赶黄草总黄酮树脂精制工艺与检测方法

本研究以总黄酮吸附量和解析率为检测指标,结合动态洗脱考察结果,考察11种树脂对赶黄草总黄酮的富集精制能力。优化树脂类型,选定了对总黄酮进行富集纯化较好的树脂HPD450;采用单因素及正交实验,确定最佳工艺条件:上样量10 mL,以6 mL/min的流速8 BV水、6 BV 80%甲醇溶液洗脱,pH值为6~7。以此方法得到90%以上的赶黄草总黄酮,实验结果良好,总黄酮精制工艺成效显著。用HPLC法精确测定赶黄草中槲皮苷、PGHG和ThA三个指标性黄酮成分,最佳色谱条件为:C18色谱柱,检测波长280 nm,进样量5μL,流动相:乙腈-0.05%磷酸溶液(0~25 min:12%~45%乙腈;25~40 min:45%~70%乙腈)梯度洗脱;精确测定赶黄草全草中3种黄酮成分的含量。采用分光光度法检测,指导优化总黄酮精制工艺,并以HPLC法精确测定三种黄酮代表成分。比较两种方法测定结果相一致,互为补充;故在工业生产时,可以分光光度法指导生产,以HPLC法精确定量测定。     

Measurement and pharmacokinetic analysis of unbound ceftazidime in rat blood using microdialysis and microbore liquid chromatography

To evaluate the biodisposition of ceftazidime in rat blood, a rapid and simple microbore liquid chromatographic technique together with a microdialysis sampling technique were developed. This method involves an on-line design for blood dialysate directly injected into a microbore liquid chromatographic system. The chromatographic conditions consisted of a mobile phase of methanol–acetonitrile–100 mM monosodium phosphoric acid (pH 3.0) (10:10:80, v/v/v) pumped through a microbore reversed-phase column at a flow-rate of 0.05 ml/min. With the detection wavelength set at 254 nm, a good linear correlation was observed between the peak area and the ceftazidime concentration at 0.1 to 50 μg/ml (r=0.999). Microdialysis probes, being custom-made, were screened for acceptable in vivo recovery while chromatographic resolution and detection were validated for response linearity, as well as intra-day and inter-day variabilities. This method was then applied to the pharmacokinetic profiling of ceftazidime in blood following intravenous 50 mg/kg administration to rats. The pharmacokinetics was calculated from the corrected data for dialysate concentrations of ceftazidime versus time. This method has been used to study ceftazidime pharmacokinetics in rats and has proven to be rapid and reproducible.     

高效液相色谱法测定银杏叶提取物中黄酮甙含量

用高效液相色谱法测定银杏叶提取物经酸水解后黄酮甙元含量．采用Ｃ１８柱,甲醇：水：磷酸（５５：４４．５：０．５）为流动相,检测波长３７０ｎｍ,方法回收率９５．１％～１０３．２％,变异系数（ＣＶ）３．１７％,１０批样品含测平均为２５．４％．     

Comparison of performance of C18 monolithic rod columns and conventional C18 particle-packed columns in liquid chromatographic determination of Estrogel and Ketoprofen gel

