柱切换HPLC技术在生物样品测定中的应用

作为一种现代分析技术 ,高效液相色谱法的应用已越来越广泛。其中 ,高效液相色谱 -高效液相色谱联用技术 (柱切换 ,columnswitchingtechnique) ,自 2 0世纪 70年代问世以来 ,经过逾30年的发展 ,以其样品预处理简单 ,样品用量少 ,可对样品在线净化、富集等优点 ,已被越来越多的分析工作者所重视 ,并广泛应用于环境保护、农药残留监测、食品检验、生化分析、体内药物分析等众多领域。柱切换高效液相色谱技术 ,就是利用多通路切换阀 ,改变进样阀与色谱柱、色谱柱与色谱柱、色谱柱与检测器之间的连接关系 ,通过改变流动相的走向 ,或改变流动相系…     

乙醇—水反相高效液相色谱法在中药及中成药分析中的应用

目的： 建立用无毒的乙醇作为反相高效液相色谱流动相溶剂分析中药及中成药有效成分的方法。方法：用乙醇－ 水系统作反相高效液相色谱流动相测定中药及中成药的主要有效成分, 并与甲醇－ 水或乙腈－ 水系统的测定结果进行比较。结果：２ 种色谱系统均达到基线分离, 测得含量基本一致, 乙醇系统与甲醇系统或乙腈系统相比, 测定相对偏差＜ ±３％ （ｎ ＝ ３）。结论： 选择合适的柱温等色谱条件, 乙醇可用作反相高效液相色谱流动相分析中药及中成药中有效成分, 安全、准确。     

药物分析中C18色谱柱的差异与选择

在药物分析中高效液相色谱法用得非常普遍,而色谱柱的选择直接影响实验结果与药物的质量控制。不同C18色谱柱的保留行为千差万别,选择适宜的色谱柱对分析样品至关重要。以C18色谱柱的内部化学键合相为切入点,介绍C18色谱柱的差异,并综述在不同的pH环境、流动相极性、药物极性等条件下以及根据色谱柱的分类系统选择合适的键合相,为如何正确快速的选择C18色谱柱提供参考。     

反相高效液相色谱法分析中药复方制剂肾宝片中淫羊藿的含量

建立了一种反相高效液相色谱梯度洗脱,使淫羊藿苷能方便的从样品的复杂成分中分离,采用Symmetry C18ODS柱(250mm×4.6mm,5 μm),以乙腈-水-冰醋酸(25:75:1)为流动相,测定复方制剂肾宝片中淫羊藿苷的含量.本文建立的方法简便、灵敏、准确、可靠,可用于中药肾宝片的质量控制.     

反相高效液相色谱法测定洛伐他汀的血浆药物浓度

采用反相高效液相色谱法测定血浆中洛伐他汀浓度，色谱柱采用Ｎｏｖａ－Ｐａｋ Ｃ１８柱，柱温控制在５０℃，流动相为乙腈－磷酸铵缓冲液，检测波长为２３８ｎｍ，血浆样品应用ＰＳＥ固相小柱萃取。     

用乙醇作为流动相测定黄芩中有效成分-黄芩苷

目的建立用乙醇作为流动相溶剂分析黄芩中有效成分的反相高效液相色谱法。方法用乙醇作为流动相溶剂分析黄芩中有效成分,并与甲醇作为流动相测定的结果进行比较。结果两种色谱系统均达到基线分离,测得含量基本一致,乙醇系统与甲醇系统相比,测定相对偏差<±1%(n=4)。结论选择合适的柱温等色谱条件,乙醇可代替甲醇作为液相色谱流动相分析黄芩中有效成分,安全、准确。     

