高效液相色譜法檢測葡萄酒中人工色素、防腐劑和甜味劑方法的研究

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

作者：張予林　　導師：王　華

西北農(nóng)林科技大學　　葡萄與葡萄酒學2011屆碩士

摘　要

食品添加劑的濫用已經(jīng)成為食品安全的重要威脅。我國國家標準GB15037－2006《葡萄酒》規(guī)定：葡萄酒中不得添加合成著色劑、甜味素、香精、增稠劑，同時對防腐劑也有限量要求。目前，國內(nèi)葡萄酒中添加劑的檢測，主要借鑒和使用飲料或配制酒的檢驗方法，但是，由于葡萄酒的化學成分遠比配制酒復雜，利用配制酒檢驗中的高效液相色譜(HPLC)法檢測葡萄酒中的合成色素、防腐劑和甜味劑往往導致檢測誤差偏大、靈敏度下降。因此，建立專門針對葡萄酒的人工合成色素、防腐劑和甜味劑檢測的HPLC方法，保證葡萄酒的質(zhì)量和安全極其必要。

本試驗通過系統(tǒng)研究HPLC檢測葡萄酒中添加的4種人工合成著色劑（檸檬黃、日落黃、胭脂紅和莧菜紅）、2種防腐劑（苯甲酸和山梨酸）和1種甜味劑（糖精鈉）的前處理方法，優(yōu)化了各自的高效液相色譜檢驗條件，并對添加合成色素的偽劣葡萄酒快速檢測方法進行了研究，主要研究結(jié)果如下：

1.　建立了變波長同時檢測葡萄酒添加4種人工合成色素的HPLC檢驗方法

通過比較樣品不同前處理方法的分離效果和優(yōu)選高效液相色譜條件，建立了利用變波長HPLC法同時檢測葡萄酒添加檸檬黃、日落黃、胭脂紅和莧菜紅的方法。葡萄酒樣品采用聚酰胺吸附法進行前處理的分離效果最好，最佳色譜條件為：Agilent ZORBAX SB-C₁₈(4.6×250mm 5μm)反相色譜柱，流動相A液為甲醇，流動相B液為醋酸銨水溶液（0.02 mol/L），流速為1.0 mL/min，梯度洗脫：A液0.0～2.0 min從22%到60%，2.0～2.2 min從60%到92%，2.2～5.0 min保持92%，變波長檢測葡萄酒樣品中檸檬黃、莧菜紅、胭脂紅、日落黃，檢測波長分別為：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），柱溫35 ℃，檢測時間為5 min，進樣量為20 μL。葡萄酒樣通過精密度及加標試驗，以上4種人工色素相對標準偏差（RSD）為0.73%～1.51%，回收率87.83%～107.42%。

2.　確定了葡萄酒中苯甲酸、山梨酸和糖精鈉的HPLC檢驗方法

基于現(xiàn)行國家標準配制酒中苯甲酸、山梨酸和糖精鈉的HPLC檢驗法（GB/T5009.29－2003），通過比較樣品不同前處理方法的分離效果和優(yōu)化高效液相色譜條件，確定了葡萄酒中苯甲酸、山梨酸和糖精鈉的HPLC檢驗方法。葡萄酒樣品采用濃縮加二次萃取進行前處理，最佳色譜條件為：Agilent TC-C₁₈(4.6×250mm 5μm)反相色譜柱，流動相A液為甲醇，流動相B液為醋酸銨水溶液（0.02 mol/L），按A:B=30:70等度洗脫，流速為1.0 mL/min，檢測波長230 nm，柱溫30 ℃，進樣量為10 μL。葡萄酒樣通過精密度及加標試驗，以上山梨酸、苯甲酸、糖精鈉的測定相對標準偏差（RSD）為0.89%～1.38%，回收率91%～103%。方法簡單快速，精確可靠，重復性好。

同時，研究了添加合成色素的劣質(zhì)葡萄酒的快速檢測方法?？赏ㄟ^改變葡萄酒的pH和添加二氧化硫的方式，能夠快速鑒定含汁量較低通過勾兌產(chǎn)生的偽劣葡萄酒?？捎酶Ａ帧たㄅc400 mg/L沒食子酸標準溶液顯色，快速檢驗含汁量在20%以下且通過添加合成色素的偽劣葡萄酒。

關(guān)鍵詞　葡萄酒　人工色素　防腐劑　甜味劑　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