The performance of monolithic HPLC columns Chromolith (made by Merck, Germany) and conventional C18 columns Discovery (Supelco, Sigma-Aldrich, Prague, Czech Republic) was tested and the comparison for two topical preparations Ketoprofen gel and Estrogel gel was made. The composition of mobile phases - for Ketoprofen analysis a mixture of acetonitrile, water and phosphate buffer adjusted to pH 3.5 (40:58:2) and for Estrogel analysis a mixture of acetonitrile, methanol, water (23:24:53) - was usually not optimal for analyses at all types of columns. Thus an adjustment of components ratio was necessary for sufficient resolution of the compounds analysed. Various flow rates (1.0-5.0 ml/min) and mobile phases (usually increasing ratio of water content) were applied. Determination of active substances, preservatives and impurities and comparison of retention times and system suitability test parameters was accomplished. For Estrogel gel, following chromatographic conditions were found: using Chromolith Flash RP-18e monolith column, mobile phase was acetonitrile, methanol, water (13:24:63, v/v/v) and flow-rate 3.0 ml/min. Using monolith column ChromolithSpeedROD RP-18e, the mobile phase was acetonitrile, methanol, water (18:24:58, v/v/v) and flow-rate 4.0 ml/min. For the monolith column Chromolith Performance RP-18e, the mobile phase was acetonitrile, methanol, water (23:24:53, v/v/v), flow-rate 3.0ml/min. Analysis of Ketoprofen gel gave the best results using following analytical conditions: for monolith column Chromolith Flash RP-18e, mobile phase as a mixture of acetonitrile, water, phosphate buffer pH 3.5 (30:68:2, v/v/v) was used, at flow-rate 2.0 ml/min. For ChromolithSpeedROD RP-18e monolith column, acetonitrile, water, phosphate buffer pH 3.5 (35:63:2, v/v/v) was used as a mobile phase at flow-rate 3.0 ml/min. Chromolith Performance RP-18e gave the best results using mobile phase acetonitrile, water, phosphate buffer pH 3.5 (30:68:2, v/v/v) at the flow-rate 5.0 ml/min. It was proved that monolith columns, due to their porosity and low back-pressure, can save analysis time by about a factor of three with sufficient separation efficiency. Thus, for example 11 min long analysis can be performed in 4 min with comparable results.     

Determination of scopoletin in rat plasma by high performance liquid chromatographic method with UV detection and its application to a pharmacokinetic study

A rapid and simple high-performance liquid chromatographic (HPLC) method has been developed and validated for determination of scopoletin in rat plasma using psoralen as internal standard. Chromatographic separation was achieved on a C(18) column using methanol and distilled water (49:51, v/v) containing 0.05% (v/v) phosphoric acid as mobile phase. The UV detector was set at 345 nm. The calibration curve was linear over the range of 0.165-9.90 microg/ml with a correlation coefficient of 0.9994. The recovery for plasma samples of 0.165, 1.32 and 6.60 microg/ml was 93.2%, 95.9% and 95.5%, respectively. The RSD of intra- and inter-day assay variations was less than 6.7%. This HPLC assay is a precise and reliable method for the analysis of scopoletin in pharmacokinetic studies.     

Liquid chromatographic separation and thermodynamic investigation of lorcaserin hydrochloride enantiomers on immobilized amylose–based chiral stationary phase

A novel liquid chromatographic method was developed for enantiomeric separation of lorcaserin hydrochloride on Chiralpak IA column containing chiral stationary phase immobilized with amylose tris (3.5‐dimethylphenylcarbamate) as chiral selector. Baseline separation with resolution greater than 4 was achieved using mobile phase containing mixture of n‐hexane/ethanol/methanol/diethylamine (95:2.5:2.5:0.1, v/v/v/v) at a flow rate of 1.2 mL/min. The limit of detection and limit of quantification of the S‐enantiomer were found to be 0.45 and 1.5 μg/mL, respectively; the developed method was validated as per ICH guideline. The influence of column oven temperatures studied in the range of 20°C to 50°C on separation was studied; from this, retention, separation, and resolution were investigated. The thermodynamic parameters ΔH°, ΔS°, and ΔG° were evaluated from van't Hoff plots,(Ink′ _versus 1/T) and used to explain the strength of interaction between enantiomers and immobilized amylose–based chiral stationary phase     

