Isolation and Purification of Unstable Iridoid Glucosides from Traditional Chinese Medicine by Preparative High Performance Liquid Chromatography Coupled with Solid-phase Extraction

An efficient preparative method was successfully developed for isolation and purification of unstable components from medicinal plant extracts, using a combined method of preparative high performance liquid chro-matography(HPLC) and solid-phase extraction(SPE). The aim of this study was to obtain an effective method with high preparative efficiency and importantly to avoid the transformation of unstable compounds. The preparative HPLC system was based on an LC/MS controlled four-channel autopurification system. The SPE method was performed with a C18 packing material to trap the target compounds and to remove the acidic additive derived from the mobile phase. Using this method, the unstable iridoid glucosides(IGs) as model compounds were successfully isolated and purified from the extract of Hedyotis diffusa Willd. Six IGs(including one new minor IG) and one nucleotide compound were simultaneously obtained, each with a purity of 91% as determined by HPLC. The structures of the isolated compounds were identified by UPLC/Q-TOF MS, UV, 1D and/or 2D NMR. It was demonstrated that the combination of preparative HPLC with SPE is a versatile tool for preparative purification of unstable compounds from complex natural products.

Advancements in the preparation of high-performance liquid chromatographic organic polymer monoliths for the separation of small-molecule drugs

The various advantages of organic polymer monoliths, including relatively simple preparation processes,abundant monomer availability, and a wide application range of pH, have attracted the attention of chromatographers. Organic polymer monoliths prepared by traditional methods only have macropores and mesopores, and micropores of less than 50 nm are not commonly available. These typical monoliths are suitable for the separation of biological macromolecules such as proteins and nucleic acids, but their ability to separate small molecular compounds is poor. In recent years, researchers have successfully modified polymer monoliths to achieve uniform compact pore structures. In particular, microporous materials with pores of 50 nm or less that can provide a large enough surface area are the key to the separation of small molecules. In this review, preparation methods of polymer monoliths for high-performance liquid chromatography, including ultra-high cross-linking technology, post-surface modification, and the addition of nanomaterials, are discussed. Modified monolithic columns have been used successfully to separate small molecules with obvious improvements in column efficiency.

Quantification of six bioactive compounds in Zhenqi Fuzheng preparation by high-performance liquid chromatography coupled with diode array detector and evaporative light scattering detector

A simple and accurate high-performance liquid chromatography（HPLC）coupled with diode array detector（DAD）and evaporative light scattering detector（ELSD）was established for the determination of six bioactive compounds in Zhenqi Fuzheng preparation（ZFP）.The monitoring wavelengths were 254,275 and 328 nm.Under the optimum conditions,good separation was achieved,and the assay was fully validated in respect of precision,repeatability and accuracy.The proposed method was successfully applied to quantify the six ingredients in 31 batches of ZFP samples and evaluate the variation by hierarchical cluster analysis（HCA）,which demonstrated significant variations on the content of these compounds in the samples from different manufacturers with different preparation procedures.The developed HPLC method can be used as a valid analytical method to evaluate the intrinsic quality of this preparation.

Studies on the Renaturation with Simultaneous Purification of Recombinant Human Proinsulin with Unit of Simultaneous Renaturation and Purification of Protein in Semi-preparative Scale

The renaturation and purification of recombinant human proinsulin (rh-proinsulin) expressed in E. coli with the unit of simultaneous renaturation and purification of protein (USRPP) in semi-preparative scale was studied. The result shows that rh-proinsulin extracted with 8.0 mol/L urea can be renatured and purified simultaneously in 45 minutes with the USRPP (1050 mm ID). The purity of rh-proinsulin was found to be more than 90% and the mass recovery to be more than 80%. The renaturation effect of rh-proinsulin with the USRPP was tested by enzyme cleavage for obtaining insulin. In addition, the result was further confirmed with RPLC, SDS-PAGE electrophoresis, and MALDI-TOF, respectively.

