High-performance liquid chromatographic analysis of arachidonic acid metabolites by pre-column derivatization using 9-anthryldiazomethane

Arachidonic acid (AA) metabolites produced by washed human platelets and rat macrophages were analyzed by high-performance liquid chromatography (HPLC) using a pre-column derivatization method. The reagent, 9-anthryldiazomethane, used in this study and AA metabolites derivatized by the reagent were purified by gel permeation chromatography (PG-pak C column), prior to normal-phase HPLC analysis. A sample containing eleven derivatives (12-, 15- and 5-hydroxyeicosatetetraenoic acid, 12-L-heptadecatrienoic acid, leukotriene B4, prostaglandins B2, D2, E2 and F2 alpha, thromboxane B2 and 6-keto-prostaglandin F1 alpha) was separated on a normal-phase column (PG-pak B); the detection limit is better than 100 pg for all components.     

High-performance liquid chromatographic method for the quantitation of quinidine and selected quinidine metabolites

A specific and sensitive assay for the separation and quantitation of quinidine, 3-hydroxyquinidine, quinidine-N-oxide, O-desmethylquinidine and dihydroquinidine is presented. The assay is shown to be sensitive to concentrations of 0.1 microgram/ml for all the above compounds when using a serum sample of 0.1 ml. The standard curve demonstrates linearity at concentrations from 0.1 to 5 micrograms/ml. The extraction procedure consists of adjusting the serum to an alkaline pH and extracting once with a mixture of methanol-dichloromethane (15:85). The organic extract is dried and the residue is solubilized in mobile phase. The chromatographic conditions are an isocratic delivery of the mobile phase 0.01 M K2HPO4-acetonitrile (96:4) through a C18 column at ambient temperature. Detection of the compounds of interest is by ultraviolet absorption at a wavelength of 210 nm. For each compound the inter-assay variation is less than 10% and the intra-assay variation is less than 15%. No interfering compounds were detected when a commercially prepared serum spiked with 28 commonly used therapeutic compounds was assayed by this method. The analytical method presented here for the isolation and quantitation of quinidine, several active metabolites, and dihydroquinidine has adequate sensitivity and specificity for monitoring the concentration of quinidine and quinidine metabolites in patient samples.     

Simultaneous reversed-phase extraction of lipoxygenase and cyclooxygenase metabolites of arachidonic acid in nasal secretions: methodological aspects

An improved analytical method for the simultaneous solid-phase extraction of arachidonic acid metabolites in biological samples is described. The major aim of the work was to define the cause of the different recoveries reported in the literature for leukotrienes and hydroxyeicosatetraenoic acids. We used nasal lavages to carry out a comparative study of solid-phase extraction parameters of practical importance in securing good recoveries of leukotrienes and hydroxyeicosatetraenoic acids from biological samples. We evaluated the influence of the pH of the sample, the pH of the water used to wash the extraction cartridge before elution of the adsorbed analytes, the comparative behaviour of commercially available octadecyl adsorbents and the influence of the concentration-evaporation step on final recoveries. Data thus obtained show that there is no significant difference in results when the samples and the water used to wash the cartridge before analyte elution are adjusted to pH values ranging between 4.0 and 7.4. Below pH 4.0, losses may be significant. Furthermore, recoveries can be very much dependent on the type of solid-phase cartridge material and on the eluent evaporation method, especially with regard to aqueous phase removal.     

High-performance liquid chromatography of arachidonic acid metabolites and its application to the determination of leukotriene B4 in stimulated leukocytes

A highly sensitive high-performance liquid chromatography with fluorescence detection for the determination of arachidonic acid metabolites is described. The metabolites are converted into corresponding fluorescent derivatives by reaction with 3-bromomethyl-6,7-methylenedioxy-1-methyl-2(1H)-quinoxalinone in the presence of potassium hydrogen carbonate and 18-crown-6 in acetonitrile. The derivatives are separated on a reversed-phase column (Inertsil ODS) with aqueous acetonitrile and detected fluorimetrically. The detection limits are 5-15 fmol at a signal-to-noise ratio of 3 in a 10-microliter injection volume. The method is applied to the determination of leukotriene B4 produced in stimulated leukocytes.     

High-performance liquid chromatographic quantitation of rhodamines 123 and 110 from tissues and cultured cells

Rhodamine 123 is a fluorescent vital dye which has potential for therapeutic use in cancer treatment. The dye concentrates in mitochondria of normal and neoplastic cells but accumulates in and is toxic to neoplastic cells. When dye-treated cells are irradiated with blue laser light at 514 nm, mitochondrial injury or cell death results. Rhodamine concentration in cultured cells and tumor tissue was quantitated to correlate cell or tumor death with drug dose. A reversed-phase separation of rhodamine 123 was accomplished using a gradient of 0.05 M phosphate buffer pH 2.85 (mobile phase A) and acetonitrile (mobile phase B), 10-80% B in 15 min with a DuPont Golden Series C8 column. Effluent was monitored with a fluorescence detector at 295 nm excitation and 520 nm emission. Stock rhodamine 123 contained approximately 6-8% of rhodamine 110, the parent compound, which eluted at 9.8 min whereas rhodamine 123 eluted at 11.7 min. Structural verification of both compounds by field desorption mass spectrometry was performed. This is the first report of the chemical separation and quantitation of rhodamine 123 from cultured tumor cells or tumor tissue.     

