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.

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHIC SEPARATION OF FULLERENES

High-performance liquid chromatographic systems for the separation of buckminsterfullerene(C_(60)) and related fullerene C_(70) were investigated with methanol/benzene or methanol/toluene (35/65-60/40 in Ⅴ/Ⅴ) as eluant in C18 reversed phase column. The system is suitable for fast analysis of fullerenes at lower percentage of methanol, and repeatly large scale perparation at higher percentage of methanol.

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHIC ANALYSIS OF KR-008 IN BIOLOGICAL FLUIDS

A high-performance liquid chromatographic method for the simultaneous determination of KR-008 and its metabolite in biological fluids was developed The samples were injected directly without further treatment,the compounds were separated and analysed on a reversed- phase column under isocratic conditions,including ion pairing.Concentrations in urine and bile were determined by an external standard method.The detection limit was 0.8μg/ml in urine.Preliminary data showed that,following i.p.administration(120mg/kg)of KR-008 to rats,approximately 2.8% of the dose was excreted in urine as unchanged compound and more than 8.9% was as a metabolite;in bile it was 0.73% excreted as unchanged form and above 0.29% as the metabolite.The peak biliary concentration of KR-008 was 0.39mg/ml at 20min after dosing.

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.

High-performance liquid chromatographic fingerprint analysis of Oxytropis falcata Bunge and Oxytropis chiliophylla Royle

A simple, sensible and reliable HPLC-DAD fingerprint analysis method for the raw materials of Oxytropisfalcata and Oxytropis chiliophylla, both of which were used as ＂Er-Da-Xia＂ in Tibetan medicines, was developed and then subsequently applied to analyze samples collected from different locations or times. 19 common fingerprint peaks for O. falcata, 24 for O. chiliophylla, and 11 for the two herbs were designated respectively, including 7 identified characteristic peaks existing in both herbs and 1 uniquely presenting in O. chiliophylla. Although there were some slight differences in the chemicals of O. falcata and O. chiliophylla, the main components of both herbs were consistent generally. The results provided scientific basis, at least from the chemical point of view, for the reasonablity of two herbs being used as the same drug in Tibetan medicines and for the necessary of further investigation on their detailed chemical and pharmacological differences.

Freshwater algae chemotaxonomy by high-performance liquid chromatographic(HPLC)analysis

The study of community composition of algae is essential for understanding the structure and dynamics of the aquatic ecosystem and for evaluating the eutrophic level of the water body.A high-performance liquid chromatographic(HPLC)method based on a reversephase C_(18) nonpolar column was developed for the main algal taxa,which includes cyanophytes,bacillariophytes,euglenophytes,dinophytes,and chlorophytes.Based on the elution order using HPLC,19 pigments were identified,and they were chlorophyllide a,19′-butanoyloxyfucoxanthin,chlorophyll c_(1)+c_(2),phephorbides a,peridinin,methyl-chlorophyllide a,fucoxanthin,neoxanthin,violaxanthin,myxoxanthophyll,diadinoxanthin,diatoxanthin,lutein,zeaxanthin,chlorophyll b allomer,chlorophyll b,chlorophyll a allomer,chlorophyll a,andβ,β-carotene.A comparison study of cell microscopic counts and accessory pigment analysis indicated that HPLC analysis could be a useful tool for monitoring phytoplankton communities and their abundance.

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.

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.

Analysis of Sucrose Esters with Long Acyl Chain by Coupling of HPLC-ELSD with ESI-MS System

The analysis of sucrose esters with long acyl chain by improved high performance liquid chromatographic method with evaporative light scattering detection （HPLC-ELSD） and electrospray ionization mass spectrum （ESI-MS） is investigated. The improved HPLC-ELSD method for the separation and quantitation of commercial and synthesized sucrose esters is described. Samples are analyzed by means of a reversed-phase （RP） HPLC using a Hypersil C8 column （250 mm× 4.6 mm, 5 μm particle size） with methanol-tetrahydrofuran （vo）ume ratio of 90 ： 10） and water under gradientcondition as the mobile phase, in which the flow rate is 1.0 ml·min^-1 and the column temperature is set at 40℃. This procedure provides a complete separation and determination ot monoester, diester, triester and higher esters with different acyl chain lengths in each fraction by a single run, in combination with the ESI-MS technology. With this method, it is possible to determine the approximate compositions of monoto polyesters in one analysis and quantitate pure positional isomers precisely using an external standard method. It is found that the method of ESI-MS coupling with HPLC system for the analysis of sucrose esters is straight forward, rapid and inexpensive, and can be readily applied in synthesis, purification and structure studies.

