柱后补偿—液相色谱—电喷雾式检测器测定饮料中的三氯蔗糖
黄超群等
1 引 言
三氯蔗糖,又名蔗糖素,属于紫外末端吸收化合物,极易溶于水,甜度为蔗糖的600倍,且甜味纯正,甜味特性曲线几乎与蔗糖重叠。它是以蔗糖等为原料经脱氧、氯化衍生而得到的半天然半合成产品,属非营养型强力甜味剂,在人体内几乎不被吸收,热量值为零。1990年联合国粮农组织和世界卫生组织(FAO/WHO)的食品添加剂专业委员(JECFA)认定其每日允许摄入量(ADL)值为15 mg/kg,即每天允许摄入量为0~15 mg/kg 体重。若超量食用,仍可能引起各种形式的毒性表现。因此必须加强对三氯蔗糖的含量控制,发挥其有利作用,防止不利影响。目前,对于食品中三氯蔗糖的检测主要采用液相色谱示差折光法[ 1]、液相色谱蒸发光散射法[ 2]、离子色谱法[ 3]、液相色谱质谱联用法[ 4]等。电喷雾式检测器(CAD),作为一种新型通用型检测器,对紫外末端吸收的化合物灵敏度相对较高,已被应用于胆汁酸[ 5]、葡萄糖、果糖[ 6]等的测定。因此,本研究采用高灵敏度及重现性的电喷雾式检测器测定饮料中三氯蔗糖。同时,在实验中采用柱后补偿技术进行有机相补偿,提高了雾化效率,增加了响应值,降低检出限。相比GB/T 222552008[ 2]的蒸发光散射法的定量限14 mg/kg,本方法将两项技术结合,其灵敏度更高,方法定量限达到1.0 mg/L。饮料样品经过简单过滤后,可以直接进样分析,无溶剂干扰,基线漂移较少。本方法操作简便、准确度高,完全能满足饮料中三氯蔗糖含量的测定。
2 实验部分
2.1 仪器与试剂 Ultimate DGP3600高效液相色谱仪,配有双三元梯度泵和电雾式检测器(美国 Thermofisher公司);超声波清洗仪(昆山超声仪器厂)。三氯蔗糖标准品(纯度>97%,美国Supelco公司);乙腈(HPLC级,美国Merck公司)。
标准溶液:称取适量三氯蔗糖,用水溶解,配制成1000 mg/L标准储备液。用水稀释成浓度为1.0, 5.0, 10, 20和50 mg/L的标准工作溶液,于4 ℃避光保存。
2.2 样品前处理 取适量样品,经超声脱气,过滤,供液相色谱检测。实际检测样品为市售某品牌可乐、功能型饮料及植物型饮料。
2.3 色谱条件 Accucore C18色谱柱(150 mm×2.1 mm,2.6 μm); 分析泵的流动相:水(A)乙腈(B),梯度洗脱:0~6.00 min,10%~30% B;6.01~10.00 min,30%~50% B; 10.00~15.00 min,50% B。补偿泵的流动相:乙腈。流速: 分析泵0.3 mL/min,补偿泵0.7 mL/min。进样体积:10 μL; 柱温箱:25 ℃。补偿泵流动相与分析泵流动相在色谱柱后三通相接, 混合后进入电雾式检测器进行定量分析。采集频率: 5 Hz; 过滤参数: 3.6; 干燥温度35 ℃; PFV值为1.2。
3 结果与讨论
3.1 柱后补偿分析 电雾式检测器的响应值较依赖于液相色谱洗脱液的雾化效率。一般通过增加有机相比例,可以提高雾化效率,从而增加响应值,降低检出限。因此,本研究将双三元高效液相色谱仪中的一套泵系统设置为补偿泵系统,流动相为100% 乙腈。结果表明,与不加补偿方法相比,增加有机相补偿后,峰高响应值增加约30%(见图1)。
3.2 电雾式检测器参数优化 电雾式检测器的原理及智能化设计,无需对多种参数进行优化,本实验仅对雾化干燥温度和采集频率进行了优化。当雾化温度分别为25, 35和50 ℃,目标化合物响应值略有增加,但基线噪音也随之增加。综合考虑, 雾化温度尽量与柱温相近,实验选择35 ℃作为雾化温度; 同时,对采集频率(2, 5和10 Hz)进行考察,同紫外检测器一样,采集频率增加,基线噪音 更明显,数据量也较大增加,在满足三氯蔗糖检测条件下,选择5 Hz作为检测器采集频率。
3.3 标准曲线, 检出限和回收率 取所配制的系列标准工作溶液,在2.3节色谱条件下测定,所得标准品谱图见图1,三氯蔗糖保留时间为5.88 min。以三氯蔗糖峰面积Y(pA min)与其对应的浓度x(mg/L)进行线性回归分析,线性方程为y=0.1412x-0.0614,相关系数r=0.9998。按信噪比S/N>10作为方法的定量限,饮料中三氯蔗糖的定量限为1.0 mg/L。相比蒸发光散射法的定量限[ 2],有很大的降低。
3.3 回收率 在3份空白样品,分别加入5.0, 10.0和15.0 mg/L的三氯蔗糖溶液1.0 mL,做标准加入回收实验。 结果表明,空白样品加标回收率在97.6%~112.1%之间。
