氨基甲酸乙酯在葡萄酒中的含量的变化及研究
摘要
本研究对氨基甲酸乙酯(EC)的HPLC-FLD和GC/MS测定方法进行了优化。应用优化后的方法对葡萄酒酿造过程中的EC含量的变化进行了测定,并且考察了不同品种和产地的赤霞珠(Cabernet Sauvignon,CS).蛇龙珠(Cabernet Gernischt,CG)和梅鹿辄(Merlot)中EC的含量,主要获得了以下结果：
1、HPLC-FLD方法的最优测定条件
利用高效液相色谱结合荧光检测器,确定的测定条件为：将白兰地样品稀释至酒精含量为25%(v/v),葡萄酒不经稀释；取200μL样品加入50μL HCl溶液及100μL的二苯吡喃醇溶液,暗反应5min后,取20μL进样；色谱条件为色谱柱(ODS-2HYPERSIL C18 4.6mm×250mm,5μm),流动相A：醋酸钠溶液(20mM,pH=7.2),流动相B：乙腈,流速为0.8mL/min;流动相梯度初始条件为流动相A：55%,流动相B：45%,10min时流动相A：50%,至35min时流动相A：0%,然后维持5min;检测器激发光波长为234nm,发射光波长为600nm。
2、GC/MS方法的最优测定条件
本实验确定的气质联用测定条件如下.,色谱条件：色谱柱为TR-WaxMs30m×0.25mm(内径)×0.25μm(膜厚)；手动进样1μL；不分流进样；程序升温起始温度80℃,再以5℃/min的速度升至180℃,后又以20℃/min升温至250℃,并在该温度下停留2min；进样口温度为250℃；载气为高纯氦气；恒流速为1mL/min。质谱条件为：EI电离方式(能量为70eV)；离子检测模式(SIM Mode)中选取m/z为44、62和74的离子碎片进行扫描,离子源及色谱和质谱连接处温度分别为200℃和220℃。
3、葡萄酒酿造和储存过程中EC的变化
在葡萄酒酿造过程中,EC的含量略微上升,但不同产地间的含量差异很大；不同产地的葡萄酒中沙城葡萄酒EC含量较其它产区低；不同品种的干红葡萄酒EC的含量依次为梅鹿辄>赤霞珠>蛇龙珠；我国大部分产区的葡萄所酿造的葡萄酒中EC的含量都在限量范围内；不同年份的白兰地随着储存时间的增加,其EC含量也在增加,但没有超过国际上对于白兰地中EC含量的限量标准；白兰地的粗馏期间EC含量维持在一个较高的水平,其变化存在一个维持不变到增大再逐渐变小的过程；精馏期间EC含量维持在一个较低的水平,其含量逐渐增大后又减小的过程。添加酸性脲酶对比实验的结果显示：在葡萄酒酿造过程中添加酸性脲酶其EC含量在增长一段时间后,维持在一个相对稳定的低水平上,可见添加一定量的酸性脲酶对于降低葡萄酒中的EC有一定作用。
In this study, the HPLC-FLD and GC/MS methods to measure ethyl carbamate (EC) have been optimized. The EC changes during the wine vintage were measured by the optimized methods for the different species and origin of the Cabernet Sauvignon (CS), Cabernet Gernischt (CG) and Merlot. The results were as follows:
1. The optimal HPLC-FLD method
The best HPLC-FLD conditions were that samples were diluted to 25% (v/v) alcohol content,200μL diluted samples were added into 50μL HC1 solution and 100μL 9-xanthydrol solution for a 5-min dark reaction, then 10μL volume was injected into ODS-2 HYPERSIL C18 (4.6mm×250mm 5μm column). The mobile phase A was sodium acetate solution (20mM, pH=7.2) while B was acetonitrile, the flow rate was 0.8mL/min. The percentage of phase B was increased from 45% to 50% during 0 to 10min, and from 50% to 100% during 10 to 35min, then maintained at 100% for 5min. For the fluorescent detector, derivative wavelength was 234nm while emissive light wavelength was 600nm.
2. The optimal GC/MS method
The optimal GC/MS measurements were performed with TR-WaxMs 30m x 0.25mm (d)×0.25μm (membrane thickness) column and 17L manual unsplit injection. The programmed temperature was 80℃at Omin and increased to 180℃with the speed of 5℃/min, then 250℃at 20℃/min, finally maintained at 250℃for 2 min. The injection temperature was 250℃. Carrier gas was high purity helium with 1mL/min flow rate. The optimal MS conditions were that, using El ionization methods (70eV), the selected m/z sizes of SIM Mode were 44,62 and 74 respectively, while the temperatures at the connection parts of ion source and GC to MS were 200℃and 220℃respectively.
3. The EC content during vintage and storage processes
In the vintage process, the content of EC changed slowly with the trend of increasing slightly, but was notably different from various producing areas; the EC in the wine from Shacheng area was the lowest among all producing areas; EC content of different grape species in dry wine was as following in the descending order: Merlot, CS and CG. It is demonstrated that the EC content in wine vinted with most domestic grape species was in international scope. The EC content of brandy, increasing with storage time, was also in international scope; the EC in brandy during raw distillation process maintained at a relatively high level with a process of stable to increasing then gradually declining, but at a lower level during the rectification with a gradual process of increasing first and decreasing afterwards. Experiments of adding acidic urease to the juice exhibited that in the vintage process, the EC content in acidic urease-added wine increased first for some time, and then maintained at a relatively stable but low level. Therefore, it was effective to reduce EC in wine by adding a certain amount of acidic urease.