穿山龙指纹图谱及其抑制人肝癌HepG2细胞增殖活性相关性研究

目的建立穿山龙的高效液相色谱指纹图谱,研究其化学成分与抑制肿瘤细胞增殖活性的相关性,为该药的质量控制提供依据。方法色谱柱为Diamonsil C18柱(250 mm×4.6 mm,5μm),流动相为水和乙腈,采用梯度洗脱方式,柱温为35℃,检测波长为210 nm。对样品进行高效液相色谱指纹图谱分析和抑制肿瘤细胞的增殖活性试验,采用相关分析和多元线性回归分析方法将活性数据与各指纹峰数据相关联。结果在HPLC指纹图谱中,确立了20个共有峰,其中10个色谱峰与抗癌活性显著相关,这10个化学成分可能是穿山龙抗肿瘤的活性成分;结合多元线性回归分析结果与系统聚类分析结果,可将样品按照活性高低分类。结论穿山龙高效液相色谱指纹图谱结合其抗癌活性研究可为评价穿山龙药材的质量提供依据。     

反相高效液相色谱法测定中药菝葜中茋类成分的含量

目的：分析反相高效液相色谱法测定中药菝葜中茋类成分含量的方法。方法：色谱柱选用Zorbax SB C18柱,流动相为乙腈-磷酸水溶液系统梯度洗脱,流速为1.0ml/min,检测波长为320nm,进样量为10μl,柱温为30℃。结果：可对菝葜中虎杖苷、氧化白藜芦醇、云杉鞣酚、三棱素等成分准确测定。结论：反相高效液相色谱法操作简单、结果准确、专属性强,值得推广。     

双黄连片高效液相色谱指纹图谱的建立及聚类分析和主成分分析

目的建立双黄连片的高效液相色谱指纹图谱,并进行聚类分析和主成分分析。方法色谱柱为Agilent Zorbax-C18柱(250 mm×4.6 mm,5μm),流动相为乙腈-0.07%甲酸溶液(梯度洗脱),流速为0.9 mL/min,检测波长为320 nm(绿原酸和木犀草苷)、220 nm(连翘酯苷A和连翘苷)、275 nm(黄芩苷、汉黄芩苷及黄芩素),柱温为30℃,进样量为10μL。以连翘酯苷A峰为参照,绘制12批双黄连片的高效液相色谱指纹图谱,采用2012年版《中药色谱指纹图谱相似度评价系统》进行相似度评价,确定共有峰,并采用SPSS 22.0统计学软件进行聚类分析和主成分分析。结果建立了12批双黄连片的高效液相指纹色谱图谱,共确定18个共有峰,方法学考察结果符合指纹图谱的技术要求,相同厂家的药品聚为一类;经主成分分析,2个主成分因子的累计方差贡献率为92.242%,样品中绿原酸、木犀草苷、连翘酯苷A、连翘苷、黄芩苷、黄芩素、汉黄芩苷、峰9、峰11、峰15对应成分的含量变化均是导致样品质量差异的重要原因。结论该方法中建立的高效液相色谱指纹图谱可为双黄连片的质量评价提供参考。     

橙皮苷的高效液相色谱定量分析

对含有橙皮苷中药材及其制剂的高效液相色谱的色谱柱、流动相及样品制备方法进行了综述。     

Recent advances in column switching high-performance liquid chromatography for bioanalysis

Bioanalysis is a relevant area of analytical chemistry for clinical studies. Biological samples are complex and diverse, so sample preparation represents a challenge when chromatographic methods are developed. According to the principles of green analytical chemistry (GAC), recent trends in sample preparation include miniaturization, automation (online coupling to the analytical instrument), and high-throughput performance. In this context, column switching liquid chromatography stands out as a multidimensional chromatographic method in which an extraction column is directly coupled to high-performance liquid chromatography (HPLC) systems. This online method consists of two steps and involves two columns, the extraction and the chromatographic columns. In the former column, the analytes are isolated from the sample and preconcentrated; in the latter column, the analytes are separated. Online systems improve the sensitivity and accuracy of analytical methods, consume lower amounts of organic solvents, and minimize sample handling. This review summarizes state-of-the-art column switching liquid chromatography and focuses on selective stationary phases for preconcentration of analytes (first dimension), including reversed phases, monolithic phases, restricted access materials (RAMs), and molecularly imprinted polymers (MIP). Principles, instrumental aspects, applications in bioanalysis, and future trends in column switching liquid chromatography are also discussed.     

