～ 40倍,控制限量对人们的身体健康有重要意义。 Summary : Cyclamate is a sweetness additive commonly used in food production, and its sweetness is 30 to 40 times that of sucrose . Controlling the limit is of great significance to people's health. 食品添加剂使用标准》中用于界定食品添加剂使用范围的食品分类系统，白酒属于蒸馏酒，不允许使用甜蜜素。 In the " GB 2760-2014 Food Additive Use Standard", a food classification system used to define the scope of use of food additives. Liquor is a distilled wine, and cyclamate is not allowed. 及相关文献资料，对白酒中的甜蜜素经提取后，在冰浴条件下，与亚硝酸钠发生重氮化反应生成衍生物环己醇亚硝酸酯，衍生物进入气相色谱质谱联用仪进行分离检测。 This method refers to GB / T 5009.97-2003 and related literatures. After extraction of cyclamate from liquor, it is subjected to diazotization reaction with sodium nitrite under ice bath conditions to generate the derivative cyclohexanol nitrite, which is derived. The compounds were separated and detected by gas chromatography-mass spectrometry.
1 Experimental part
1.1 Instruments and reagents
质谱联用仪： GC-MS 6800 Jiangsu Tianrui Instrument Co., Ltd. GC-MS 6800
％）于干燥容量瓶中，用水配成1000m ｇ /kg标准储备溶液。 Cycladin standard stock solution: Weigh a certain amount of Cyclamate standard (purity 99.0 %) into a dry volumetric flask, and prepare 1000mg / kg standard stock solution with water.
The reagents are all pure, and the test water is secondary distilled water.
1.2 Instrument working conditions
1.2.1 Gas chromatography conditions
×0.25mm,0.25um）；载气：氦气（纯度为99.999 ％），载气流量1.0 mL/min；进样口温度250℃ ，进样量1.0uL ，进样方式为分流进样,分流比10:1 ；程序升温：初始温度为40℃ ，保持时间10min ，以25℃/min速率升至270℃ ，保持时间5min 。 DB-5 capillary chromatography ( 30m × 0.25mm, 0.25um); carrier gas: helium ( 99.999 % purity), carrier gas flow rate 1.0 mL / min; inlet temperature 250 ℃ , injection volume 1.0uL , injection The method is split injection with a split ratio of 10: 1 ; programmed temperature rise: initial temperature is 40 ° C , holding time is 10min , and the rate is increased to 270 ° C at 25 ° C / min , and the holding time is 5min .
1.2.2 Mass spectrometry conditions
），四级杆温度150℃ ，接口温度280℃ ；采集方式为选择离子监测扫描（ SIM ），溶剂延迟时间6min,选择3个监测离子质荷比（ m/z ）为55,81,83 。 Electron bombardment ion source ( EI ), quadrupole temperature 150 ° C , interface temperature 280 ° C ; acquisition method is selected ion monitoring scan ( SIM ), solvent delay time is 6min, three monitoring ion mass-to-charge ratios ( m / z ) are selected as 55,81,83 .
1.3 Experimental method (omitted)
2 results and discussion
2.1 Selection of features and ions
。 Cyclohexanol nitrite, the main derivative of cyclamate, is qualitative and quantitative, as shown in Figure 1 . 定性离子（ m/z ）为55,81,83 。 Retention time was 8.511min. Qualitative ions ( m / z ) were 55,81,83 . 。 The quantitative ion was 55 . The mass spectrum is shown in Figure 2.
Figure 1.The chromatogram of cyclamate derivatives in total ion current.
、 Cyclamate derivatives total ion chromatogram Figure 1 , Cyclamate derivatives total ion chromatogram
2 、甜蜜素衍生物的质谱图 Figure 2 Mass spectrum of cyclamate derivative
、 Cyclamate derivative spectrum Figure 2 , Cyclamate derivative spectrum
3 、甜蜜素衍生物在 SIM 扫描下的离子流色谱图 Figure 3.Ion chromatogram of cyclamate derivative under SIM scan
、 Figure 3 Cyclamate derivative SIM under scanning ion chromatogram
4 、甜蜜素衍生物在 SIM 扫描下的质谱图 Figure 4 Mass spectrum of cyclamate derivative under SIM scan
、 Cyclamate derivative SIM under scanning spectrum Figure 4 、 Cyclamate derivative SIM under scanning spectrum
2.2 Pretreatment condition selection
2.2.1 Derivative time selection
甜蜜素储备液溶液按照上述衍生步骤进行衍生化，甜蜜素衍生物浓度为200ug/mL ，衍生反应时间分别设置为10min 、 20min 、 30min 、 40min 、 50min ，考察反应时间对反应的影响。 1ml cyclamate stock solution was accurately derivatized according to the above derivatization steps. The concentration of cyclamate derivative was 200ug / mL , and the derivatization reaction time was set to 10min , 20min , 30min , 40min , and 50min , respectively. The effect of reaction time on the reaction was examined. . 升至30min ，衍生物信号响应值逐渐增加，随着反应时间的延长，响应值反而出现降低，因此，衍生反应温度30min最佳。 The results show that the response value of the derivative signal gradually increases with the increase of time from 10min to 30min , and the response value decreases with the extension of the reaction time. Therefore, the derivatization reaction temperature is the best at 30min .
