Creation of reversed phase high-performance liquid chromatographic technique to assay platelet-activating factor

Objective: To establish a new assay for platelet-activating factor (PAF), to compare it with bio-assay; and to discuss its significance in some elderly people diseases such as cerebral infarction and coronary heart disease. Methods: To measure PAF levels in 100 controls, 23 elderly patients with cerebral infarction and 65 cases with coronary heart disease by reversed phase high-performance liquid chromatographic technique (rHPLC). Results:rHPLC is more convenient, sensitive,specific, and less confusing, compared with bio-assay. The level of plasma PAF in patients with cerebral infarction was higher than that in the controls (P<0.01), and in patients with coronary heart disease. Conclusion: Detection of PAF with rHPLC is more reliable and more accurate. The new assay has important significance in PAF research.

A Sensitive Reversed-Phase High-Performance Liquid Chromatography Method for the Quantitative Determination of Milk Xanthine Oxidase Activity

A new reversed-phase high performance liquid chromatography method was developed to quantitate the activity of xanthine oxidase involved in milk fat globule membrane with xanthine as the substrate and the separation of product (uric acid). The increment of uric acid in the reaction system was used to calculate the total activity of XO. The optimized assay conditions, linearity of detection, recovery of uric acid and chromatogram were developed in text, indicating this method is simple, rapid and efficient. It is an alternative potential method for the determination of the activity of XO in milk.

Simultaneous Determination of Amlodipine with H₁-Receptor Antagonists by Reversed Phase High Performance Liquid Chromatography and Application to Interaction Studies

A rapid, fast and precise method has been developed and validated for the simultaneous determination of amlodipine with H1-receptor antagonists (cetirizine, fexofenadine, and buclizine) from dosage forms. The chromatography was performed on a Purospher? Star, C18 (5 mm, 250 × 4.6 mm) column using acetonitrile: buffer (0.01 mM) (40:60, v/v, pH adjusted to 3.0), as a mobile phase. The mobile phase was pumped at a flow rate of 1.0 mL·min-1 and UV detection was performed at 240 nm. The method was validated for linearity, accuracy, precision and specificity. The method was applied to study the interaction between amlodipine and H1-receptor antagonists. These interactions were carried out in simulated gastric juice (pH 1), simulated full stomach (pH 4), blood pH (pH 7.4) and simulating GI (pH 9). The interacting drugs were heated at 37℃ with intermit-tent shaking and the samples were withdrawn every thirty minutes for three hours and drug contents were analyzed by RP-HPLC techniques. In most cases the in vitro availability of amlodipine was decreased. It was observed that the change in in vitro availability was pH dependent.

Quantitative analysis by reversed-phase high-performance liquid chromatography and retinal neuroprotection after topical administration of moxonidine

AIM:To determine moxonidine in aqueous humor and iris-ciliary body by reversed-phase high performance liquid chromatography(RP-HPLC),and to evaluate the retinal neuroprotective effect after topical administration with moxonidine in a high intraocular pressure(IOP)model.METHODS:The eyes of albino rabbits were administered topically and ipsilaterally with 0.2%moxonidine.A RPHPLC method was employed for the identification and quantification of moxonidine between 2 and 480 min,which presented in the aqueous humor and iris-ciliary body.Flash electroretinography(F-ERG)amplitude and superoxide dismutase(SOD)level were measured between day 1 and day 15 after topical administration with moxonidine in a rabbit model of high IOP.Histological and ultrastructural observation underwent to analyze the changes of retinal morphology,the inner retinal layers(IRL)thickness,and retinal ganglion cell(RGC)counting.RESULTS:Moxonidine was detectable between 2 and 480 min after administration,and the peak concentration developed both in the two tissues at 30 min,0.51μg/m Lin aqueous humor and 1.03μg/g in iris-ciliary body.In comparison to control,F-ERG b-wave amplitude in moxonidine eyes were significantly differences between day 3 and day 15(P<0.01)in the high IOP model;SOD levels were significantly higher at all time-points(P<0.01)with a maximum level of 20.29 U/mgprot at day 15;and RGCs were significantly higher(P<0.05).CONCLUSION:Moxonidine is a viable neuroprotective agent with application to high IOP model.All layers of retina,including RGC layer,retinal nerve fiber layer and INL,are more preserved after moxonidine administration.SOD plays a neuroprotective role in ocular hypertension-mediated RGC death.