3.4 样品分析 取市售某品牌可乐、植物饮料、功能饮料样品,经超声脱气后,过滤,进样。检测结果表明,某功能饮料中三氯蔗糖含量为68.4 mg/L。功能饮料的色谱图见图2。
References
1 MA SiNa, DENG Ken. Modern Food Science and Technology, 2009, 25(8): 982-984
马思娜, 邓 垦. 现代食品科技, 2009, 25(8): 982-984
2 GB/T 222552008, Determination of Sucralose in Foods . National Standards of the People′s Republic of China.
食品中三氯蔗糖(蔗糖素)的测定. 中华人民共和国国家标准. GB/T 222552008
3 LIN Li, WANG LinLin, QIAN Cong. China Food Additives, 2013, 5: 214-218
林 立, 王琳琳, 钱 聪. 中国食品添加剂, 2013, 5: 214-218
4 ZHOU LiLi, YANG Ying, LIU YanMing. Journal of Instrumental Analysis, 2011, 30(10): 1175-1178
周莉莉, 杨颖, 刘艳明. 分析测试学报, 2011, 30(10): 1175-1178
5 WANG YiBo, WANG ChunYu, QU FanNa, ZHANG LiYing, LIANG LiNa, YU YanHai, PAN YuanYuan. Chinese J. Anal. Chem., 2014, 42(1): 109-112
王一博, 王春雨, 曲范娜, 张丽英, 梁丽娜, 余彦海, 潘媛媛. 分析化学, 2014, 42(1): 109-112
6 Grembecka M, Lebiedzinska A, Szefer P. Microchemical Journal, 2014, 117: 77-82
7 LI Geng, REN ZhiLin, DAI Jie, ZHANG RongWen, ZHAO Ya, LAI XiaoPing. Journal of Chinese Medicinal Materials, 2013, 36(1): 73-75
李 耿, 任志麟, 戴 洁, 张榕文, 赵 亚, 赖小平. 中药材, 2013, 36(1): 73-75
Determination of Sucralose with PostColumn Compensation
and Liquid ChromatographyCharged Aerosol
Detection Technique in Beverage
HUANG ChaoQun*1, LIU LüYe2, L ChunHua1, LOU ChengJie1, JIN Yan2
1(Zhejiang Academy of Science and Technology for Inspection and Quarantine, Hangzhou 310016, China)
2(Thermofisher Scientific Corporation, Shanghai 201203, China)
Abstract A method was developed for the determination of sucralose in soft drink by liquid chromatography (LC) tandem a charged aerosol detector(CAD) with post column compensation. The results showed that the linearity was good and the correlation coefficient was above 0.999. The limit of quantitation of the method was 1.0 mg/L, and the recoveries of spiked samples were 97.6%-112%. The method was simple and accurate, and can be used for the determination of sucralose in soft drink.