Narrow-bore high performance liquid chromatographic method for the determination of cetirizine in human plasma using column switching

An improved column switching high performance liquid chromatographic (HPLC) method was developed for determination of cetirizine in human plasma. Plasma samples were prepared by liquid-liquid extraction using methylene chloride. The samples extracted were initially injected into a clean-up Capcell Pak MF C8 column and the peaks of cetirizine and internal standard were separated to an analytical C18 micro-column via column switching device. This analysis showed highly sensitive and selective results. Also, it was successfully applied to evaluate the pharmacokinetics of cetirizine in human volunteers after single oral administration.     

Direct injection analysis of melphalan in plasma using column-switching high-performance liquid chromatography.

An automated column-switching high-performance liquid chromatographic (HPLC) method with ultraviolet detection is described for a simple and rapid determination of melphalan, an alkylating agent, in human plasma following direct sample injection. The system consists of a pretreatment column and an analytical column connected in series via a switching valve. Plasma samples are loaded onto a pretreatment column with an aqueous mobile phase, with which the sample to be analyzed is retained and the solubilized plasma proteins are flushed to be discarded. The retained compound is eluted from the pretreatment column onto the analytical column by using the chromatographic mobile phase with a higher elution capacity. The column-switching technique can be used to achieve an automated assay. Analytical recovery of the compound was 99.1%, and the coefficients of both intra- and interday variations did not exceed 3.5%. The detection limit was 10 ng/ml for the compound. Recovery of melphalan in plasma was only 2.1% after 4 weeks at 25 degrees C, as compared to 24.5% at 5 degrees C and 100.1% at -20 degrees C.     

Direct simultaneous determination of drug discovery compounds in monkey plasma using mixed-function column liquid chromatography/tandem mass spectrometry.

A direct injection method based on a single column and high-performance liquid chromatography (HPLC) with tandem mass spectrometry (MS/MS) was developed for the simultaneous determination of two drug candidates in monkey plasma samples in support of pharmacodynamic studies. Each diluted monkey plasma sample containing internal standard was directly injected into a mixed-function column for sample cleanup, enrichment and chromatographic separation. The proteins and macromolecules first passed through the column while the drug molecules were retained on the bonded hydrophobic phase. The analytes retained on the column with an aqueous liquid mobile phase were then chemically eluted by switching to a strong organic mobile phase at a constant flow rate of 1.0 ml/min. The column effluent was also diverted from waste to mass spectrometer for analyte detection. Samples from two different analyte studies were assayed in one analytical procedure and the calibration curves were prepared using both analytes. The calibration curves were linear over the range of 5-2500 ng/ml for both analytes. The retention times for analytes and the internal standard were found to be consistent and no column deterioration was observed after 200 injections. The apparent on-column recoveries for the test compounds in monkey plasma samples were greater than 90% with 6% CV (N=5). The total analysis time was less than 5 min per sample.     

Determination of famotidine in human plasma by high performance liquid chromatography with column switching

A rapid, sensitive and robust reverse-phase high performance liquid chromatographic (HPLC) method with column switching and an internal standard for the quantitative determination of famotidine in human plasma is described. Famotidine and the internal standard were isolated from plasma samples by cation exchange solid phase extraction with SCX cartridges. The chromatographic separation was accomplished by an Inertsil C4 column with a mobile phase of acetonitrile/phosphate aqueous solution, connected by a switching valve to a BDS Hypersil C8 column with a mobile phase of acetonitrile/sodium dodecyl sulfate and phosphate aqueous solution. UV detection was set at 267 nm. The standard curve was linear in the concentration range of 1–100 ng ml⁻¹. The intraday coefficients of variation at all concentration levels were less than 10%. The interday consistency was assessed by running QC samples during each daily run. The limit of quantification for famotidine in human plasma was 1 ng ml⁻¹. The method has been utilized to support clinical pharmacokinetic studies in healthy volunteers who received famotidine 10 mg orally.     

