WO2022061637A1 - Method for determining content of low-molecular-weight fucosylated glycosaminoglycan - Google Patents
Method for determining content of low-molecular-weight fucosylated glycosaminoglycan Download PDFInfo
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- the invention belongs to the technical field of medicine, and relates to a method for measuring the content of low-molecular-weight fucosified glycosaminoglycans, in particular to a method for measuring the content of low-molecular-weight fucosified glycosaminoglycans by volumetric method.
- Fucosylated Glycosaminoglycan is a glycosaminoglycan with a special chemical structure and pharmacological activity that has only been found in echinoderms so far. It has a main chain similar to chondroitin sulfate and contains fucose. Fucosyl (Fucosyl, Fuc) side chain substituted glycosaminoglycan analogs (Yoshida et.al, Tetrahedron Lett, 1992, 33:4959-62; et.al, J Biol Chem, 1996, 271:23973-84).
- LFG Low Molecular Weight Fucosylated Glycosaminoglycan, low molecular weight fucosylated glycosaminoglycan, hereinafter referred to as "LFG" is a glycosaminoglycan derivative obtained from sea cucumber body wall extraction and depolymerization. Lactose and glucuronic acid are polymerized. LFG is a polysaccharide drug. Due to the complex structure and polydispersity of the polysaccharide itself, quality control is difficult. Determination of sugar content is a necessary step in the research of polysaccharide drugs, and it is also an important part of quality control and product standard formulation.
- polysaccharide drugs Due to the diverse sources of polysaccharide drugs and different structural types, there are many methods for the determination of polysaccharides. Common sugar groups in polysaccharide drugs include neutral hexose, pentose and deoxyhexose, negatively charged uronic acid, sialic acid, positively charged amino sugar, etc. Hydrolysis to monosaccharides establishes specificity for the assay.
- LFG is a macromolecular polysaccharide substance, it is composed of a series of oligosaccharide chains of different molecular weights, which is relatively complicated and is not suitable for the above-mentioned conventional monosaccharide content determination method.
- gel size exclusion chromatography has many influencing factors, such as poor column durability and relatively high peak spread.
- the baseline is not easy to balance, and the reproducibility is poor.
- a new content determination method needs to be established. There is no report on the determination of fucosylated glycosaminoglycans by volumetric method in the existing reports.
- the purpose of the present invention is to provide a method for measuring the content of low molecular weight fucosylated glycosaminoglycan, which is used to monitor the quality of a low molecular weight fucosylated glycosaminoglycan and ensure the product of low molecular weight fucosylated glycosaminoglycan.
- the stability and controllability of quality promote the application of low molecular weight fucosylated glycosaminoglycans in medicine, food and other fields.
- the present invention provides a method for determining the content of low molecular weight fucosylated glycosaminoglycan, wherein the fucosylated glycosaminoglycan has the following structure:
- R 1 , R 2 , R 3 , R 4 , R 5 are optionally independently -H or -SO 3 H;
- R 6 is optionally -H, substituted or unsubstituted C1-C6 hydrocarbyl or C7-C12 aryl;
- R 7 is optionally -H, -SO 3 H, C2-C5 acyl
- R 8 is optionally a group represented by formula (II), formula (III) or formula (IV):
- R 1 , R 2 , R 3 , R 4 , R 5 , R 6 and R 7 are as defined above;
- R 9 and R 10 are optionally -H, substituted or unsubstituted C1-C6 hydrocarbyl or C7 - C12 aryl;
- R 11 is optionally -NHR 12 , -OR 13 , wherein R 12 and R 13 are optionally -H, substituted or unsubstituted C 1 -C 6 hydrocarbyl, or C 7 -C 12 aryl; and n is optionally is a natural number from 0 or 1 to 8;
- the measurement method is to decationize the low-molecular-weight fucosylated glycosaminoglycan reference substance, add it to titration, record the change of the conductance value during the titration, and measure the volume of the titrant consumed by the reference substance; On the ordinate, draw the titration curve with the volume of the titrant as the abscissa; draw the first, second, and third best straight lines for the three linear parts of the sudden drop, slight climb, and sharp rise of the titration curve respectively; The intersection of the second straight line and the third straight line is a vertical line to the abscissa, and the intersection of the vertical line and the abscissa is the volume of the titrant consumed by sulfate and carboxylate.
- the titration solution is an alkaline solution, preferably a hydroxide solution.
- a sodium solution more preferably a sodium hydroxide solution with a concentration of 0.02-0.1 mol/L.
