纤维素衍生物手性固定相高效液相色谱法分离盐酸川丁特罗对映体

以纤维素三-(3,5-二甲基苯基氨基甲酸酯)为手性固定相(Lux Cellulose-1),建立了在正相色谱条件下直接分离盐酸川丁特罗对映体的高效液相色谱法。考察了乙醇、异丙醇等有机改性剂,三氟乙酸、二乙胺等流动相添加剂和柱温对对映体分离的影响。结果显示,酸性和碱性添加剂对对映体分离的影响最为显著: 添加二乙胺时两对映体无分离趋势;添加三氟乙酸时对映体保留强,且分离趋势明显;而同时添加三氟乙酸和二乙胺则两对映体分离显著改善,分离度可达4.0。优化后的色谱条件: 色谱柱为Lux Cellulose-1手性柱(250 mm×4.6 mm, 5 μm),流动相为正庚烷-乙醇-三氟乙酸-二乙胺(88:12:0.3:0.05, v/v/v/v),流速为1.0 mL/min,紫外检测波长为246 nm,柱温为25 ℃。该方法简便,快速,可用于左旋盐酸川丁特罗原料中右旋异构体杂质的检查。     

Novel imidazolium stationary phase for high-performance liquid chromatography

A new imidazolium anion-exchange phase immobilized on silica is synthesized. HPLC separations of common inorganic anions (IO3-, Cl-, NO2-, Br-, NO3-, I-, SCN-) have been performed using a HPLC column (200 mm x 4.6 mm I.D.) packed with this stationary phase, with a phosphate buffer solution as the mobile phase and UV detection at 200 nm. The effects of pH and concentration of eluent on the separation of anions have been studied. Chromatographic parameters are calculated and the results show that the new stationary phase is of significant potential for the analysis of these anions. Successful separations of some ordinary organic anions have also been achieved with the said stationary phase. Meaningfully, organic and inorganic anions can be determined simultaneously and satisfactorily with several neutral compounds using the column. The separation of some organic compounds including hydroxybenzenes, bases and amines by this stationary phase with only water as the eluent has been investigated.     

A novel stability-indicating HPLC method for the determination of enantiomeric purity of eluxadoline drug: Amylose tris(3,5-dichlorophenyl carbamate) stationary phase

A simple and sensitive stability-indicating chiral HPLC method has been developed and validated per International Conference on Harmonization guidelines for the determination of enantiomeric purity of eluxadoline (Exdl). The impact of different mobile phase compositions and chiral stationary phases on the separation of Exdl enantiomer along with process- and degradation-related impurities has been studied. Homogeneity of Exdl and stable results of Exdl enantiomer in all degraded samples reveal the fact that the proposed method was specific (stability indicating). Amylose tris(3,5-dichlorophenyl carbamate) stationary phase column Chiralpak IE-3 (150 × 4.6 mm, 3 μm) provided better resolution with polar organic solvents than cellulose derivative, crown ether, and zwitterion stationary phases and nonpolar solvents. The mobile phase consisted of acetonitrile, tetrahydrofuran, methanol, butylamine, and acetic acid in the ratio of 500:500:20:2:1.5 (v/v/v/v/v). Isocratic elution was performed at a flow rate of 1.0 mL/min, column temperature of 35°C, injection volume of 10 μL, and UV detection of 240 nm. The United States Pharmacopeia (USP) resolution of the Exdl enantiomer was found to be more than 4.0 within a 65-min run time. Exdl enantiomer detector response linearity over the concentration range of 0.859–4.524 μg/mL was found to be R² = 0.9985. The limit of detection, limit of quantification, and average percentage recovery values were established as 0.283 μg/mL, 0.859 μg/mL, and 96.0, respectively.     

On-chip electrochromatography using sol-gel immobilized stationary phase with UV absorbance detection

A chromatography column on a chip was fabricated by immobilizing reversed-phase stationary phase particles (5 microm, C4) using sol-gel technology. Channels were fabricated in quartz using photolithography and wet etching. Localization of the stationary phase was achieved by immobilizing the stationary phase at the desired location in the separation channel prior to bonding of the cover plate. Cross channel design was employed for gated injection. An optical fiber setup was developed for carrying out on-chip UV absorbance detection. The effective optical path length was theoretically determined for the trapezoidal shaped channel and the result was shown to match closely with the experimentally determined value. The effect of applied voltage on velocity was evaluated using thiourea as an unretained marker. Separation performance of the stationary phase was demonstrated by separation of three peptides (Trp-Ala, Leu-Trp and Trp-Trp) under isocratic chromatographic conditions.     

