CN1908005A - Composite protein precipitator - Google Patents
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Abstract
一种复合蛋白沉淀剂，它涉及一种蛋白沉淀剂。它解决了现有单一组分蛋白沉淀剂蛋白沉淀率低、提取分离效果差，10％的三氯醋酸与6％的高氯酸酸性过强对色谱柱损害大及采用液－液萃取法提取生物样本中药物耗时长、操作繁琐、样本损耗大的缺陷。复合蛋白沉淀剂按重量份数比由1～1.5份浓度为分析纯的5－磺基水杨酸、75～85份浓度为分析纯的甲醇和15～25份蒸馏水制成。所发明的复合蛋白沉淀剂的蛋白沉淀率达99％以上，pH呈弱酸性对色谱柱损害小。使用本发明的复合蛋白沉淀剂，蛋白沉淀与生物样本中的药物提取分离一步完成，操作简单，用时少，样本中药物成分损耗少，药物提取回收率达70％以上，大大提高了体内药物分析的效率与准确性，能够满足药物研究的需要。A compound protein precipitating agent relates to a protein precipitating agent. It solves the problem of the low protein precipitation rate and poor extraction and separation effect of the existing single-component protein precipitant, the excessive acidity of 10% trichloroacetic acid and 6% perchloric acid is too strong to damage the chromatographic column, and the liquid-liquid extraction method is used for extraction. Drugs in biological samples have the defects of long time-consuming, cumbersome operation, and large sample loss. The composite protein precipitant is prepared from 1-1.5 parts of analytically pure 5-sulfosalicylic acid, 75-85 parts of analytically pure methanol and 15-25 parts of distilled water in parts by weight. The protein precipitation rate of the invented composite protein precipitation agent reaches over 99%, and the pH is weakly acidic, causing little damage to the chromatographic column. Using the composite protein precipitation agent of the present invention, protein precipitation and drug extraction and separation in biological samples are completed in one step, the operation is simple, the time is less, the loss of drug components in the sample is less, and the recovery rate of drug extraction is more than 70%, which greatly improves drug analysis in vivo The efficiency and accuracy can meet the needs of drug research.
Description
技术领域technical field
本发明涉及一种蛋白沉淀剂。The invention relates to a protein precipitation agent.
背景技术Background technique
现有的有机溶剂蛋白沉淀剂、酸性或碱性蛋白沉淀剂与重金属蛋白沉淀剂多为单一组分，蛋白沉淀率低，提取分离效果差。虽然，10％的三氯醋酸与6％的高氯酸有较高的蛋白沉淀率，与样本等体积的情况下蛋白沉淀率达98.5％～99.5％，但存在酸性过强，对色谱柱损害大，成本高的问题；而采用液-液萃取法进行生物样本中的药物提取，耗时长、操作繁琐、样本中药物成分损耗大，极大的影响了体内药物分析的效率与准确性，不能满足快速、准确提供实验数据的需要。Most of the existing organic solvent protein precipitants, acidic or alkaline protein precipitants and heavy metal protein precipitants are single components, the protein precipitation rate is low, and the extraction and separation effect is poor. Although 10% trichloroacetic acid and 6% perchloric acid have a high protein precipitation rate, and the protein precipitation rate reaches 98.5% to 99.5% in the case of the same volume as the sample, but the acidity is too strong, which will damage the chromatographic column. However, the use of liquid-liquid extraction to extract drugs from biological samples is time-consuming, cumbersome, and the loss of drug components in the sample is large, which greatly affects the efficiency and accuracy of drug analysis in vivo. Meet the needs of providing experimental data quickly and accurately.