- V is the volume of the titrant that sulfate and carboxylate consume together in the test sample
- R is the ratio of the volume of the titrant and the quality of the reference substance consumed by sulfate radicals and carboxylate radicals in the fucosylated glycosaminoglycan reference substance;
- m is the weight of the fucosylated glycosaminoglycan to be tested, calculated as a dry product.
- the method for rapidly measuring a low molecular weight fucosified glycosaminoglycan provided by the invention has the advantages of simple operation, good accuracy and economical efficiency. It is very suitable for the promotion and application of product batch inspection and quality control in industrial scale production.
- Embodiment 1 LFG content determination method verification
- Table 1 Main equipment information table
- Table 2 Main reagent information table
- the content of the reference substance is expressed in terms of HPLC purity (differential detector, area normalized), calculated in terms of dry product, and the titration coefficient is obtained after calibration.
- the low molecular weight fucosylated glycosaminoglycan test substance used in the examples was extracted from sea cucumber according to the method disclosed in Chinese Patent No. 201410007855.
- Reference substance solution Weigh an appropriate amount of LFG reference substance, accurately weigh, dissolve in water and dilute to make a solution containing about 5mg per 1ml, shake well, prepare 2 copies in parallel, and set aside.
- Test solution Weigh an appropriate amount of LFG test product, accurately weigh it, dissolve in water and dilute to make a solution containing about 5mg per 1ml, shake well, prepare 6 copies in parallel, and set aside.
- the coefficient described in Table 4 is the volume V1 of the sodium hydroxide titration solution consumed by the LFG reference substance, and the amount of sodium hydroxide titration solution consumed per unit volume is calculated, which is equivalent to a certain quality of LFG (calculated as dry product).
- the average value of the content determination results of this method is 104.16%, and the repeatability RSD value is 2.90%.
- the method is used as a method for the determination of multi-component samples of fucosylated glycosaminoglycans, and the accuracy of the determination results is good.
- An example of a titration curve is shown in Figure 1.
- Embodiment 2 Determination of test product
- the reagents, consumables and instruments required by the method are all conventional laboratory equipment, and the measurement cost is lower than that of the prior art HPLC measurement method, and the operation is simpler.
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Abstract
Disclosed is a volumetric method for determining the content of a low-molecular-weight fucosylated glycosaminoglycan. A reference sample and a test sample are separately subjected to cation exchange treatment, and an alkaline titrant is added for titration. The volume of the titrant collectively consumed by sulfate and carboxylate is recorded. The content of fucosylated glycosaminoglycan in the low-molecular-weight fucosylated glycosaminoglycan test sample is obtained by means of comparison and calculation. The operation is simple, economical and efficient, such that the method is suitable for large-scale process production.
Description
本发明属于医药技术领域，涉及一种低分子量岩藻糖化糖胺聚糖的含量测定方法，具体地说是一种容量法测定低分子量岩藻糖化糖胺聚糖含量测定的方法。The invention belongs to the technical field of medicine, and relates to a method for measuring the content of low-molecular-weight fucosified glycosaminoglycans, in particular to a method for measuring the content of low-molecular-weight fucosified glycosaminoglycans by volumetric method.
岩藻糖化糖胺聚糖(Fucosylated Glycosaminoglycan,FG)是迄今仅见于棘皮动物中的一种具有特殊化学结构及药理活性的糖胺聚糖，其具有类似硫酸软骨素主链，并存在岩藻糖基(Fucosyl,Fuc)侧链取代的糖胺聚糖类似物(Yoshida et.al,Tetrahedron Lett,1992,33:4959-62；
et.al,J Biol Chem,1996,271:23973-84)。LFG(Low Molecular Weight Fucosylated Glycosaminoglycan，低分子量岩藻糖化糖胺聚糖，以下简称“LFG”)是自海参体壁提取解聚所得糖胺聚糖衍生物，由岩藻糖、N-乙酰氨基半乳糖和葡萄糖醛酸聚合而成。LFG为多糖类药物，由于多糖本身复杂的结构和多分散性，所以质量控制比较困难。糖含量测定是多糖类药物研究的必要步骤，也是质量控制和产品标准拟定的重要内容，由于多糖类药物来源多样，结构类型不同，故而多糖的测定方法有很多种。多糖类药物中常见的糖基有中性的已糖、戊糖和脱氧已糖，负电型的糖醛酸、唾液酸，正电性的氨基糖等，可针对不同多糖化学结构的不同或水解为单糖建立专属性的方法进行测定。
Fucosylated Glycosaminoglycan (FG) is a glycosaminoglycan with a special chemical structure and pharmacological activity that has only been found in echinoderms so far. It has a main chain similar to chondroitin sulfate and contains fucose. Fucosyl (Fucosyl, Fuc) side chain substituted glycosaminoglycan analogs (Yoshida et.al, Tetrahedron Lett, 1992, 33:4959-62; et.al, J Biol Chem, 1996, 271:23973-84). LFG (Low Molecular Weight Fucosylated Glycosaminoglycan, low molecular weight fucosylated glycosaminoglycan, hereinafter referred to as "LFG") is a glycosaminoglycan derivative obtained from sea cucumber body wall extraction and depolymerization. Lactose and glucuronic acid are polymerized. LFG is a polysaccharide drug. Due to the complex structure and polydispersity of the polysaccharide itself, quality control is difficult. Determination of sugar content is a necessary step in the research of polysaccharide drugs, and it is also an important part of quality control and product standard formulation. Due to the diverse sources of polysaccharide drugs and different structural types, there are many methods for the determination of polysaccharides. Common sugar groups in polysaccharide drugs include neutral hexose, pentose and deoxyhexose, negatively charged uronic acid, sialic acid, positively charged amino sugar, etc. Hydrolysis to monosaccharides establishes specificity for the assay.
