离子色谱法和高效液相色谱法测定奶粉中胆碱含量的研究
摘要
胆碱(choline)是强有机碱性化合物,以游离态胆碱、乙酰胆碱和复合磷脂的形式广泛存在于生物机体中。它是动物生长过程中不可缺少的一种水溶性维生素。[1]研究表明,[2-3]它在脑发育和开发认知、记忆与学习能力方面有很重要的作用。对于脑发育关键时期的婴儿,在婴儿配方及奶粉中添加胆碱是十分必要的。[4-5]它的另一个主要作用是促进肝脏机能。如果缺乏胆碱,特别是老年人可能引起脂肪肝、肝硬化、动脉硬化,也可能引起老年痴呆症。[6]
以往测定胆碱的方法有高氯酸非水滴定法[7]、雷氏盐比色法[7-9]和雷氏盐重量法。[10]现行的国家标准[11]GB/T 5413.20-1997采用的是酶反应显色分光光度计法检测胆碱含量。这些传统方法操作繁琐,选择性差,干扰因素多,无法鉴别掺入的廉价无机氯化物和胺类等物质。[12]近些年可参见的文献中有高效液相色谱-电化学检测法、[11]化学发光法、[13]离子色谱-电导检测法[5、11、15-17]等,但尚未见高效液相色谱-蒸发光散射检测法及高效液相色谱-质谱法对胆碱含量检测的报道。
本论文建立了离子色谱法、高效液相色谱-蒸发光散射检测法及高效液相色谱-质谱法三种方法对奶粉中胆碱含量的测定,并将此三种方法进行对比讨论。通过正交试验设计法L(933),固定奶粉质量5.00g,盐酸体积30mL,设计盐酸浓度(0.5mol/L、0.8mol/L、1.0mol/L三水平),反应温度(60℃、70℃、80℃三水平),反应时间(3h、4h、5h三水平),寻找到奶粉前处理的最佳酸水解条件是1mol/L盐酸70℃下反应3小时。最后还对离子色谱测定奶粉中胆碱含量的方法做出不确定度的分析和评定。
三种方法对胆碱含量测定的最低检测限分别为0.1mg/L、0.2mg/L和0.01mg/L,相对标准偏差分别为1.13%、2.67%和3.86%,添加回收率分别为98.78%～100.72%、96.72%～102.03%和91.67%～100.02%。方法操作简单,灵敏度高,选择性强,能克服传统方法中繁琐,准确度低等缺陷和不足。本论文丰富了奶粉中胆碱的含量测定的方法同时对其他同类产品中胆碱含量的测定提供了参考。
Choline is a strong organic alkaline compounds which widespread in the living organisms in the form of free choline, acetylcholine and phospholipid complex. It is an indispensable water-soluble vitamin for growth of animal. Research shows that it plays a very important role in brain growth and the development of cognitive, memory and learning ability. It’s another main role to promote liver function. The lack of choline may cause fatty liver, cirrhosis of the liver andarteriosclerosis especially for the elderly, and it can also caused Alzheimer's disease. It is very necessary that adding choline in infant formula and dry milk. Ever country has provision of adding content of choline.
The previous methods of detecting choline are perchlorate non-aqueous titration method, Leishi salt colorimetry and Leishi salt weight method. The current national standard GB/T 5413.20-1997 uses the enzyme reaction color spectrophotometer to measure the content of choline. These traditional methods are complex, bad selectivity, having many interfering factors, and they can not identify incorporation of cheap inorganic chloride and amine. In recent years' literatures we can find some new methods such as the high performance liquid chromatography-electrochemical detection method, the chemiluminescence method, the ion chromatography-conductivity detection method etc. But there is not any report about HPLC-ELSD method and HPLC-MSD method to detect the content of choline.
This paper establishes ion chromatography (IC), high-performance liquid chromatography-evaporative light scattering detection (HPLC-ELSD) and high-performance liquid chromatography-mass spectrometry (HPLC-MSD) three methods to detect the content of choline in dry milk, and discusses the comparison of these three methods. By orthogonal experiment design L9 (33), fix dry milk quality of 5.00g and hydrochloric acid volume of 30mL, design the concentration of hydrochloric acid (0.5mol/L, 0.8mol/L, 1.0mol/L three levels), the reaction temperature (60℃, 70℃, 80℃three levels), the reaction time (3h, 4h, 5h three levels), and get the best pretreatment condition is 1mol/L HCl at 70℃responsing three hours. Finally, I give the uncertainty evaluation of the determination of choline in dry milk by IC method.
The minimum detection limits of three methods of detecting choline are 0.1mg/L, 0.2mg/L and 0.01mg/L. The relative standard deviations are 1.13%, 2.67% and 3.86%. The add recovery are 98.78%～100.72%,96.72%～102.03% and 91.67%～100.02%. The method is simple, high sensitivity, good selectivity while traditional methods can not. This paper adds methods of determination of choline in dry milk, and also gives a reference about determination of choline on other similar products.
