High-performance liquid chromatographic determination of vinblastine, 4-O-deacetylvinblastine and the potential metabolite 4-O-deacetylvinblastine-3-oic acid in biological fluids.

Procedures for the determination of vinblastine (VBL), 4-O-deacetylvinblastine (DVBL) and 4-O-deacetylvinblastine-3-oic acid (DVBLA) in biological samples using high-performance liquid chromatography (HPLC) combined with selective sample clean-up are presented. VBL and DVBL were determined in plasma and urine using ion-exchange normal-phase HPLC with fluorescence detection. The limit of detection was 1 microgram/l for both compounds using a 500-microliter sample. Successful chromatographic analyses of DVBLA were achieved by using a glass column packed with 5-microns Hypersil ODS and acetonitrile-0.05 M phosphate buffer (pH 2.7) (23:77, v/v). Positive identification was supported by the use of diode-array detection. The limit of detection (at 270 nm) was 10 micrograms/l using 1-ml samples.     

BIO-Fully Automated Sample Treatment high-performance liquid chromatography and radioimmunoassay for leukotriene E4 in human urine from asthmatics

BIO-Fully Automated Sample Treatment (BIO-FAST) high-performance liquid chromatography (HPLC) is a sophisticated column-switching technique in which a fresh pre-column is used for each sample prior to reversed-phase HPLC. The pre-columns, Varian Advanced Automated Sample Processor (AASP) cartridges, are held and automatically advanced by the Varian AASP. A rapid and efficient extraction and separation for leukotrienes C4 and E4 from human urine has been developed using a C8 cartridge and subsequent C18 analytical HPLC column. Quantitation of leukotriene E4, accomplished by post-column radioimmunoassay, shows significantly increased leukotriene E4 concentrations in urine samples from asthmatics after antigen challenge. This further confirms an active role for leukotrienes in the pathogenesis of bronchial asthma.     

Determination of dopamine-3- and 4-O-sulphate in human plasma and urine by anion-exchange high-performance liquid chromatography with fluorimetric detection

A high-performance liquid chromatographic (HPLC) method is reported for the determination of dopamine-3- and -4-O-sulphate isomers in human plasma and urine using an anion exchanger coupled with post-column hydrolysis and fluorimetric detection. Samples of plasma or urine are partially purified on Dowex 1 and Dowex 50 columns and separated using HPLC. These compounds are then hydrolysed and determined automatically by the p-aminobenzoic acid method in a continuous-flow reaction system. As the p-aminobenzoic acid method is very specific for dopamine, it is also possible to determine the isomers by injecting 5-20 microliter of urine or 100-200 microliter of deproteinized plasma directly into the HPLC system without clean-up. The detection limit of the method for both isomers is 0.3 pmol. In normal subjects, the plasma levels of dopamine-3- and -4-O-sulphate are 26.5 (S.D. 11.1) and 2.68 (S.D. 0.34) pmol/ml, and their urinary excretion rates are 1.73 (S.D. 0.56) and 0.27 (S.D. 0.04) nmol/min, respectively. Thus the two isomers are present in both plasma and urine and their urinary excretions reflect directly their plasma levels.     

Photolytic interface for high-performance liquid chromatography--chemiluminescence detection of non-volatile N-nitroso compounds

A photolytic interface between high-performance liquid chromatography (HPLC) and a chemiluminescence detector has been developed for the trace detection of non-volatile N-nitroso compounds in biological matrices. A chromatographic effluent containing separated N-nitrosoamino acids and N-nitrosamides is introduced into a glass coil with a purge stream of He and irradiated with ultraviolet light. Nitrogen oxide, cleaved by photolysis, is separated rapidly from the solvent through a series of cold traps and carried by the He into the reaction chamber of a chemiluminescence detector. The method is compatible with most types of HPLC, especially reversed-phase, and yields low-nanogram sensitivity for underivatised N-nitrosoamino acids and N-nitrosamides. The detection of a model N-nitrosamide, trimethylnitrosourea, in spiked porcine gastric fluid (42 micrograms l-1), and of N-nitrosoproline and N-nitroso-1,3-thiazolidine-4-carboxylic acid, in spiked human urine (7-8 micrograms l-1), is demonstrated.     