High-performance liquid chromatographic detection and quantitation of amines in must and wine

Three fluorigenic reagents were tried in order to increase the sensitivity of the detection of various amines. The derivatives formed were then used to develop a reversed-phase high-performance liquid chromatographic (HPLC) procedure for the separation of at least five amines. Dns-C1 and fluorescamine were rejected. The chromatogram of Dns-amines from red wine was overcrowded with unidentifiable peaks. It was then postulated that ammonia or phenol derivatives or other by-products of the Dns derivatization reaction interfered with the separation of amines. Fluorescamine, although it produced highly fluorescent derivatives, had the drawback of reacting with di- and polyamines to give more than one derivative and this interfered with the resolution. o-Phthaldialdehyde (OPT) was used successfully for the derivatization of amines in red must and wine. The method involved the reaction of amines with OPT in the presence of mercaptoethanol followed by extraction of the derivatives with ethyl acetate. A reversed-phase HPLC system was developed for the separation of OPT derivatives of agmatine, cadaverine, ethanolamine, histamine, phenylethylamine, putrescine, tryptamine, tyramine, spermine and spermidine within 40 min.     

High-performance liquid chromatography of microbial acid metabolites

The use of high-performance liquid chromatography with a cation-exchange column and effluent monitoring at 210 nm has been evaluated for the profiling of selected microbial metabolites including aliphatic, dicarboxylic, and phenolic acids, as an adjunct to the identification of selected bacteria, detection of bacterial metabolites in foods, and the monitoring of industrial microbial fermentations. Advantages of the technique include the simultaneous profiling of different classes of organic acids without derivatization. Most applications require only qualitative or semi-quantitative data. For others, data are given on the day-to-day reproducibility for several acids.     

High-performance liquid chromatographic determination of the stereoisomeric metabolites of ibuprofen

A stereospecific reversed-phase high-performance liquid chromatographic (HPLC) method has been developed to simultaneously quantitate the stereoisomers of the two major metabolites of ibuprofen: hydroxyibuprofen and carboxyibuprofen. The metabolites were derivatized with S-(alpha)-methylbenzylamine to form diastereomeric amides which were separated and quantified on a C8 column. The validity of the stereoselective assay was confirmed by comparison with a non-stereoselective HPLC method. The stereoselective assay was applied to the quantification of all the stereoisomeric ibuprofen metabolites in urine from human volunteers dosed with racemic ibuprofen or the individual enantiomers of ibuprofen. Significant substrate and product stereo-selectivities were observed in the formation of carboxyibuprofen.     

High-performance liquid chromatographic separation of femtomolar quantities of endogenous carboxylic acids, including arachidonic acid metabolites, as 4-bromomethyl-7-acetoxycoumarin derivatives

A major limitation of high-performance liquid chromatographic techniques for measuring biologically active eicosanoids has been the inadequate sensitivity of most on-line detection systems. In addition, the availability of a technique suitable for measuring small quantities of non-esterified fatty acids (NEFAs) in plasma would allow longitudinal studies of plasma levels of these lipids in small animals. To improve the sensitivity of detection, the compounds with acyl groups containing carboxylic acids were derivatized with the highly fluorescent compound, 4-bromomethyl-7-acetoxycoumarin. All classes of NEFA and arachidonic acid metabolites, including the cyclooxygenase and lipoxygenase products, and hydroxy acid compounds could be derivatized with this reagent. The derivatized metabolites were separated with a reversed-phase high-performance liquid chromatographic system using a radial compression column and a gradient elution technique. Reproducible measurements of plasma NEFAs from as little as 5 microliters of plasma, and femtomolar concentrations of eicosanoids, could be detected using an on-line fluorescent spectrometer. This improvement in sensitivity should permit the quantification of all eicosanoids, including the leukotrienes, in biologic fluids and the longitudinal measurement of changes in plasma NEFA levels in small animals.     

