Metabolite analysis by supercritical fluid chromatography

Owing to its favorable properties, such as low viscosity and high diffusivity, a supercritical fluid can be used as the mobile phase in chromatography. Supercritical fluid chromatography (SFC) can provide high-speed and high-resolution separation. Since supercritical carbon dioxide (SCCO(2)), which is generally used as the mobile phase in SFC, is automatically emitted at room temperature, SFC is most commonly used as a preparative method. However, SFC can also be used to perform high-precision biomolecular analysis, especially for hydrophobic metabolites, because of the low polarity of SCCO(2). The use of a mass spectrometer with SFC can widen the scope of application of SFC to bioanalysis. In this review, we summarize practical application of SFC as a tool for the analysis of metabolites in real biological samples.     

Packed column supercritical fluid chromatography of sodium stearyl fumarate aqueous suspension

A method for the determination of sodium stearyl fumarate aqueous suspension is described. This straightforward method is based on homogenisation of the sample, dilution of a known aliquot with methanol to a suitable clear solution and mixing with an internal standard; (S)-naproxen. Separation and quantification is performed by packed column supercritical fluid chromatography on a commercial tartaric acid network polymeric column (tertbutylbenzoyl) with UV-detection at 214 nm. The precision of the presented method upon repeated analysis of a 20 mg/ml suspension is 0.5% (n = 8), and the yield is near 100%. Less than 5 min is required for the chromatographic separation with a resolution of about 3 to the internal standard. With some modification of the chromatographic conditions water samples can also be analysed.     

超临界流体萃取法毛细管气相色谱法分析牡丹皮及制剂中丹皮酚的含量

采用超临界流体萃取法(supercritical fluid extraction,SFE)提取中药牡丹皮及其成方制剂中丹皮酚,以氯仿作改性剂,在温度90℃,压力28MPa下,二氧化碳动态萃取体积3ml;静态萃取时间5min。此法简便快速,萃取完全。用大孔径毛细管柱气相色谱法作含量监测,结果:相关性好(γ=0.9999),中药与制剂的回收率分别为97.8%,RSD=2.35%(n=3);100.3%,RSD=1.89%(n=3)。为中药有效成分的提取和质量控制提供了一种有效可靠的方法。     

超临界流体色谱拆分齐留通对映体

目的研究超临界流体色谱对齐留通对映体的分离效果。方法考察确定5种手性柱中分离效果最佳的柱子,在该柱上考察改性剂的种类、浓度、温度及背压对分离度的影响。结果采用Chiralpak AD-H柱、改性剂为异丙醇,异丙醇在流动相中比例为18%、背压为140 bar、温度为303 K时,齐留通对映体达到最佳分离,分离度为11.5,出峰时间分别为4.59、7.35min。结论所用超临界流体色谱的方法简单、快捷、经济有效,适用于齐留通对映体的分离。     

超临界CO2萃取猪肺表面活性物质的工艺研究

目的：用超临界CO2流体萃取技术提纯猪肺表面活性物质，并对萃取工艺条件进行优化。方法：通过单因素对比实验以及正交试验设计对其萃取过程中的主要影响因素进行了考察，以优化萃取工艺。结果：确定最佳萃取条件为：压力30MPa，温度50℃，动态萃取时间5h，0．3mL无水乙醇为夹带剂。结论：该提纯方法简便、高效、有机溶剂污染少。     

Analysis of mono-, di- and triglycerides in pharmaceutical excipients by capillary supercritical fluid chromatography.

Mono-, di- and triglycerides are important components of oils, fats and other natural products. Since in general fatty acids are mixtures and glycerol can be differently substituted, finger-prints of the composition are suitable for better characterization. Since capillary supercritical fluid chromatography (SFC) employing carbon dioxide as mobile phase is compatible with flame ionization detection, it is possible to analyse many solutes at trace levels. Supercritical carbon dioxide offers higher solute diffusivity compared with the inert carrier gas conventionally used in gas chromatography and has a lower viscosity than the liquid solvents used in HPLC. Thus, glycerides of fatty acids can be separated and eluted at a lower temperature and with shorter analysis time in SFC. In this study the analysis of mono-, di- and triglyceride mixtures in several pharmaceutical excipients is reported using capillary SFC. Quantitative analysis is possible on the basis of a response factor established for each analyte. The accuracy of the method and its advantages are demonstrated.     

Separation of naproxen enantiomers by supercritical/subcritical fluid chromatography

An isocratic supercritical/subcritical fluid chromatography method for the separation of naproxen enantiomers on the Kromasil CHI-TBB column was investigated. The mobile phase was composed of supercritical CO2 with 2-propanol as modifier. The experimental conditions were temperature 293 K-323 K, pressure 9.4 MPa-21.3 MPa, and 2-propanol concentration 6%-15% (by mass), respectively. The enthalpic contribution to the overall enantiomer transfer energy was more important than the entropic contribution in the temperature range examined. The preferred operation conditions were 293 K, 9.4 MPa, and the concentration of 2-propanol in the mobile phase 11% (by mass).     

