佳提取和检测条件如下:黑果枸杞花青素提取溶剂为盐酸-80%(体积分数)乙醇(3:97,体积比),料液比为1:100(g:mL),提取温度为50 ℃,提取时间为30 min,缓冲溶液稀释5倍后静置平衡20 min。液相色谱-三重四级杆串联质谱法鉴别黑果枸杞中主要以矮牵牛素类花青素为主(占97.96%),黑果枸杞特有的混合花青素平均摩尔质量为912.7 g/mol,平均摩尔消光系数为29 591 L/(mol·cm)。pH示差法改进后能够满足方法学验证要求,固体样品和液体样品最低检出限分别为28.2 mg/100 g、0.282 mg/100 mL。方法改进后花青素提取增长率均大于20%,静置平衡20 min后单次检测结果精密度小于0.3%。以矮牵牛素类花青素代替矢车菊素-3-O-葡萄糖苷计算花青素含量平均提高了2.41倍,能真实地反映黑果枸杞及其制品中花青素的含量。
An improved pH differential method for determination of anthocyanidins in Lycium ruthenicum Murr.and products based on the Association of Official Analytical Chemists (AOAC) method was established.The optimal extraction and detection conditions of anthocyanidins in Lycium ruthenicum Murr.and products were investigated.The specific chemical structures of anthocyanidins from Lycium ruthenicum Murr.were identified by liquid chromatography-triple quadrupole tandem mass spectrometry, and the average molar mass of mixed anthocyanidins was calculated.The average molar extinction coefficient of mixed anthocyanidins was measured by spectrophotometry.The method validation and determination of anthocyanidins used by the improved pH differential method were conducted.Results showed that the optimum extraction and detection conditions were as follows:the hydrochloric acid-80% (volume fraction) ethanol (3:97, volume ratio) was the extraction solvent of anthocyanidins in Lycium ruthenicum Murr., the solid-liquid ratio was 1:100, the extraction temperature was 50 ℃, the extraction time was 30 minutes and the let stand equilibration time was 20 minutes after the buffer solution was diluted 5 times.The anthocyanidins identified by liquid chromatography-triple quadrupole tandem mass spectrometry were mainly petunidins (97.96%) in Lycium ruthenicum Murr.The average molar mass of mixed anthocyanidins that was unique to Lycium ruthenicum Murr.was 912.7 g/mol and the average molar extinction coefficient was 29 591 L/(mol·cm).The pH differential method could meet the requirements of methodology validation after improvement, the minimum detection limits for solid and liquid samples were 28.2 mg/100 g and 0.282 mg/100 mL, respectively.The extraction growth rate of anthocyanidins was all greater than 20%, and the precision of a single detection result after standing equilibration for 20 minutes was less than 0.3%.The content of anthocyanidins had been increased by an average of 2.41 times when replacing cyanidin-3-O-glucoside with petunidins to calculate, which could truly reflect the content of anthocyanidins in Lycium ruthenicum Murr.and products.
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