Determination of Trace Amount of Polycyclic Aromatic Hydrocarbons in Urban Sewage by Solid-phase Extraction Coupled with High Performance Liquid Chromatograph

[Method] This study aimed to determine trace amount of polycyclic aromatic hydrocarbons(PAHs) in urban sewage by using solid-phase extraction(SPE) coupled with high performance liquid chromatograph(HPLC).[Method] From the aspects of solid-phase extraction column,elution solvent,elution volume,elution speed and so forth,the test conditions of SPE-HPLC method were optimized,and trace amount of PAHs in urban sewage was determined.[Result] The optimized solid-phase extraction conditions were SUPELCLEAN LC-18 solid-phase extraction column,methylene dichloride as elution solvent,15 ml elution volume,2 ml/min elution speed,5 ml/min loading speed,1 000 ml water with 200 ml methanol loading volume.Under the optimized extraction conditions,the recovery was high,namely 76.3%-105.2%;relative standard deviation was 3.8%-6.0%,showing good precision;detection limit was low,only 0.000 8-0.048 0 μg/L.[Conclusion] This method is user-friendly,with high sensitivity and good precision,and suitable for continuous determination of a large volume of water samples.

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.

THYMINE BONDED-STATIONARY PHASE FOR HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

A new type of HPLC stationary phase containing thymine derivative was successfully prepared.It was found to give selective separation of nucleic acid bases and several purine derivatives,such as caffeine and theophylline.The retention behaviour and elution order of the solutes were interpreted in terms of molecular structure.

Determination of seven active components in Salvia miltiorrhiza herb by matrix solid phase dispersion combined with ion liquid extraction followed by high performance liquid chromatography

A low cost,rapid and sensitive preparation method of silica gel supported ionic liquid(SGSIL)combined with matrix solid phase dispersion(MSPD)followed by high performance liquid chromatography(HPLC)with ultraviolet detection(UV)is proposed,and it was applied to determine the seven active compounds in Salvia Miltiorrhiza herb.SGSIL and ionic liquid[BMIM]BF4 were used as the adsorbent and the green elution reagent in the MSPD procedure.Several extraction conditions including type of filler and elution solvent,the volume of elution solvent,material liquid ratio were optimized.Under the optimum conditions,the SGSIL-MSPD-HPLC method showed a low limit of detection(LOD,S/N=3)of 0.0122-0.8788μg/mL for standard solution,limit of quantification(LOQ,S/N=10)of 0.0406-2.9292μg/mL for standard solution,wide linear range from 1.56 to 2000μg/mL for all compounds for standard solution,correlation coefficients(r)of more than 0.9990,acceptable reproducibility(relative standard deviations,RSDs<3.54%),and precision of RSDs<3.36%for intra-day,RSDs<3.50%for inter-day.The satisfactory recoveries ranged from 96.4 to 102.5,with RSDs less than 3.45%.The developed SGSIL-MSPD method is easier and more suitable for the determination of the seven active compounds in Salvia Miltiorrhiza herb than the traditional ultrasonic extraction.It was an effective and efficient method for the extraction and quantification of the seven active compounds in traditional Chinese herbal samples.

High Performance Liquid Chromatographic Determination of Phenolic Acids in Fruits and Vegetables

A simple isocratic HPLC technique has been developed for the quantitative analysis of phenolic acids (PAs) in fruits and vegetables. Nine benzoic and cinnamic acid derivatives were separated in less than 30 min, and the resolution was all more than 1.23. The ranges of linearity for PAs standards were 0.2-100 ng, even up to 600 ng (r = 0.983-1.000) and the detection limits were 0.02-0.24 mg/kg. Samples of fresh vegetables and fruits were extracted with 80% mcthanol and ethyl acetate, then purified with C18 Sep-Pak cartridge and determined by HPLC. This method was applied to the determination of PAs in 7 kinds of fruits and vegetables, i.e., apple, pear, Chinese cabbage, cauliflower, turnip, soybean sprout and white grape wine. The content of the 9 PAs varied widely in the 7 kinds ol'foods studied. The average concentrations ofchlorogenic acid in apple (100.2 mg/kg) and pear (30.8 mg/kg) were quite high, and sinapinic acid was remarkable (42.5 mg/kg) in Chinese cabbage, and protocatechuic acid had the highest concentration of all the PAs in white wine.