紫茎泽兰9-羰基-10,11-去氢泽兰酮分布积累动态

9-羰基-10,11-去氢泽兰酮为紫茎泽兰（Eupatorium adenophorum）的主要致肝脏毒性成分及杀虫的生物活性成分。从紫茎泽兰叶片中分离提纯得到9-羰基-10,11-去氢泽兰酮（Euptox A）标准品,建立了高效液相色谱法测定紫茎泽兰中Euptox A含量的分析方法。采用C18反相色谱柱,柱温30°C,以甲醇-水（60：40,v/v）为流动相、流速为0.8 mL.min–1、检测波长为255 nm进行测定。Euptox A在紫茎泽兰中的添加回收率为97.3%–103.7%,检测限为0.4μg.g–1。利用建立的方法测定Euptox A在紫茎泽兰体内分布与积累的动态变化规律。结果表明,Euptox A主要分布在紫茎泽兰的叶片中,且在营养生长期积累量高,生殖生长期积累量低。该方法快速、简捷,可用于紫茎泽兰原料及其产品中Euptox A成分的测定。     

A preliminary pharmacokinetic study of the enantiomers of the terfenadine acid metabolite in humans

A stereoselective and sensitive achiral/chiral method for the determination of terfenadine acid metabolite in human plasma was developed. The metabolite was separated and quantitated using an achiral chromatographic procedure with a cyano column. The mobile phase was 1 mM sodium acetate buffer (pH 4.0) and acetonitrile (25:75% v/v) at a flow rate of 2 ml/min, at ambient temperature. The stereospecific resolution was accomplished using a chiral-AGP column and a mobile phase consisting of sodium acetate (0.01 M): methanol (98.7:1.3% v/v), and 20 mM di-n-butylamine at a flow rate of 1.2 ml/min. The column temperature was maintained at 32°C. The eluent was monitored at 230 nm (excitation) and 300 nm (emission) with a cut-off filter at 270 nm. This assay was used for a pharmacokinetic study in five subjects after administration of a single dose of 60 mg of terfenadine. The t_½ values of the two enantiomers were similar, but the AUC values of the (+)-enantiomer were 2.05–2.35 times higher than those of (?)-enantiomer. © 1994 Wiley-Liss, Inc.     

DL－氨基酸拆分条件的优化

本文利用高效液相色谱,应用两种不同类型的方法拆分Ｄ、Ｌ－氨基酸,对拆分条件进行了优化。当利用脲衍生型手性色谱柱时,流动相组成为：正已烷／二氯乙烷／乙醇＝７５：１８：７拆分迅速、有效;当利用柱前衍生反相色谱拆分时,在甲醇／ＮａＡｃ缓冲溶液流动相中加入适量ＴＨＦ得到很好的分离效果。     

Central composite design as a powerful optimisation technique for enantioresolution of the rac-11-dihydrooracin--the principal metabolite of the potential cytostatic drug oracin

Three types of chiral stationary phase were used to achieve chromatographic resolution of enantiomers of rac-11-dihydrooracin (DHO), the principal metabolite of a potential cytostatic drug oracin. Chiralcel OD-R as a chiral stationary phase with mobile phase comprising acetonitrile (modifier) and sodium perchlorate (buffering component) proved to be the most suitable system. Chemometric optimisation based on the Box-Wilson central composite design was employed to find the optimum resolution. The optimum factor space was defined by three parameters: temperature, modifier concentration and buffer concentration. Newly designed chromatographic response functions based on a combination of resolution R(S) and retention time of the last component eluted t(RL) were employed to evaluate the resolution with regard to quality and quantity. Optimum values predicted from those models of response surfaces were in excellent agreement with the experimental results. The chromatographic resolution of DHO enantiomers is suitable for xenobiochemical studies on stereoselectivity and stereospecificity of biotransformation enzymes.     

Enantiomeric separation of vilanterol trifenatate by chiral liquid chromatography

Vilanterol trifenatate is a novel chiral long‐acting β2‐agonist developed. Vilanterol combined with inhaled corticosteroids can treat COPD and asthma. A simple liquid chromatographic method is developed for the quantitative determination of R‐vilanterol and S‐vilanterol (impurity S). HPLC separation was achieved on Chiralpak ID (250 × 4.6 mm; particle size 5 μm) column using hexane‐ethanol‐ethanolamine (75:25:0.1, v/v/v) as mobile phase at a flow rate of 1.0 mL/min. The resolution is greater than 3.3. Ethanolamine in the mobile phase is vital to enhance chromatographic efficiency and resolution between the isomers. The method was validated with respect to accuracy, specificity, precision, LOD, LOQ, linearity, and robustness as ICH guidelines.     