2,6-萘二甲酸的高效液相色谱分析

建立了一种2,6-二异丙基萘液相氧化制备2,6-萘二甲酸反应产物的反相高效液相色谱分析方法。色谱柱为Zorbax eclipse XDBC8,流动相为乙腈(A)和体积分数O．001％三氟乙酸水溶液(B),y(A)：V(B)=55：45,流速为O．85mL/min,紫外检测波长为210nm,苯甲酸为内标物。该方法相对标准偏差为0．049％,可用于2,6-萘二甲酸的合成工艺以及2,6-二异丙基萘的氧化动力学研究。     

High-performance liquid chromatographic fractionation and characterization of fulvic acid

High-performance immobilized metal ion affinity chromatography (HP-IMAC) was used to fractionate humic substances (HS) based on their affinity for the immobilized copper(II) ion using acidic and glycine eluents. The work was carried out with two naturally occurring aqueous fulvic acids and commercially available Suwannee River fulvic acid. The IMAC-fractionated HS were then characterized by reversed-phase high-performance liquid chromatography (RP-HPLC) and size exclusion chromatography. The results showed that the affinity HS fraction eluted first using an acidic pH=2 eluent exhibited a relatively high hydrophilic character, whereas the fraction eluted later using a glycine eluent exhibited both a higher hydrophobic character and larger molecular size. On the other hand, the HS fraction with no affinity for the immobilized copper had low molecular size. The affinity of the HS fraction for copper(II) increased with increasing molecular weight. Based on the composite results of three different HS, it is evident that strong relationships exist between affinity, molecular weight, and hydrophilic/hydrophobic properties during the HP-IMAC fractionation. The results presented here have significance for understanding the nature of chemical interactions at the molecular level between dissolved organic matter and trace metals. IMAC, coupled with other liquid chromatographic strategies, is a promising tool for chemical fractionation and characterization of HS.     

High-performance liquid chromatographic analysis of tiaprofenic acid and its metabolites in plasma and urine by direct injection

A rapid, convenient, sensitive and selective reversed-phase high-performance liquid chromatographic method was developed to measure tiaprofenic acid, its reduced and oxidized metabolites and their conjugates in biological fluids. The method involved direct injections of plasma and urine samples into the chromatograph before and after alkaline hydrolysis of the conjugates. Concentrations as low as 0.5 micrograms/ml of the drug in plasma and urine were quantifiable. The method was suitable for analysis of tiaprofenic acid and its metabolites in biological fluids after administration of therapeutic doses. Several other non-steroidal anti-inflammatory drugs which were applied to the system did not interfere with the assay.     

High-performance liquid chromatographic separation of peptide and amino acid stereoisomers

Several high-performance liquid chromatographic (HPLC) methods are described for separation of peptide stereoisomers which are not well resolved by traditional reversed-phase chromatography. These chiral HPLC methods include investigations with a beta-cyclodextrin column, a Pirkle D-Phenyl Glycine column and a Chiral-Pak WH column. A method based on derivatization of dipeptides with a chiral reagent, N-acetyl-L-cysteine and o-phthalaldehyde, is also discussed. A series of linear and cyclic dipeptides and modified amino acids were chromatographed on the four systems. Resolution varied for the four different systems depending on the types of compounds that were chromatographed.     

High-performance liquid chromatographic determination of pyridostigmine bromide, nicotine, and their metabolites in rat plasma and urine

This study reports on the development of a rapid and simple method for the determination of the antinerve agent drug pyridostigmine bromide (3-dimethylaminocarbonyloxy-N-methyl pyridinium bromide) (PB), its metabolite N-methyl-3-hydroxypyridinium bromide, nicotine (S-1-methyl-5-(3-pyridyl)-2-pyrrolidine), and its metabolites nornicotine (2-(3-pyridyl)pyrrolidine) and cotinine (S-1-methyl-5-(3-pyridyl)-2-pyrrolidone) in rat plasma and urine. The compounds are extracted and eluted by methanol and acetonitrile using C18 Sep-Pak cartridges and separated using high-performance liquid chromatography by a gradient of methanol, acetonitrile, and water (pH 3.2) at a flow rate of 0.8 mL/min in a period of 14 min. UV detection was at 260 nm for nicotine and its metabolites and at 280 nm for PB and its metabolite. The limits of detection ranged between 20 and 70 ng/mL, and the limits of quantitation were 50-100 ng/mL. The average percent recovery of five spiked plasma samples were 85.7 +/- 7.3%, 80.4 +/- 5.8%, 78.9 +/- 5.4%, 76.7 +/- 6.4%, and 79.7 +/- 5.7% and for urine were 85.9 +/- 5.9%, 75.5 +/- 6.9%, 82.6 +/- 7.9%, 73.6 +/- 5.9%, and 77.7 +/- 6.3% for nicotine, nornicotine, cotinine, PB, and N-methyl-3-hydroxypyridinium bromide, respectively. The calibration curves for standard solutions of the compounds of peak areas and concentration are linear for a range between 100 and 1,000 ng/mL. This method is applied in order to analyze the previously mentioned chemicals and metabolites following their oral administration in rats.