Porous Monoliths: Stationary Phases of Choice for High Performance Liquid Chromatography in Various Formats

Modern porous monoliths have been conceived as a new class of stationary phases for high performance liquid chromatography(HPLC) in classical columns in the early 1990s and later extended to the capillary format.These monolithic materials are prepared using simple processes carried out in an external mold(inorganic monoliths) or within the confines of the column(organic monoliths and all capillary columns).These methods afford macroporous materials with large through-pores that enable applications in a rapid flow-through mode.Since all the mobile phase must flow through the monolith,the convection considerably accelerates mass transport within the monolithic separation medium and improves the separations.As a result,the monolithic columns perform well even at very high flow rates.The applications of monolithic capillary columns are demonstrated on numerous separations in the HPLC mode.

Determination of Quinolone Antibiotics in Water Using Solid Phase Extraction-High Performance Liquid Chromatography-Fluorescence Method

[Objective] To develop a solid phase extraction-high performance liquid chromatography-fluorescence method for determination of quin- olone antibiotics in water. [ Metbod] The standard curves of four quinolones （norfloxacin, ciprofloxacin, Iomefloxacin and enrofloxacin） were pre- pared. The detection limit in water and recovery were determined. The water samples collected from different areas, river and tap water were trea- ted using solid-phese extraction method and analyzed by high performance liquid chromatography. Then the concentration of quinolones antibiotics was determined by fluorescence method. [ Result] The detection limit of quinolone antibiotics in water was 0.083 -0.248 μg/L, and their recovery was 63.7% -134.1%. The four quinolone antibiotics at different levels were detected in various water samples, and the total concentration of quin- olone antibiotics was 0.045 -3.969 μg/L. The total concentration of quinolone antibiotics was higher in the water samples collected from rivers in Shenzhen area than in the sewage samples. The four quinolone antibiotics could be detected in all tap water samples. [ CoaduLsion ] The solid phase extraction-high performance liquid chromatography-fluorescence method is feasible and effective to detect quinolones in water. In addition, this method needs low cost and can meet requirements of daily monitorina and analysis.

Trace Determination of Tamoxifen in Biological Fluids Using Hollow Fiber Liquid-Phase Microextraction Followed by High-Performance Liquid Chromatography-Ultraviolet Detection

The applicability of hollow fiber liquid-phase microextraction (HF-LPME) combined with high-performance liquid chromatography-ultraviolet detection (HPLC-UV) was evaluated for the extraction and determination of tamoxifen (TAM) in biological fluids including human urine and plasma. The drug was extracted from a 15 mL aqueous sample (source phase;SP) into an organic phase impregnated in the pores of the hollow fiber (membrane phase;MP) followed by the back-extraction into a second aqueous solution (receiving phase;RP) located in the lumen of the hollow fiber. The effects of several factors such as the nature of organic solvent, compositions of SP and RP solutions, extraction time, ionic strength and stirring rate on the extraction efficiency were examined and optimized. An enrichment factor of 360 along with substantial sample clean up was obtained under the optimized conditions. The calibration curve showed linearity in the range of 1 - 500 ng?mL–1 and the limit of detection was found to be 0.5 ng?mL–1 in aqueous medium. A reasonable relative recovery (≥89%) and satisfactory intra-assay (3.7% - 4.2%, n = 3) and inter-assay (7.5% - 7.8%, n = 3) precision illustrated good performance of the analytical procedure in spiked human urine and plasma samples.

Molecularly Imprinted Micro-Solid-Phase Extraction for the Selective Determination of Phenolic Compounds in Environmental Water Samples with High Performance Liquid Chromatraphy

2,4,6-trichlorophenol molecularly imprinted suspension polymer has been prepared and applied to the molecularly imprinted micro-solid-phase extraction procedure for selective preconcentration of phenolic compounds from environmental water samples. The influence of functional monomer, cross-linker, polymerization condition, porogen, and the ratio of template molecule and functional monomer to cross-linker on the size of the obtained particles were investigated. It was found that methyacrylic acid as functional monomer, divinylbenzene as cross-linker, the molar ratio of template molecule and functional monomer to cross-linker was 1:4:20, the amount of AIBN was 100 mg, ultraviolet radiation at 365 nm were the optimal conditions, and at these conditions, the polymers had the best adsorption efficiency and had the monodispersity of 2 - 3 μm microsphere particles. The characteristics of the MIMSPE method were valid by high performance liquid chromatography. This MIMSPE-HPLC method has been successfully applied to the direct preconcentration and determination of phenolic compounds (phenol, 4-chlorophenol, 2,4-dichlorophenol, 2,4,6-trichlorophenol, pentachlorophenol) in environmental water samples.