高效液相色谱手性固定相法拆分阿折地平对映体

建立了阿折地平对映体的高效液相色谱拆分方法。采用Chiralpak AD-H (250 mm×4.6 mm, 5.0 μm, Daicel公司)手性色谱柱在正相条件下直接拆分阿折地平对映体,考察了固定相种类、流动相组成及柱温等对阿折地平对映体分离的影响。确定了最佳的拆分条件: 流动相为正己烷-异丙醇(90:10, v/v),流速为0.8 mL/min,检测波长为254 nm;柱温为20 ℃;在此条件下阿折地平对映体的分离度为3.3。该法简单快速,重现性好。     

苯并芘DNA加合物anti-BPDE-N^2-dG四种立体异构体的合成及色谱分离

体外合成了苯并芘DNA加合物-邻二醇环氧苯并芘-脱氧鸟苷加合物(anti-BPDE-N2-dG)四种立体异构体(两对手性异构体)。通过优化体外反应条件,anti-BPDE-N2-dG四种异构体的合成产量较现有合成方法提高了2倍多,为定量检测生物体中anti-BPDE-N2-dG提供了标准品。并首次将五氟苯基色谱柱应用于该立体异构体的色谱分离提纯,通过优化色谱条件,采用常规的五氟苯基色谱柱(250 mm×4.6 mm,5μm),以乙腈-0.1%甲酸水(22.5∶77.5)为流动相,流速1.2 mL/min条件下,45 min内即可分离提纯四种立体异构体。该方法与常规C18柱(250 mm×4.6 mm,5μm)需要160 min,苯基柱(250 mm×4.6 mm,5μm)需要85~100 min才能将四种立体异构体实现色谱分离相比,缩短了分离时间,提高了提纯效率。通过紫外吸收光谱、质谱、圆二色谱对四种立体异构体进行表征,确定出峰顺序为trans(-)、trans(+)、cis(+)、cis(-)-anti-BPDE-N2-dG。此外,利用高效液相色谱-串联质谱(HPLC-MS/MS)检测anti-BPDE-N2-dG四种立体异构体标准品时,使用常规的五氟苯基色谱柱可在30 min内完成分离检测,与相同规格的苯基柱需要60 min相比提高了检测效率。     

Use of a polar-embedded stationary phase for the separation of tocopherols by CEC

A polar-embedded stationary phase (ULTIMA C18) has been investigated for the separation of alpha-, beta-, gamma- and delta-tocopherols by CEC in comparison with commercially available C(18) and C(30) n-alkyl RPs. The behavior of this stationary phase was tested for different mobile phases based on methanol, ACN, or mixtures thereof and different separation parameters such as retention factors and resolution were evaluated. The main feature of this stationary phase is the improved selectivity for the separation of beta- and gamma-tocopherols (positional isomers) when compared with the pure n-alkyl C(18) material, which was unable to resolve these compounds. Additionally, it is possible to observe a reversal in the elution order of the beta- and gamma-tocopherol isomers with respect to that obtained on the C(30) column. The resulting data indicate that the enhanced selectivity obtained with the polar-embedded stationary phase, with respect to the conventional C(18) material, is due to the participation of both hydrophobic and polar interactions: these latter are of the hydrogen bridge type with the amide group of the polar-embedded stationary phase, which increases the retention of the tocopherols and facilitates the discrimination between the beta- and gamma-isomers. Adequate separation of the four tocopherols was obtained by CEC using the polar-embedded stationary phase and 95:5 v/v methanol/water (5 mM Tris, final concentration) as the mobile phase.     

