A rapid miniature thin layer chromatography system for analysis of prostaglandins an lipoxygenase products

We have developed a miniature thin layer chromatography system for rapidly identifying the major arachidonate metabolites in a radiolabeled form elaborated by cells or tissues. This system separates the total spectrum of cyclooxygenase products and several of the most commonly found lipoxygenase pathway metabolites, while retaining the fine resolution of larger-scale and more time-consuming procedures. It requires less than one hour for extraction of metabolites, chromatography, and counting.     

Column-switching high-performance liquid chromatographic detection of pholcodine and its metabolites in urine with fluorescence and electrochemical detection

A sensitive and selective method for the detection of pholcodine and its metabolite morphine in urine using high-performance liquid chromatography is described. It involves on-line clean-up of urine on a trace enrichment column packed with a polymeric strong cation-exchange material. Pholcodine and its metabolites were separated on two analytical columns with different selectivities. Pholcodine was detected by a fluorescence detector and morphine was detected electrochemically. One system, based on reversed-phase chromatography, applied a polystyrene—divinylbenzene column and gradient elution. The other system was based on normal-phase chromatography with a silica column and isocratic elution. Morphine was confirmed to be a metabolite of pholcodine by reversed-phase chromatography and electrochemical detection. Two unidentified metabolites of pholcodine were separated from pholcodine by normal-phase chromatography and detected by fluorescence detection.     

The metabolism of 3-methylcholanthrene by rat liver microsomes - a reinvestigation

Metabolites of 3-methylcholanthrene (3-MC) formed by rat liver microsomes were analyzed by high pressure liquid chromatography. The metabolic profile is significantly different from previous studies using thin layer chromatography. The major metabolites include 1-and 2-hydroxy-3-MC. Use of the high pressure liquid chromatographic system allows for the separation of at least seven new metabolites. The amounts of three of these new metabolites are substantially decreased when the potent epoxide hydrase inhibitor 3,3,3-trichloropropene oxide is added to the incubation system. These results then suggest the formation of epoxides of 3-methylcholanthrene other than the K-region oxide.     

The use of high-performance liquid chromatography and diode array detection in fungal chemotaxonomy based on profiles of secondary metabolites

FRISVAD, J. C, 1989. The use of high-performance liquid chromatography and diode array detection in fungal chemotaxonomy based on profiles of secondary metabolites. Fungal chemotaxonomy (that part dealing with secondary metabolites) has often been based on thin layer chromatography (TLC) and visual or UV inspection of separated spots, before and after different chemical treatments. The identity of a small proportion of the spots can be suggested based on known internal and external standards. In most chemotaxonomical studies it is impossible to isolate, purify and identify all secondary metabolites produced, due to restraints of time and resources. High performance liquid chromatography (HPLC) of fungal extracts may have some advantages over TLC, but the problems mentioned above remain. These problems have been approached by using an alkylphenone retention time index in a reversed phase HPLC system combined with the use of a diode array UV-VIS detector. High performance thin layer chromatography is used for further confirmation of identity of the secondary metabolites. A particular advantage of this method is that the number of biosynthetic families or groups ('chemosyndromes') can be detected, as biosynthetically related metabolites usually have the same chromophores and UV-VIS spectra. Results obtained from Penicillium, Aspergillus and Fusanum species have shown that each species produces 5 to 15 different biosynthetic families of secondaiy metabolites, indicating that good chromatography data may be sufficient to identify species in the three genera. The use of the technique is exemplified by data on Aspergillus and Talaromyces species.     

High-performance liquid chromatographic separation and isolation of quinidine and quinine metabolites in rat urine

A procedure for the separation and isolation of the urinary metabolites of quinidine and quinine by reversed-phase high-performance liquid chromatography is described. Nine metabolites of quinidine and eight metabolites of quinine were detected in the urine of male Sprague-Dawley rats after a single dose of quinidine or quinine (50 mg kg^?1). Following extraction from urine, the metabolites were separated on either an analytical or a semi-preparative reversed-phase column by gradient elution. After isolation and derivatization, the metabolites were analyzed by gas chromatography and gas chromatography—mass spectrometry.     

An analytical method for the separation of dopamine metabolites in cellular extracts by high-pressure liquid chromatography

An analytical scheme using high-performance liquid chromatography (HPLC) has been developed to separate radiolabeled catecholamines in cell extracts derived from mammalian cells grown in tissue culture. Four different types of chromatographic systems have been employed to effect separations of groups of metabolites that possess similar organic functional groups. Data obtained by thin-layer chromatography are also presented and it is demonstrated that HPLC is the system of choice for the separation and quantitative analysis of metabolites of dopamine in physiological fluids.     