Determination of Organic Acids in Root Exudates by High Performance Liquid Chromatography:Ⅱ.Influence of Several Testing Conditions

Effects of column temperature and flow rate on separation of organic acids were studied by determining nine low-molecular-weight organic acids on reversed- phase C18 column, using high performance liquid chromatography (HPLC) with a wavelength of UV (ultraviolet) 214 urn and a mobile phase of 18 mmol L-1 KH2PO4 buffer solution (pH 2.1). The thermal stability of organic acids was determined by comparing the recoveries of organic acids in different temperature treatments. The relationships between column temperature, flow rate or solvent pH and retention time were analyzed. At low solvent pH, separation efficiency of organic acids was increased by raising the flow rate of the solvent because of lowering the retention time of organic acids. High column temperature was unfavorable for the separation of organic acids. The separating effect can be enhanced through reducing column temperature in organic acid determination due to increasing retention time. High thermal stability of organic acids with low concentrations was observed at temperature of 40 ℃-45℃. Sensitivity and separation effect of organic acid determination by HPLC were clearly improved by a combination of raising flow rate and lowering column temperature at low solvent pH.

Study and ICH validation of a reverse-phase liquid chromatographic method for the quantification of the intact monoclonal antibody cetuximab

Cetuximab （CTX） is a potent chimeric mouse/human monoclonal antibody （mAb） approved worldwide for treatment of metastatic colorectal cancer. Among the various biological and physical analyses per- formed for full study on this biopharmaceutic, the determination of the concentration preparations throughout manufacturing and subsequent handling in hospital is particularly relevant. In the present work, the study and validation of a method for quantifying intact CTX by reverse-phase high-perfor- mance liquid chromatography with diode array detection （（RP）HPLC/DAD） is presented. With that end, we checked the performance of a chromatographic method for quantifying CTX and conducted a study to validate the method as stability-indicating in accordance with the International Conference on Harmo- nization guidelines （ICH） for biotechnological drugs; therefore, we evaluated linearity, accuracy, preci- sion, detection and quantification limits, robustness and system suitability. The specificity of the method and the robustness of the mAb formulation against external stress factors were estimated by compre- hensive chromatographic analysis by subjecting CTX to several informative stress conditions. As de- monstrated, the method is rapid, accurate, and reproducible for CTX quantification. It was also suc- cessfully used to quantify CTX in a long-term stability study performed under hospital conditions.

Preparative Optimization of Cellulose Microspheres Applied as Supports for High-Performance Liquid Chromatography

The aim of this work is optimizing the techniques to prepare pure cellulose microspheres, which are used as packing adsorbents for high-performance liquid chromatography. Thereupon, cellulose was dissolved in a pre-cooled NaOH/urea solution, from which various-size microspheres were prepared. The volume-average diameters were controlled approximately at 30 p,m, 8 ~tm and 4 pm grades when cyclohexane, liquid paraffin and pump oil were used as dispersants, respectively. The present investigations reveal that higher viscosity dispersant is suitable for the preparation of smaller-size microspheres, while larger size microspheres are prepared preferably using lower-viscosity dispersant. The chiral stationary phase derived from 8 μm grade microspheres can separate the enantiomers of efavirenz.

Rapid determination of atrazine in environmental water samples by a novel liquid phase microextraction

A novel method was described for the rapid determination of atrazine using dispersive liquid phase microextraction in combination with high performance liquid chromatography （HPLC）. Possible impact parameters such as sample pH, extraction and disperser solvents, salting-out effect, and extraction time were investigated. The experimental results indicated that proposed method possessed an excellent analytical performance, The linear range, detection limit, and precision （R.S.D.） were 0.1- 50 ng mL- 1 （R2 = 0.9955）, 0.601 ng mL- 1 and 6,4%, respectively. The proposed method was validated with the real water samples, and the spiked recoveries were in the range of 69.9-89.8%, respectively. These results indicated that the established method with high enrichment factor, short extraction time was an excellent alternative for the routine analysis of atrazine in environmental samples. 2007 Qing Xiang Zhou. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.