Derivatization of isolated endogenous butyrobetaine with 4'-bromophenacyl trifluoromethanesulfonate followed by high-performance liquid chromatography.

A method for the isolation and chromatography of butyrobetaine from plasma, urine, and liver is described. The recovery of [3H-methyl]butyrobetaine from spiked biological samples was from 76-80%. Spiked samples then were derivatized with 4'-bromophenacyl trifluoromethanesulfonate and the butyrobetaine 4'-bromophenacyl ester was isolated by high-performance liquid chromatography (HPLC). Radioactivity eluted in a single peak which co-chromatographed with authentic butyrobetaine 4'-bromophenacyl ester. Two identical liver specimens were treated according to this isolation procedure. Prior to derivatization, one specimen was treated with butyrobetaine hydroxylase. After derivatization, there was no butyrobetaine 4'-bromophenacyl ester peak in the specimen treated with butyrobetaine hydroxylase. The HPLC detection sensitivity to butyrobetaine 4'-bromophenacyl ester was 1 pmol injected with a signal-to-noise greater than 2:1.     

Determination of selenium species in human urine by high performance liquid chromatography and inductively coupled plasma mass spectrometry

A method developed to determine organic and inorganic selenium species in human urine samples is presented in detail. After a simple sample treatment based on elimination of non-charged organic compounds, selenium species were separated by high performance liquid chromatography (HPLC) on a Spherisorb 5 ODS/AMINO column using two different chromatographic conditions: phosphate buffers at pH 2.8 and 6.0. Detection was carried out using an on-line inductively coupled plasma mass spectrometer (ICP-MS). Trimethylselenonium ion and two unknown selenium species in urine samples were found. Selenium species were shown to have stability problems, with the maximum allowed storage time of 1 week.     

Separation of 4-dimethylamino-6-(4-methoxy-1-naphthyl)-1,3,5-triazine-2-hydrazine derivatives of carbonyl compounds by reversed-phase capillary electrochromatography

4-Dimethylamino-6-(4-methoxy-1-naphthyl)-1,3,5-triazine-2-hydrazine (DMNTH) is a novel derivatizing reagent specially designed for the determination of carbonyl compounds. In this work, we describe the separation of DMNTH-derivatized carbonyl compounds by reversed-phase capillary electrochromatography (CEC). After systematic investigations of the effects of experimental conditions viz. pH and concentration of buffer, type of stationary phase, injection volume of sample, organic modifier, and temperature, optimal conditions were found. The sample compounds, which were separated with gradient high performance liquid chromatography (HPLC), were separated by CEC under isocratic elution due to the high efficiency. Comparisons of separations by CEC and micellar electrokinetic chromatography (MEKC) were made.     

A methodological and metabolite identification study of the metabolism of S-carboxymethyl-L-cysteine in man

Summary The analysis of 08:00–16:00 (0–8) hour urine collections following oral S-carboxymethyl-L-cysteine administration of 750 mg to 30 individuals identified S-carboxymethyl-L-cysteine, S-carboxymethyl-L-cysteine S-oxide, S-methyl-L-cysteine and S-methyl-L-cysteine S-oxide as the major urinary drug related compounds. No S-(carboxymethylthio)-L-cysteine mixed disulphide metabolite was found in the 08:00–16:00 hour urine collection but the metabolite was detected in the 16:00–00:00 (8–16) hour urune collection by paper chromatography, TLC and HPLC. The production of the S-oxide metabolites 08:00–16:00 hour urine collection) and the mixed disulphide metabolite (16:00–00:00 hour urine collection) were both shown to be biomodally distributed. A significant linear correlation of the S-oxides recovered following 08:00–16:00 hour urine collection as analysed by paper chromatography, TLC and HPLC is reported.     

