Analysis of Monosaccharide Composition of Pu-erh Tea Polysaccaride by Pre-column Derivatization HPLC

[Objective] This study aimed to establish a pre-column derivatization HPLC method for the identification and analysis of monosaccharide composition of Pu-erh tea polysaccharide. [Method] Pu-erh tea polysaccharide was extracted using the wa- ter extraction method, further isolated and purified by DEAE cellulose-52 columns. The obtained tea polysaccharide and four components TPS1, TPS2, TPS3 and TPS, were first derived by 1-phenyl-3-methyl-5-pyrazolone （PMP）, and then the PMP derivatives of monosaccharide were analyzed by high performance liquid chromatog- raphy （HPLC）. [Result] Pu-erh tea polysaccharide contained eight kinds of monosac- chaddes （mannose, rhamnose, glucuronic acid, galacturonic acid, grucose, galactose, arabinose, fucose）, without xylose; so it was the same with TPS1; each of TPS2, TPS3 and TPS4 contained seven monosaccharides, while no fucose. [Conclusion] This method is simplified and rapid, which can be used to determine the monosac- charide composition of Pu-erh tea polysaccharide and monosaccharide content.

Identification of the starting reaction position in the hydrogenation of (N-ethyl)carbazole over Raney-Ni

Hydrogenation of carbazole and N-ethylcarbazole over Raney-Ni catalyst were realized in the temperature range of 393-503 K. 4[H] adduct dominated the hydrogenation products and the formation of 2[H] adduct was the rate-limiting step during the period, in which the conversion of carbazole was less than 40%. The hydrogenation process followed pseudo-first-order kinetics and the hydrogenation activation energies of carbazole and N-ethylcarbazole were 90 kJ/mol and 115 kJ/mol, respectively. The reaction starting position as well as the pathway of the hydrogenation of （N-ethyl）carbazole were investigated by comparing the kinetic characteristics of hydrogen uptake of carbazole and N- ethylcarbazole. The results showed that the reaction was a stepwise hydrogenation process and the first H_2 was added to the C1 = C10 double bond in the hydrogenation.

Determination of Sparfloxacin in Human Urine by Reversed-Phase High Performance Liquid Chromatography With Nitrous Acid and Hydroiodic Pre-Column Derivatization

Sparfloxacin can be oxidized by nitrous acid, then react with hydroiodic acid to form a fluorescent derivative. Based on this, a reversed-phase high performance liquid chromatographic pre-column derivatization new method is described for the determination of sparfloxacin in human urine. The linear range is 0.05 mg/L to 4.0 mg/L, the recoveries are 91.5%similar to 95.7% and the RSD is 1.2%similar to4.2%. The results showed that this method is suitable for the determination of sparfloxacin in human urine.

Determination of 18 Kinds of Amino Acids in Fresh Tea Leaves by HPLC Coupled with Pre-column Derivatization

A rapid and accurate quantitative method of high performance liquid chromatography( HPLC) with fluorescence detector has been developed for the analysis of 18 kinds of amino acids in fresh tea leaves. The samples were minced and mixed,and extracted with ultra pure water at 90℃ for 20 min. The 6-aminoquinolyl N-hydroxy-succinimidyl carbamate( AQC) was used as pre-column derivatization reagent. Gradient HPLC separation was performed on a C_(18) column( Symmetry C_(18),3. 9 mm × 15 cm,4 μm). Good linearity between concentrations and peak areas was achieved in the concentration range of 5. 0-250 μmol/L for 18 kinds of amino acids. The method was validated by the analysis of five replicates. The 18 kinds of amino acid standards were spiked in fresh tea leaf samples and the average recovery rate was 86. 25%-109. 05% with relative standard deviations( n = 5) ranging from 6. 03% to 10. 56%. The limit of detection( LOD) for the analytes was0. 05-1. 27 μmol/L. The method was successfully applied to the analysis of the 18 kinds of amino acids in fresh tea leaves from east Dongting and west Dongting mountains in Suzhou. The results indicate that the method is simple,rapid,precise and reliable.

Preparative Resolution of Gatifloxacin Enantiomers with Pre-Column Esterification Strategy and Comparing Their Enantioselectivity to Bacteria and Antibody

Gatifloxacin （GFX） is a kind of chiral fluoroquinolones compound due to the methyl group at the C-3 position of the piperazine ring[1]. Although the enantiomers of GFX show similar levels of antimicrobial activity and pharmacokinetics[2], the other biological activities （i.e., toxicity or enantioselective recognition to various receptors in vivo） of GFX enantiomers have not yet been studied. With this in mind, we developed a rapid and cost-effective high performance liquid chromatographic （HPLC） separation procedure for GFX enantiomers with a pre-column esterification strategy.

