Chromatographic determination of high-molecular weight impurities in amoxicillin

The separation of high molecular weight impurities in amoxicillin has been examined by three methods of gel filtration chromatography (GFC) and reversed phase liquid chromatography (RPLC). Specificity of GFC was checked by applying the method for the determination of amoxicillin and its related impurities and linking of GFC and RPLC. The result shows that for the determination of polymers the gel filtration chromatography is a rather simple separation mode as compared to RPLC. Regarding the three gel filtration chromatographic methods, the separation on the Superdex peptide column is advantageous over other methods of determining amoxicillin polymers.     

胰高血糖素分离纯化的研究

作者研究了自胰岛素生产过程中分离纯化胰高血糖素的方法。以胰岛素盐析物为起始物,经大孔强酸离子交换树脂柱层析,胰岛素不被吸附,随流出液流出(A),以NH₄OH稀溶液洗脱获得含有胰高血糖素的蛋白质溶液(B)。B液经凝胶过滤法分离,峰Ⅲ为富含胰高血糖素部分(C)。C液经DEAE—纤维素柱层析分离,其峰Ⅱ为电泳纯胰高血糖素(D)。标准品与D样品电泳行为相一致。兔血糖检查证明D保留有全部活性。以D浓缩物在pH 8.5下结晶,获得菱形十二面体结晶。以A制备胰岛素也同时获得胰岛素结晶。以上经离子交换树脂、凝胶过滤及DEAE—纤维素柱层析所得样品均经一种新型吸附树脂脱盐、浓缩。胰高血糖素收率为1 mg/kg猪胰。为自胰岛素生产过程中同时获得胰岛素及胰高血糖素提供了新的方法。     

High-performance liquid chromatographic analysis of chrysin derivatives on a Nova-Pak C18 column

A high-performance liquid chromatographic method has been developed for the separation and quantification of chrysin and synthetic chrysin derivatives (12 chrysin alkyl and 7 chrysin acyl derivatives). The chromatography was performed using a Nova-Pak C18 column. A RP-HPLC was performed by using a binary mixture (MeOH-10 mM H3PO4) as a mobile phase, and the column temperature was maintained at room temperature. A flow rate was 1.0 ml/min, and the effluent was monitored at a wavelenth of 280 nm. The retention times for chrysin acyl and alkyl derivatives were within 10 minutes and 20 minutes, respectively. The absolute recovery of samples were all over 96%. The detection limits were 0.1-18 ng at S/N = 3 ratio.     

吡柔比星的分离与纯化

目的确定用硅胶柱层析纯化吡柔比星粗品的工艺条件。方法采用薄层层析-硅胶柱层析法考察不同展开剂的层析效果,最优洗脱剂为二氯甲烷-甲醇;以吡柔比星的回收率和平均含量为评价指标,考察了流速、进样量与层析柱再生条件对层析粗分和精分的影响。结果硅胶柱层析粗分吡柔比星的最佳工艺条件:以梯度洗脱方式洗脱,洗脱剂流速5.0mL.min-1,上样量16.6mg.mL-1柱体积,0.5g.L-1氢氧化钠溶液离线再生硅胶。吡柔比星质量分数经粗分后由原来的35.7%提高到90%左右,回收率为96.2%。硅胶柱层析精分吡柔比星的最佳工艺条件为洗脱剂二氯甲烷-甲醇比例为90∶10,流速3.5mL.min-1,上样量16.6mg.mL-1柱体积,甲醇再生3倍柱体积。吡柔比星质量分数经精分后由粗分的90%提高到99.6%左右,回收率为97.5%。结论硅胶柱层析纯化吡柔比星所得产品符合质量要求。     

Inflammatory action of 8-methoxypsoralen-spermine photoproduct (8-MOP-Spm-P(GFC)) and effects of various drugs on rat paw edema induced by 8-MOP-Spm-P(GFC)

