CN109632986B - 一种糖及糖醇类化合物的柱后衍生检测方法 - Google Patents
一种糖及糖醇类化合物的柱后衍生检测方法 Download PDFInfo
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Abstract
本发明涉及一种糖及糖醇类化合物的柱后衍生检测方法，涉及葡萄糖、乳糖、蔗糖、甘露醇等糖及糖醇类化合物的检测技术领域。本发明将糖或糖醇类物质采用高效液相色谱‑十八烷基硅胶色谱柱分离技术，将糖类进行分离纯化，再通过强氧化剂将其分子上的羟基经氧化转化为羰基，并与荧光传感化合物在一定温度、时间条件下发生衍生反应，生成含荧光显色基团的衍生化合物，最后采用荧光检测器实现快速定量定性检测的一种检测方法。采用此法测定糖及糖醇类化合物，数据稳定可靠，检测限可提高至ppm级。
Description
技术领域
本发明属于化学分析技术领域，具体涉及糖及糖醇类化合物高效液相色谱（HPLC）分离-化学衍生制备-荧光光谱检验的方法，本发明的方法可用于临床医学、药品与食品检测领域。
背景技术
糖类化合物是自然界中存在数量最多、分布最广且具有重要生物功能的有机化合物，从细菌到高等动物的机体都含有糖类化合物。糖是除蛋白质和核酸外的又一类非常重要的生命物质，糖化学在生命科学、药物研究中占有很重要的地位和广泛的用途，对糖类物质的检测方法研究，一直是糖化学的重要研究内容。
糖类物质由于极性较强，通常以乙腈-水为流动相，采用氨基键合色谱柱进行分离，同时由于糖类物质化学结构中缺乏紫外或荧光等显色基团，因而多采用示差检测器进行定性定量测定，如《GB/T 22221-2008 食品中果糖、葡萄糖、蔗糖、麦芽糖、乳糖的测定 高效液相色谱法》中采用此方法进行检测，但实际应用存在许多缺陷，如灵敏度低，选择性差，受环境温度的干扰大，不适合应用梯度洗脱等。为了克服这些方法的缺点并实现高灵敏度检测和痕量分析，目前开发了很多衍生化测定法，通过将糖类物质进行衍生化反应，标记成荧光或紫外发光化合物，从而提高灵敏度，并根据高效液相色谱进行分离前后衍生顺序的不同，分为柱前衍生或柱后衍生，本发明即属于高效液相色谱-柱后衍生-荧光检测技术，但本发明与其他文献研究的方法相比有很多优势。
目前糖类衍生常选择1-苯基-3-甲基-5-吡唑啉酮(PMP)、2-氨基吡啶(2-AP)、芴甲氧基羰基-肼(FMOC-肼)、2-氨基苯甲酰胺(2-AB)以及2-氨基吖啶酮(2-AMAC)等试剂进行柱前衍生，采用最多为选择1-苯基-3-甲基-5-吡唑啉酮(PMP)为衍生试剂，如专利号为201210592158.3的《一种具有降血糖功能制剂的检测方法》，即采用此衍生方法，但此类方法只能对还原性的糖类进行衍生，如鼠李糖,阿拉伯糖,葡萄糖,半乳糖和葡萄糖醛酸等，但对非还原糖如蔗糖等，糖醇类如甘露醇等则无法进行测定。同时此类方法由于衍生反应时间长，只能进行柱前衍生，前处理较为复杂。
由于乙腈强毒性试剂，费用高，用量大，对环境十分不友好，同时键合氨基色谱柱价格贵、寿命短，使用成本高，本发明研究使用一种离子对试剂-磷酸盐缓冲液混合纯水相、非有机溶剂为流动相，选择十八烷基硅烷键合硅胶色谱柱为分析色谱柱对糖类化合物进行高效液相分离，不仅避免使用了强毒性的乙腈试剂，改善环境影响，还大大降低了检测成本。
有文献报道使用1-萘硼酸作为糖类的衍生试剂进行柱后衍生-荧光检测含量测定，如《赵鑫. 1-萘硼酸柱后衍生高效液相色谱荧光法测定单糖的研究[D]. 河北师范大学, 2013.》中的研究，但根据文献内容此方法只适用于单糖测定，对于双糖或多糖如乳糖、蔗糖，则无法测定，同时使用1-萘硼酸衍生反应对pH较为敏感，需在特定pH值下进行反应，限定了液相分离中流动相选择范围。
本发明申请的一种糖及糖醇类化合物的柱后衍生检测方法，可以很好的克服上述测定过程中的缺陷，可作为糖类及糖醇类物质的通用型快速测定方法。
发明内容
本发明主要提供一种糖类及糖醇类化合物的检测方法，该方法操作简单、重复性好、分析结果准确。为了实现上述发明目的，本发明采用的技术方案包含三个步骤。
第一步，采用十八烷基硅烷键合硅胶色谱柱对糖类及糖醇类化合物进行高效液相分离，其流动相为离子对试剂-磷酸盐缓冲液。
流动相所使用的离子对试剂包括戊烷磺酸钠、己烷磺酸钠、庚烷磺酸钠、辛烷磺酸钠、十二烷基磺酸钠，浓度为0.01~1%，磷酸盐缓冲液浓度为0.01~0.5moL/L，pH范围为2~8。