Enantioresolution of a Series of Chiral Benzyl Alcohols by HPLC on a Dinitrobenzoylphenylglycine Stationary Phase after Achiral Pre-Column Derivatization

High performance liquid chromatography method for the separation of a series of chiral benzyl alcohols on N-(3,5-dinitrobenzoyl)-D-phenylglycine stationary phase (Macherey Nagel, Chiral-2) after pre-column achiral derivatization was developed. Cheap and easy available aromatic acid chlorides were used as derivatization agents. Good to excellent separations of the enantiomers were achieved in all cases in relatively short analytical runs. It was shown that the enantiorecognition depends on the substituents both in the starting alcohol and in the acid chloride. The method presents an efficient alternative to the direct analyses on polysaccharide and cyclodextrine-derived stationary phases.

Pre-column Derivatization-High Performance Liquid Chromatography for the Detection of Monensin in Livestock and Poultry Meat

[Objectives]A method for the detection of monensin in poultry and livestock meat by pre-column derivatization-high performance liquid chromatography was established.[Methods]The sample was extracted with chloroform,derivatized with trichloroacetic acid and 2,4-dinitrophenylhydrazine,and centrifuged to obtain a purified solution.A C18 chromatographic column(4.6 mm×150 mm,5μm)was used for separation with(1.5%)acetic acid water∶methanol(volume ratio)=1∶9 as the mobile phase using a DAD detector for detection,and the external standard method was adopted for peak area quantification.[Results]Monensin had good linearity in the concentration range of 5.00-200 mg/L,with the linear correlation coefficient r 2>0.999;the detection limit was 5.00 mg/kg;the relative standard deviation was smaller than 10%;and the recoveries of standard addition experiment were in the range of 75%-110%.[Conclusions]The method has the advantages of simple pretreatment operation,good derivatization effect and fast detection speed,and is suitable for detecting monensin in poultry and livestock meat.

Coupling of on-Line Pre-Column Oxidative Cleavage and Solid-Phase Enrichment with Liquid Chromatography Using an Eco-Friendly Analytical Procedure to Determine Low Levels of Methotrexate

A simple, sensitive and precise green high-performance liquid chromatographic method including on-line pre-column oxidation combined by column switching with a short Hypersil ODS analytical column (100 mm × 4.0 mm i.d.) for enrichment and separation was developed and validated to determine low levels of methotrexate (MTX). The method was based on oxidative cleavage of MTX into highly fluorescence products, 2,4-diaminopteridine-6-carboxaldehyde and the corresponding 2,4-diaminopteridine-6-carboxylic acid, during the flow of phosphate buffer (0.04 M, pH 3.4) containing the analyte through the packed reactor of cerium (IV) trihydroxyhydroperoxide (CTH) at a flow-rate of 0.2 mL/min and 40℃. The fluorescent products were enriched on the head of ODS analytical column for the final separation. The separation was performed at room temperature using an environmentally friendly mobile phase consisting of ethanol and phosphate buffer (0.04 M, pH 3.4) in the ratio of 10:90 (v/v). The eluent was monitored at emission and excitation wavelengths of 463 and 367 nm, respectively. The method was successfully applied, without any interference from the excipients, for the determination of drug in tablets and vials with a detection limit of 0.06 ng/mL from 500 ?L of sample MTX.

HPLC of Amino Acids and Oligopeptides by Pre-Column Fluorescence Derivatization with 9-Acridine Formyl Chloride

A highly sensitive HPLC method for the detection of amino acids and oligopeptides with 9-acridine formyl chloride by pre-column fluorescence derivatization has been developed. Glycine, glycylglycine, histidine, triglycine and glutathione were separated on a reversed-phase C-18 column with methanol-water-triethylamine eluent, derivatization and chromatographic conditions were optimized. The five derivatives were eluted in 28 min with a good reproducibility. Linear range of the calibration graph was 0.08-260 nmol/ml(-1). The relative standard deviations(n=6) are < 5%. Detection limits (signal-to-noise ratio=3) for the five derivatives are 20-40 fmol.