The photoproducts produced by irradiating 8-methoxypsoralen (8-MOP) in the presence of spermine (Spm) were fractionated using gel filtration chromatography (GFC) on a Sephadex G-25 column. As a result, two bands which were characterized by the effects on hyaluronidase activity were obtained. The first band strongly activated the hyaluronidase, but a second band did not exhibit any effect on the enzyme activity. The first and second bands contained photoproducts with molecular weights (MW)>2700 and MW<728, respectively, determined by the GFC method. The photoproducts, 8-MOP-Spm-P(GFC) obtained from the first band, but not the photoproducts with lower MW from the second band, showed enzyme activating action. 8-MOP-Spm-P(GFC) induced paw edema, which was stronger in the first phase than the second one in rats, differing from that induced by carrageenin. This photoproduct was a substance with lower cell toxicity because it did not cause hemolysis on red blood cells or the release of lactic dehydrogenase from mast cells in rats. The effects of various drugs on 8-MOP-Spm-P(GFC)-induced edema were investigated. As a result, edema formation was inhibited by drugs with an anti-histaminic action, such as alimemazine, dl-chlorpheniramine, promethazine, ketotifen and azelastine, and with anti-serotonin action such as cyproheptadine. On the other hand, tranilast did not show significant inhibition and indomethacin showed a tendency to increase its formation. These results suggested that 8-MOP-Spm-P(GFC) is a new inflammatory substance and is very useful as an agent to develop new anti-inflammatory drugs without cyclooxygenase inhibitory action.     

Bauhinia bauhinioides plasma kallikrein inhibitor: interaction with synthetic peptides and fluorogenic peptide substrates related to the reactive site sequence

A serine proteinase inhibitor was purified from Bauhinia bauhinioides seeds after extraction with 0.15M NaCl by ion-exchange column chromatography on DEAE-Sephadex, gel filtration on Superose 12 column, Mono Q chromatography or alternatively by affinity chromatography on trypsin- Sepharose. The inhibitor is a single polypeptide chain with molecular mass 20 kDa by gel filtration on Superose 12, but was resolved into two peaks by ion - exchange chromatography on Mono Q (FPLC system). The main eluted peak inhibits trypsin (Ki = 0.6 nM), plasma kallikrein (Ki = 0.35 nM), plasmin (Ki = 33.1 nM), and weakly chymotrypsin (Ki = 2,700 nM), being the most effective plasma kallikrein inhibitor isolated from Bauhinia seeds. Therefore, it was denominated Bauhinia bauhinioides kallikrein inhibitor (BbKI). Activity is thermolabile and on trypsin inhibition optimum pH is 8.0. BbKI displays high homology to other plant Kunitz inhibitors, except for the absence of disulfide bridges, and the only cysteine residue is at the C-terminal position (residue 154) characterizes a distinct member of the Kunitz family. The affinity of the inhibitor to trypsin was confirmed by adsorption to trypsin-Sepharose resin and by isolation of the trypsin-inhibitor complex by gel filtration. Peptides with variations around the reactive site of BbKI (GLPVRFESPLRINIIKESY) were synthesized containing a quenched fluorogenic group. Trypsin but not plasma kallikrein substrates, these peptides strongly inhibited plasma kallikrein.     

蚯蚓新型脱氧核糖核酸酶的相对分子质量测定

目的测定蚯蚓组织中一种新型脱氧核糖核酸酶(EWD3)的相对分子质量。方法采用凝胶过滤层析法、十二烷基磺酸钠-聚丙烯酰氨凝胶电泳法(SDS-PAGE)和基质辅助激光解吸电离飞行时间质谱法(MALDI-TOF-MS)3种方法测定一种新型蚯蚓脱氧核糖核酸酶(EWD3)的相对分子质量。结果凝胶过滤层析法、SDS-PAGE法和质谱法测定其相对分子质量分别为55080、58270和63460。结论凝胶过滤层析法和SDS-PAGE法可以常规粗略测定蛋白的相对分子质量,质谱法可以比较准确测定蛋白的相对分子质量,EWD3为单亚基蛋白,其相对分子质量约在55000￣65000之间。     