Lysophosphatidylcholine Biomarkers of Lung Cancer Detected by Ultra-performance Liquid Chromatography Coupled with Quadrupole Time-of-flight Mass Spectrometry

Centrifugal ultrafiltration after methanol extraction of whole plasma was used as an optimal condition for the preparation of blood plasma before metabonomic studies. The plasma samples from 102 lung cancer patients and 34 healthy volunteers were prepared with this approach. With ultra-performance liquid chromatography（UPLC） coupled with quadrupole time-of-flight mass spectrometry（Q-TOF MS） analysis, the samples were investigated in order to find potential disease biomarkers. After data acquisition, orthogonal signal correction partial least squares models were built to differentiate the healthy volunteers from lung cancer patients and to identify metabolites that showed significantly different expression between the two groups. Several metabolite ions were identified as potential biomarkers according to the variable importance in the project（VIP） value in both ion modes. Five lysophosphatidylcholines were further identified as specifically lysoPC 16：0, isomer of lysoPC 16：0, lysoPC 18：0, lysoPC 18：1 and lysoPC 18：2. These results suggest that UPLC coupled with Q-TOF MS is an effective technique for the analysis of plasma metabolites in metabonomic studies.

Modifying current thin-film microextraction(TFME)solutions for analyzing prohibited substances:Evaluating new coatings using liquid chromatography

For identifying and quantifying prohibited substances,solid-phase microextraction(SPME)continues to arouse interest as a sample preparation method.However,the practical implementation of this method in routine laboratory testing is currently hindered by the limited number of coatings compatible with the ubiquitous high-performance liquid chromatography(HPLC)systems.Only octadecyl(C18)and polydimethylsiloxane/divinylbenzene ligands are currently marketed for this purpose.To address this situation,the present study evaluated 12 HPLC-compatible coatings,including several chemistries not currently used in this application.The stationary phases of SPME devices in the geometry of thin filmcoated blades were prepared by applying silica particles bonded with various functional ligands(C18,octyl,phenyl-hexyl,3-cyanopropyl,benzenesulfonic acid,and selected combinations of these),as well as unbonded silica,to a metal support.Most of these chemistries have not been previously used as microextraction coatings.The 48 most commonly misused substances were selected to assess the extraction efficacy of each coating,and eight desorption solvent compositions were used to optimize the desorption conditions.All samples were analyzed using an HPLC system coupled with triple quadrupole tandem mass spectrometry.This evaluation enables selection of the best-performing coatings for quantifying prohibited substances and investigates the relationship between extraction efficacy and the physicochemical characteristics of the analytes.Ultimately,using the most suitable coatings is essential for trace-level analysis of chemically diverse prohibited substances.

HPLC fingerprint-oriented preparative separation of major flavonoids from safflower extract by preparative pressurized liquid chromatography

The aim of this study was to rapidly isolate the major effective flavanoids from the extract of safflower（Carthamus tinctorius） using ODS medium pressure liquid chromatography（MPLC） and semi-preparative HPLC, guided by a developed fingerprint. Twelve compounds were isolated and their structures were elucidated as kaempferol 3-O-β-D-rutinoside（1）, kaempferol 3-O-β-D-glucoside（2）, rutin（3）, quercetin 3-O-β-D-glucoside（4）, 6-hydroxykaempferol 3,6,7-tri-O-β-D-glucoside（5）, 6-hydroxykaempferol 3-O-β-D-glucoside（6）, 6-hydroxykaempferol 6,7-di-O-β-D-glucoside（7）, 6-hydroxykaempferol 3-O-β-Drutinoside（8）, 6-hydroxykaempferol 3,6-di-O-β-D-glucosyl 7-O-β-D-glucuronide（9）, isosafflomin C（10）, safflomin C（11） and hydroxysafflor yellow A（12） by spectroscopic analysis and comparing with the literature. Our results demonstrated that preparative pressurized liquid chromatography combined with HPLC fingerprint guide is an efficient tool to isolate the target compounds quickly.