Evaluation of Baishao(Radix Paeoniae Alba)and Chishao(Radix Paeoniae Rubra)from different origins based on characteristic spectra of amino acids

Objective To evaluate the difference between Baishao(Paeoniae Radix Alba,PRA)and Chishao(Paeoniae Radix Rubra,PRR)from different regions based on the characteristic spectra of amino acids(AAs).Methods Fingerprints of the 21 standard AAs were established using O-phthalaldehyde-9-fluorenylmethyl chloroformate(OPA-FMOC)pre-column derivation method.The AA components in PRA and PRR were characterized qualitatively and quantitatively,and different AAs were screened using pattern recognition technology.Results Twelve AAs were identified in both PRA and PRR.Meanwhile,aspartic acid,glutamic acid,arginine,alanine,and gamma-aminobutyric acid were screened as characteristic components,and their concentrations could effectively distinguish PRA from PRR.Conclusion The established characterization method,which is based on the characteristic spectra of AAs,is accurate,efficient,and sensitive and can effectively distinguish between PRA and PRR from different producing areas,thus providing a reference for the overall characterization and evaluation of Chinese medicinal materials.

Contents Variation Analysis ofγ-Amino Butyric Acid in Semen sojae praeparatum Fermentation Using Online Pre-Column Derivatization-HPLC

This paper reported the contents variation analysis ofγ-amino butyric acid(GABA)in Semen sojae praeparatum(SSP)which is a famous traditional Chinese medicine.High performance liquid chromatography(HPLC)was used and GABA was derivatized by online pre-column derivatization with o-phthalaldehyde(OPA).To validate this method,the precision,stability,repeatability and recovery were discussed.In the concentration range from 0.0125 to 0.400 mg/m L,the calibration curve for GABA was linear and the regression equation was obtained with correlation coefficient(R2)of 0.9999.Relatively high levels of GABA exist in SSP and the content changes of GABA at different time points during the fermenting process were detected.At the"yellow cladding"stage,GABA level was very low or even undetectable;the"secondary fermentation"stage witnessed a rapid increase of GABA content to 1.39-5.52 mg/g,which remained stable after 18 days of"secondary fermentation".This study demonstrated that GABA was generated at the"secondary fermentation"stage,revealing the significance and rationality of the"secondary fermentation"stage in the fermenting process of SSP.On the other hand,it suggested the downside of taking soy isoflavones as the only measurement in existing quality assessment and optimization approach for the fermenting process of SSP.

Application of liquid pre-column capillary electrophoresis technique to the study of interaction between drug enantiomers and human serum albumin

Based on the chiral separation of several basic drugs, dimetindene, tetryzoline, theodrenaline and verapamil, the liquid pre-column capillary electrophoresis (LPC-CE) technique was established. It was used to determine free concentrations of dmg enantiomers in mixed solutions with human serum albumin (HSA). To prevent HSA entering the CE chiral srparation zone, the mobility differences between HSA and drugs under a specific pH condition were employed in thr LPC. Thus, the detection confusion caused by protein was totally avoided. Further study of binding constants determination and protein binding competitions was carried out. The study proves that the LPC technique could be used for complex mrdia, particularly the matrix of protein coexisting with a variety of dmgs.

Reversed-phase high-performance liquid chromatographic analysis of seven pairs of chiral drug enantiomers in transport medium after chiral derivatization

A reversed-phase high-performance liquid chromatographic （RP-HPLC） method was established for the determination of the enantiomers of 7 aryloxy aminopropanol drugs （atenolol, sotalol, celiprolol, carvedilol, metoprolol, propranolol and propafenone） in transport medium. The method involved liquid-liquid extraction of chiral drugs from transport medium, and employed 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl isothiocyanate （GITC, 1.0 mg/mL in acetonitrile） as a pre-column chiral derivatization reagent. After derivatization, the products were separated on an Agilent Zorbax C8 column （150 min×4.6 mm, 5 μm） at 25 ℃. The mobile phase consisted of a mixture of acetonitrile and 0.01 M phosphate buffer （pH 3.5）. The present methods were specific for the determination of enantiomers of each chiral drug. The absolute recoveries of the enantiomers and internal standards were 〉78%. The relative recoveries of the enantiomers were approximately 100%. The intra- and inter-day variations were 〈 15%. The method was reproducible and sufficiently sensitive to determine the enantiomers of seven aryloxy aminopropanol drugs in transport medium. The method could be used to study the transport of atenolol, sotalol, celiprolol, carvedilol, metoprolol, propranolol and propafenone.