长白山白眉蝮蛇蛇毒纤溶酶的纯化

从长白山白眉蝮蛇蛇毒中纯化具有溶栓功效的纤溶酶。方法：用 ＤＥＡＥ Ｓｅｐｈａｄｅｘ Ａ５０离子交换、Ｓｅｐｈａｄｅｘ Ｇ７５凝胶过滤层析和 ＤＥＡＥ Ｓｅｐｈａｄｅｘ Ａ ５０三步柱层析分离方法,对长白山白眉蝮蛇蛇毒进行分离纯化,得到了一种纤溶酶活性组分。结果：经ＳＤＳ－聚丙烯酰胺凝胶电泳和基质辅助激光解吸电离飞行时间质谱表征该酶为单一蛋白,其分子量为 ２３．３６７ ｋＤ。结论：为进一步研究其结构和功能提供了可靠依据。     

A new macromolecular antitumor antibiotic, C-1027. II. Isolation and physico-chemical properties

A new macromolecular antibiotic C-1027 was obtained from the broth filtrate of Streptomyces globisporus C-1027 by precipitation with ammonium sulfate, DEAE-cellulose column chromatography and gel filtration chromatography on a Sephadex G-75 column. This antibiotic, prepared as a white powder, is an acidic polypeptide having an isoelectric point of pH 3.5-3.7 and a molecular weight of 15,000 as determined by SDS-polyacrylamide gel electrophoresis and gel filtration chromatography. The acid hydrolysate of the purified antibiotic C-1027 contained no methionine or tryptophan. From the physico-chemical data, it may be considered to possess a very labile non-protein chromophore.     

Preparative separation of high-purity troxerutin and related substances from mother liquor of troxerutin by silica gel column chromatography and semi-preparative liquid chromatography

Troxerutin (TRO) is a mixture of semi-synthetic flavonoids prepared by hydroxyethylation of rutin, and it is commonly used for the treatment of cerebrovascular diseases. The main active ingredient is trishydroxyethyl rutin. The mother liquor of TROcontains a lot of TRO and other derivatives of hydroxyethylated rutin. In order to make full use of the mother liquor of TRO, an efficient method was developed for recovering high-purity TRO from mother liquor of TRO by combining silica gel column chromatography with semi-preparative liquid chromatography. In the silica gel column chromatographic separation, the ratio of silica gel to sample and eluent composition were investigated to obtain optimum separation effect. The results showed that when the ratio of silica gel to sample was 50, and acetone–ethyl acetate–water–glacial acetic acid (10:10:3:1, v/v/v/v) was used as the eluent, the separation effect of TRO and adjacent impurities was good. Moreover, 150 g ofTRO with a purity of 80% could be obtained from 1 kg of mother liquor of TRO by the silica gel column chromatographic separation, and the results were consistent with the quality standard of TRO raw material. Subsequently, the semi-preparative HPLC was performed, and 100 g TRO with a purity of up to 98% (w/w) was obtained. Meanwhile, tetrahydroxyethylrutin and tetrahydroxyethylquercetin with purity greater than 98% were obtained. This work proposed the separation and preparation of TRO with high-purity from the production waste of TRO for the first time, which had certain environmental benefits and economic benefits.     

凝胶过滤分离蛋白质实验条件的研究

目的研究凝胶过滤各影响因素对蛋白质分离的影响。方法应用一般色谱方法,选择葡聚糖凝胶Sephadex G-75和几种蛋白质对装柱、洗脱液、流速和操作压等条件进行试验。结果选用不同浓度的凝胶悬液进行装柱,不同离子成分、pH的洗脱液,不同流速和操作压对几种蛋白质进行色谱分离,得到不同的结果。结论凝胶悬液较稀利于装柱,洗脱液的离子成分不直接影响分离效果,而洗脱液的pH对几种蛋白质分离产生明显影响,酸性条件下分离的效果好,适当的流速和操作压有利于提高色谱柱的分辨率,保证色谱柱的反复正常使用。     