Pharmacokinetics and bioequivalence of ranitidine and bismuth derived from two compound preparations

AIM： To evaluate the bioequivalence of ranitidine and bismuth derived from two compound preparations. METHODS： The bioavailability was measured in 20 healthy male Chinese volunteers following a single oral dose （equivalent to 200 mg of ranitidine and 220 mg of bismuth） of the test or reference products in the fasting state. Then blood samples were collected for 24 h. Plasma concentrations of ranitidine and bismuth were analyzed by high-performance liquid chromatography and inductively coupled plasma-mass spectrometry （ICPMS）, respectively. The non-compartmental method was used for pharmacokinetic analysis. Log-transformed Cmax, AUC（0-t） and AUC（0-∞） were tested for bioequivalence using ANOVA and Schuirmann two-one sided t-test. Tmax was analyzed by Wilcoxon＇s test. RESULTS： Various pharmacokinetic parameters of ranitidine derived from the two compound preparations, including Cmax, AUC（0-t）, AUC（0-∞）, Tmax and T1/2, were nearly consistent with previous observations. These parameters derived from test and reference drug were as follows： Cmax（0.67 ± 0.21 vs 0.68 ± 0.22 mg/L）, AUC（0-t）（3.1 ± 0.6 vs 3.0 ± 0.7 mg/L per hour）, AUC（0-∞）（3.3 ± 0.6 vs 3.2 ± 0.8 mg/L per hour）, Tmax （2.3 ± 0.9 VS 2.1 ± 0.9 h） and T1/2 （2.8 ± 0.3 vs 3.1 ± 0.4 h）. In addition, double-peak absorption profiles of ranitidine were found in some Chinese volunteers. For bismuth, those parameters derived from test and reference drug were as follows： Cmax （11.80 ± 7.36 vs 11.40 ± 6.55 μg/L）, AUC（0-t） （46.65 ± 16.97 vs 47.03 ± 21.49 μg/L per hour）, Tmax （0.50 ± 0.20 vs 0.50 ± 0.20 h） and T1/2 （10.2 ± 2.3 vs 13.0 ± 6.9 h）. Ninety percent of confidence intervals for the test/reference ratio of Cmax, AUC（0-t） and AUC（0-∞） derived from both ranitidine and bismuth were found within the bioequivalence acceptable range of 80%-125%. No significant difference was found in Tmax derived from both ranitidine and bismuth. CONCLUSION： The two compound preparations are bioequivalent and may be prescribed interchangeably.

全自动固相萃取—高效液相色谱法测定现制饮料中7种合成着色剂

目的:建立一种全自动固相萃取—高效液相色谱双波长法测定现制饮料中7种合成着色剂的方法。方法:样品经纯水提取2次,离心后,合并上清液,调节pH至3~4。利用全自动固相萃取装置活化Poly-sery色素专用固相萃取小柱,并上样、淋洗、洗脱。洗脱液氮吹浓缩至200μL,用50%流动相A+50%流动相B混合液定容至1.0 mL。色谱柱采用C 18柱,流动相为20 mmol/L乙酸铵—甲醇,梯度洗脱,流速1.0 mL/min,进样量10μL,检测波长254,628 nm。结果:各物质在0.5~50.0μg/mL范围内相关系数均大于0.9999,方法检出限为0.023~0.179 mg/kg,定量限为0.078~0.598 mg/kg,加标回收率为92.4%~96.8%,相对标准偏差(RSD)为1.2%~4.2%。结论:该方法试剂使用量小,操作步骤简便,自动化程度高,回收率高,重复性好,适合于批量样品测定。

宠物用丹参咀嚼片制备工艺研究及质量标准建立

旨在确定宠物用丹参咀嚼片的制备工艺并建立其质量标准。本试验通过湿法制粒辅料种类、制粒工艺、压片工艺进行考察,确定其制备工艺,采用薄层色谱法、高效液相色谱法(HPLC)对宠物用丹参咀嚼片中丹参酮ⅡA、丹酚酸B分别开展定性和定量研究,建立其质量标准。结果:将丹参喷雾粉加辅料混合均匀,混合时间5 min,转速100 r/min,以10%淀粉浆为黏合剂制成颗粒,干燥温度40~50℃,0.3%硬脂酸镁作为润滑剂,压片速率10~35 r/min;薄层鉴别显示丹参酮ⅡA斑点清晰,分离度良好且专属性强;高效液相色谱显示丹酚酸B在8.0~256.0μg/mL范围内线性关系良好(R^(2)=0.999 9),系统适用性、专属性、精密度、稳定性、重复性、系统耐用性良好,加样回收率为95.0%~105.0%;对6批样品中丹酚酸B进行含量测定,设定含量限度为11.484 1 mg/g。综上,本试验确定的宠物用丹参咀嚼片制备工艺稳定可行,建立的薄层鉴别方法和高效液相色谱含量测定方法可用于宠物用丹参咀嚼片的质量控制。