A highly sensitive SPE-liquid/liquid extraction-RPLC analytical method for the determination of 6β-hydroxycortisol and cortisol in cancer patients' urine

Objective: A highly sensitive SPE-liquid/liquid extraction RPLC method has been developed for the analysis of 6β-hydroxycortisol and cortisol in the urine of cancer patients. Methods: After SPE column purification and liquid-liquid extraction, the sample test solutions were analyzed with RPLC using a C18 analytical column. This improved analytical method has been validated for linearity, accuracy (recovery from urine), repeatability (within-day and between-day precision), specificity, sensitivity, and stability. This SPE-liquid/liquid extraction-RPLC is rapid, simple, accurate and reproducible. The technique is particularly useful for monitoring the CYP3A activity of cancer patients in clinical settings. The results are expressed as the ratio of 6β-hydroxycortisol to cortisol. Results: The CYP3A activity from a total of 153 samples was measured using this improved method. Considerable variation in the CYP3A activity of different cancer patients has been documented. Thus, personalized medical treatment based on the individual metabolic enzyme activity level is necessary. Conclusion: This new analytical method facilitates such individualized medical treatments.

Comparison of the performance of chiral stationary phase for separation of fluoxetine enantiomers

Separation of fluoxetine enantiomers on five chiral stationary phases (chiralcel OD-H, chiralcel OJ-H, chiralpak AD-H, cyclobond ? 2000 DM and kromasil CHI-TBB) was investigated. The optimal mobile phase compositions of fluoxetine separation on each column were hexane/isopropanol/diethyl amine (98/2/0.2, v/v/v), hexane/isopropanol/diethyl amine (99/1/0.1, v/v/v), hexane/isopropanol/diethyl amine (98/2/0.2, v/v/v), methanol/0.2% triethylamine acetic acid (TEAA) (25/75, v/v; pH 3.8) and hexane/isopropanol/diethyl amine (98/2/0.2, v/v/v), respectively. Experimental results demonstrated that baseline separation (RS>1.5) of fluoxetine enantiomers was obtained on chiralcel OD-H, chiralpak AD-H, and cyclobond ? 2000 DM while the best separation was obtained on the last one. The eluate orders of fluoxetine enantiomers on the columns were determined. The first eluate by chiralcel OJ-H and kromasil CHI-TBB is the S-enantiomer, while by chiralpak AD-H and cyclobond I 2000 DM is the R-enantiomer.

分子印迹固相萃取-高效液相色谱法测定果蔬中4种有机磷类农药的残留量

以4种目标物丙溴磷、毒死蜱、二嗪磷、辛硫磷的混合物作为模板分子,乙二醇二甲基丙烯酸酯为交联剂、甲基丙烯酸为功能单体,通过水热聚合法制备分子印迹聚合物(MIPs),将MIPs填充在聚丙烯空柱中制备MIPs固相萃取(MIPs-SPE)柱。将果蔬样品洗净、晾干、粉碎后,分取5.00 g,加入2 g无水硫酸钠,研磨均匀。加入30 mL乙腈,超声1.5 h,离心10 min。分取1 mL上清液过活化好的MIPs-SPE柱,用4 mL正己烷淋洗,6 mL体积比9∶1的甲醇-乙酸混合溶液洗脱。收集洗脱液,于35℃氮气吹干,用1 mL乙腈定容,用高效液相色谱法分析。结果显示:MIPs可特异性识别4种目标物,对目标物的吸附量约非分子印迹聚合物(NIPs)的2.5倍,对硫磷、马拉硫磷、甲拌磷、甲基毒死蜱的吸附量显著低于目标物的;经MIPs-SPE柱净化后,样品大部分基质成分被去除,目标物测定无干扰。4种目标物的浓度在0.005~2.0μmol·L^(−1)内和对应的峰面积呈线性关系,检出限(3S/N)为0.0015~0.0040μmol·L^(−1);在0.01,0.5,1.0,2.0 mg·kg^(−1)加标浓度水平下,4种目标物的回收率为85.9%~102%,测定值的相对标准偏差(n=5)为3.0%~7.1%。方法用于4种果蔬样品的分析,在草莓样品中检出了丙溴磷(检出量为0.07 mg·kg^(−1)),甘蓝样品中检出了辛硫磷(检出量为0.05 mg·kg^(−1)),其他样品中均未检出这4种目标物。