High-performance liquid chromatographic enantioseparation of N-protected α-amino acids using nonporous silica modified by a quinine carbamate as chiral stationary phase

In this study, tert-butyl carbamoylated quinine as chiral selector was immobilized on nonporous silica (NPS) 1.5 μm particles developed by MICRA, and this new chiral stationary phase (CSP) was packed into a 3.3 cm column (4.6 mm ID). A series of various N-protected α-amino acids was chosen as chiral selectands, including 3.5-dinitrobenzyloxycarbonyl amino acids (DNZ-AAs). In order to optimize the chromatographic conditions with this novel CSP and to apply it to the resolution of acidic analytes the following parameters have been varied and studied: pH of the mobile phase, buffer concentration, and percentage of methanol or acetonitrile in the mobile phase. DryLab^R software was applied to optimize enantioseparation by simulating chromatographic functions of experimental conditions for isocratic and/or gradient runs. Thus, we were able to resolve a set of test compounds within several minutes, whereby our attention was particularly drawn to the resolution of DNZ-AA derivatives. Chirality 9:157–161, 1997. © 1997 Wiley-Liss, Inc.     

HPLC测定7种龙胆科植物花中龙胆苦苷与獐牙菜苦苷的含量

目的:建立7种龙胆花中龙胆苦苷和獐牙菜苦苷含量测定的HPLC方法。方法:采用微波辅助动态回流法进行提取,色谱条件:Fusion-RP 80 A C18柱(150 mm×4.6 mm,5μm);流动相:甲醇-0.2%磷酸溶液梯度洗脱(0~25 min:15%~30%);流速:1 mL/min;柱温:30℃;检测波长240 nm。结果:7种龙胆花中獐牙菜苦苷和龙胆苦苷的色谱峰与共存组分完全达到基线分离,线性范围分别为0.105~0.945μg(r=0.999 9),0.3~0.7μg(r=0.999 9),平均加样回收率分别为97.8%(RSD=1.02%),98.9%(RSD=1.51%)。结论:所建立的方法测定快速,结果准确可靠。     

Fully automated high-performance liquid chromatography of ciprofloxacin with direct injection of plasma and Mueller–Hinton broth for pharmacokinetic/pharmacodynamic studies

An isocratic high-performance liquid chromatographic method with column switching and direct injection has been developed to determine ciprofloxacin in plasma and Mueller–Hinton broth. An on-line dilution of the sample was performed with a loading mobile phase consisting of 173 mM phosphoric acid. The analyte was retained on a LiChrocart 4-4 precolumn filled with a LiChrospher 100 RP18, 5 μm. An electric-actuated system with two six-port valves allowed a clean-up step with a mixture 20 mM phosphate buffer (pH 3.5)–methanol (97: 3, v/v) and the transfer of the analyte by a back-flush mode to a 150×4.6 mm I.D. column packed with a Kromasil C₈ 5 μm, using a mobile phase of 20 mM phosphate buffer (pH 3.5)–acetonitrile (85:15, v/v). Fluorescence detection allowed a quantification limit of 0.078 μg/ml with a 40-μl sample size. The method was evaluated to determine its usefulness in studying the pharmacokinetic/pharmacodynamic behaviour of ciprofloxacin in an in vitro model.     