普通念珠藻中多糖的分离纯化研究

目的 研究普通念珠藻多糖分离纯化方法,并测定其相对分子质量.方法 采用水提醇沉法制备粗多糖,Molish反应和硫酸苯酚法对其进行定性和定量检测,二乙胺基乙基纤维素离子交换柱色谱法进行分离纯化,高效凝胶渗透色谱法测定其相对分子质量.结果 普通念珠藻粗多糖的提取率为8.6％,分离获得一个组成均一的酸性多糖组分NSP,其重均相对分子质量为402 717.结论 NSP为首次从普通念珠藻中分离得到一个酸性多糖组分.     

膝沟藻毒素Gonyautoxins的分离纯化膝沟藻毒素Gonyautoxins的分离纯化

目的从微小亚历山大藻(Alexandriun minutum Halim)Amtk 2中分离纯化膝沟藻毒素gonyautoxins。 方法 用凝胶色谱、离子交换色谱等方法从人工培养的微小亚历山大藻Amtk 2酸酒精萃取物中分离纯化膝沟藻毒素。结果从100 L Amtk 2培养液中获得(6.74±0.31)×109个藻细胞,其酸酒精萃取物经凝胶色谱分离首次在国内得到膝沟藻毒素GTX-4,GTX-1,GTX-3和GTX-2混合物(29.59±0.28) mg。从此混合物中取出4.06 mg经两次离子交换色谱纯化得到纯GTX-4(0.40±0.002) mg, GTX-1(5.95±0.03)×10^-2 mg,GTX-3(6.92±0.05)×10^-4 mg和GTX-2(0.11±0.005) mg。结论人工培养的微小亚历山大藻Amtk 2酸酒精萃取物通过凝胶色谱和两次离子交换色谱分离能够得到纯的膝沟藻毒素GTX-4,GTX-1,GTX-3和GTX-2。     

Purification and preliminary characterization of a plasma kallikrein inhibitor isolated from sea hares Aplysia dactylomela Rang, 1828.

An inhibitor active against pancreatic trypsin was found in the crude extract from the sea hares Aplysia dactylomelaRang, 1828. A stronger inhibitory activity against human plasma kallikrein was detectable after treating this extract at 60 degrees C, for 30 min. The plasma kallikrein inhibitor (AdKI) purification was achieved by acetone fractionation (80%) v/v, ion-exchange chromatography on Mono Q column and gel filtration chromatography on Superdex 75 column (FPLC system). By the latter a molecular mass of 2900 Da was estimated. The purified inhibitor strongly inhibits human plasma kallikrein with a K(i) value of 2.2 x 10(-10)M, while human plasmin and pancreatic trypsin were inhibited with K(i) values of 1.8 x 10(-9) and 4.7 x 10(-9)M, respectively. Chymotrypsin, pancreatic elastase, pancreatic kallikrein and thrombin are not inhibited. The effect of AdKI on plasma kallikrein was confirmed by the prolongation of activated partial thromboplastin time, using a clotting time assay. The inhibitor did not affect prothrombin time or thrombin time. AdKi is a more specific inhibitor than other serine proteinase inhibitors from marine invertebrates.     

常压柱色谱组合法制备石杉碱甲

目的寻求一条经济实用、便于产业化的石杉碱甲制备工艺路线。方法以薄层色谱法选择柱色谱分离系统后,以回收率和含量为考察指标,对影响柱色谱分离效果的各因素进行选择与优化,寻找最佳工艺路线。结果采用碱性氧化铝柱和硅胶柱的组合进行分离纯化,碱性氧化铝柱的工艺参数为:填料粒度100～200目,干法装柱(径高比1:10),干法上样(上样比例1:50),三氯甲烷-甲醇(47:3,V/V)洗脱;硅胶柱的工艺参数为:填料粒度200～300目,湿法装柱(径高比1:10),湿法上样(上样比例1:30),三氯甲烷-甲醇(48:2,V/V)洗脱。所得产物溶于丙酮,静置过夜后,即可获得符合《中国药典》要求的结晶产物,本工艺条件下的石杉碱甲总回收率达到76.1%。结论本工艺设备要求简单,样品处理量大,生产成本低,适合工业化生产。     

