CN112114068A - 一种同时测定蜜柚中6种糖苷类香气前体的方法 - Google Patents
一种同时测定蜜柚中6种糖苷类香气前体的方法 Download PDFInfo
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Abstract
本发明公开了一种同时测定蜜柚中香叶醇β‑葡萄糖苷、香叶醇β‑樱草糖苷、芳樟醇β‑樱草糖苷、苯甲醇β‑樱草糖苷、2‑苯乙醇β‑樱草糖苷和橙花叔醇β‑樱草糖苷等6种糖苷类香气前体的UPLC‑MS/MS检测方法，包括提取、净化、配制基质标准工作溶液和采用UPLC‑MS/MS法进行测定步骤。本发明的测定方法能够有效提取蜜柚中的香叶醇β‑葡萄糖苷、香叶醇β‑樱草糖苷、芳樟醇β‑樱草糖苷、苯甲醇β‑樱草糖苷、2‑苯乙醇β‑樱草糖苷和橙花叔醇β‑樱草糖苷等6种糖苷类香气前体，并能进行定性定量测定。该方法快速、准确，灵敏度高，重现性好。
Description
技术领域
本发明属于分析化学领域，具体涉及一种同时测定柚果中香叶醇β-葡萄糖苷、香叶醇β-樱草糖苷、芳樟醇β-樱草糖苷、苯甲醇β-樱草糖苷、2-苯乙醇β-樱草糖苷和橙花叔醇β-樱草糖苷等6种糖苷类香气前体的UPLC-MS/MS检测方法。
背景技术
植物中的风味化合物实质上是以非挥发性和无味糖配合物的形式存在的，而且在一些植物中发现的糖苷结合芳香化合物的浓度比游离的化合物高出几倍。为了长时间保持香味，许多水果或花卉可以通过糖苷酶的水解从糖苷香味前体中长期释放香气化合物。香叶醇、芳樟醇、橙花叔醇、苯甲醇和2-苯乙醇是各种植物的糖苷类香气前体。
柚子是我国南方广泛种植的柑桔栽培品种。柚子因其芳香可口、有益健康和药用价值而被认为是很有吸引力的水果，并被用作果酱和糖果的原料。柚子皮也加工成精油，因为会产生各种芳香化合物。香叶醇、芳樟醇、橙花叔醇、苯甲醇和2-苯乙醇是柚子香气的主要成分。然而，关于柚子中糖苷类成分的测定，尤其是糖苷类成分的测定，却鲜有报道。建立测定柚子中糖苷类香气前体的方法，研究糖苷类香气前体产生芳香化合物的机理，进而对柚子加工过程中的增香行为具有重要意义。
目前，植物叶、花和果实中糖苷类香气前体的检测方法主要有通过气相色谱-质谱（GC-MS）技术检测糖苷水解释放的苷元、气相色谱-质谱法通过检测三氟乙酸转化的糖苷类芳香前体衍生物。水解法可能会因为在水解过程中芳香异构体的重排以及缺乏糖基信息等因素而影响对糖苷类香气前体的定性定量分析。衍生法也存在着因糖苷类香气前体的衍生化不具有选择性而导致产品质量的差异的缺点。因此，建立一种直接测定蜜柚中糖苷类香气前体的检测方法尤为重要。
发明内容
本发明提供一种同时测定水果中香叶醇β-葡萄糖苷、香叶醇β-樱草糖苷、芳樟醇β-樱草糖苷、苯甲醇β-樱草糖苷、2-苯乙醇β-樱草糖苷和橙花叔醇β-樱草糖苷等6种糖苷类香气前体的UPLC-MS/MS检测方法。该方法首次利用超高压液相色谱-质谱联用仪，通过优化质谱条件和流动相，实现香叶醇β-葡萄糖苷、香叶醇β-樱草糖苷、芳樟醇β-樱草糖苷、苯甲醇β-樱草糖苷、2-苯乙醇β-樱草糖苷和橙花叔醇β-樱草糖苷的分离和同时测定。
本发明的目的是通过以下技术方案来实现的。
一种同时测定蜜柚中糖苷类香气前体的方法，包括如下步骤：