Separation and determination of astaxanthin from microalgal and yeast samples by molecularly imprinted microspheres

In this work, molecularly imprinted microspheres (MIMs) were synthesized by aqueous microsuspension polymerization using astaxanthin (3,3'-dihydroxy-beta,beta'-carotene-4,4'-dione) as imprinting molecule. The MIMs obtained were subsequently packed into the stainless steel column and the chromatographic characterization of the column was investigated. The effects of pH and composition of the mobile phase on the retention factor (k') were investigated in detail. The mixture of methanol and dichloromethane (DCM) (8:2, v/v) was used as mobile phase A while the mixture of methanol and water (5:5, v/v) as mobile phase B. The separation of astaxanthin and zeaxanthin (3,3'-dihydroxyl-beta-carotene) was obtained when the concentration of mobile phase B was higher than 30% (v/v) due to their strong lipophilicity. The method developed was successfully applied to separate astaxanthin in the saponified samples of the microalga Haematococcus pluvialis and the yeast Phaffia rhodozyma. The recovery of adding 40 mg astaxanthin to 1.0 g microalgal sample was 95.5% with an R.S.D. (n =5) of 5.3%. The results of determination of astaxanthin in the microalga and the yeast were 3.7% (R.S.D (n = 1.5%, n = 9) and 0.041% (R.S.D n= 7.3%, n = 9), respectively.     

Enantiomer separation of imidazo-quinazoline-dione derivatives on quinine carbamate-based chiral stationary phase in normal phase mode

The normal phase mode liquid chromatographic enantiomer separation capability of a quinine tert-butyl-carbamate-type chiral stationary phase (CSP) has been investigated for a set of polar [1,5-b]-quinazoline-1,5-dione derivatives. This class of chiral heterocycles is currently under development as potential alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and/or N-methyl-D-aspartic acid (NMDA) receptor antagonists. The effect of the nature and concentration of polar modifier, i.e., ethanol and isopropanol, in n-hexane-based mobile phases, as well as the substituent pattern of the phenyl ring attached to the quinazolone framework on retention factor, enantioselectivity, and resolution was investigated. The Soczewiński competitive adsorption model was used to describe the relationship between the retention and the binary mobile phase compositions. According to this model, linear plots of the logarithms of retention factor versus molar fractions of the polar modifiers were obtained over a wide concentration range (X(B) between 0.15 and 0.35). Addition of equimolar ethanol yields higher resolution than isopropanol, R(S) values ranging between 1.54 and 2.75, whereas the latter allows to achieve moderately increased enatioselectivity. The resolution was further improved by using a ternary mixture of n-hexane:methanol:isopropanol/85:5:10 (v/v). The most pronounced selectivity factor alpha and resolution R(S) values were obtained for the para-hydroxy substituted compound, indicating that chiral recognition is sensitive to steric and stereoelectronic factors. In the course of optimization, the temperature-dependence on the chiral separation was also investigated. It turned out that the enantiomer separation is predominantly enthalpically driven in normal phase mode.     

Determination of unbound 20(S)-camptothecin in rat bile by on-line microdialysis coupled to microbore liquid chromatography with fluorescence detection

To evaluate the biliary excretion of unbound camptothecin, a flow-through microdialysis probe was constructed for bile sampling. The shunt linear probe was connected from the bile duct, between the liver side to the duodenum to avoid obstruction of the bile duct or bile salt waste. For automatic analysis of microdialysate, an on-line injector was connected to a microbore high-performance liquid chromatographic column with fluorescence detection. Samples were eluted with a mobile phase containing methanol–100 mM monosodium phosphoric acid (35:65, v/v, pH 2.5, adjusted with orthophosphoric acid). The limit of quantification was 1 ng/ml for camptothecin. Following camptothecin administration (5 mg/kg, i.v.), it was found in the bile microdialysate. It was concluded that the in vivo microdialysis technique yields useful data on the biliary excretion of camptothecin. This method is suitable for additional pharmacokinetic studies in rat bile.     