超临界流体色谱在药物分析中的研究进展

超临界流体色谱是以超临界流体为流动相,依靠流动相的溶剂化能力来进行分离分析的一种色谱,具有分离速度快、效能高、绿色环保等优点,是气相色谱和液相色谱的有力补充,具有广阔的应用前景。本文利用中国知网（CNKI）、ScienceDirect、PubMed等数据库检索了近几年来超临界流体色谱在药物分析中的研究文献,归纳了其在手性药物、天然产物、药物代谢和杂质检测中的应用进展,并对今后的进一步研究提出思考。     

A feasibility study on direct assay of an aqueous formulation by chiral supercritical fluid chromatography (SFC)

Supercritical fluid chromatography (SFC) has gained considerable importance in the area of Separation Science in pharmaceutical analysis over the past few years. The synthesis of chiral compounds is of particular significance in the pursuit of new drug entities. SFC is rapidly replacing high performance liquid chromatography (HPLC) in many pharmaceutical and biotechnological companies as the standard screening and method development tool for chiral compounds. Analysis of pharmaceutical formulations of research compounds is an area where SFC is recently being explored as a possible alternate or complementary technique to HPLC in limited scope. A feasibility study was carried out to perform direct assay of a chiral drug compound AZM in 100% aqueous formulations by SFC. The results indicated that this approach has the potential to significantly reduce the typical sample processing time prior to analysis. The method was reproducible, linear over a wide dynamic range, and sensitive enough to detect the minor enantiomeric impurity in the chiral drug compound investigated here. Further application will be pursued for other research compounds in the future to illustrate the broader applicability of this approach.     

超临界萃取法提取川芎中阿魏酸的研究及其HPLC测定

目的考察影响超临界萃取川芎中有效成分的各因素并确定最佳提取条件。方法采用超临界二氧化碳萃取法,以提取液中阿魏酸含量为考察指标,对影响阿魏酸提取工艺的因素进行了研究。结果优选出川芎中阿魏酸的最佳超临界萃取条件。结论提取阿魏酸的最佳SFE条件是:萃取罐的温度70℃、萃取压力35MPa、萃取时间2h。以甲醇-水-冰醋酸=30∶70∶0.2为流动相,测定波长为320nm。     

超临界流体萃取法净化熟地中有机氯农药的研究

目的去除熟地中残留的有机氯农药.方法采用超临界CO2流体萃取法去除熟地中残留的有机氯农药,用毛细管气相色谱法测定除毒前后熟地中农药残留量,评价除毒率.用高效液相色谱法测定除毒前后熟地中梓醇及相关组分的变化,评价除毒后熟地药材的药用价值.结果正交设计L9(34)选出最佳萃取条件为压力15 MPa,温度60℃,萃取时间30 min,流速35 kg·h-1.残留农药去除率达84.6%(RSD为3.7%)熟地中梓醇的相对含量101.1%(RSD为2.7%),相关组分含量没有显著性变化.结论采用超临界CO2流体萃取法去除熟地中残留的有机氯农药,方法可行.     

Purification of pharmaceutical excipients with supercritical fluid extraction

Supercritical fluid extraction (SFE), with carbon dioxide as the solvent, was tested for its ability to remove common reactive impurities from several pharmaceutical excipient powders including starch, microcrystalline cellulose (MCC), hydroxypropylcellulose (HPC), polyethylene oxide (PEO), and polyvinylpyrrolidone (PVP). Extraction of the small molecule impurities, formic acid and formaldehyde, was conducted using SFE methods under conditions that did not result in visible physical changes to polymeric excipient powders. It could be shown that spiked, largely surface-bound, impurities could be removed effectively; however, SFE could only remove embedded impurities in the excipient particles after significant exposure times due to slow diffusion of the impurities to the particle surfaces. Attempts at hydrogen peroxide extraction were hindered by its low solubility in CO2, thereby effectively precluding SFE for removal of hydrogen peroxide from excipients. This work suggests that SFE will only be commercially useful for removal of low molecular weight impurities in polymeric excipients when migration of the impurities to the particle surfaces is sufficiently rapid for extraction to be completed in a reasonable time frame.     