制备高效液相色谱用于纽莫康定杂质B1和B5的纯化

目的开展低含量成分的富集和纯化研究,从Glarea lozoyensis菌体的发酵产物(即纽莫康定B0粗品)中制备出杂质B1和B5,并对其进行结构鉴定。方法建立了基于制备高效液相色谱(prep-HPLC)技术的两步纯化法,第一步使用亲水(HILIC)模式固定相对目标杂质进行富集,第二步采用反相(RPLC)模式固定相对杂质进行制备。得到的两个杂质采用质谱(MS)、核磁(NMR)进行鉴定,确定化合物结构。结果两个杂质为B1和B5,相对分子质量均为1049 Da,色谱纯度分别为97.83%和98.13%。经核磁鉴定,B1为B0的高酪氨酸类似物,B5为B0的鸟氨酸类似物。结论本研究发展的基于prep-HPLC的两步纯化方法为低含量成分制备提供参考,第一步分离实现目标成分富集,第二步利用正交性的色谱柱提高分离选择性,成功制备出杂质B1与B5,有助于B0杂质谱的建立和进一步的质量控制研究。

低渗储层渗吸剂界面性能与组分分离

对石油磺酸盐WPS-D和十八烷基二甲基甜菜碱复配的渗吸剂进行界面张力测试,得出0.3%浓度8∶2比例下的渗吸剂达到超低界面张力(<1.0×10^(-3)mN/m),0.4%和0.5%浓度比例为9∶1的体系接近超低界面张力。采用制备色谱与质谱相结合,对石油磺酸盐WPS-D进行组分分离,并结合界面张力测试结果,考察不同组分的界面活性。结果表明,石油磺酸盐分离出5种结构组成差异较大的不同组分;以重烷基苯磺酸盐为主,C_(17)~C_(19)烷基苯磺酸盐达到超低界面张力并维持,C_(18)~C_(24)的烷基苯磺酸盐接近超低界面张力后反弹,得出提供界面活性的主要组分是C_(17)~C_(19)磺酸盐。

黑果分散片的制备、质量及体外活性研究

为了探索黑果分散片的制备工艺、质量及其体外活性,通过单因素与正交试验相结合的方式考察了黑果分散片的制备工艺,采用UV法考察了黑果分散片总三萜的含量及其体外活性,采用超高效液相色谱法测定了黑果分散片中没食子酸、儿茶素、表儿茶素的含量.结果发现黑果分散片的优选处方为微晶纤维素作稀释剂、交联聚乙烯吡咯烷酮作崩解剂、75%乙醇为黏合剂、微粉硅胶作润滑剂.分散片中总三萜的含量测定方法显色前后稳定性符合测定要求、仪器精密度和重复性良好,总三萜的加样回收率为99.48%;体外活性试验结果表明,黑果分散片具有一定的清除DPPH和抑制脂质体的能力;超高效液相色谱法测定没食子酸、儿茶素、表儿茶素的加样回收率分别为99.83%、100.69%和99.61%.优选的制剂工艺制备的分散片,其崩解时限符合要求,总三萜的含量为1.42 mg/片,没食子酸、儿茶素、表儿茶素的含量分别为111.15μg/g、940.19μg/g、598.25μg/g.试验结果为黑果分散片的应用提供了理论依据.