发明内容Contents of the invention
本发明的目的是为了解决现有单一组分蛋白沉淀剂蛋白沉淀率低、提取分离效果差，10％的三氯醋酸与6％的高氯酸酸性过强对色谱柱损害大、成本高以及采用液-液萃取法提取生物样本中药物耗时长、操作繁琐、样本损耗大的缺陷，从而提供了一种复合蛋白沉淀剂。复合蛋白沉淀剂按重量份数比由1～1.5份浓度为分析纯的5-磺基水杨酸、75～85份浓度为分析纯的甲醇和15～25份蒸馏水制成。本发明复合蛋白沉淀剂配制简单，组分少，蛋白沉淀率高，达99％以上。本发明复合蛋白沉淀剂pH呈弱酸性，对色谱柱损害小(样本PH＜2.5对色谱柱损害大)，成本低。使用本发明的复合蛋白沉淀剂，蛋白沉淀与生物样本中的药物提取分离一步完成，操作简单，用时少，样本中药物成分损耗少，药物提取回收率达70％以上，大大提高了体内药物分析的效率与准确性，能够满足药物研究的需要。The purpose of the present invention is to solve the low protein precipitation rate of the existing single component protein precipitant, the poor extraction and separation effect, the excessive acidity of 10% trichloroacetic acid and 6% perchloric acid are too strong to the chromatographic column, the cost is high and the The liquid-liquid extraction method is used to extract the defects of long time-consuming, cumbersome operation and large sample loss of drugs in biological samples, thereby providing a composite protein precipitation agent. The composite protein precipitant is prepared from 1-1.5 parts of analytically pure 5-sulfosalicylic acid, 75-85 parts of analytically pure methanol and 15-25 parts of distilled water in parts by weight. The composite protein precipitation agent of the invention is simple to prepare, has few components, and has a high protein precipitation rate of over 99%. The pH of the composite protein precipitant of the present invention is weakly acidic, has little damage to the chromatographic column (sample pH<2.5 has great damage to the chromatographic column), and has low cost. Using the composite protein precipitation agent of the present invention, protein precipitation and drug extraction and separation in biological samples are completed in one step, the operation is simple, the time is less, the loss of drug components in the sample is less, and the recovery rate of drug extraction is more than 70%, which greatly improves drug analysis in vivo The efficiency and accuracy can meet the needs of drug research.
附图说明Description of drawings
图1是经浓度为6％的高氯酸处理的空白血浆色谱图，图2是经复合蛋白沉淀剂处理的空白血浆色谱图。Fig. 1 is the chromatogram of blank plasma treated with 6% perchloric acid, and Fig. 2 is the chromatogram of blank plasma treated with complex protein precipitant.
具体实施方式Detailed ways
具体实施方式一：本实施方式为复合蛋白沉淀剂按重量份数比由1～1.5份浓度为分析纯的5-磺基水杨酸、75～85份浓度为分析纯的甲醇和15～25份蒸馏水制成。Specific embodiment one: this embodiment is that the composite protein precipitant is analytically pure 5-sulfosalicylic acid by 1～1.5 parts by weight ratio, 75～85 parts concentration is analytically pure methanol and 15～25 parts by weight. made with distilled water.
本实施方式将浓度为分析纯的5-磺基水杨酸、浓度为分析纯的甲醇和蒸馏水按重量份数比混合、溶解后即可使用。In this embodiment, 5-sulfosalicylic acid with an analytically pure concentration, methanol with an analytically pure concentration, and distilled water are mixed in parts by weight and dissolved before use.
本实施方式复合蛋白沉淀剂应用于盐酸塞利洛尔等心血管类药物生物样本的前处理，以便进行反相高效液相色谱分析。生物样本(血浆)的处理步骤：(一)按1∶1的体积比将生物样本与本实施方式复合蛋白沉淀剂混合、振荡；(二)离心，蛋白沉淀，取上清液进行液相色谱分析，分析生物样本中的药物成分。In this embodiment, the composite protein precipitant is applied to the pretreatment of biological samples of cardiovascular drugs such as celiprolol hydrochloride, so as to conduct reversed-phase high-performance liquid chromatography analysis. The processing steps of the biological sample (plasma): (1) mix and shake the biological sample and the composite protein precipitation agent of this embodiment according to the volume ratio of 1:1; (2) centrifuge, protein precipitate, and take the supernatant for liquid chromatography Analysis, analysis of pharmaceutical ingredients in biological samples.
生物样本经本实施方式复合蛋白沉淀剂沉淀后高效液相色谱分析的精密度高于现有其它蛋白沉淀剂采用液-液萃取后高效液相色谱分析的精密度。对几种蛋白沉淀剂进行比较，结果如表1所示。The precision of HPLC analysis of biological samples after being precipitated by the composite protein precipitant of this embodiment is higher than that of other existing protein precipitants after liquid-liquid extraction. Several protein precipitation agents were compared, and the results are shown in Table 1.