Reversed-phase porous silica rods, an alternative approach to high-performance liquid chromatographic separation using the sequential injection chromatography technique

A commercially available porous silica rod column was used as a separation tool for the sequential injection analysis (SIA). A porous solid monolithic column showed high performance at a low pressure, allowing sequential injection analysis to be used for the first time for separation in HPLC fashion. In this contribution, we tried to demonstrate a new separation concept with SIA manifold for the simultaneous determination of four different compounds (methylparaben (MP), propylparaben (PP), triamcinolone acetonide (TCA) and internal standard ketoprofen (KP)) in a pharmaceutical triamcinolon cream 0.1% formulation. A Chromolith Flash RP-18e, 25 mm x 4.6 mm column with a 10 mm pre-column (Merck, Germany) and a FIAlab 3000 system (USA) with an 8-port selection valve and 10 ml syringe were used for sequential injection chromatographic separations in our study. The mobile phase used was acetonitrile-methanol-water (35:5:65, v/v/v) + 0.05% nonylamine, pH 2.5, flow rate 0.6 ml min(-1). The analysis time was <6 min. A novel sequential injection chromatography (SIC) technique with UV spectrophotometric detection was optimised and validated.     

衍生化β-环糊精手性固定相高效液相色谱法拆分米那普仑对映体及其分离机制

建立了新型抗抑郁药米那普仑在环糊精手性固定相上的高效液相色谱拆分方法。在反相色谱条件下采用未衍生化β-环糊精(Cyclobond I 2000)、乙酰基-β-环糊精(AC-β-CD)、2,3-二甲基-β-环糊精(DM-β-CD)、3,5-二甲基苯基氨基甲酸酯-β-环糊精(DMP-β-CD)4种手性柱分离米那普仑对映体。考察了固定相、流动相比例、pH、流速和柱温对拆分的影响。利用分子对接和结合能计算方法,研究米那普仑分子与AC-β-CD的对接过程,探讨其可能的分离机制。优化后的拆分条件如下:固定相为乙酰基-β-环糊精手性柱Astec CYCLOBONDTMI 2000 AC(25 cm×4.6 mm,5μm),流动相为乙腈-0.1%(体积分数)pH 5.0醋酸三乙胺溶液(TEAA)(5∶95,v/v),流速为0.4mL/min,柱温为25℃,检测波长为220 nm。在此条件下,米那普仑对映体获得快速拆分,分离度(Rs)为1.74,理论塔板数为10 125。分子模拟结果表明引起手性识别的作用力主要是环糊精衍生化的乙酰基导致的氢键作用差异。该方法快速、高效、重现性好。     

Direct separation of the enantiomers of ramosetron on a chlorinated cellulose‐based chiral stationary phase in hydrophilic interaction liquid chromatography mode

Ramosetron is an enantiopure active pharmaceutical ingredient marketed in Japan since 1996 and later in a few Southeast Asian countries predominantly as an antiemetic for patients receiving chemotherapy. In this study, a simple and rapid high‐performance liquid chromoatography method for the separation of ramosetron and its related enantiomeric impurity by using hydrophilic interaction liquid chromatography mode is presented. Chiral resolution was performed on an analytical column (100 mm × 4.6 mm id) packed with 3 μm particles of cellulose‐based Chiralpak IC‐3 chiral stationary phase. Using a mobile phase containing acetonitrile–water–diethylamine (100:10:0.1, v/v/v) and setting the column temperature at 35°C, the resolution value was 7.35. At a flow rate of 1 mL/min, the enantioseparation was completed within 5 min. The proposed method was partially validated and it has proven to be sensitive with limit of detection and limit of quantitation of the (S)‐enantiomer impurity of 44.5 and 133.6 ng/mL.     

Separation and identification of chiralN-acylcalix[4]arene amino acid derivatives by use of reversed-phase HPLC

Summary This work focuses on the separation and identification of p-tert butyl calix[4]arene derivatives. An isocratic mixture of 65% (v/v) acetonitrile and 35% (v/v) water with 0.1% (v/v) phosphoric acid was used as the mobile phase. A Bio-Rad Bio-Sil ODS-5S (250 mm×4 mm) column was used as the stationary phase with UV detection of the analytes at 274 nm. The reversed-phase high performance liquid chromatographic method which was developed provided baseline separation of five p-tertbutyl calix[4]arene derivatives in twenty minutes.     

Quantitation of glucosamine from shrimp waste using HPLC

This work presents a high-performance liquid chromatography (HPLC) method for the quantitation of glucosamine in chitin. The method includes an acid hydrolysis of chitin. The chromatographic separation is achieved using a Hypersil ODS 5-microm column (250 x 4.6 mm) at 38 degrees C, with precolumn derivatization with 9-fluorenylmethyl-chloroformate and UV detection (lambda = 264 nm). The mobile phase is a mixture of mobile phase A [30 mM ammonium phosphate (pH 6.5) in 15:85 methanol-water (v/v)], mobile phase B [15:85 methanol-water (v/v)], and mobile phase C [90:10 acetonitrile-water (v/v)], with a flow rate of 1.2 mL/min. The HPLC method proposed showed adequate repeatability (relative standard deviation, 5.8%), accuracy (92.7% recovery), and sensitivity, with a detection limit of 2 microg/mL. The method is successfully applied to the quantitation of glucosamine for the determination of the purity of chitin from shrimp waste.     