2.2.2 Selection of sulfuric acid concentration
甜蜜素储备液溶液按照上述衍生步骤进行衍生化，甜蜜素衍生物浓度为200ug/mL ，冰浴反应条件下，加入50g/L的亚硝酸钠和分别为50g/L ， 75g/L ， 100g/L ， 125g/L ， 150g/L 。 1mL cyclamate stock solution was accurately derivatized according to the above derivatization steps. The concentration of cyclamate derivatives was 200ug / mL . Under ice-bath reaction conditions, 50g / L sodium nitrite and 50g / L , 75g / L were added . L , 100g / L , 125g / L , 150g / L. 至100g/L ，反应衍生物信号响应值明显增加，浓度升高时，响应值出现减少，因此，最终衍生反应酸度为100g/L 。 The results showed that from 50g / L to 100g / L , the response value of the reaction derivative signal increased significantly. When the concentration increased, the response value decreased. Therefore, the final derivative reaction acidity was 100g / L.
2.2.3 Effect of sodium nitrite concentration on derivatization reaction
甜蜜素储备液溶液按照上述衍生步骤进行衍生化衍生化，甜蜜素衍生物浓度为200ug/mL ，冰浴反应条件下，加入100g/L的硫酸，和分别为30g/L ， 50g/L ， 70g/L ， 90g/L亚硝酸钠。 Accurately draw 1ml cyclamate stock solution to perform derivatization according to the above derivatization steps. The concentration of cyclamate derivative is 200ug / mL . Under ice bath reaction conditions, add 100g / L sulfuric acid, and 30g / L , 50g respectively. / L , 70g / L , 90g / L sodium nitrite. 至50g/L ，反应衍生物信号响应值明显增加，浓度升高时，响应值出现减少，因此，最终亚硝酸钠衍生反应为50g/L 。 The results show that from 30g / L to 50g / L , the response value of the reaction derivative signal increases significantly, and the response value decreases when the concentration increases. Therefore, the final sodium nitrite derivative reaction is 50g / L.
3 Experimental data
3.1 Precision and repeatability experiments
于离心管中，用水定容20mL按照上述标样衍生步骤进行衍生化，衍生物浓度为100ug/mL ，平行制备反应液6次，用GC-MS测定。 500 uL of the stock solution was accurately transferred to a centrifuge tube, and the volume was adjusted to 20 mL with water to perform derivatization according to the above standard derivatization step. The derivative concentration was 100 ug / mL , and the reaction solution was prepared 6 times in parallel and measured by GC-MS . 个平行样的谱。 The reproducibility of the derivatization method and the relative standard deviations of component retention times and peak areas were examined. Spectra of 6 parallel samples. 为0.98% ，峰面积RSD为6.00%, ，具体实验数据如下表1所示。 The results show that the retention time RSD is 0.98% and the peak area RSD is 6.00% . The specific experimental data are shown in Table 1 below.
Table 1. Repeatability test results
Table 1, repeatable test results
RSD% Retention time RSD%
RSD% Peak area RSD%
3.2 Linear relationship and detection limit
的甜蜜素标准溶液10ul于50ml离心管中对应衍生物浓度分别为2ug/mL 、 10ug/mL 、 20ug/mL 、 100ug/mL 、 200ug/mL ，按照上述标样衍生化步骤进行衍生化，进行线性测试。 Accurately draw 10ul of the standard solution of cyclamate at a concentration of 10ug / mL in a 50ml centrifuge tube. The corresponding derivative concentrations are 2ug / mL , 10ug / mL , 20ug / mL , 100ug / mL , 200ug / mL , and derivatized according to the above standards. The steps are derivatized and tested for linearity. ，线性相关系数为0.9984 ，如图5所示。 The experimental linear equation is: y = 940.43x-3525.57 , and the linear correlation coefficient is 0.9984 , as shown in Figure 5 .
线性关系图 Figure 5. Linear diagram
， Linear Figure 5 , Linear
3.3 Recovery rate test
、 50mg/kg 、 200mg/kg的标样添加浓度，对某品牌白酒进行回收率实验，每个浓度分别做三次平行样，按处理样品的方法处理加标后的样品并测试，采用外标法定量，测试结果如下表2所示。 Select 2mg / kg , 50mg / kg , 200mg / kg standard samples to add concentration, perform recovery experiments on a certain brand of liquor, each concentration is made three parallel samples, and the spiked samples are processed and tested according to the method of processing samples. The external standard method was used for quantification. The test results are shown in Table 2 below.
/mg/kg Concentration / mg / kg
1/% Recovery rate 1 /%
2/% Recovery rate 2 /%
3/% Recovery rate 3 /%
/% Average recovery rate /%
Table 2. Recovery test results
Table 2, the recovery test results
3.4 actual sample test
进行测试，测试结果如下表3所示： Pretreatment and derivatization of liquor samples of different types according to the above sample processing methods, and use GC-MS6800 to test.
/mg/kg Cyclamate content / mg / kg
1 Wine sample 1
2 Wine sample 2
3 Wine sample 3
4 Wine sample 4
5 Wine sample 5
表示“未检出” Note: "ND" means " Not Checked Out "
、酒中甜蜜素含量测试结果 Table 3. Test results of cyclamate content in wine
，在SIM扫描模式下对酒中的甜蜜素进行测试和分析，方法稳定可靠，线性范围好，灵敏度高，受杂质峰的干扰小，能够有效地对酒中的甜蜜素进行定性定量分析。 In this experiment, GCMS-6800 was used to test and analyze the cyclamate in wine under the SIM scanning mode. The method is stable and reliable, with good linear range, high sensitivity, and little interference from impurity peaks. It can effectively control the cyclamate in wine. Perform qualitative and quantitative analysis.