Keywords Sucralose; Post column compensation; High performance liquid chromatography; Charged aerosol detection; Beverage
(Received 31 August 2014; accepted 10 October 2014)
林 立, 王琳琳, 钱 聪. 中国食品添加剂, 2013, 5: 214-218
4 ZHOU LiLi, YANG Ying, LIU YanMing. Journal of Instrumental Analysis, 2011, 30(10): 1175-1178
周莉莉, 杨颖, 刘艳明. 分析测试学报, 2011, 30(10): 1175-1178
5 WANG YiBo, WANG ChunYu, QU FanNa, ZHANG LiYing, LIANG LiNa, YU YanHai, PAN YuanYuan. Chinese J. Anal. Chem., 2014, 42(1): 109-112
王一博, 王春雨, 曲范娜, 张丽英, 梁丽娜, 余彦海, 潘媛媛. 分析化学, 2014, 42(1): 109-112
6 Grembecka M, Lebiedzinska A, Szefer P. Microchemical Journal, 2014, 117: 77-82
7 LI Geng, REN ZhiLin, DAI Jie, ZHANG RongWen, ZHAO Ya, LAI XiaoPing. Journal of Chinese Medicinal Materials, 2013, 36(1): 73-75
李 耿, 任志麟, 戴 洁, 张榕文, 赵 亚, 赖小平. 中药材, 2013, 36(1): 73-75
Determination of Sucralose with PostColumn Compensation
and Liquid ChromatographyCharged Aerosol
Detection Technique in Beverage
HUANG ChaoQun*1, LIU LüYe2, L ChunHua1, LOU ChengJie1, JIN Yan2
1(Zhejiang Academy of Science and Technology for Inspection and Quarantine, Hangzhou 310016, China)
2(Thermofisher Scientific Corporation, Shanghai 201203, China)
Abstract A method was developed for the determination of sucralose in soft drink by liquid chromatography (LC) tandem a charged aerosol detector(CAD) with post column compensation. The results showed that the linearity was good and the correlation coefficient was above 0.999. The limit of quantitation of the method was 1.0 mg/L, and the recoveries of spiked samples were 97.6%-112%. The method was simple and accurate, and can be used for the determination of sucralose in soft drink.
Keywords Sucralose; Post column compensation; High performance liquid chromatography; Charged aerosol detection; Beverage
(Received 31 August 2014; accepted 10 October 2014)
林 立, 王琳琳, 钱 聪. 中国食品添加剂, 2013, 5: 214-218
4 ZHOU LiLi, YANG Ying, LIU YanMing. Journal of Instrumental Analysis, 2011, 30(10): 1175-1178
周莉莉, 杨颖, 刘艳明. 分析测试学报, 2011, 30(10): 1175-1178
5 WANG YiBo, WANG ChunYu, QU FanNa, ZHANG LiYing, LIANG LiNa, YU YanHai, PAN YuanYuan. Chinese J. Anal. Chem., 2014, 42(1): 109-112
王一博, 王春雨, 曲范娜, 张丽英, 梁丽娜, 余彦海, 潘媛媛. 分析化学, 2014, 42(1): 109-112
6 Grembecka M, Lebiedzinska A, Szefer P. Microchemical Journal, 2014, 117: 77-82
7 LI Geng, REN ZhiLin, DAI Jie, ZHANG RongWen, ZHAO Ya, LAI XiaoPing. Journal of Chinese Medicinal Materials, 2013, 36(1): 73-75
李 耿, 任志麟, 戴 洁, 张榕文, 赵 亚, 赖小平. 中药材, 2013, 36(1): 73-75
Determination of Sucralose with PostColumn Compensation
and Liquid ChromatographyCharged Aerosol
Detection Technique in Beverage
HUANG ChaoQun*1, LIU LüYe2, L ChunHua1, LOU ChengJie1, JIN Yan2
1(Zhejiang Academy of Science and Technology for Inspection and Quarantine, Hangzhou 310016, China)
2(Thermofisher Scientific Corporation, Shanghai 201203, China)
Abstract A method was developed for the determination of sucralose in soft drink by liquid chromatography (LC) tandem a charged aerosol detector(CAD) with post column compensation. The results showed that the linearity was good and the correlation coefficient was above 0.999. The limit of quantitation of the method was 1.0 mg/L, and the recoveries of spiked samples were 97.6%-112%. The method was simple and accurate, and can be used for the determination of sucralose in soft drink.