Rapid HPLC Method for Monitoring Relevant Residues of Pharmaceuticals Products in Environmental Samples

This work presents a multi-residue analytical method based on solid phase extraction (SPE) followed by high-performance liquid chromatographic (HPLC) with diode array (DAD) detection for the simultaneous determination of a group of pharmaceutical products that include ten antidepressants and three anticanceri- genic in environmental samples (water and soil). Baseline separation of the studied compounds was obtained on an ultrabase C18 (4.6 mm i.d. × 150 mm, 5 μm particle) column using acetonitrile:phosphate buffer pH 2.5 (35:65 v/v) as mobile phase with a flow rate of 1.5 mL/min. Different aspects including linearity, accuracy, precision and detection and quantification limits were examined in order to validate the proposed method. Detection limits between 1 and 50 ng/mL were obtained for all the target compounds. This method was ap- plied to the analysis of environmental samples as waters and soils of different precedence. Prior, the HPLC determination the samples were purified and enriched using SPE or liquid-liquid extraction (LLE) of the tar- get compounds.

Simultaneous Determination of Three Components in Herba Taxilli from Different Host Trees by HPLC

[Objectives] To establish a HPLC method for the simultaneous determination of three components( namely,quercetin,quercitrin and avicularin) in Herba Taxilli. [Methods]Agilent C18 liquid chromatography column( column size: 150 mm × 4. 6 mm,5 m) was used;mobile phase Dacetonitrile∶ water = 22∶ 78; flow rate: 1. 0 m L/min; column temperature: 35℃; injection volume: 10 μL; detection wavelength: 254 nm. [Results]The injection volume of quercetin,quercitrin and avicularin was in a good linear relationship with the peak area in the range of 0. 22-4. 4 μg( R = 1),0. 096-3. 84 μg( R = 0. 999 6),and 0. 106-5. 3 μg( R = 0. 999 9),and the average sample recovery was 99. 32%,100. 53%,and 98. 96%,respectively. [Conclusions]The stability and reproducibility of the established method for determining the content of Herba Taxilli in this experiment are good,and it is expected to provide reference for its quality control and evaluation.

Study of Phytochemical Screening and Antimicrobial Activity of Citrus aurantifolia Seed Extracts

Citrus aurantifolia, the family Rutaceae, which consists of 150 genera and 900 species, is gaining grounds as important source for treatment in complementary medicine, and commonly called limone. Chloroform, methanol and ethanol extract exhibited significant antimicrobial activity and highlighted the biological monitoring of activity from the methanol extract of seeds. Phytochemical screening of the diethyl ether, methanol and aqueous extracts of seeds confirmed the presence of alkaloids, saponins, sterols and triterpenes, carotenoids, coumarins, tannins and carbohydrates. Chloroform extract was fractionated by sing liquid solid column chromatographic technique and column fractions eluted with chloroform and methanol. The antibacterial secondary metabolites were achieved by fractionation of the active. The seed extracts were monitored analytically by TLC preparative and some chemical analyses of seeds have been done such as determination of ash, nitrogen, protein content.

Determination of Amantadine Residue in Honey by Solid-phase Extraction and High-performance Liquid Chromatography with Pre-column Derivatization and Fluorometric Detection

Amantadine （AMA） is an anti-viral drug used in apiculture to protect honeybee against the sacbrood virus （Morator aetatulae）. This study described a reliable high-performance liquid chromatographic （HPLC） method for analyzing AMA in honey using a solid-phase extraction （SPE） cartridge （Plexa PCX） for purification, 4-fluoro-7- nitro-2,1,3-benzoxadiazole （NBD-F） as a pre-column derivatization agent, and fluorometric detection （λex =470 nm, λem=530 nm）. The chromatographic separation was performed on an XDB C18 column （150×4.6 mm i.d.） using 0.1% trifluoroacetic acid/acetonitrile （35 ; 65, V/V） as the mobile phase at a flow rate of 1.0 mLomin 1 with a run time of 20 min. Under these optimal conditions, a linear relationship was observed in the range of 0.025--1.0μg·mL-1 with a good correlation coefficient （0.998） and low limit of detection （0.0080 μg·g-1）, the recoveries were all above 90%, and the intra-day and inter-day precision （RSD） ranged from 3.4%--5.1%.

Determination of Ciprofloxacin in Human Plasma and Urine by Precolumn Derivatization High Performance Liquid Chromatography with Fluorescence Detection

A sensitive, simple and selective high-performance liquid chromatographic （HPLC） method was developed for the determination of ciprofloxacin in biological fluids. The method is based on the reaction between the drug and 4-chloro-7-nitrobenzofurazan （NBD-C1） in borate buffer of pH 9.0 to yield a highly fluorescent derivative that is measured at 535 nm after excitation at 464 nm. The calibration curves were linear over the concentration ranges of 25--3000 and 50--3000 ng·mL -1 for plasma and urine, respectively. The mean recovery of ciprofloxacin from plasma and urine was 98.37% and 98.40%, respectively. The method was found to be sensitive, precise, accurate, and reproducible. All of the validation parameters were within the acceptance range.