High-performance liquid chromatographic assay for xanomeline, a specific M-1 agonist, and its metabolite in human plasma

A reversed-phase high-performance liquid chromatographic assay (HPLC) was utilized for monitoring xanomeline (LY246708/NNC 11–0232) and a metabolite, desmethylxanomeline, in human plasma. Xanomeline, desmethylxanomeline and internal standard were extracted from plasma with hexane at basic pH. The organic solvent extract was evaporated to dryness with nitrogen and the dried residue was reconstituted with 0.2 M HCl-methanol (50:50, v/v). A Zorbax CN 150 × 4.6 mm I.D., 5-μm column and mobile phase consisting of 0.5% (5 ml/l) triethylamine (TEA) adjusted to pH 3.0 with concentrated orthophosphoric acid-tetrahydrofuran (THF) (70:30, v/v) produced consistent resolution of analytes from endogenous co-extracted plasma components. Column effluent was monitored at 296 nm/0.008 a.u.f.s. and the assay limit of quantification was 1.5 ng/ml. A linear response of 1.5 to 20 ng/ml was sufficient to monitor plasma drug/metabolite concentrations during clinical trials. HPLC assay validation as well as routine assay quality control (QC) samples indicated assay precision/accuracy was better than ±15%.     

The simultaneous separation and determination of six flavonoids and troxerutin in rat urine and chicken plasma by reversed-phase high-performance liquid chromatography with ultraviolet-visible detection

The method of high-performance liquid chromatography (HPLC) with UV-vis detection was used and validated for the simultaneous determination of six flavonoids (puerarin, rutin, morin, luteolin, quercetin, kaempferol) and troxerutin in rat urine and chicken plasma. Chromatographic separation was performed using a VP-ODS column (150 mm x 4.6 mm, 5.0 microm) maintained at 35.0 degrees C. The mobile phase was a mixture of water, methanol and acetic acid (57:43:1, v/v/v, pH 3.0) at the flow rate of 0.8 mL/min. Six flavonoids and troxerutin were analyzed simultaneously with good separation. On optimum conditions, calibration curves were found to be linear with the ranges of 0.10-70.00 microg/mL (puerarin, rutin, morin, luteolin, quercetin, kaempferol) and 0.50-350.00 microg/mL (troxerutin). The detection limits were 0.010-0.050 microg/mL. The method was validated for accuracy and precision, and it was successfully applied to determine drug concentrations in rat urine and chicken plasma samples from rat and chicken that had been orally administered with six flavonoids and troxerutin.     

Determination of total mycophenolic acid and its glucuronide metabolite using liquid chromatography with ultraviolet detection and unbound mycophenolic acid using tandem mass spectrometry

Two simple, sensitive and reproducible methods for determination of total mycophenolic acid (MPA) and its glucuronide metabolite (MPAG) as well as unbound MPA (fMPA) was developed by the use of HPLC-UV and LC-MS/MS methods, respectively. For the total MPA/MPAG method, the analytes were extracted using Isolute C(2) solid-phase extraction (SPE) cartridges and analyzed at 254 nm over a Zorbax Rx C(8) column (150 mm x 4.6 mm, 5 microm). The mobile phase was a gradient mixture of methanol and water (containing 0.1% (v/v) phosphoric acid). The total run time was 18 min and the extraction recovery was 77% for MPA and 84% for MPAG. The method was precise and accurate with a lower limit of quantification (LLOQ) of 0.5 mg/l for MPA and 5.0 mg/l for MPAG. For the fMPA method, plasma was subjected to ultrafiltration followed by SPE using C(18) cartridges. Analytical column was the same as the HPLC-UV method and the mobile phase was a gradient composition of methanol:0.05% formic acid with a flow rate of 0.6 ml/min for the first 3 min and 0.7 ml for the last 4 min. The chromatographic method separated MPA from its metabolites MPAG and Acyl-MPAG. Mass transitions in negative ionization mode for MPA and the internal standard, indomethacin were m/z: 319-->190.9 and m/z: 356-->312.2, respectively. The assay was linear in the concentration range of 1-1000 microg/l for fMPA with a LLOQ of 1 microg/l and an accuracy of >95%. The two methods reported have an adequate degree of robustness and dynamic concentration range for the measurement of MPA, MPAG and fMPA for therapeutic drug monitoring purposes or pharmacokinetics investigations.