Biodegradation studies of 4-fluorobenzoic acid and 4-fluorocinnamic acid: an evaluation of membrane inlet mass spectrometry as an alternative to high performance liquid chromatography and ion chromatography

The biodegradation of 4-fluorobenzoic acid (4-FBA) and 4-fluorocinnamic acid (4-FCA) has been monitored by membrane inlet mass spectrometry (MIMS) using a hollow-fibre silicone membrane. A novel in-membrane pre-concentration/thermal desorption (IMP-MIMS) technique was employed for MIMS analysis using an oven temperature profile that allowed semi-volatile organic compounds to be accumulated in the membrane and then released by rapid heating. Air drying of the membrane between the analyte pre-concentration and thermal desorption stages improved mass spectrometric performance by removing residual water from the membrane. The concentrations of 4-FBA and 4-FCA determined by MIMS compare well with data obtained by high performance liquid chromatography (HPLC). Stoichiometric amounts of fluoride were monitored using ion chromatography (IC). Intermediates in the biodegradation pathway were identified by liquid chromatography/mass spectrometry (LC/MS). These data establish the potential of MIMS as an alternative to chromatographic methods for monitoring the biodegradation of semi-volatile organic compounds.     

Determination of paraquat and diquat in human body fluids by high-performance liquid chromatography/tandem mass spectrometry

Paraquat (PQ) and diquat (DQ) in human whole blood and urine were analyzed by high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) with positive ion electrospray ionization (ESI). The compounds were extracted with Sep-Pak C18 cartridges from whole blood and urine samples containing ethyl paraquat as an internal standard. The separation of PQ and DQ was carried out using ion-pair chromatography with heptafluorobutyric acid in 20 mM ammonium acetate and acetonitrile gradient elution for successful coupling with MS. Both compounds formed base peaks due to [M-H]+ ions by HPLC/ESI-MS and the product ions produced from each [M-H]+ ion by HPLC/MS/MS. Selective reaction monitoring (SRM) showed much higher sensitivity for both body fluids. Therefore, a detailed procedure for the detection of compounds by SRM with HPLC/MS/MS was established and carefully validated. The recoveries of PQ and DQ were 80.8-95.4% for whole blood and 84.2-96.7% for urine. The calibration curves for PQ and DQ showed excellent linearity in the range of 25-400 ng ml(-1) of whole blood and urine. The detection limits were 10 ng ml(-1) for PQ and 5 ng ml(-1) for DQ in both body fluids. The intra- and inter-day precision for both compounds in whole blood and urine samples were not greater than 13.0%. The data obtained from the determination of PQ and DQ in rat blood after oral administration of the compounds are also presented.     

锦灯笼果实的化学成分研究

从锦灯笼中分离得到5个甾体类化合物,分别鉴定为酸浆素A(1,alkekengilin A)、酸浆素B(2,alkekengilin B)、酸浆苦素D(3,physalin D)、酸浆苦素P(4,physalin P)、4,7-二去氢新酸浆苦素B(5,4,7-didehydro-neophysalin B).其中酸浆素A(1,alkekengilin A)和酸浆素B(2,alkekengilin B)为新的天然产物.运用现代波谱技术对上述化合物的结构进行了确证.     

HPLC-based measurement of the chelator 1,2-dimethyl-3-hydroxy-pyrid-4-one (L1) and its iron complex for pharmacokinetic studies in humans

An improved HPLC based method to assay the oral active iron chelator 1,2-dimethyl-3-hydroxypyrid-4-one (L1, CP20) in serum and urine is described. The L1 peak has been well separated from other endogenous compounds, allowing the exact determination of the drug in both biological fluids. Moreover urinary iron excretion due to L1 therapy has been monitored by measuring urine Fe-(L1)₃ complex concentrations using reverse phase HPLC and subsequent detection at 450 nm. In patients and normal volunteers receiving this drug there is a good correlation between urine iron excretion measured by AAS and by the HPLC based method.     

Rapid preparative isolation of a new phenylpropanoid glycoside and four minor compounds from Sparganium stoloniferum using high-speed counter-current chromatography as a fractionation tool