优化法结合制备型高效液相色谱分离纯化虾青素

以雨生红球藻中的虾青素为研究对象,研究其虾青素的皂化工艺条件,通过单因素试验和响应面优化法进行NaOH-甲醇溶液皂化工艺优化,并通过制备型高效液相色谱纯化。最终得出的最佳工艺条件为皂化时间12 h、皂化温度32℃、NaOH-甲醇溶液浓度2.5%,在此条件下测得虾青素含量为(19.423±0.186)μg/mL。进一步通过制备型高效液相色谱对皂化得到的虾青素纯化处理,经液相色谱测定虾青素的纯度达94%以上,利用红外光谱和核磁共振对其纯化产物进行定性分析,通过图谱分析发现制备得到的虾青素与标准品虾青素图谱基本一致。

头孢克肟制剂中有关物质结构鉴定与定量分析

目的旨在建立头孢克肟制剂中有关物质结构鉴定与定量分析方法。方法采用HPLC-Q-TOF-MS/MS法对主要杂质结构进行分析。建立HPLC法,采用C18色谱柱(250 mm×4.6 mm,5μm),以乙腈-四丁基氢氧化铵溶液为流动相进行梯度洗脱,检测波长为254 nm,柱温为40℃,对头孢克肟制剂进行有关物质检测。结果初步鉴定了头孢克肟制剂中9个杂质,分别为杂质A、B、D、E、F、G、H、头孢克肟亚砜和头孢克肟叔丁酯。在建立的HPLC色谱条件下,头孢克肟及各杂质均能达到基线分离,各有关物质的质量浓度在0.1934~20.03μg·mL^(-1)内线性关系良好,r>0.9990;回收率为98.4%~101.8%(RSD<2.0%)。结论HPLC-Q-TOF-MS/MS技术能初步鉴定头孢克肟制剂的杂质结构,该方法能检测不同厂家头孢克肟片剂、颗粒剂、胶囊剂及干混悬剂中的有关物质。

基于UPLC-MS/MS和感官评价对人参提取物中主要苦味物质分析研究

采用高效液相色谱法、高效制备液相色谱法、超高效液相色谱-串联质谱法(UPLC-MS/MS)以及感官评价对人参提取物中的主要苦味物质进行分离、纯化和鉴定。通过滋味稀释分析法对人参提取物中的主要苦味物质进行贡献度排序。结果表明,人参皂苷Rg1、人参皂苷Rf、人参皂苷Rb1、20(S)-人参皂苷Rg2和人参皂苷Rb3是人参提取物样品中的苦味物质,其中人参皂苷Rb1和20(S)-人参皂苷Rg2滋味稀释因子最高,是其主要的苦味物质,且与20(S)-人参皂苷Rg2相比,人参皂苷Rb1具有更小的苦味识别阈值,是人参提取物中最关键的苦味物质。

复方参芪颗粒的制备工艺及其质量控制

目的:优选复方参芪颗粒的最佳配方和制备工艺,并对其质量控制进行初步研究。方法:以颗粒的吸湿率、成型率、流动性和口感为评价指标,按不同权重综合评分,以综合得分为响应值,结合响应面法分析,优选配方和制备工艺;用高效液相色谱法测定黄芪甲苷的含量。结果:浸膏与辅料比例为1∶4,甘露醇与乳糖比例为4∶3,矫味剂用量为0.20%,成型率、吸湿率、休止角分别为96.86%、2.52%、27.60°,颗粒微甜,可接受;黄芪甲苷的线性回归方程为y=1.321 9x+1.113 3,R^(2)=0.999 6。黄芪甲苷在1.994~7.976μg范围内具有良好的线性关系。结论:此方法重现性好,优选的复方参芪颗粒制备工艺切实可行;黄芪甲苷含量测定方法线性关系良好,可用于其质量控制。