（1） 提取：称取打碎后的蜜柚样品2 g至50 mL离心管中，加入20 mL甲醇，在10000 r/min条件下均质2分钟；然后超声提取20 min，再4500 r/min下离心5 min，上清液转移至另一新的离心管中；上清液通过0.22μm有机膜过滤至进样小瓶，供高效液相色谱----串联质谱仪测定；
（2）配制基质标准工作溶液在甲醇中以1 mg/mL的浓度制备香叶醇β-葡萄糖苷、香叶醇β-樱草糖苷、芳樟醇β-樱草糖苷、苯甲醇β-樱草糖苷、2-苯乙醇β-樱草糖苷和橙花叔醇β-樱草糖苷的储备溶液；将这些储备溶液混合，然后稀释以产生连续校准标准溶液；在甲醇中制备标准混合物，配制至少五个浓度的糖苷系列基质标准工作溶液储备溶液和校准标准溶液在-18℃下保存；
（3）液相色谱-串联质谱法LC-MS/MS测定
质谱检测使用多反应监测扫描模式，香叶醇β-葡萄糖苷的母离子361.1，子离子分别为361.1；香叶醇β-樱草糖苷的母离子493.1，子离子分别为149.1和89.1；芳樟醇β-樱草糖苷的母离子493.1， 子离子分别为315.1和161.1；苯甲醇β-樱草糖苷的母离子447.0,子离子分别为401.1和269.1；2-苯乙醇β-樱草糖苷的母离子461.1,子离子分别为415.1和149.1；橙花叔醇β-樱草糖苷的母离子561.1,子离子分别为515.2和383.2；
定量测定：将步骤(2)中系列基质标准工作液进行LC-MS/MS测定，以基质标准工作液的色谱峰面积对其相应浓度进行回归分析，得到标准工作曲线;在相同条件下将步骤(1) 中样品液注入LC-MS/MS进行测定，测得样品液中糖苷类化合物的色谱峰面积，代入标准曲线，得到样品液中糖苷类药物含量，然后根据样品液所代表试样的质量计算得到样品中糖苷类药物残留量；
定性测定：检测步骤(1)中样品液中目标化合物母离子和子离子对，若其离子色谱峰保留时间与标准工作溶液一致；且样品液中目标化合物的两个子离子的相对丰度与浓度相当，则判断该样品中存在该种目标化合物；若上述两个条件不能同时满足，则判断不含该种目标化合物；所述的6种糖苷类香气前体是香叶醇β-葡萄糖苷、香叶醇β-樱草糖苷、芳樟醇β-樱草糖苷、苯甲醇β-樱草糖苷、2-苯乙醇β-樱草糖苷和橙花叔醇β-樱草糖苷；
液相色谱条件为，色谱柱：Shim-pack GIST C18柱（2.0um，2.1mm×100mm）；流速：0.3mL/min；柱温：40℃；样品盘温度：15℃；进样量：2μL；流动相A为0.1%（V/V）甲酸水溶液，流动相B为乙腈；
梯度洗脱条件为：0 min，10% B；3 min，25% B；9 min，40% B；11 min，90% B；12 min，90% B；12 min，10% B，16 min，10% B；
质谱条件如下：离子源：电喷雾离子源ESI；扫描方式：负离子扫描；检测方式：多反应监测扫描模式；气压电离（API）气体：氮气（纯度>95%，压力600-900kpa）；碰撞气体：高纯氩；界面电压：3.0 kv；界面温度：300 ℃；脱溶剂温度：250 ℃；干燥气：10 L/min，雾化气：3 L/min，加热气：10 L/min，热块温度：400 ℃。