Keywords Sucralose; Post column compensation; High performance liquid chromatography; Charged aerosol detection; Beverage
(Received 31 August 2014; accepted 10 October 2014)
1 引 言
三氯蔗糖,又名蔗糖素,属于紫外末端吸收化合物,极易溶于水,甜度为蔗糖的600倍,且甜味纯正,甜味特性曲线几乎与蔗糖重叠。它是以蔗糖等为原料经脱氧、氯化衍生而得到的半天然半合成产品,属非营养型强力甜味剂,在人体内几乎不被吸收,热量值为零。1990年联合国粮农组织和世界卫生组织(FAO/WHO)的食品添加剂专业委员(JECFA)认定其每日允许摄入量(ADL)值为15 mg/kg,即每天允许摄入量为0~15 mg/kg 体重。若超量食用,仍可能引起各种形式的毒性表现。因此必须加强对三氯蔗糖的含量控制,发挥其有利作用,防止不利影响。目前,对于食品中三氯蔗糖的检测主要采用液相色谱示差折光法[ 1]、液相色谱蒸发光散射法[ 2]、离子色谱法[ 3]、液相色谱质谱联用法[ 4]等。电喷雾式检测器(CAD),作为一种新型通用型检测器,对紫外末端吸收的化合物灵敏度相对较高,已被应用于胆汁酸[ 5]、葡萄糖、果糖[ 6]等的测定。因此,本研究采用高灵敏度及重现性的电喷雾式检测器测定饮料中三氯蔗糖。同时,在实验中采用柱后补偿技术进行有机相补偿,提高了雾化效率,增加了响应值,降低检出限。相比GB/T 222552008[ 2]的蒸发光散射法的定量限14 mg/kg,本方法将两项技术结合,其灵敏度更高,方法定量限达到1.0 mg/L。饮料样品经过简单过滤后,可以直接进样分析,无溶剂干扰,基线漂移较少。本方法操作简便、准确度高,完全能满足饮料中三氯蔗糖含量的测定。
2 实验部分
2.1 仪器与试剂 Ultimate DGP3600高效液相色谱仪,配有双三元梯度泵和电雾式检测器(美国 Thermofisher公司);超声波清洗仪(昆山超声仪器厂)。三氯蔗糖标准品(纯度>97%,美国Supelco公司);乙腈(HPLC级,美国Merck公司)。
标准溶液:称取适量三氯蔗糖,用水溶解,配制成1000 mg/L标准储备液。用水稀释成浓度为1.0, 5.0, 10, 20和50 mg/L的标准工作溶液,于4 ℃避光保存。
2.2 样品前处理 取适量样品,经超声脱气,过滤,供液相色谱检测。实际检测样品为市售某品牌可乐、功能型饮料及植物型饮料。
2.3 色谱条件 Accucore C18色谱柱(150 mm×2.1 mm,2.6 μm); 分析泵的流动相:水(A)乙腈(B),梯度洗脱:0~6.00 min,10%~30% B;6.01~10.00 min,30%~50% B; 10.00~15.00 min,50% B。补偿泵的流动相:乙腈。流速: 分析泵0.3 mL/min,补偿泵0.7 mL/min。进样体积:10 μL; 柱温箱:25 ℃。补偿泵流动相与分析泵流动相在色谱柱后三通相接, 混合后进入电雾式检测器进行定量分析。采集频率: 5 Hz; 过滤参数: 3.6; 干燥温度35 ℃; PFV值为1.2。
3 结果与讨论
3.1 柱后补偿分析 电雾式检测器的响应值较依赖于液相色谱洗脱液的雾化效率。一般通过增加有机相比例,可以提高雾化效率,从而增加响应值,降低检出限。因此,本研究将双三元高效液相色谱仪中的一套泵系统设置为补偿泵系统,流动相为100% 乙腈。结果表明,与不加补偿方法相比,增加有机相补偿后,峰高响应值增加约30%(见图1)。
3.2 电雾式检测器参数优化 电雾式检测器的原理及智能化设计,无需对多种参数进行优化,本实验仅对雾化干燥温度和采集频率进行了优化。当雾化温度分别为25, 35和50 ℃,目标化合物响应值略有增加,但基线噪音也随之增加。综合考虑, 雾化温度尽量与柱温相近,实验选择35 ℃作为雾化温度; 同时,对采集频率(2, 5和10 Hz)进行考察,同紫外检测器一样,采集频率增加,基线噪音 更明显,数据量也较大增加,在满足三氯蔗糖检测条件下,选择5 Hz作为检测器采集频率。
3.3 标准曲线, 检出限和回收率 取所配制的系列标准工作溶液,在2.3节色谱条件下测定,所得标准品谱图见图1,三氯蔗糖保留时间为5.88 min。以三氯蔗糖峰面积Y(pA min)与其对应的浓度x(mg/L)进行线性回归分析,线性方程为y=0.1412x-0.0614,相关系数r=0.9998。按信噪比S/N>10作为方法的定量限,饮料中三氯蔗糖的定量限为1.0 mg/L。相比蒸发光散射法的定量限[ 2],有很大的降低。