氢化松香酯键合硅胶制备柱的研制及性能研究

将氢化松香丙烯酸乙二醇酯(EGHRA)通过点击化学反应键合到巯基化硅胶(SH@SiO_(2))表面,制备了一种新型的氢化松香丙烯酸乙二醇酯键合硅胶(EGHRA@SiO_(2))固定相。对合成的固定相进行元素分析、红外光谱、热失重分析、N 2吸附-脱附实验和粒径分布测试,结果显示:EGHRA@SiO_(2)固定相中的C元素较SiO_(2)增加了10.72个百分点,比表面积270.06 m 2/g,平均孔径6.16 nm,平均粒径10μm。使用匀浆法填装了250 mm×30 mm制备柱并对其进行了一系列色谱评价,证明EGHRA@SiO_(2)制备柱具有较好的流通性和典型的反相色谱特性,且表现出良好的稳定性和可重复性。Tanaka测试表明:由于键合单体具有类菲环骨架,使得EGHRA@SiO_(2)柱的空间选择性优于C18柱。反相色谱模式下,EGHRA@SiO_(2)制备柱对红豆杉树皮粗提物有较理想的分离效果,筛选出乙腈/水(体积比47∶53),在流速15 mL/min时进样1.0 mL,一次纯化将粗提物中紫杉醇的纯度提高了91.52个百分点,10-去乙酰基紫杉醇的纯度提高了83.24个百分点,7-木糖-10-去乙酰基紫杉醇的纯度提高了62.99个百分点,三尖杉宁碱的纯度提高了55.85个百分点。

香砂平胃凝胶贴膏制备工艺及质量标准的研究

目的 将香砂平胃丸改制为凝胶贴膏,筛选最佳处方与制备工艺,建立其质量标准。方法 采用水醇双提法提取挥发油与浸膏;采用正交实验法,以初黏力、持黏力和综合感官为评价指标筛选最佳处方;用薄层色谱法定性鉴别处方中木香、陈皮、甘草、苍术;用高效液相色谱法测定指标性成分厚朴酚、和厚朴酚的含量。结果 最佳处方为混合浸膏10.0 g、水16.0 g,甘油18.0 g、聚维酮K90 0.2 g、聚丙烯酸钠2.4 g,羧甲基纤维素钠0.6 g、甘羟铝0.5 g、柠檬酸0.5 g、微粉硅胶1.2 g,综合评分高;木香、陈皮、甘草、苍术薄层斑点清晰,分离度好,阴性样品无干扰;建立的厚朴酚与和厚朴酚含量测定方法专属性好,线性范围分别为9.98~149.70μg·mL^(-1)(r=0.9991)、10.10~150.60μg·mL^(-1)(r=0.9999),平均回收率分别为100.03%、102.52%,RSD值分别为1.74%、1.23%。每片中厚朴酚与和厚朴酚的总平均含量为3.8 mg。结论 按照优选的处方及制备工艺,该品成型性和黏附性较好,载药量适宜,质量稳定,满足临床及生产的需要。

苦杏仁抑菌肽的筛选富集及结构鉴定

为探究苦杏仁抑菌肽的制备、富集及结构分析,本实验以苦杏仁为原料,在脱脂后提蛋白,使用酶法制备苦杏仁蛋白抑菌肽,优化制备工艺,检测抑菌肽的抑菌活性,进一步纯化富集,分离出抑菌活性最强的抑菌肽组分并对其进行结构分析。结果表明,木瓜蛋白酶酶解物对于革兰氏阳性菌尤其是金黄色葡萄球菌的抑菌作用最强。优化苦杏仁抑菌肽的制备工艺最佳条件为:酶解温度为74℃,酶底比为2.5%,pH为7。得到抑菌肽的蛋白浓度为19.21%,并测得最低抑菌浓度为3.13 mg/mL。利用超滤和凝胶过滤色谱(Sephadex G-25)对抑菌肽进行分离纯化,从中筛选出肽组分A-II-b对金黄色葡萄球菌的抑制活性最强。将凝胶过滤条件优化为:样品浓度9.38 mg/mL,洗脱速度0.68 mL/min、洗脱剂为纯水。在此条件下测得肽组分A-II-b的峰面积比为13.15%。采用液相色谱-质谱联用技术对肽组分A-II-b进行结构分析,将结果导入Uniprot数据库与APD3数据库进行匹配,筛选出7种分子量在1500~2500 Da的潜在具有抗菌活性的苦杏仁肽序列,分别是ALPDEVLQNAFRIS、ESWNPRDPQFQWAGVA、VAYWSYNNGEQPLVA、FLDLSNDQNQLQLDQVPR、GENDNRNQIIRVR、RNLQGE NDNRNQIIRVR和RALPDEVLQNAFRIS。本研究促进了杏仁活性肽的深度开发和资源再利用,同时也为其作为抑菌肽的研究和进一步开发利用提供理论依据。