表1
分别以浓度为6％的高氯酸和本实施方式复合蛋白沉淀剂为蛋白沉淀剂对空白血浆生物样本进行沉淀，然后再进行液相色谱分析，检测波长：254nm，流动相：甲醇∶H2O(65∶35，色谱柱：C18柱，流速：1mL/min，运行时间：10min，进样量：20μL。色谱分析结果如图1和图2所示，血浆杂质峰均在开始后4.5分钟内出现，其后无任何杂质峰干扰，说明复合蛋白沉淀剂的应用有利于药物样品与杂质的分离测定。Precipitate the blank plasma biological sample with perchloric acid with a concentration of 6% and the composite protein precipitant of this embodiment as the protein precipitant respectively, and then carry out liquid chromatography analysis, detection wavelength: 254nm, mobile phase: methanol: H 2 O(65:35, chromatographic column: C 18 column, flow rate: 1mL/min, running time: 10min, injection volume: 20 μ L. The chromatographic analysis results are shown in Figure 1 and Figure 2, and the peaks of plasma impurities are all at 4.5 It appeared within minutes, and there was no interference from any impurity peaks thereafter, indicating that the application of the composite protein precipitant is beneficial to the separation and determination of drug samples and impurities.
具体实施方式二：本实施方式与具体实施方式一的不同点是：复合蛋白沉淀剂按重量份数比由1.1～1.4份浓度为分析纯的5-磺基水杨酸、76～84份浓度为分析纯的甲醇和16～24份蒸馏水制成。其它步骤与实施方式一相同。Specific embodiment two: the difference between this embodiment and specific embodiment one is: the composite protein precipitating agent is analytically pure 5-sulfosalicylic acid, 76～84 parts concentration by weight ratio by 1.1～1.4 parts It is made of analytically pure methanol and 16-24 parts of distilled water. Other steps are the same as those in Embodiment 1.
具体实施方式三：本实施方式与具体实施方式一的不同点是：复合蛋白沉淀剂按重量份数比由1.2～1.3份浓度为分析纯的5-磺基水杨酸、78～82份浓度为分析纯的甲醇和18～22份蒸馏水制成。其它步骤与实施方式一相同。Specific embodiment three: the difference between this embodiment and specific embodiment one is: the composite protein precipitant is analytically pure 5-sulfosalicylic acid, 78～82 parts concentration by weight and number ratio by 1.2～1.3 parts concentration It is made of analytically pure methanol and 18-22 parts of distilled water. Other steps are the same as those in Embodiment 1.
具体实施方式四：本实施方式与具体实施方式一的不同点是：复合蛋白沉淀剂按重量份数比由1.2份浓度为分析纯的5-磺基水杨酸、80份浓度为分析纯的甲醇和20份蒸馏水制成。其它步骤与实施方式一相同。Specific embodiment four: the difference between this embodiment and specific embodiment one is: the composite protein precipitant is analytically pure 5-sulfosalicylic acid by 1.2 parts of concentration, and 80 parts of concentration is analytically pure by weight. Methanol and 20 parts distilled water. Other steps are the same as those in Embodiment 1.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101717756B (en) * | 2008-10-09 | 2012-05-09 | 哈尔滨仁皇药业股份有限公司 | Preparation method of superoxide dismutase |
| CN101717755B (en) * | 2008-10-09 | 2012-06-27 | 哈尔滨仁皇药业股份有限公司 | Composite protein precipitator, preparation method and application thereof |
| CN114624362A (en) * | 2022-03-17 | 2022-06-14 | 广东省中医院（广州中医药大学第二附属医院、广州中医药大学第二临床医学院、广东省中医药科学院） | Kit for detecting advanced glycosylation end products in serum and application thereof |
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| CN1110279A (en) * | 1994-04-11 | 1995-10-18 | 余国华 | Method for extracting albumen from animal's blood by precipitating method |
| CA2306629A1 (en) * | 1997-10-17 | 1999-04-29 | Harvest Technologies Corporation | Precipitation of growth-factor-enriched fibrinogen concentrate from platelet rich plasma |
| EP1170593A3 (en) * | 2000-06-19 | 2002-04-10 | Felix Levine | Reagent, kit and method for the determination of total proteins and globulines such as hemoglobin in biological fluids |
| CN1570633A (en) * | 2003-07-17 | 2005-01-26 | 赵青华 | Biochemical detection liquid for urine protein |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101717756B (en) * | 2008-10-09 | 2012-05-09 | 哈尔滨仁皇药业股份有限公司 | Preparation method of superoxide dismutase |
| CN101717755B (en) * | 2008-10-09 | 2012-06-27 | 哈尔滨仁皇药业股份有限公司 | Composite protein precipitator, preparation method and application thereof |
| CN114624362A (en) * | 2022-03-17 | 2022-06-14 | 广东省中医院（广州中医药大学第二附属医院、广州中医药大学第二临床医学院、广东省中医药科学院） | Kit for detecting advanced glycosylation end products in serum and application thereof |
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