3.3 回收率 在3份空白样品,分别加入5.0, 10.0和15.0 mg/L的三氯蔗糖溶液1.0 mL,做标准加入回收实验。 结果表明,空白样品加标回收率在97.6%~112.1%之间。
3.4 样品分析 取市售某品牌可乐、植物饮料、功能饮料样品,经超声脱气后,过滤,进样。检测结果表明,某功能饮料中三氯蔗糖含量为68.4 mg/L。功能饮料的色谱图见图2。
References
1 MA SiNa, DENG Ken. Modern Food Science and Technology, 2009, 25(8): 982-984
马思娜, 邓 垦. 现代食品科技, 2009, 25(8): 982-984
2 GB/T 222552008, Determination of Sucralose in Foods . National Standards of the People′s Republic of China.
食品中三氯蔗糖(蔗糖素)的测定. 中华人民共和国国家标准. GB/T 222552008
3 LIN Li, WANG LinLin, QIAN Cong. China Food Additives, 2013, 5: 214-218
林 立, 王琳琳, 钱 聪. 中国食品添加剂, 2013, 5: 214-218
4 ZHOU LiLi, YANG Ying, LIU YanMing. Journal of Instrumental Analysis, 2011, 30(10): 1175-1178
周莉莉, 杨颖, 刘艳明. 分析测试学报, 2011, 30(10): 1175-1178
5 WANG YiBo, WANG ChunYu, QU FanNa, ZHANG LiYing, LIANG LiNa, YU YanHai, PAN YuanYuan. Chinese J. Anal. Chem., 2014, 42(1): 109-112
王一博, 王春雨, 曲范娜, 张丽英, 梁丽娜, 余彦海, 潘媛媛. 分析化学, 2014, 42(1): 109-112
6 Grembecka M, Lebiedzinska A, Szefer P. Microchemical Journal, 2014, 117: 77-82
7 LI Geng, REN ZhiLin, DAI Jie, ZHANG RongWen, ZHAO Ya, LAI XiaoPing. Journal of Chinese Medicinal Materials, 2013, 36(1): 73-75
李 耿, 任志麟, 戴 洁, 张榕文, 赵 亚, 赖小平. 中药材, 2013, 36(1): 73-75
Determination of Sucralose with PostColumn Compensation
and Liquid ChromatographyCharged Aerosol
Detection Technique in Beverage
HUANG ChaoQun*1, LIU LüYe2, L ChunHua1, LOU ChengJie1, JIN Yan2
1(Zhejiang Academy of Science and Technology for Inspection and Quarantine, Hangzhou 310016, China)
2(Thermofisher Scientific Corporation, Shanghai 201203, China)
Abstract A method was developed for the determination of sucralose in soft drink by liquid chromatography (LC) tandem a charged aerosol detector(CAD) with post column compensation. The results showed that the linearity was good and the correlation coefficient was above 0.999. The limit of quantitation of the method was 1.0 mg/L, and the recoveries of spiked samples were 97.6%-112%. The method was simple and accurate, and can be used for the determination of sucralose in soft drink.