鉴于LFG为大分子多糖类物质，由一系列不同分子量的寡糖链组成，较为复杂，不适合上述常规单糖的含量测定方法。曾有研究采用高效凝胶色谱法进行了定量研究，但考虑到多糖本身为聚合物，实际分析测定中，凝胶排阻色谱的影响因素较多，比如色谱柱耐用性较差，峰展较宽，基线不易平衡，重现性较差，鉴于此，需建立一种新的含量测定方法。现有报道中未有采用容量法测定岩藻糖化糖胺聚糖的报道。Considering that LFG is a macromolecular polysaccharide substance, it is composed of a series of oligosaccharide chains of different molecular weights, which is relatively complicated and is not suitable for the above-mentioned conventional monosaccharide content determination method. There have been studies using high-efficiency gel chromatography to conduct quantitative research, but considering that polysaccharides themselves are polymers, in actual analysis and determination, gel size exclusion chromatography has many influencing factors, such as poor column durability and relatively high peak spread. wide, the baseline is not easy to balance, and the reproducibility is poor. In view of this, a new content determination method needs to be established. There is no report on the determination of fucosylated glycosaminoglycans by volumetric method in the existing reports.
发明内容SUMMARY OF THE INVENTION
本发明的目的是提供一种低分子量岩藻糖化糖胺聚糖的含量测定方法，用于监控一种低分子量岩藻糖化糖胺聚糖的质量，保障低分子量岩藻糖化糖胺聚糖 产品质量的稳定性和可控性，推动低分子量岩藻糖化糖胺聚糖在医药、食品等领域的应用。The purpose of the present invention is to provide a method for measuring the content of low molecular weight fucosylated glycosaminoglycan, which is used to monitor the quality of a low molecular weight fucosylated glycosaminoglycan and ensure the product of low molecular weight fucosylated glycosaminoglycan. The stability and controllability of quality promote the application of low molecular weight fucosylated glycosaminoglycans in medicine, food and other fields.
本发明提供一种低分子量岩藻糖化糖胺聚糖的含量测定方法，所述岩藻糖化糖胺聚糖具有如下的结构：The present invention provides a method for determining the content of low molecular weight fucosylated glycosaminoglycan, wherein the fucosylated glycosaminoglycan has the following structure:
式中，In the formula,
R
1、R
2、R
3、R
4、R
5任选为相互独立的-H或-SO
3H；
R 1 , R 2 , R 3 , R 4 , R 5 are optionally independently -H or -SO 3 H;
R
6任选为-H、取代或未取代的C1-C6烃基或C7-C12芳基；
R 6 is optionally -H, substituted or unsubstituted C1-C6 hydrocarbyl or C7-C12 aryl;
R
7任选为-H、-SO
3H、C2-C5酰基；
R 7 is optionally -H, -SO 3 H, C2-C5 acyl;
R
8任选为式(II)、式(III)或式(IV)所示基团：
R 8 is optionally a group represented by formula (II), formula (III) or formula (IV):
其式(II)、(III)和式(IV)中，In its formula (II), (III) and formula (IV),
R
1、R
2、R
3、R
4、R
5、R
6和R
7均同上定义；
R 1 , R 2 , R 3 , R 4 , R 5 , R 6 and R 7 are as defined above;
R
9和R
10任选为-H、取代或未取代的C1-C6烃基或C
7-C
12芳基；
R 9 and R 10 are optionally -H, substituted or unsubstituted C1-C6 hydrocarbyl or C7 - C12 aryl;
R
11任选为-NHR
12、-OR
13，其中R
12和R
13任选为-H、取代或未取代的C
1-C
6烃基或C
7-C
12芳基；且n是任选为0或1～8的自然数；
R 11 is optionally -NHR 12 , -OR 13 , wherein R 12 and R 13 are optionally -H, substituted or unsubstituted C 1 -C 6 hydrocarbyl, or C 7 -C 12 aryl; and n is optionally is a natural number from 0 or 1 to 8;