Quantification of huperzine A in Huperzia serrata by HPLC-UV and identification of the major constituents in its alkaloid extracts by HPLC-DAD-MS-MS

A rapid, simple and reliable high performance liquid chromatography (HPLC) method has been established for the analysis of the major alkaloids in Huperzia serrata, a traditional Chinese medicine (TCM) herb. After chromatographic separation on a reversed-phase C18 column with methanol-ammonium acetate (pH 6.0; 80 mM, 30/70, v/v) as the mobile phase, nine alkaloid compounds in the alkaloid extracts of H. serrata were identified by online diode array detection-MS and by comparing with data from literature and standard samples. One compound in the extract, huperzine A, is a drug for treating Alzheimer's disease. Its content was quantified by HPLC coupled with UV-vis. The method was the validated. The recovery rates were 96.8-97.7% with R.S.D <2.44%. The intra- and inter-day precisions, expressed as R.S.D., ranged from 0.53% to 1.51%. Good linear regression was observed in the concentration range of 5-100 microg/ml (r = 0.9997). The results demonstrate that this method is simple, selective, and suitable for the quality control of this TCM herb.     

生血宝合剂中二苯乙烯苷的含量测定

目的 建立测定生血宝合剂中君药制何首乌的有效成分二苯乙烯苷（2,3,5,4’-四羟基二苯乙烯-2-O-β-D-葡萄糖苷）含量的高效液相色谱（HPLC）法,为生血宝合剂的质量控制提供依据。方法 Waters alliance高效液相色谱仪,色谱柱为Suntek Kromasil C18柱（250 mm×4.6 mm,5μm）,流动相为乙腈-水（20∶80）,流速为1.0 mL/min,检测波长320 nm。结果 二苯乙烯苷在0.054 60~1.092 00μg范围内与峰面积积分值线性关系良好（R2=0.999 9）。结论 该方法灵敏度高、定量准确、方法简便、重现性好,能有效控制生血宝合剂的质量。     

Evaluation of high-performance liquid chromatography and gas chromatography for quantitation of dextromethorphan hydrobromide in cough-cold syrup preparations

A rapid and sensitive high-performance liquid chromatographic (HPLC) procedure is described for the analysis of the antitussive dextromethorphan hydrobromide in several cough-cold syrup preparations and compared with a gas chromatographic (GC) procedure. In the HPLC procedure, the active ingredient is analyzed as the hydrobromide salt by dilution in the mobile phase and separation on a reverse-phase cyano column. In the GC method, the active ingredient is analyzed as the free base, in which an aqueous solution of the antitussive is made alkaline and extracted with dichloromethane before injection onto the GC column. Excellent resolution of the antitussive agent was obtained by both systems; however, the HPLC assay is preferred for routine analysis (RSD 1%), as compared with the GC assay (RSD 4%).     

芩连片HPLC指纹图谱研究

目的研究芩连片高效液相色谱指纹图谱,建立整体特征的质量评价方法。方法采用DAD-ELSD串联检测技术,Zorbax色谱柱,以乙腈-1.5%冰醋酸为流动相,梯度洗脱,检测波长为280nm。结果本色谱条件下芩连片各成分得到了较好的分离,标准指纹图谱均由10个特征峰组成。结论所建立的HPLC指纹图谱表达了芩连片可测组分的整体特征。具有重复性好、特征性强、方法简便等特点,可用于芩连片的质量评价。     

余苣降酸茶的HPLC指纹图谱研究

目的:建立余苣降酸茶HPLC指纹图谱,以控制该中药复方茶剂的质量。方法:采用HPLC梯度洗脱法测定余苣降酸茶的指纹图谱,色谱柱为Waters Sun Fire C18色谱柱(250 mm×4.6 mm,5μm);流动相为0.2%磷酸水溶液-乙腈,柱温:30℃,检测波长:308 nm,进样量:10μl,流速:1.0 ml·min^-1。结果:测得10批余苣降酸茶供试品HPLC色谱图,并标示出20个共有峰,确定其中2个已知峰分别为没食子酸和葛根素;并明确了20个共有峰的原药材归属。结论:该方法建立的余苣降酸茶HPLC指纹图谱重复性好,色谱峰信息量大,为全面控制该制剂的质量提供了可靠的方法。