漆酚酯键合硅胶液相色谱固定相的制备与应用

以甲基丙烯酸漆酚酯为色谱配体,制备了一种新型色谱固定相。首先以漆酚和甲基丙烯酰氯为原料制备得到甲基丙烯酸漆酚酯,并通过物理吸附涂覆到由3-（甲基丙烯酰氧）丙基三甲氧基硅烷化学修饰的硅胶上,再通过自由基引发与硅烷化硅胶的双键聚合制得漆酚酯键合硅胶固定相（USP）。对固定相进行傅里叶红外光谱（FT-IR）、热重分析（TGA）、扫描电子显微镜（SEM）和元素分析（EA）表征,结果表明通过共聚反应成功地将漆酚酯固定在硅烷化硅胶上,且制备出的固定相具有良好的单分散性。采用匀浆法装柱,以乙腈-0.05%磷酸溶液（3：97,v/v）为流动相,流速为0.4 mL/min,检测波长为220 nm,考察固定相对天麻浸膏的分离性能。以乙腈-水（50：50,v/v）为流动相,流速为0.5 mL/min,检测波长为290 nm,考察固定相对吴茱萸浸膏的分离性能。结果表明该固定相对天麻浸膏和吴茱萸浸膏均具有良好的分离性能,从天麻浸膏中分离出5个色谱峰,从吴茱萸浸膏中分离出2个色谱峰。与商品化C₁₈ 柱相比,USP柱可以从天麻浸膏中分离出更多的有效组分并实现基线分离,分离吴茱萸浸膏的色谱条件更为环保和安全。采用低流速对天麻浸膏和吴茱萸浸膏进行分离,减少了流动相的使用量,分离结果令人满意。以天然产物漆酚制备色谱固定相,既为分离纯化天麻素和吴茱萸碱提供了一种新的方法,又为液相色谱固定相制备提供了新的思路,还拓展了生漆在色谱分离材料方面的应用。     

Determination of rotenone in river water utilizing packed capillary column switching liquid chromatography with UV and time-of-flight mass spectrometric detection

Fast and sensitive packed capillary column switching liquid chromatography methodology has been developed for the determination of the pesticide rotenone in river water. Sample volumes of up to 1 ml are loaded onto a 23 x 0.25 mm, 5 microm Kromasil C18 packed capillary precolumn using a noneluting solvent composition of water-acetonitrile (99:1, v/v) at flow-rates up to 100 microl/min prior to solute backflushing onto a 200 x 0.32 mm, 3.5 microm Kromasil C18 packed capillary analytical column using a mobile phase of water-acetonitrile (30:70, v/v) at a flow-rate of 5 microl/min. The method was evaluated using river water samples spiked with rotenone in the concentration range 0.5-50 ng/ml using UV detection. The within-assay precision was between 5.0 and 7.7% relative standard deviation (RSD, n = 6) and the between assay precision was between 7.5 and 8.9% RSD (n = 6). The method was linear within the investigated mass range displaying a calibration curve correlation factor of 0.997. The mass limit of detection was 10 pg corresponding to a concentration limit of detection of 10 pg/ml, using time-of-flight mass spectrometry.     

直链淀粉手性固定相高效液相色谱法拆分比索洛尔对映体

采用直链淀粉手性固定相高效液相色谱法在正相条件下直接拆分了比索洛尔对映异构体。分别以异丙醇、乙醇为有机改性剂,考察了流动相的组成与配比、流速及柱温等因素对比索洛尔对映体分离的影响。确定了比索洛尔对映体的最佳拆分条件:流动相正己烷-乙醇-二乙胺(体积比为88∶12∶0.1),流速0.6 mL/min,检测波长270 nm,柱温20 ℃。该方法可快捷、简便地拆分比索洛尔对映体。     

High performance liquid chromatography analysis of 9-(2',3'-dideoxy-2'beta-fluoro-D-threo-penta furanosyl) adenine and its metabolite in human plasma using solid-phase extraction on a polyfluorinated reversed stationary phase.