第二步，将糖类及糖醇类化合物进行氧化反应后键合荧光基团。
氧化反应通过氧化试剂将糖化合物中的邻二醇氧化为羰基后，再与荧光化合物键合。氧化试剂包括四醋酸铅、高碘酸或高碘酸盐（钠/钾）等。荧光化合物为胺类化合物，包括对氨基苯磺酸、氨基乙基磺酸、酚试剂(MBTH)、丹磺酰肼等。衍生反应的温度为50~100℃。
以高碘酸为氧化剂，对氨基苯磺酸为荧光试剂为例，其反应原理如下：
第三步，采用荧光检测器进行进行荧光定量检测。
荧光检测的激发波长为200~400nm，发射波长为400~500nm。
附图说明
图1为本发明实施例一中葡萄糖与果糖标准衍生物色谱图，其中葡萄糖衍生物质出峰时间为18.548分钟，果糖衍生物质出峰时间为6.820分钟。
具体实施方式
实施例一 血糖浓度柱后衍生-高效液相分离-荧光色谱检测方法：
血糖监测是临床中最常做的监测项目，其检测方法通常有葡萄糖氧化酶法、己糖激酶法，但此类检测方法干扰较多，如葡萄糖氧化酶法其原理是利用氧化酶将葡萄糖氧化为葡萄糖酸，并同时产生过氧化氢，再利用过氧化氢酶使其与4-氨基安替比林和酚类物质进行缩合为红色醌亚胺类化合物，这类物质在可见光波长下有吸收峰，采用比色法进行测定，得出葡萄糖浓度，但血液中的的血脂成分，如乳糜微粒和极低密度脂蛋白对葡萄糖检测有极大干扰，影响结果真实性。本实施例采用柱后衍生-高效液相分离-荧光色谱技术，提供了一种专属性强、精密度高、灵敏度高的血糖测定方法，特别适用于药物临床测试等准确度要求很高的研究场合。方法包括下述内容。
1色谱条件。
仪器：设备应具有2个泵分别控制流动相与衍生反应液，样品进样器，色谱柱，反应管，冷却管，检测器和记录设备，其中反应管与冷却管应放于恒温装置中。
检测器：荧光检测器，激发波长：368nm，发射波长：417nm，灵敏度为1。
反应管：不锈钢管，0.3mm×15m（内径×长度）。
冷却管：不锈钢管，0.3mm×1.5m（内径×长度）。
色谱柱：C18色谱柱，4.6mm×25cm粒径5mm。
流动相：以0.15%十二烷基磺酸钠的磷酸盐缓冲液（取磷酸二氢钾3.02g与磷酸氢二钾6.2g，加水溶解成1000ml，pH值约为7.0）为流动相。
流速：调节流动相流速使得葡萄糖峰保留时间约为18分钟，并调节荧光反应试剂的流速与流动相的流速一致。
反应温度：85~90℃、冷却温度：5~10℃。
2.溶液配制。
对照品储备液：精密称取葡萄糖对照品25mg，用甲醇溶解，配制成0.25mg/ml的对照品储备液，低温避光保存。
内标溶液：精密称取果糖对照品25mg，用甲醇溶解，配制成25μg/ml的内标储备液，低温避光保存。
荧光反应试剂：取对氨基苯磺酸5g与高碘酸1.5g，加水溶解并稀释至1000ml，既得。
3.血清样品处理方法。
精密吸取血清样品200μl 置于1.5 ml离心管中，精密加入10 μl内标溶液，涡旋30s，精密加入150μl乙腈，涡旋3 min，14000 r/min 离心10 min，取上清液100 μl，取20 μl注入液相色谱仪，记录色谱图。按标准曲线计算浓度，即得。
4.标准曲线的制备。
取适量葡萄糖标准溶液，氮气吹干，加入100 μl空白血清配制成血清中含待测物葡萄糖浓度分别为：25，50，100，150，200 μg/100μl（同时做空白对照），按“3”项下操作，进样分析。以待测物浓度（Y，μg/100μl）为纵坐标，待测物峰面积减去空白本底药物峰面积（As）与内标峰面积（Ai）的比值（X）为横坐标，用最小二乘法进行线性回归，回归方程：Y=8.541X＋0.412(r=0.999 9)。葡萄糖在25~200 μg/100μl范围内线性良好，定量下限为1.875 μg/100μl。
Claims (1)
1.一种糖及糖醇类化合物的柱后衍生检测方法，其特征在于其包括如下步骤：将葡萄糖和果糖经过高效液相色谱-十八烷基硅胶色谱柱分离后，在以0.15%十二烷基苯磺酸钠的磷酸盐缓冲液为流动相条件下分离后，以高碘酸为氧化剂，对氨基苯磺酸为荧光试剂，在85-90℃下衍生后，进行荧光检测，定量测定糖类化合物质，荧光检测的激发波长为200-400nm，发射波长为400-500nm；所述磷酸盐缓冲液为磷酸二氢钾3.02g与磷酸氢二钾6.2g，加水溶解成1000ml,pH值为7.0。
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