本发明的有益效果：本发明的测定方法能够有效提取蜜柚中香叶醇β-葡萄糖苷、香叶醇β-樱草糖苷、芳樟醇β-樱草糖苷、苯甲醇β-樱草糖苷、2-苯乙醇β-樱草糖苷和橙花叔醇β-樱草糖苷等6种糖苷类香气前体，首次利用超高压液相色谱-质谱联用仪，通过优化色谱条件和质谱条件，实现蜜柚中香叶醇β-葡萄糖苷、香叶醇β-樱草糖苷、芳樟醇β-樱草糖苷、苯甲醇β-樱草糖苷、2-苯乙醇β-樱草糖苷和橙花叔醇β-樱草糖苷等6种糖苷类香气前体的提取、分离和快速检出，方法快速、准确，灵敏度高，重现性好。
附图说明
图1为6种糖苷类香气前体的结构式；
图2为本发明6种糖苷类香气前体的色谱图；1.香叶醇β-葡萄糖苷，2.香叶醇β-樱草糖苷，3.芳樟醇β-樱草糖苷，4.苯甲醇β-樱草糖苷，5.2-苯乙醇β-樱草糖苷，6.橙花醇β-樱草糖苷；
图3为本发明6种糖苷类香气前体测定的超高液相质谱图；1.香叶醇β-葡萄糖苷，2.香叶醇β-樱草糖苷，3.芳樟醇β-樱草糖苷，4.苯甲醇β-樱草糖苷，5.2-苯乙醇β-樱草糖苷，6.橙花醇β-樱草糖苷。
具体实施方式
本发明以下将结合实施例作进一步描述，但并不限制本发明：
实施例1
①样品提取：取已研磨的新鲜柚子花样品2.0 g置于50 mL离心管中，加入20 mL甲醇作为溶剂，在10000 r/min条件下均质2 min，超声提取20 min后，在4500 r/min离心分离，以0.22μm的尼龙滤膜过滤，制得试样。
②测定：采用UPLC-MS/MS法进行测定，仪器条件如下：
液相色谱条件为，色谱柱：Shim-pack GIST C18柱（2.0um，2.1mm×100mm）；流速：0.3mL/min；柱温：40℃；样品盘温度：15℃；进样量：2μL；流动相A为0.1%（V/V）甲酸水溶液，流动相B为乙腈；
梯度洗脱条件为：0 min，10% B；3 min，25% B；9 min，40% B；11 min，90% B；12 min，90% B；12 min，10% B，16 min，10% B；
质谱条件如下：离子源：电喷雾离子源ESI；扫描方式：负离子扫描；检测方式：多反应监测扫描模式；气压电离（API）气体：氮气（纯度>95%，压力600-900kpa）；碰撞气体：高纯氩；界面电压：3.0 kv；界面温度：300 ℃；脱溶温度：250 ℃；干气：10 L/min，雾化气：3 L/min，热气：10 L/min，热块温度：400 ℃。
测定结果显示：柚花中香叶醇β-葡萄糖苷的含量为470.8 µg/kg、香叶醇β-樱草糖苷的含量为1252.9 µg/kg、芳樟醇β-樱草糖苷的含量为65.0 µg/kg、苯甲醇β-樱草糖苷241.3的含量为 µg/kg、2-苯乙醇β-樱草糖苷的含量为30.8 µg/kg和橙花叔醇β-樱草糖苷的含量为134.2 µg/kg。
表1 糖苷类香气前体物的质谱参数
表2 6种糖苷香气前体测试的工作曲线相关参数表
Claims (5)
1.一种同时测定蜜柚中6种糖苷类香气前体的方法，其特征在于：包括如下步骤：
（1） 提取：称取打碎后的蜜柚样品2 g至50 mL离心管中，加入20 mL甲醇，在10000 r/min条件下均质2分钟；然后超声提取20 min，再4500 r/min下离心5 min，上清液转移至另一新的离心管中；上清液通过0.22μm有机膜过滤至进样小瓶，供高效液相色谱----串联质谱仪测定；