A quick and sensitive reversed-phase HPLC method has been developed for the analysis of 2'-beta -fluoro-2',3'-dideoxy adenosine (F-ddA), the acid-stable anti-AIDS drug, and its metabolite 2'-fluoro-2',3'-dideoxy inosine (F-ddI) in human plasma using polyfluorinated stationary phase column (Fluo fix, 15 cm, 4.0 mm i.d., 5 microm particle size). The mobile phase consisted of ammonium phosphate buffer solution (10 mM) adjusted with phosphoric acid 85% to pH 6.8:dimethyl formamide (97:3, v/v). F-ddA and F-ddI were monitored by UV-visible detector at 258 and 247 nm, respectively. The recoveries of F-ddA and F-ddI from plasma using a C(18) solid-phase extraction cartridge were 99.2% and 99.7%, respectively.     

HPLC analysis of organic acids using a novel stationary phase

In the present work, a high performance liquid chromatographic method with UV detection for the separation of six organic acids including, tartaric, malic, acetic, lactic, citric and succinic is described.The separation was performed on a novel stationary phase TEKNOKROMA, Tr-010065 Mediterranea sea₁₈ (15 cm × 0.4 cm, i.d. 3 μm) and using water with a 0.1% (v/v) of formic acid as mobile phase. The advantages of this packing over a conventional octadecylsilane (ODS2) column are reported.The method was validated with respect to linearity, limits of detection and repeatabilities within day and between days and satisfactory results were obtained.The proposed method was applied for the determination of these compounds in commercially available white wines. The samples were injected directly without previous treatment. LC-MS was used as a confirmatory technique.     

反相高效液相色谱法测定生物转化体系中的甘草酸

采用高效液相色谱法在Hypersil C18色谱柱(4.6 mm i.d.×250 mm,5 μm)上以甲醇-水-冰醋酸(70∶30∶1, 体积比)为流动相分离测定了甘草酸单铵盐生物(酶)转化体系中的甘草酸,流动相流速为1.0 mL/min,紫外检测波长254 nm。实验结果表明,该方法在进样量为0.2~20 μg时具有良好的线性;样品的加标回收率为98%～103％,相应的相对标准偏差为0.16％～1.58％。方法简便、快速、可靠。     

Online trace enrichment to determine pyrethroids in river water by HPLC with column switching and photochemical induced fluorescence detection

The potential of online trace enrichment on a highly apolar short column in LC was evaluated for the determination of pyrethroids in river water. Twelve millilitres of water samples, modified with 8 mL ACN (ACN/water 40:60, v/v), were passed through 50 x 4.6 mm ID first separation column packed with 5 microm Hypersil Elite C18. Pesticides were preconcentrated in this column while the matrix background was eluted to waste. Separation of pesticides was performed on a 3.5 microm symmetric C18 column (250 x 4.6 mm ID) with an ACN step gradient as mobile phase and fluorescence detection was used after postcolumn derivatization by using UV light. The use of photochemically induced fluorescence for detection improved sensitivity and selectivity. Quantification limits ranged from 0.05 to 0.1 microg/L and pesticide recoveries at two concentration levels (0.1 and 0.5 microg/L) were between 93.1 and 118.6%, with RSD between 2.5 and 7.5% (n = 3) in river water samples. No matrix effect was detected.     

Determination of erythromycin ethylsuccinate by liquid chromatography.

A method is described for the determination of erythromycin ethylsuccinate by liquid chromatography. A C18 reversed-phase column (25 cm x 4.6 mm I.D.) was used with acetonitrile-0.2 M tetrabutylammonium sulphate (pH 6.5)-0.2 M phosphate buffer (pH 6.5)-water [x:5:5:(90-x)] as mobile phase. The proportion of acetonitrile (x) has to be adapted to the type of stationary phase used. For RSil C18 LL, 42.5% was used. The column was heated at 35 degrees C, the flow-rate was 1.5 ml/min and UV detection was performed at 215 nm. The main component, erythromycin A ethylsuccinate, was separated from all other components which were present in commercial samples. The main impurities were erythromycin A and the ethylsuccinate esters of erythromycin B and C. The amide N-ethylsuccinyl-N-demethylerythromycin A was shown to be present in all the samples examined. The method was successfully applied to the analysis of specialities.