防腐剂和甜味剂方法的研究

高效液相色谱法检测葡萄酒中人工色素、防腐剂和甜味剂方法的研究

HPLC METHODS FOR DETERMINING SYNTHETIC COLORANT,PRESERVATIVE AND SWEETENER ADDED INTO WINE

作者：张予林　　导师：王　华

西北农林科技大学　　葡萄与葡萄酒学2011届硕士

摘　要

食品添加剂的滥用已经成为食品安全的重要威胁。我国国家标准GB15037－2006《葡萄酒》规定：葡萄酒中不得添加合成着色剂、甜味素、香精、增稠剂，同时对防腐剂也有限量要求。目前，国内葡萄酒中添加剂的检测，主要借鉴和使用饮料或配制酒的检验方法，但是，由于葡萄酒的化学成分远比配制酒复杂，利用配制酒检验中的高效液相色谱(HPLC)法检测葡萄酒中的合成色素、防腐剂和甜味剂往往导致检测误差偏大、灵敏度下降。因此，建立专门针对葡萄酒的人工合成色素、防腐剂和甜味剂检测的HPLC方法，保证葡萄酒的质量和安全极其必要。

本试验通过系统研究HPLC检测葡萄酒中添加的4种人工合成着色剂（柠檬黄、日落黄、胭脂红和苋菜红）、2种防腐剂（苯甲酸和山梨酸）和1种甜味剂（糖精钠）的前处理方法，优化了各自的高效液相色谱检验条件，并对添加合成色素的伪劣葡萄酒快速检测方法进行了研究，主要研究结果如下：

1.　建立了变波长同时检测葡萄酒添加4种人工合成色素的HPLC检验方法

通过比较样品不同前处理方法的分离效果和优选高效液相色谱条件，建立了利用变波长HPLC法同时检测葡萄酒添加柠檬黄、日落黄、胭脂红和苋菜红的方法。葡萄酒样品采用聚酰胺吸附法进行前处理的分离效果最好，最佳色谱条件为：Agilent ZORBAX SB-C₁₈(4.6×250mm 5μm)反相色谱柱，流动相A液为甲醇，流动相B液为醋酸铵水溶液（0.02 mol/L），流速为1.0 mL/min，梯度洗脱：A液0.0～2.0 min从22%到60%，2.0～2.2 min从60%到92%，2.2～5.0 min保持92%，变波长检测葡萄酒样品中柠檬黄、苋菜红、胭脂红、日落黄，检测波长分别为：430 nm（0～2.2 min），230 nm（2.50～3.95 min），480 nm（3.95～5.00 min），480 nm（3.95～5.00 min），柱温35 ℃，检测时间为5 min，进样量为20 μL。葡萄酒样通过精密度及加标试验，以上4种人工色素相对标准偏差（RSD）为0.73%～1.51%，回收率87.83%～107.42%。

2.　确定了葡萄酒中苯甲酸、山梨酸和糖精钠的HPLC检验方法

基于现行国家标准配制酒中苯甲酸、山梨酸和糖精钠的HPLC检验法（GB/T5009.29－2003），通过比较样品不同前处理方法的分离效果和优化高效液相色谱条件，确定了葡萄酒中苯甲酸、山梨酸和糖精钠的HPLC检验方法。葡萄酒样品采用浓缩加二次萃取进行前处理，最佳色谱条件为：Agilent TC-C₁₈(4.6×250mm 5μm)反相色谱柱，流动相A液为甲醇，流动相B液为醋酸铵水溶液（0.02 mol/L），按A:B=30:70等度洗脱，流速为1.0 mL/min，检测波长230 nm，柱温30 ℃，进样量为10 μL。葡萄酒样通过精密度及加标试验，以上山梨酸、苯甲酸、糖精钠的测定相对标准偏差（RSD）为0.89%～1.38%，回收率91%～103%。方法简单快速，精确可靠，重复性好。

同时，研究了添加合成色素的劣质葡萄酒的快速检测方法。可通过改变葡萄酒的pH和添加二氧化硫的方式，能够快速鉴定含汁量较低通过勾兑产生的伪劣葡萄酒。可用福林—肖卡与400 mg/L没食子酸标准溶液显色，快速检验含汁量在20%以下且通过添加合成色素的伪劣葡萄酒。