A rapid high-speed counter-current chromatography (HSCCC) method was used to isolate five minor compounds from rhizome of Sparganium stoloniferum namely San Leng in Chinese, including two phenylpropanoid glycosides, sparganiaside A (1) and 1-O-feruloyl-3-p-coumaroylglycerol (2), and three aromatic acids, vanillic acid (3), p-hydroxylcinnamic acid (4), and p-hydroxybenzoic acid (5), of which, compound 1 was a new one. Five compounds were preparatively enriched at top efficiency by one-step HSCCC operation in the isolation procedure. A suitable solvent system composed of chloroform-methanol-water (4:3.5:1.8, v/v/v) was used. And the operation time was less than 4 h. The purities of compounds (1-5) in the enriched fractions were determined to be 75.8%, 66.3%, 90.6%, 79.9%, and 98.2%, respectively. The mean recoveries of the five compounds were 84.8%, 87.3%, 81.8%, 90.3%, and 92.7%, respectively. Compounds 1-4 were further purified by semi-preparative high-performance liquid chromatography (HPLC). This is the first report on the use of HSCCC as a fractionation tool for preparative isolation of minor compounds from S. stoloniferum. The method was proved to be rapid, convenient, high yield, and low cost. HSCCC was shown to be a quick and effective tool in isolation of natural products even though the compounds were not abundant.     

Separation of capsular polysaccharide K4 and defructosylated K4 derived disaccharides by high-performance capillary electrophoresis and high-performance liquid chromatography

A rapid, highly sensitive and reproducible high-performance capillary electrophoresis (HPCE) method (electrokinetic chromatography with sodium dodecyl sulfate) is described for the determination of disaccharides present in the polysaccharide from the uropathogenic Escherichia coli K4 bacteria (05:K4:H4) and its defructosylated product. Following chondroitinase digestion of K4 and its derivative, the two disaccharides, DeltaHexAFrc-GalNAc for K4 and deltaHexA-GalNAc for defructosylated K4, are separated and readily determined within 20 min on an uncoated fused-silica capillary using normal polarity at 20 kV and detection at 230 nm. Comparison was made by separation of these two disaccharides in isocratic strong-anion exchange HPLC. A linear relationship was found for the two unsaturated disaccharides over a wide range of concentrations, from approximately 0.5 to 5 micro g for high-performance liquid chromatography (HPLC) and from approximately 0.06 to 0.3 micro g for HPCE. The HPCE separation produced a greater detection sensitivity (about 10 times greater) than HPLC. The described methods were used to evaluate the defructosylation process of K4 under drastic acid conditions. Good correspondence was found for the amount of unsaturated disaccharides for the two techniques.     

Enzymic preparation of dioxygen-18 labelled leukotriene E4 and its use in quantitative gas chromatography-mass spectrometry.

A simple and rapid method is described for the preparation of a stable isotope oxygen-18 labelled leukotriene E4 (LTE4). Oxygen-18 labelling of LTE4 methyl ester in oxygen-18 water catalysed by a pig liver esterase resulted in the incorporation of two oxygen-18 atoms in the carboxylic group of LTE4 to the extent of 89.8% ([18O2]LTE4) and one oxygen-18 atom to the extent of 9.4% ([16O18O]LTE4), with only 0.7% remaining unchanged ([16O2]LTE4). [18O2]LTE4 was found not to back-exchange following incubation in acidified urine (pH 4.0) at 4 degrees C for up to 20 h. [18O2]LTE4 was demonstrated to be a useful internal standard in a method for the quantitative determination of LTE4 in human urine involving high-performance liquid chromatography and gas chromatography with negative-ion chemical ionization tandem mass spectrometry: the concentration of LTE4 in a 24-h urine sample of a healthy subject was determined to be 68.1 pg/ml.     

An Analytical Approach to Metabolic Profiling of Aromatic Compounds Using Liquid Chromatography

Abstract

An analytical approach to metabolic profiling of aromatic compounds is described for both conjugated and “free” metabolites in biological systems. Initially, an ethyl acetate extraction removes the less polar metabolites. A salting-out procedure using Sephadex G-10 is combined with reverse-phase high-performance liquid chromatography (HPLC) to analyze the water-soluble conjugates directly using sequential uv and fluorescence detection. The “free” metabolites are also derived from individual conjugate peaks by enzymatic or hydrolytic procedures and then re-chromatographed by HPLC. Metabolites in the ethyl acetate fraction are similarly analyzed by reverse-phase HPLC. The utility of this method is demonstrated for 2-acetylaminofluorene (2-AAF and 4-ethylsulfonyl-naphthalene-1-sulfonamide (ENS), two compounds of interest in the study of murine bladder carcinogenesis.     