测量方法为将所述低分子量岩藻糖化糖胺聚糖对照品进行去阳离子处理，加入滴定液滴定，滴定过程中记录电导值的变化，并测定对照品消耗滴定液的体积；以电导率为纵坐标，以滴定液的体积为横坐标，绘制滴定曲线；分别对滴定曲线的突然下降，稍微爬升，急剧上升的3个线性部分图形作出第一、第二、第三条最佳直线；在第二条直线和第三条直线的交点对横坐标作垂直线，该垂线与横坐标的交点即为硫酸根和羧酸根共同消耗滴定液的体积。根据滴定曲线计算对照品中硫酸根和羧酸根共同消耗滴定液的体积，进而计算出对照品的质量与对照品中硫酸根和羧酸根共同消耗滴定液体积的比值R。同时对待测低分子量岩藻糖化糖胺聚糖供试品进行滴定，并根据R计算出其中低分子量岩藻糖化糖胺聚糖的含量，所述滴定液为碱性溶液，优选地为氢氧化钠溶液，更优选地为浓度为0.02～0.1mol/L的氢氧化钠溶液。The measurement method is to decationize the low-molecular-weight fucosylated glycosaminoglycan reference substance, add it to titration, record the change of the conductance value during the titration, and measure the volume of the titrant consumed by the reference substance; On the ordinate, draw the titration curve with the volume of the titrant as the abscissa; draw the first, second, and third best straight lines for the three linear parts of the sudden drop, slight climb, and sharp rise of the titration curve respectively; The intersection of the second straight line and the third straight line is a vertical line to the abscissa, and the intersection of the vertical line and the abscissa is the volume of the titrant consumed by sulfate and carboxylate. According to the titration curve, calculate the volume of the titrant consumed by sulfate and carboxylate in the reference substance, and then calculate the ratio R of the mass of the reference substance and the volume of the titrant consumed by sulfate and carboxylate in the reference substance. At the same time, the low molecular weight fucosylated glycosaminoglycan to be tested is titrated, and the content of the low molecular weight fucosylated glycosaminoglycan is calculated according to R. The titration solution is an alkaline solution, preferably a hydroxide solution. A sodium solution, more preferably a sodium hydroxide solution with a concentration of 0.02-0.1 mol/L.
在本发明的一种具体实施方式中，采用如下步骤：In a specific embodiment of the present invention, the following steps are adopted:
(1)取本品适量，加水溶解并定量稀释制成每1ml中约含5mg的溶液，作为供试品溶液；(1) Take an appropriate amount of this product, dissolve it in water and quantitatively dilute it to make a solution containing about 5mg per 1ml, as the test solution;
(2)取2ml供试品溶液加入阳离子交换树脂柱上样，用15ml水缓慢洗入小烧杯中，置电磁搅拌器上，浸入电极，搅拌，待电导读数稳定后，记录初始读数；(2) Take 2ml of the test solution and add it to the cation exchange resin column for sample loading, slowly wash it into a small beaker with 15ml of water, place it on an electromagnetic stirrer, immerse the electrode, stir, and record the initial reading after the conductance reading is stable;
(3)用移液枪加入滴定液50μL，当电导读数稳定后，记录并继续滴定，直至电导值变化变缓，加入10μL滴定液，当电导值又剧烈变化时，加入50μL滴定液，直至电导值有规律的升高，至终点(电导值恢复到开始滴定时)，记录下滴定液消耗的体积。(3) Add 50 μL of titrant with a pipette. When the conductance reading is stable, record and continue titration until the conductance value changes slowly, add 10 μL of titrant, and when the conductance value changes drastically again, add 50 μL of titrant until The conductance value rises regularly, to the end point (when the conductance value returns to the start of titration), the volume consumed by the titrant is recorded.