Determination of Polysaccharide in Radix pseudostellariae Extract by Size-Exclusion High-Performance Liquid Chromatography

A simple size-exclusion high-performance liquid chromatographic method was developed for rapid molecular mass screening to determine the average molecular mass, polydispersity, and quantity of Taizishen polysaccharides extract. The screening used a TOSOH TSK-GEL G3000SW high-performance liquid chromatographic column with 0.1 mol/L NaNO3 buffer as the mobile phase and refractive index detector. The molecular mass calibration curve was linear for polysaccharide standards from 10 to 100 kDa with a correlation coefficient of 0.9991. The method can be used to analyze the quantity, average molecular mass, and polydispersity of polysaccharides. In addition, the screening method is suitable for quality control of polysaccharide preparations in Chinese medicines and pharmaceutical products.

Ultra-High-Performance Liquid Chromatograph with Triple-Quadrupole Mass Spectrometer Quantitation of Twelve Phenolic Components in Different Parts of Sarcandra glabra

Objective:The study objective was to determine phenolic components for the quality control(QC)of cultivated Sarcandra glabra(Thunb.)Makino(S.glabra).Materials and Methods:A sensitive,ultra-high-performance liquid chromatography-tandem mass spectrometric method for the simultaneous determination of 12 phenolic components has been developed.Six caffeoylquinic acids,two caffeoylshikimic acids,and four flavanonol glucosides were selected for the comprehensive analysis of distribution in different parts(root,stem,and leaf).Results:Twelve phenolic components were linear in the concentration range of 0.005–5.0μg/mL(R^(2)>0.995).The relative standard deviation of intra-day and inter-day precision across three validation runs over the entire concentration range was<5%.The recovery determined was within 5%in terms of relative error.Our results showed that the 12 phenolic compounds were mainly distributed in leaves and stems far more than those in roots.Conclusions:This study provided an ultra-high-performance liquid chromatograph with triple-quadrupole mass spectrometer quantitative method of 12 phenolic components for the QC of cultivated S.glabra.It was found that the phenolic components were significantly accumulated in the aerial parts(stems and leaves)of cultivated S.glabra.

Control System for Low Pressure Gradient Mixer in High Performance Liquid Chromatograph

A control system used in high performance liquid chromatograph(HPLC) was described.The control system adopting low pressure gradient elution was tested with different initial and end volume fractions,and four types of gradient elution curves.The experimental results verified the theoretical analyses of the applied method.This self-designed control system can achieve approving accuracy,repeatability and low cost,which has a bright outlook for domestic applications.

Studies of the intestinal absorption of the alkaloids in the Wu-tou decoction combined with different incompatible medicinal herbs in a Caco-2 cell culture system using UPLC-MS/MS

In this study, seven alkaloids were detected in Wu-tou decoction using ultra performance liquid chromatography coupled with tandem mass spectrometry （UPLC-MSn）. The aim of this study was to investigate the effect of Fritillariae Cirrhosae Bulbus, Fritillariae Thunbergii Bulbus, Pinelliae Rhizoma in different ratios with Wu-tou decoction （2：1, 1：1, 1：2） by measuring the therapeutic effects in Wu-tou decoction of main seven alkaloids including benzoylaconitine （BA）, benzoylmesaconitine （BM）, benzoylhypaconitine （BH）, hypaconitine （HA）, fuziline （FU）, niaolin （NE） and deoxyaconitine （DA）. The permeability of aconitum alkaloids extract through a Caco-2 cell monolayer was analyzed in the absence and presence of Fritillariae Cirrhosae Bulbus, Fritillariae Thunbergii Bulbus, and Pinelliae Rhizoma, respectively. The results showed that Pinelliae Rhizoma could reduce the absorption of the alkaloids and increase the excretion of the alkaloids, which would attenuate the therapeutic effects of Wu-tou decoction. Therefore, Pinelliae Rhizoma is an incompatible herb of Wu-tou decoction because of the inhibition of the absorption of alkaloids in the intestine. And that Fritillariae Cirrhosae Bulbus and Fritillariae rhunbergii Bulbus showed the effects to improve the permeability of the alkaloids in Wu-tou decoction. These effects of these two herbs were similar, but the former was stronger than the latter, which most likely is due to the fact that the compositions of these two traditional Chinese medicines are similar. The in vitro data suggests that the compounds such as fritillary presented in alkaloids in the formula maybe improve the therapeutic function caused by the increased bioavailability of alkaloids in intestine.