1、HPLC-FLD方法的最优测定条件
利用高效液相色谱结合荧光检测器,确定的测定条件为：将白兰地样品稀释至酒精含量为25%(v/v),葡萄酒不经稀释；取200μL样品加入50μL HCl溶液及100μL的二苯吡喃醇溶液,暗反应5min后,取20μL进样；色谱条件为色谱柱(ODS-2HYPERSIL C18 4.6mm×250mm,5μm),流动相A：醋酸钠溶液(20mM,pH=7.2),流动相B：乙腈,流速为0.8mL/min;流动相梯度初始条件为流动相A：55%,流动相B：45%,10min时流动相A：50%,至35min时流动相A：0%,然后维持5min;检测器激发光波长为234nm,发射光波长为600nm。
2、GC/MS方法的最优测定条件
本实验确定的气质联用测定条件如下.,色谱条件：色谱柱为TR-WaxMs30m×0.25mm(内径)×0.25μm(膜厚)；手动进样1μL；不分流进样；程序升温起始温度80℃,再以5℃/min的速度升至180℃,后又以20℃/min升温至250℃,并在该温度下停留2min；进样口温度为250℃；载气为高纯氦气；恒流速为1mL/min。质谱条件为：EI电离方式(能量为70eV)；离子检测模式(SIM Mode)中选取m/z为44、62和74的离子碎片进行扫描,离子源及色谱和质谱连接处温度分别为200℃和220℃。
3、葡萄酒酿造和储存过程中EC的变化
在葡萄酒酿造过程中,EC的含量略微上升,但不同产地间的含量差异很大；不同产地的葡萄酒中沙城葡萄酒EC含量较其它产区低；不同品种的干红葡萄酒EC的含量依次为梅鹿辄>赤霞珠>蛇龙珠；我国大部分产区的葡萄所酿造的葡萄酒中EC的含量都在限量范围内；不同年份的白兰地随着储存时间的增加,其EC含量也在增加,但没有超过国际上对于白兰地中EC含量的限量标准；白兰地的粗馏期间EC含量维持在一个较高的水平,其变化存在一个维持不变到增大再逐渐变小的过程；精馏期间EC含量维持在一个较低的水平,其含量逐渐增大后又减小的过程。添加酸性脲酶对比实验的结果显示：在葡萄酒酿造过程中添加酸性脲酶其EC含量在增长一段时间后,维持在一个相对稳定的低水平上,可见添加一定量的酸性脲酶对于降低葡萄酒中的EC有一定作用。
In this study, the HPLC-FLD and GC/MS methods to measure ethyl carbamate (EC) have been optimized. The EC changes during the wine vintage were measured by the optimized methods for the different species and origin of the Cabernet Sauvignon (CS), Cabernet Gernischt (CG) and Merlot. The results were as follows:
1. The optimal HPLC-FLD method
The best HPLC-FLD conditions were that samples were diluted to 25% (v/v) alcohol content,200μL diluted samples were added into 50μL HC1 solution and 100μL 9-xanthydrol solution for a 5-min dark reaction, then 10μL volume was injected into ODS-2 HYPERSIL C18 (4.6mm×250mm 5μm column). The mobile phase A was sodium acetate solution (20mM, pH=7.2) while B was acetonitrile, the flow rate was 0.8mL/min. The percentage of phase B was increased from 45% to 50% during 0 to 10min, and from 50% to 100% during 10 to 35min, then maintained at 100% for 5min. For the fluorescent detector, derivative wavelength was 234nm while emissive light wavelength was 600nm.
2. The optimal GC/MS method
The optimal GC/MS measurements were performed with TR-WaxMs 30m x 0.25mm (d)×0.25μm (membrane thickness) column and 17L manual unsplit injection. The programmed temperature was 80℃at Omin and increased to 180℃with the speed of 5℃/min, then 250℃at 20℃/min, finally maintained at 250℃for 2 min. The injection temperature was 250℃. Carrier gas was high purity helium with 1mL/min flow rate. The optimal MS conditions were that, using El ionization methods (70eV), the selected m/z sizes of SIM Mode were 44,62 and 74 respectively, while the temperatures at the connection parts of ion source and GC to MS were 200℃and 220℃respectively.
3. The EC content during vintage and storage processes
In the vintage process, the content of EC changed slowly with the trend of increasing slightly, but was notably different from various producing areas; the EC in the wine from Shacheng area was the lowest among all producing areas; EC content of different grape species in dry wine was as following in the descending order: Merlot, CS and CG. It is demonstrated that the EC content in wine vinted with most domestic grape species was in international scope. The EC content of brandy, increasing with storage time, was also in international scope; the EC in brandy during raw distillation process maintained at a relatively high level with a process of stable to increasing then gradually declining, but at a lower level during the rectification with a gradual process of increasing first and decreasing afterwards. Experiments of adding acidic urease to the juice exhibited that in the vintage process, the EC content in acidic urease-added wine increased first for some time, and then maintained at a relatively stable but low level. Therefore, it was effective to reduce EC in wine by adding a certain amount of acidic urease.
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