然后按照如下方法计算供试品中岩藻糖化糖胺聚糖含量：Then calculate the content of fucosylated glycosaminoglycans in the test product according to the following method:
其中，V为供试品中硫酸根和羧酸根共同消耗滴定液的体积；Wherein, V is the volume of the titrant that sulfate and carboxylate consume together in the test sample;
R为岩藻糖化糖胺聚糖对照品中硫酸根和羧酸根共同消耗滴定液体积与对照品 质量的比值；R is the ratio of the volume of the titrant and the quality of the reference substance consumed by sulfate radicals and carboxylate radicals in the fucosylated glycosaminoglycan reference substance;
m为岩藻糖化糖胺聚糖供试品称样量，以干燥品计。m is the weight of the fucosylated glycosaminoglycan to be tested, calculated as a dry product.
本发明提供的快速测量一种低分子量岩藻糖化糖胺聚糖的方法，操作简单，准确度好，经济高效。非常适合工业规模化生产中对产品批次检验，质量控制的推广和应用。The method for rapidly measuring a low molecular weight fucosified glycosaminoglycan provided by the invention has the advantages of simple operation, good accuracy and economical efficiency. It is very suitable for the promotion and application of product batch inspection and quality control in industrial scale production.
图1低分子量岩藻糖化糖胺聚糖滴定曲线。Figure 1. Low molecular weight fucosylated glycosaminoglycan titration curve.
实施例1 LFG含量测定方法验证Embodiment 1 LFG content determination method verification
1、设备、试剂、对照品和样品1. Equipment, reagents, controls and samples
1.1主要设备信息1.1 Main equipment information
表1主要设备信息表Table 1 Main equipment information table
1.2主要试剂信息1.2 Main reagent information
表2主要试剂信息表Table 2 Main reagent information table
1.3对照品信息1.3 Reference substance information
表3对照品信息表Table 3 Reference Substance Information Sheet
注：对照品含量以HPLC纯度(示差检测器，面积归一化)表示，以干燥品计，经过标定后得到滴定系数。Note: The content of the reference substance is expressed in terms of HPLC purity (differential detector, area normalized), calculated in terms of dry product, and the titration coefficient is obtained after calibration.
实施例所使用低分子量岩藻糖化糖胺聚糖供试品根据中国专利201410007855公开的方法，从海参中提取得到。The low molecular weight fucosylated glycosaminoglycan test substance used in the examples was extracted from sea cucumber according to the method disclosed in Chinese Patent No. 201410007855.
2.测定方法2. Measurement method
2.1树脂处理2.1 Resin treatment
取一定量732强酸性阳离子交换树脂，用蒸馏水(新沸放冷)漂洗干净，浸泡1h使其充分膨胀，用4倍树脂体积的1mol/L HCl溶液搅拌浸泡1h，蒸馏水洗至中性；用4倍树脂体积的1mol/L NaOH溶液搅拌浸泡1h，蒸馏水洗至中性；最后用4倍量1mol/L HCl溶液搅拌浸泡1h，蒸馏水洗至中性，备用。Take a certain amount of 732 strong acid cation exchange resin, rinse with distilled water (newly boiled and let cool), soak for 1 hour to fully expand, stir and soak with 1 mol/L HCl solution of 4 times the resin volume for 1 hour, and rinse with distilled water until neutral; Stir and soak in 1 mol/L NaOH solution of 4 times the resin volume for 1 h, and wash with distilled water until neutral; finally, stir and soak with 4 times the volume of 1 mol/L HCl solution for 1 h, wash with distilled water until neutral, and set aside.
2.2样品溶液配制2.2 Preparation of sample solution
对照品溶液：称取LFG对照品适量，精密称定，加水溶解并稀释制成每1ml中约含5mg的溶液，摇匀，平行配制2份，待用。Reference substance solution: Weigh an appropriate amount of LFG reference substance, accurately weigh, dissolve in water and dilute to make a solution containing about 5mg per 1ml, shake well, prepare 2 copies in parallel, and set aside.
供试品溶液：称取LFG供试品适量，精密称定，加水溶解并稀释制成每1ml中约含5mg的溶液，摇匀，平行配制6份，待用。Test solution: Weigh an appropriate amount of LFG test product, accurately weigh it, dissolve in water and dilute to make a solution containing about 5mg per 1ml, shake well, prepare 6 copies in parallel, and set aside.