Development of HPLC-MS/MS method for the simultaneous determination of environmental phenols in human urine

We present a sensitive method for simultaneous determination of bisphenol A (BPA), benzophenone-3 (BP-3), 4-tert-octylphenol (t-OP), ortho-phenylphenol (OPP), four parabens (methyl, ethyl, propyl, butyl parabens) and five chlorophenols (2,4-dichlorophenol (2,4-DCP), 2,5-dichlorophenol (2,5-DCP), 2,4,5-trichlorophenol (2,4,5-triclorophenol), 2,4,6-trichlorophenol (2,4,6-TCP), and triclosan (TCS)), in human urine by high-pressure liquid chromatography (HPLC) mass spectrometry (MS). Samples were processed using enzymatic deconjugation of glucuronides followed by solid phase extraction (SPE) on a C18 cartridge and the eluate was concentrated. Analytes were separated by reversed-phase HPLC and then detected by atmospheric pressure chemical ionisation (APCI) MS and quantified by isotope dilution method. We describe details for optimisation of each step of the procedure. The sample treatment steps are straightforward and not labour-intensive and, therefore, permit a high sample throughput with excellent prospects for automation. This method shows low inter-day variation, and detection limits for most of the compounds are below 1 ng/mL in 1 mL of urine. The method accuracy was also verified by the analysis of proficiency testing urine samples.     

Determination of 4-amino-5-ethyl-3-thiophenecarboxylic acid methyl ester and its acid metabolite in plasma and urine by high-performance liquid chromatography

Two separate, rapid, sensitive and selective high-performance liquid chromatographic (HPLC) assays were developed for the determination of 4-amino-5-ethyl-3-thiophene-carboxylic acid methyl ester (I) and its acid metabolite, 4-amino-5-ethyl-3-thiophene-carboxylic acid (II), in plasma and urine. The analysis of I is performed directly on a hexane extract of plasma or urine (buffered to pH 11) by normal-phase HPLC analysis using a 10-micron silica gel column with an eluting solvent of hexane-ethanol (95:5) and UV detection of the effluent at 254 nm. A methyl analogue, 4-amino-5-methyl-3-thiophenecarboxylic acid methyl ester, was used as the internal standard. The analysis of II is performed on the residue of either a diethyl-ether-washed protein-free filtrate of plasma or a methylene chloride-isopropanol (95:5) extract of urine (buffered to pH 5.3) using a 10-micron alkyl phenyl (reversed-phase) column with an eluting solvent of water-methanol-1 M phosphoric acid, pH 2.5 (70:30:0.05) with UV detection of the effluent at 254 nm. An isopropyl analogue, 4-amino-5-isopropylthiophene-3-carboxylic acid (IV), was used as the internal standard. The assay of compounds I and II were applied to the determination of plasma and urine concentrations of I and II in the dog and in man following oral administration of I X HCl. The data obtained demonstrated the extremely rapid and virtually complete deesterification of I (ester) to II (acid) in both species.     

Simultaneous determination of 3,4-dihydroxymandelic acid, 4-hydroxy-3-methoxymandelic acid, 3,4-dihydroxyphenylglycol, 4-hydroxy-3-methoxyphenylglycol, and their precursors, in human urine by HPLC with electrochemical detection

Summary A method has been developed for the quantification of urinary 3,4-dihydroxymandelic acid (DOMA), 4-hydroxy-3-methoxymandelic acid (VMA), 3,4-dihydroxyphenylglycol (DHPG), and 4-hydroxy-3-methoxyphenyglycol (MHPG). Separation and determination of these compounds in biological samples was previously thought to be very difficult. In this work the separation has been achieved by reversed-phase high-performance liquid chromatography with step-wise gradient elution with three mobile phases. The conditions for coulometric detection have been optimized for effective determination of these compounds. In analysis of a sample of human urine, after a simple deproteinization proceudre, DOMA, VMA, DHPG, and MHPG were separated from interferences and quantified successfully; the average levels of these compounds in six different samples were 33.87±1.03, 1202±41.3, 31.3±1.92, and 80.6±2.15 μg (24 h)⁻¹, respectively. Their precursors E, MN, DOPA, DA, NE, DOPAC, HVA, 3MT, and NMN, and the indolamine 5HT and its metabolite 5HIAA (a list of abbreviations is given at the end of the paper) can also be determined simultaneously in the same chromatographic run. The overlapping peak of DHPG was resolved by deconvolution.