Drug encapsulation using supercritical fluid extraction of emulsions

The current work was aimed at evaluating a new method, supercritical fluid extraction of emulsions (SFEE), for the production of composite (e.g., polymer-drug) micro- and nanoparticles, intended for application in sustained-release drug delivery formulations. Using the proposed method, composite particles were obtained, both in a continuous or batch manner by supercritical carbon dioxide extraction of oil-in-water (o/w) emulsions. Model drugs indomethacin and ketoprofen and biodegradable polymers poly(lactic/glycolic) acid and Eudragit RS were used in order to demonstrate the effectiveness of the SFEE process for producing these particles. Stable aqueous suspensions of composite micro and nanoparticles, having sizes ranging between 0.1 and 2 microm were consistently obtained. Emulsion droplet diameter was found to be the major size control parameter. Other parameters investigated included polymer and drug concentrations in solvent and emulsion solvent fraction. The residual solvent content in the particle suspension obtained was consistently below 50 ppm. Standard dissolution tests were used to observe the sustained release phenomenon of the composite particles. The dissolution profile was characterized in terms of the intrinsic dissolution kinetic coefficients taking into account the specific surface area and solubility of the particles. It was observed that the kinetic coefficient parameter for encapsulated drugs was reduced by 2-4 orders of magnitude when compared to the unprocessed drug particles.     

pSFC色谱法分离夏天无中的原阿片碱和延胡索乙素

目的 建立一种采用色谱柱串联的填充柱超临界流体色谱法(pSFC)分离夏天无中原阿片碱和延胡索乙素,为生物碱类化合物的分离、制备提供新的方法.方法 考察单用2-乙基吡啶柱(Pyr)时,改性剂比例、压力及温度对分离的影响.再将Pyr柱分别与二醇柱(Diol)、氰基柱(Cyano)串联.结果 采用Pyr柱与Cyano柱串联,夏天无中原阿片碱和延胡索乙素能较好地分离.结论 色谱柱串联运用于pSFC分离中药成分特别是生物碱,具有良好的应用前景.     

Validation of direct assay of an aqueous formulation of a drug compound AZY by chiral supercritical fluid chromatography (SFC)

Supercritical fluid chromatography (SFC) is increasingly being recognized as a powerful technique for analysis of pharmaceutical compounds in various dosage forms. Assay of aqueous formulations of research compounds by SFC is, however, a relatively unexplored area primarily due to the potential problems associated with it. This work describes the development of a direct assay of a chiral drug compound AZY in a 100% aqueous formulation by SFC, and its qualification following ICH and FDA validation guidelines on chromatographic methods. The results indicated that SFC has the potential for assaying aqueous formulations of research compounds with high degree of selectivity, accuracy, precision, robustness, sensitivity, and linearity over a wide range of concentrations. This work also confirmed a previous hypothesis that direct formulation assay by SFC approach is applicable to both acidic and basic pharmaceutical compounds with equal degree of success.     

超临界萃取技术在中药研究中的应用

超临界萃取(Supercritical Fluid Extraction,简称SFE)技术因其工艺特点和超临界CO2流体的特性,备受研究工作者的重视。近年,在SFE实施中草药现代化的进程中,SFE-CO2萃取又列为高效提取分离的新技术。笔者参考了近5年的文献,对超临界萃取技术在中药研究方面的应用进行了综述。1超临界萃取技术在中药研究中的应用1.1萃取挥发油等挥发性成分SFE-CO2萃取挥发油等挥发性成分即就是对脂溶性、低沸点、热敏性成分的萃取。近几年,有关这方面报道较多。刘红梅〔1〕等采用超临界萃取技术对白芷中香豆素类成分研究发现,在最佳萃取条件下,白芷…     

Direct injection of aqueous samples in packed column supercritical fluid chromatography of isosorbide-5-mononitrate from drug release testing

A method for the analysis of aqueous samples of isosorbide-5-mononitrate (5-ISMN) is presented. It is based on packed column supercritical fluid chromatography (SFC) using 20% of 2-propanol in carbon dioxide as the mobile phase and a diol silica column as the stationary phase. Using the described conditions it is possible to quantitate 5-ISMN released from Imdur tablets in gastric media. The precision upon repeated injections was 2% (RSD) at the 20 microg/ml level (n=8), using peak height measurements, when the solution was circulated through the sample loop of the injector. Samples from drug release testing that had been analyzed with reversed phase LC were analyzed with the present method and the results agreed well. It is also possible to monitor the drug released in a dissolution-testing vessel through direct on-line continuous loading (recirculation) of the sample loop of the SFC instrument.     

超临界CO2流体萃取丹参素的工艺研究

目的研究萃取丹参素的最佳工艺条件。方法通过正交设计,用超临界CO2流体萃取,优化出合理工艺条件,并与传统溶剂提取工艺相对照。结果超临界CO2流体萃取率为传统工艺萃取率的1．1倍。结论超临界CO2流体萃取丹参素具有开发价值。