2.3试验方法2.3 Test method
制备新鲜煮沸的纯水2000ml，迅速冷却(当天使用)；将处理好的树脂缓缓倒入层析柱，填充时打开下口阀开关，避免产生气泡，留一部分水在树脂上面，树脂应填充得均匀、无间隙、无气泡，层析柱用装有碎冰的冰袋包裹，填充体积约10cm×1cm。取对照品溶液2ml，沿壁缓缓加入遇冷层析柱中，当对照品加入柱子后，关闭层析柱开关，静置约20min，以使离子交换更加充分。然后打开层析柱开关，用15ml水缓缓洗入50ml小烧杯中(保存在冰浴中)，洗脱液应用氢氧化钠滴定液(0.1mol/L)立即滴定。将电导电极、温度探头、磁子放入小烧杯中，小烧杯置于冰浴中，调节磁力搅拌器，使磁子正常搅拌，待电导读数稳定后，记录读数。用移液枪加入0.1mol/L氢氧化钠滴定液50μL， 当电导读数稳定后，记录数值并继续滴定，直至电导值变化变缓，加入10μL滴定液，当电导值又剧烈变化时，加入50μL滴定液，直至电导值有规律的升高，至终点(电导值恢复到开始滴定时)，记录下氢氧化钠滴定液消耗的体积。Prepare 2000ml of freshly boiled pure water and cool it quickly (use on the same day); slowly pour the treated resin into the chromatography column, turn on the lower valve switch when filling to avoid air bubbles, and leave a part of the water on the resin, the resin should be filled To obtain uniformity, no gaps and no bubbles, the chromatography column was wrapped in an ice bag filled with crushed ice, and the filling volume was about 10cm×1cm. Take 2ml of the reference solution and slowly add it to the cooling column along the wall. When the reference is added to the column, turn off the switch of the column and let it stand for about 20min to make the ion exchange more sufficient. Then turn on the switch of the chromatographic column, slowly wash with 15ml of water into a 50ml small beaker (stored in an ice bath), and titrate the eluate immediately with sodium hydroxide titration solution (0.1mol/L). Put the conductance electrode, temperature probe and magnetron into a small beaker, place the small beaker in an ice bath, adjust the magnetic stirrer to make the magnetron stir normally, and record the reading after the conductance reading is stable. Add 50μL of 0.1mol/L sodium hydroxide titration solution with a pipette. When the conductance reading is stable, record the value and continue titration until the change of the conductance value becomes slow. Add 10μL of titration solution. When the conductance value changes drastically, add 50 μL of titration solution, until the conductance value rises regularly, to the end point (when the conductance value returns to the beginning of titration), record the volume consumed by the sodium hydroxide titration solution.
按照上述同法滴定LFG供试品溶液，记录下氢氧化钠滴定液消耗的体积。Titrate the LFG test solution according to the same method as above, and record the volume consumed by the sodium hydroxide titrant.
2.4试验结果2.4 Test results
表4容量法重复性实验结果表Table 4 Volumetric method repeatability test result table
注：表4中所述系数为根据LFG对照品消耗氢氧化钠滴定液的体积V1，计算出每消耗单位体积氢氧化钠滴定液的量，相当于一定质量的LFG(以干燥品计)。Note: The coefficient described in Table 4 is the volume V1 of the sodium hydroxide titration solution consumed by the LFG reference substance, and the amount of sodium hydroxide titration solution consumed per unit volume is calculated, which is equivalent to a certain quality of LFG (calculated as dry product).
2.5实验结论2.5 Experimental conclusion
根据实验结果，本方法含量测定结果平均值为104.16％，重复性RSD值为2.90％。本方法作为岩藻糖化糖胺聚糖类多组分样品的测定方法，测定结果准确度良好。滴定曲线实例见图1所示。According to the experimental results, the average value of the content determination results of this method is 104.16%, and the repeatability RSD value is 2.90%. The method is used as a method for the determination of multi-component samples of fucosylated glycosaminoglycans, and the accuracy of the determination results is good. An example of a titration curve is shown in Figure 1.
实施例2 供试品的测定Embodiment 2 Determination of test product
分别称取四个批次LFG供试品(20170531、20170612、20170616、20170623)适量，精密称定，加水溶解并稀释制成每1ml中约含5mg的溶液，摇匀，待用。Weigh four batches of LFG test samples (20170531, 20170612, 20170616, 20170623) in appropriate amounts, accurately weigh them, dissolve and dilute them with water to make a solution containing about 5mg per 1ml, shake well, and set aside.