Keywords Sucralose; Post column compensation; High performance liquid chromatography; Charged aerosol detection; Beverage
(Received 31 August 2014; accepted 10 October 2014)
林 立, 王琳琳, 钱 聪. 中国食品添加剂, 2013, 5: 214-218
4 ZHOU LiLi, YANG Ying, LIU YanMing. Journal of Instrumental Analysis, 2011, 30(10): 1175-1178
周莉莉, 杨颖, 刘艳明. 分析测试学报, 2011, 30(10): 1175-1178
5 WANG YiBo, WANG ChunYu, QU FanNa, ZHANG LiYing, LIANG LiNa, YU YanHai, PAN YuanYuan. Chinese J. Anal. Chem., 2014, 42(1): 109-112
王一博, 王春雨, 曲范娜, 张丽英, 梁丽娜, 余彦海, 潘媛媛. 分析化学, 2014, 42(1): 109-112
6 Grembecka M, Lebiedzinska A, Szefer P. Microchemical Journal, 2014, 117: 77-82
7 LI Geng, REN ZhiLin, DAI Jie, ZHANG RongWen, ZHAO Ya, LAI XiaoPing. Journal of Chinese Medicinal Materials, 2013, 36(1): 73-75
李 耿, 任志麟, 戴 洁, 张榕文, 赵 亚, 赖小平. 中药材, 2013, 36(1): 73-75
Determination of Sucralose with PostColumn Compensation
and Liquid ChromatographyCharged Aerosol
Detection Technique in Beverage
HUANG ChaoQun*1, LIU LüYe2, L ChunHua1, LOU ChengJie1, JIN Yan2
1(Zhejiang Academy of Science and Technology for Inspection and Quarantine, Hangzhou 310016, China)
2(Thermofisher Scientific Corporation, Shanghai 201203, China)
Abstract A method was developed for the determination of sucralose in soft drink by liquid chromatography (LC) tandem a charged aerosol detector(CAD) with post column compensation. The results showed that the linearity was good and the correlation coefficient was above 0.999. The limit of quantitation of the method was 1.0 mg/L, and the recoveries of spiked samples were 97.6%-112%. The method was simple and accurate, and can be used for the determination of sucralose in soft drink.
Keywords Sucralose; Post column compensation; High performance liquid chromatography; Charged aerosol detection; Beverage
(Received 31 August 2014; accepted 10 October 2014)
林 立, 王琳琳, 钱 聪. 中国食品添加剂, 2013, 5: 214-218
4 ZHOU LiLi, YANG Ying, LIU YanMing. Journal of Instrumental Analysis, 2011, 30(10): 1175-1178
周莉莉, 杨颖, 刘艳明. 分析测试学报, 2011, 30(10): 1175-1178
5 WANG YiBo, WANG ChunYu, QU FanNa, ZHANG LiYing, LIANG LiNa, YU YanHai, PAN YuanYuan. Chinese J. Anal. Chem., 2014, 42(1): 109-112
王一博, 王春雨, 曲范娜, 张丽英, 梁丽娜, 余彦海, 潘媛媛. 分析化学, 2014, 42(1): 109-112
6 Grembecka M, Lebiedzinska A, Szefer P. Microchemical Journal, 2014, 117: 77-82
7 LI Geng, REN ZhiLin, DAI Jie, ZHANG RongWen, ZHAO Ya, LAI XiaoPing. Journal of Chinese Medicinal Materials, 2013, 36(1): 73-75
李 耿, 任志麟, 戴 洁, 张榕文, 赵 亚, 赖小平. 中药材, 2013, 36(1): 73-75
Determination of Sucralose with PostColumn Compensation
and Liquid ChromatographyCharged Aerosol
Detection Technique in Beverage
HUANG ChaoQun*1, LIU LüYe2, L ChunHua1, LOU ChengJie1, JIN Yan2
1(Zhejiang Academy of Science and Technology for Inspection and Quarantine, Hangzhou 310016, China)
2(Thermofisher Scientific Corporation, Shanghai 201203, China)
Abstract A method was developed for the determination of sucralose in soft drink by liquid chromatography (LC) tandem a charged aerosol detector(CAD) with post column compensation. The results showed that the linearity was good and the correlation coefficient was above 0.999. The limit of quantitation of the method was 1.0 mg/L, and the recoveries of spiked samples were 97.6%-112%. The method was simple and accurate, and can be used for the determination of sucralose in soft drink.
Keywords Sucralose; Post column compensation; High performance liquid chromatography; Charged aerosol detection; Beverage
(Received 31 August 2014; accepted 10 October 2014)