一种新型蚯蚓脱氧核糖核酸酶相对分子质量的测定

目的测定从蚯蚓组织中提取纯化的一种新型脱氧核糖核酸酶的相对分子质量。方法采用凝胶过滤层析法、十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)法和基质辅助激光解吸附电离飞行时间质谱法三种方法测定一种新型蚯蚓脱氧核糖核酸酶(EWD1)的相对分子质量。结果凝胶过滤层析法、SDS-PAGE法和质谱法测定其相对分子质量分别为126440、136320和157190。结论EWD1由两个亚基组成,其相对分子质量最终确定为157190。     

凝胶色谱法测定美罗培南中的聚合物

目的建立凝胶色谱法测定美罗培南中聚合物的方法。方法采用SephadexG-10柱（10mm×300mm）,流动相A为pH8.0的0.05mol/L磷酸盐缓冲液,流动相B为水。流速为0.3mL/min,检测波长为254nm,进样量为500μL,柱温为30℃,对照品采用法罗培南钠工作标准品。结果美罗培南在4.202～16.552mg/mL范围内与聚合物峰面积呈良好的线性关系（r=0.9999）。结论该方法准确、可靠、灵敏度高,适用于美罗培南聚合物的控制。     

白豆中α-淀粉酶抑制剂的分离及其活性研究

采用乙醇分级沉淀、CMII纤维素离子交换柱色谱及凝胶柱色谱，从白豆中纯化得到一种α- 淀粉酶抑制剂(α-AI)，经SDS-PAGE及Sepharose CL-6B柱色谱鉴定其为结构均一的糖蛋白。该糖蛋白中蛋白质含量为88.2%，氨基酸组成主要为天冬氨酸、谷氨酸、苏氨酸及丝氨酸。糖链部分单糖组成为甘露糖、葡萄糖、半乳糖和木糖，其摩尔比为2.42∶1.50∶1.52∶1.00。糖和蛋白质结合的糖肽键类型为O-糖肽键。白豆α-AI使用剂量为150 mg·kg^-1体重，连续使用7 d时，α-AI可明显降低高血糖大鼠的空腹血糖；使用剂量为300 mg·kg^-1时，对高血糖大鼠的糖耐量具有明显的改善作用。研究结果表明，从白豆中分离得到的淀粉酶抑制剂对高血糖大鼠具有明显的降血糖功能，其作为一种安全、天然的降糖药物具有良好的开发前景。     

阿莫西林颗粒中高分子杂质含量和生产工艺的相关性评价

目的 评价海南先声药业阿莫西林颗粒的生产工艺和其中高分子杂质的相关性。方法采用反相色谱和Superdex Peptide凝胶色谱分别测定阿莫西林颗粒和原料中的高分子杂质。结果6批阿莫西林颗粒中均无高聚物，其高分子杂质种类与原料一致，均为阿莫西林闭环二聚体和阿莫西林噻唑酸，且二者的含量均一、稳定。结论该阿莫西林颗粒制剂工艺较好，且水平稳定可控。     

川芎挥发油中藁本内酯的制备分离

目的：建立硅胶柱层析法分离川芎挥发油中的藁本内酯。方法：将川芎挥发油以石油醚稀释后上硅胶柱，用石油醚：乙酸乙酯（100：1）进行洗脱，将分离制得的藁本内酯采用光谱分析进行结构鉴定，并用气相色谱-质谱联合分析（GC-MS）面积归一化法进行纯度检查。结果：从川芎挥发油中分离得纯度≥98％的藁本内酯，可作为对照品使用。结论：本研究建立的硅胶柱层析法简单、方便，能从川芎挥发油中分离得纯度高的藁本内酯单体。