分别取各供试品溶液2ml加入阳离子交换树脂柱上样，用15ml水缓慢洗入小烧杯中，置电磁搅拌器上，浸入电极，搅拌，待电导读数稳定后，记录读数。 用移液枪加入0.1mol/L氢氧化钠滴定液50μL，当电导读数稳定后，记录并继续滴定，直至电导值变化变缓，加入10μL滴定液，当电导值又剧烈变化时，加入50μL滴定液，直至电导值有规律的升高，至终点(电导值恢复到开始滴定时)，记录下氢氧化钠滴定液消耗的体积，以电导率为纵坐标，以滴定液的体积为横坐标，绘制曲线；分别对突然下降，稍微爬升，急剧上升的3个线性部分图形作出第一、第二、第三最佳直线；在第二条直线和第三条直线的交点对横坐标作垂直线，该垂线与横坐标的交点即为硫酸根和羧酸根共同消耗滴定液的体积V。Take 2ml of each test solution and add it to the cation exchange resin column for sample loading, slowly wash it into a small beaker with 15ml of water, place it on an electromagnetic stirrer, immerse the electrode, stir, and record the reading after the conductance reading is stable. Add 50 μL of 0.1mol/L sodium hydroxide titration solution with a pipette. When the conductance reading is stable, record and continue titration until the conductance value changes slowly, add 10 μL of titration solution, and when the conductance value changes drastically, add 50 μL Titrate until the conductance value rises regularly, to the end point (when the conductance value returns to the start of titration), record the volume consumed by the sodium hydroxide titrant, with the conductivity as the ordinate and the volume of the titrant as the abscissa , draw a curve; draw the first, second, and third best straight lines for the three linear part graphs that suddenly drop, climb slightly, and rise sharply; at the intersection of the second straight line and the third straight line, make a vertical to the abscissa Line, the intersection of the vertical line and the abscissa is the volume V of the titrant consumed by sulfate and carboxylate.
根据用LFG对照品计算出的每消耗1ml氢氧化钠滴定液(0.1mol/L)相当于31.16mg的LFG，计算出LFG样品的含量。计算结果如下：Calculate the content of LFG sample according to the consumption of 1 ml of sodium hydroxide titration solution (0.1 mol/L) calculated by LFG reference substance, which is equivalent to 31.16 mg of LFG. The calculation results are as follows:
表5四批LFG容量法测定含量结果表Table 5 Four batches of LFG volumetric determination results table
且本方法所需要的试剂、耗材与仪器均为实验室常规设备，测定成本与现有技术HPLC测定法相比，成本更加低廉，操作更加简便。In addition, the reagents, consumables and instruments required by the method are all conventional laboratory equipment, and the measurement cost is lower than that of the prior art HPLC measurement method, and the operation is simpler.
Claims (6)
- 一种低分子量岩藻糖化糖胺聚糖的含量测定方法，所述岩藻糖化糖胺聚糖具有如下的结构：A method for determining the content of low-molecular-weight fucosylated glycosaminoglycans, wherein the fucosylated glycosaminoglycans have the following structure:
式中，In the formula,R 1、R 2、R 3、R 4、R 5任选为相互独立的-H或-SO 3H； R 1 , R 2 , R 3 , R 4 , R 5 are optionally independently -H or -SO 3 H;R 6任选为-H、取代或未取代的C1-C6烃基或C7-C12芳基； R 6 is optionally -H, substituted or unsubstituted C1-C6 hydrocarbyl or C7-C12 aryl;R 7任选为-H、-SO 3H、C2-C5酰基； R 7 is optionally -H, -SO 3 H, C2-C5 acyl;R 8任选为式(II)、式(III)或式(IV)所示基团： R 8 is optionally a group represented by formula (II), formula (III) or formula (IV):
其式(II)、(III)和式(IV)中，In its formula (II), (III) and formula (IV),R 1、R 2、R 3、R 4、R 5、R 6和R 7均同上定义； R 1 , R 2 , R 3 , R 4 , R 5 , R 6 and R 7 are as defined above;R 9和R 10任选为-H、取代或未取代的C1-C6烃基或C7-C12芳基； R 9 and R 10 are optionally -H, substituted or unsubstituted C1-C6 hydrocarbyl or C7-C12 aryl;R 11任选为-NHR 12、-OR 13，其中R 12和R 13任选为-H、取代或未取代的C1-C6烃基或C7-C12芳基；且n是任选为0或1～8的自然数； R 11 is optionally -NHR 12 , -OR 13 , wherein R 12 and R 13 are optionally -H, substituted or unsubstituted C1-C6 hydrocarbyl, or C7-C12 aryl; and n is optionally 0 or 1 ~8 natural numbers;其特征在于：将所述低分子量岩藻糖化糖胺聚糖对照品进行阳离子交换处理，以碱性溶液为滴定液进行滴定，滴定过程中记录电导值的变化，并绘出滴定曲线；根据滴定曲线计算对照品中硫酸根和羧酸根共同消耗滴定液的体积，进而计算出对照品的质量与对照品中硫酸根和羧酸根共同消耗滴定液体积的比值R；用同样的方法，滴定测量并计算出低分子量岩藻糖化糖胺聚糖供试品中硫酸根和羧酸根共同消耗滴定液的体积V，根据对照品的R计算出供试品中低分子量岩藻糖化糖胺聚糖的含量。It is characterized in that: the low molecular weight fucosylated glycosaminoglycan reference substance is subjected to cation exchange treatment, and the titration is carried out with an alkaline solution as a titration solution, the change of the conductance value is recorded during the titration process, and a titration curve is drawn; The curve calculates the volume of titrant consumed by sulfate and carboxylate in the reference substance, and then calculates the ratio R between the mass of the reference substance and the volume of titrant consumed by sulfate and carboxylate in the reference substance; Calculate the volume V of the titrant consumed by sulfate and carboxylate in the low-molecular-weight fucosylated glycosaminoglycan to be tested, and calculate the content of low-molecular-weight fucosylated glycosaminoglycans in the test sample according to the R of the reference substance . - 根据权利要求1所述的测定方法，其特征在于：所述碱性溶液为氢氧化钠溶液。The assay method according to claim 1, wherein the alkaline solution is a sodium hydroxide solution.