（2）配制基质标准工作溶液在甲醇中以1 mg/mL的浓度制备香叶醇β-葡萄糖苷、香叶醇β-樱草糖苷、芳樟醇β-樱草糖苷、苯甲醇β-樱草糖苷、2-苯乙醇β-樱草糖苷和橙花叔醇β-樱草糖苷的储备溶液；将这些储备溶液混合，然后稀释以产生连续校准标准溶液；在甲醇中制备标准混合物，配制至少五个浓度的糖苷系列基质标准工作溶液储备溶液和校准标准溶液在-18℃下保存；
（3）液相色谱-串联质谱法LC-MS/MS测定
定量测定：将步骤(2)中系列基质标准工作液进行LC-MS/MS测定，以基质标准工作液的色谱峰面积对其相应浓度进行回归分析，得到标准工作曲线；在相同条件下将步骤(1) 中样品液注入LC-MS/MS进行测定，测得样品液中糖苷类化合物的色谱峰面积，代入标准曲线，得到样品液中糖苷类药物含量，然后根据样品液所代表试样的质量计算得到样品中糖苷类药物残留量；
定性测定：检测步骤(1)中样品液中目标化合物母离子和子离子对，若其离子色谱峰保留时间与标准工作溶液一致；且样品液中目标化合物的两个子离子的相对丰度与浓度相当，则判断该样品中存在该种目标化合物；若上述两个条件不能同时满足，则判断不含该种目标化合物；所述的6种糖苷类香气前体是香叶醇β-葡萄糖苷、香叶醇β-樱草糖苷、芳樟醇β-樱草糖苷、苯甲醇β-樱草糖苷、2-苯乙醇β-樱草糖苷和橙花叔醇β-樱草糖苷。
2.根据权利要求1所述的一种同时测定蜜柚中6种糖苷类香气前体的方法，其特征在于：质谱检测使用多反应监测扫描模式，香叶醇β-葡萄糖苷的母离子361.1，子离子分别为361.1；香叶醇β-樱草糖苷的母离子493.1，子离子分别为149.1和89.1；芳樟醇β-樱草糖苷的母离子493.1， 子离子分别为315.1和161.1；苯甲醇β-樱草糖苷的母离子447.0,子离子分别为401.1和269.1；2-苯乙醇β-樱草糖苷的母离子461.1,子离子分别为415.1和149.1；橙花叔醇β-樱草糖苷的母离子561.1,子离子分别为515.2和383.2。
3.根据权利要求1所述的一种同时测定蜜柚中6种糖苷类香气前体的方法，其特征在于：液相色谱条件为，色谱柱：Shim-pack GIST C18柱，2.0um，2.1mm×100mm；流速：0.3 mL/min；柱温：40℃；样品盘温度：15℃；进样量：2μL；流动相A为0.1%（V/V）甲酸水溶液，流动相B为乙腈。
4.根据权利要求1所述的一种同时测定蜜柚中6种糖苷类香气前体的方法，其特征在于：梯度洗脱条件为：0 min，10% B；3 min，25% B；9 min，40% B；11 min，90% B；12 min，90%B；12 min，10% B，16 min，10% B。
5.根据权利要求1所述的一种同时测定蜜柚中6种糖苷类香气前体的方法，其特征在于：质谱条件如下：离子源：电喷雾离子源ESI；扫描方式：负离子扫描；检测方式：多反应监测扫描模式；气压电离API气体：氮气纯度>95%，压力600-900kpa；碰撞气体：高纯氩；界面电压：3.0 kv；界面温度：300 ℃；脱溶剂温度：250 ℃；干燥气：10 L/min，雾化气：3 L/min，加热气：10 L/min，热块温度：400 ℃。
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