关键词　葡萄酒　人工色素　防腐剂　甜味剂　HPLC

Abstract

The abuse of food additives has already been a serious threat to food safety. In Chinese national standard GB15037-2006 of “wine”,synthetic colorant,sweetener,essence and/or thickener are not permitted to add into wine,and the added amounts of preservative in wine are also strictly limited. At present,the detection of additives in wine primarily refers to the methods used in beverage and imitation wine. However,considering the chemical components in wine are more complex than those in imitation wine,direct detection of synthetic colorant,preservative and sweetener in wine using the high-performance liquid chromatography (HPLC) method used in imitation wine,always leads to the increase of the detection error and the decrease of sensitivity. Therefore,it is essential to establish new HPLC methods,specially used in wine for detection of synthetic colorant,preservative and sweetener,so as to ensure the quality and safety of wine.

In this study,the pre-treatment methods of 4 synthetic colorants (tartrazine,sunset yellow,carmine and amaranth),2 preservatives (benzoic acid and sorbic acid) and saccharin sodium were comprehensively examined,their HPLC detection conditions were optimized respectively,and some inferior wines were also investigated. The main results obtained are as following:

1.　A method was established for simultaneous determination of four synthetic colorants in wine by a HPLC method with variable wavelength detector

Comparing the separation effects of different wine sample pre-treatment methods and optimizing the HPLC detection conditions,a HPLC method was established for simultaneous determination of tartrazine,sunset yellow,carmine and amaranth in wine with variable wavelength detector. The best wine samples pre-treatment method is by polyamide absorption. The optical chromatographic conditions are: 5 μm particle size reversed phase column Agilent ZORBAX SB-C₁₈ (4.6 × 250 mm) was used and the mobile phase consisted of solution A (methanol) and solution B (ammonium acetate 0.02 mol/L) with gradient elution and variable wavelength detection,and the detection wavelengths for tartrazine,sunset yellow,carmine and amaranth were 430 nm (0 ～ 2.2 min),230 nm (2.50 ～ 3.95 min),480 nm (3.95 ～ 5.00 min),480 nm (3.95 ～ 5.00 min),respectively. The flow rate was 1.0 mL/min,column temperature 35 ºC,detection time 5 min,and the injection volume was 20 μL. When the four standards of the colorants were added to the wine respectively and precision test,the average relative standard deviation (RSD) was 0.73% ～ 1.51%,and the average recovery was 87.83% ～ 107.42%.

2.　A HPLC method for simultaneous determination of benzoin acid,sorbic acid and saccharin sodium in wine was determined

Based on Chinese national HPLC method for detection of benzoin acid,sorbic acid and saccharin sodium used in imitation wine (GB/T5009.29—2003),the separation effects of different wine sample pre-treatment methods were compared and the HPLC detection conditions were optimized,and a HPLC method was determined for simultaneous determination of benzoin acid,sorbic acid and saccharin sodium in wine. Wine samples were best pre-treated by means of concentration and re-extraction. The optimal chromatographic conditions are: 5 μm particle size reversed phase column Agilent TC-C₁₈ (4.6 × 250 mm) was used,using solution A (methanol) and solution B (ammonium acetate 0.02 mol/L) (A : B = 30 : 70,v/v) as the mobile phase with isocratic elution. The flow rate was 1.0 mL/min,detection wavelength was 230 nm,column temperature 30 ºC,and the injection volume was 10 μL. When the standards of benzoin acid,sorbic acid and saccharin sodium were added to the wine respectively and precision test,the average relative standard deviation (RSD) was 0.89% ～ 1.38%,and the average recovery was 91% ～ 103%. The developed method is simply fast,reliable and reproducible.

On the other hand,some useful methods were investigated for fast determining some fake and inferior wines. The fake and inferior wines made with low juice content can be identified by means of changing the pH value of wine samples or the adding ways of sulfur dioxide. The method that uses the color reaction developed by Folin-Ciocalteu and gallic acid standard solution (400 mg/L) to distinguish inferior wines may only be applied to inferior wines with grape juice content below 20%.

Key words　Wine　Synthetic colorant　Preservative　Sweetener　HPLC