- 根据权利要求1所述的测定方法，其特征在于：所述氢氧化钠溶液的浓度为0.02～0.1mol/L。The assay method according to claim 1, wherein the concentration of the sodium hydroxide solution is 0.02-0.1 mol/L.
- 根据权利要求1所述的测定方法，其特征在于所述硫酸根和羧酸根共同消耗滴定液体积的测定方法为以电导率为纵坐标，以滴定液的体积为横坐标，绘制滴定曲线；分别对曲线的突然下降，稍微爬升，急剧上升的3个线性部分图形作出第一、第二和第三条直线；在第二条直线和第三条直线的交点对横坐标作垂直线，该垂直线与横坐标的交点即为硫酸根和羧酸根共同消耗滴定液的体积V。assay method according to claim 1, it is characterized in that the assay method that described sulfate radical and carboxylate group consumes titrant volume together is to take electrical conductivity as ordinate, take the volume of titration solution as abscissa, draw titration curve; respectively; The first, second and third straight lines are drawn for the 3 linear parts of the graph of sudden descent, slight climb, and sharp rise of the curve; The intersection of the line and the abscissa is the volume V of the titrant consumed by sulfate and carboxylate.
- 根据权利要求1所述的测定方法，其特征在于包括如下步骤：assay method according to claim 1, is characterized in that comprising the steps:(1)取低分子量岩藻糖化糖胺聚糖供试品适量，加水溶解并定量稀释制成5mg/ml的溶液，作为供试品溶液；(1) Take an appropriate amount of the low molecular weight fucosylated glycosaminoglycan test product, dissolve in water and quantitatively dilute to make a solution of 5 mg/ml, as the test solution;(2)取2ml供试品溶液加入阳离子交换树脂柱上样，用15ml水缓慢洗入小烧杯中，置电磁搅拌器上，浸入电极，搅拌，待电导读数稳定后，记录初始读数；(2) Take 2ml of the test solution and add it to the cation exchange resin column for sample loading, slowly wash it into a small beaker with 15ml of water, place it on an electromagnetic stirrer, immerse the electrode, stir, and record the initial reading after the conductance reading is stable;(3)用移液枪加入滴定液50μL，当电导读数稳定后，记录并继续滴定，直至电导值变化变缓，加入10μL滴定液，当电导值又剧烈变化时，加入50μL滴定液，直至电导值有规律的升高，至电导值恢复到开始滴定时结束滴定过程。(3) Add 50 μL of titrant with a pipette. When the conductance reading is stable, record and continue titration until the conductance value changes slowly, add 10 μL of titrant, and when the conductance value changes drastically again, add 50 μL of titrant until The conductance value rises regularly, and the titration process ends when the conductance value returns to the beginning of the titration.
- 根据权利要求1所述的测定方法，其特征在于所述低分子量岩藻糖化糖胺聚糖含量的计算公式如下：The assay method according to claim 1, wherein the calculation formula of the low molecular weight fucosylated glycosaminoglycan content is as follows:
其中，供试品中硫酸根和羧酸根共同消耗滴定液的体积V；Wherein, in the test sample, sulfate and carboxylate consume the volume V of the titrant together;R为低分子量岩藻糖化糖胺聚糖对照品的质量与对照品中硫酸根和羧酸根共同消耗滴定液体积的比值；R is the ratio of the mass of the low-molecular-weight fucosylated glycosaminoglycan reference substance to the volume of the titrant consumed by sulfate and carboxylate in the reference substance;m为低分子量岩藻糖化糖胺聚糖供试品称样量，以干燥品计。m is the weight of the low-molecular-weight fucosylated glycosaminoglycan to be tested, calculated as a dry product.
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