以往测定胆碱的方法有高氯酸非水滴定法[7]、雷氏盐比色法[7-9]和雷氏盐重量法。[10]现行的国家标准[11]GB/T 5413.20-1997采用的是酶反应显色分光光度计法检测胆碱含量。这些传统方法操作繁琐,选择性差,干扰因素多,无法鉴别掺入的廉价无机氯化物和胺类等物质。[12]近些年可参见的文献中有高效液相色谱-电化学检测法、[11]化学发光法、[13]离子色谱-电导检测法[5、11、15-17]等,但尚未见高效液相色谱-蒸发光散射检测法及高效液相色谱-质谱法对胆碱含量检测的报道。
本论文建立了离子色谱法、高效液相色谱-蒸发光散射检测法及高效液相色谱-质谱法三种方法对奶粉中胆碱含量的测定,并将此三种方法进行对比讨论。通过正交试验设计法L(933),固定奶粉质量5.00g,盐酸体积30mL,设计盐酸浓度(0.5mol/L、0.8mol/L、1.0mol/L三水平),反应温度(60℃、70℃、80℃三水平),反应时间(3h、4h、5h三水平),寻找到奶粉前处理的最佳酸水解条件是1mol/L盐酸70℃下反应3小时。最后还对离子色谱测定奶粉中胆碱含量的方法做出不确定度的分析和评定。
三种方法对胆碱含量测定的最低检测限分别为0.1mg/L、0.2mg/L和0.01mg/L,相对标准偏差分别为1.13%、2.67%和3.86%,添加回收率分别为98.78%～100.72%、96.72%～102.03%和91.67%～100.02%。方法操作简单,灵敏度高,选择性强,能克服传统方法中繁琐,准确度低等缺陷和不足。本论文丰富了奶粉中胆碱的含量测定的方法同时对其他同类产品中胆碱含量的测定提供了参考。
Choline is a strong organic alkaline compounds which widespread in the living organisms in the form of free choline, acetylcholine and phospholipid complex. It is an indispensable water-soluble vitamin for growth of animal. Research shows that it plays a very important role in brain growth and the development of cognitive, memory and learning ability. It’s another main role to promote liver function. The lack of choline may cause fatty liver, cirrhosis of the liver andarteriosclerosis especially for the elderly, and it can also caused Alzheimer's disease. It is very necessary that adding choline in infant formula and dry milk. Ever country has provision of adding content of choline.
The previous methods of detecting choline are perchlorate non-aqueous titration method, Leishi salt colorimetry and Leishi salt weight method. The current national standard GB/T 5413.20-1997 uses the enzyme reaction color spectrophotometer to measure the content of choline. These traditional methods are complex, bad selectivity, having many interfering factors, and they can not identify incorporation of cheap inorganic chloride and amine. In recent years' literatures we can find some new methods such as the high performance liquid chromatography-electrochemical detection method, the chemiluminescence method, the ion chromatography-conductivity detection method etc. But there is not any report about HPLC-ELSD method and HPLC-MSD method to detect the content of choline.
This paper establishes ion chromatography (IC), high-performance liquid chromatography-evaporative light scattering detection (HPLC-ELSD) and high-performance liquid chromatography-mass spectrometry (HPLC-MSD) three methods to detect the content of choline in dry milk, and discusses the comparison of these three methods. By orthogonal experiment design L9 (33), fix dry milk quality of 5.00g and hydrochloric acid volume of 30mL, design the concentration of hydrochloric acid (0.5mol/L, 0.8mol/L, 1.0mol/L three levels), the reaction temperature (60℃, 70℃, 80℃three levels), the reaction time (3h, 4h, 5h three levels), and get the best pretreatment condition is 1mol/L HCl at 70℃responsing three hours. Finally, I give the uncertainty evaluation of the determination of choline in dry milk by IC method.
The minimum detection limits of three methods of detecting choline are 0.1mg/L, 0.2mg/L and 0.01mg/L. The relative standard deviations are 1.13%, 2.67% and 3.86%. The add recovery are 98.78%～100.72%,96.72%～102.03% and 91.67%～100.02%. The method is simple, high sensitivity, good selectivity while traditional methods can not. This paper adds methods of determination of choline in dry milk, and also gives a reference about determination of choline on other similar products.
引文
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[7] 常碧影,郭吉原,宋荣.氯化胆碱市场的掺假新动向及应对措施[J].饲料广角,2002,16:3-4.
[8] 李英等.松花粉中胆碱的提取及测定研究[J].光谱实验室,2006,1(1):44-46.
[9] 冯振兴,任爱农.柿子中胆碱含量测定[J].天津中医药,2004,21(3):248-249.
[10]宋荣,常碧影.雷氏盐重量法测定饲料级氯化胆碱[J].饲料广角,2001,23:17-19.
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[12]丁永胜,牟世芬.离子色谱法测定饲料中氯化胆碱和三甲胺的含量[J].色谱,2004,22(2):174-176.
[13]Greaney M D,Marshall D L,Bailey B A,Acworth I N.[J].Chromatogr Biomed Appl,1993,622(2):l25.
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[15]Determination of Choline in Dry Milk and Infant Formula[Z].Dionex Corporation,2002.
[16]Wong K S,Jan J.Effect of Pushing Agents on the Separation and Detection of Debranched Amylopectin by High Performance Anion Exchange Chromatography With Pulsed Amperometric Detection [J].J Liq Chromatogr.1995,18(1):63-68.
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