Screening of eltanolone metabolites in dog urine by anion-exchange/reversed-phase liquid chromatography and mass spectrometry

Strong anion-exchange (SAX) chromatography and reversed-phase liquid chromatography (RPLC) followed by different mass spectrometric techniques for the separation and identification of conjugated and unconjugated ¹⁴C-labelled eltanolone (5β-Pregnan-3α-ol-20-one) metabolites in biological fluids are presented. Conjugates of estradiol were used as model compounds for the development of a SAX based group separation of neutral steroids, glucuronides, sulfates and di-conjugated steroids. The usefulness of the technique is demonstrated by the analysis of ¹⁴C-labelled eltanolone metabolites in dog urine. The analytical SAX column used prior to RPLC improved the capacity to separate the metabolites from each other and from endogenous components, compared to a single reversed-phase system. Liquid chromatography negative ion electrospray-mass spectrometry (LC–ESI-MS) was used for the molecular mass determination of conjugated eltanolone metabolites. Unconjugated metabolites and hydrolysed conjugates were identified using gas chromatography–mass spectrometry with an electron impact ion source (GC–MS) after trimethylsilyl (TMS) derivatization. An unexpected finding in dog urine was the diglucuronide formation of eltanolone (presumably after enolisation of its carbonyl group).     

Rapid analysis of naphthalene and its metabolites in biological systems: Determination by high-performance liquid chromatography/fluorescence detection and by plasma desorption/chemical ionizations mass spectometry

A rapid procedure for the determination of naphthalene and its metabolites in bile of rainbow trout and mice is described. The integrated analytical techniques combine high-performance liquid chromatography/ultraviolet fluorescence detection and plasma desotption/chemical ionization mass spectrometry for identification and quantitation. After separation by reverse-phase liquid chromatography, naphthalene and its metablolites are detected and quantitated by ultraviolet fluoresence spectometry. Identification of two metabolites is confirmed by mass spectometry. A direct insertion probe tip for a conventional chemical ionization mass spectometer was modified to obtain spectra of thermally labile compounds. A spectrum of less than 100 ng of naphthyl glucuronide, a labile glucuronic acid conjugate of 1-naphthol, was obtained with this system.     

Rapid determination of doxorubicin and its fluorescent metabolites by high pressure liquid chromatography.

An original method for the separation and quantitation of doxorubicin (DOX) and its metabolites by high-pressure liquid chromatography and fluorometry is described. Doxorubicin and its derivatives are extracted from biological samples in a rapid, non-destructive manner, with a recovery close to 100%. The different compounds are rapidly separated by high-pressure liquid chromatography using an eluant system containing magnesium chloride, and detected quantitatively by fluorometry down to a concentration of 1.5 ng/ml in less than 5 min. Using this method, we have determined doxorubicin and its metabolites in plasma and urine, after an intravenous injection into DBA₂ and NMRI mice.     

Human myeloid leukemia cells metabolize 25-hydroxyvitamin D3 in vitro

Human promyelocytic leukemia cells (HL-60) converted 25-hydroxyvitamin D3 to two more polar metabolites during in vitro incubations. A two-step high pressure liquid chromatography system revealed two unique elution positions of those leukemic cell-derived metabolites that exactly co-migrated with the elution positions of 5(Z)-19-nor-10-oxo-25-hydroxyvitamin D3 and 5(E)-19-nor-10-oxo-25-hydroxyvitamin D3, respectively. These unique metabolites did not bind specifically to a protein receptor for 1,25-dihydroxyvitamin D3.     

Methods for the complete analysis of 5-fluorouracil metabolites in cell extracts

Methodology that permits complete analysis of the intracellular metabolites of 5-fluorouracil (FUra) has been developed. A high-pressure liquid chromatography system that is capable of separating all metabolites of FUra found in acid-soluble cell extracts is described. In addition to the expected FUra metabolites, FUDP-hexoses were found to be present in large amounts in L121O cells treated with FUra. Improved procedures that permit quantitation of the FdUMP which is covalently bound to dTMP synthetase, as well as the total intracellular FdUMP levels are described; the latter is accomplished by dissociation of the FdUMP-dTMP synthetase complex in sonicated cell extracts followed by phosphatase treatment and subsequent high-pressure liquid chromatography analysis of FdUrd. An example of the integrated methodology in which all metabolites of FUra metabolism are analyzed over a 6-h exposure period of L1210 cells to [6-³H]FUra is provided.     

Isolation and identification of 4-hydroxy- and 4-oxoretinoic acid. In vitro metabolites of all-trans-retinoic acid in hamster trachea and liver.

Incubation of [3H]retinoic acid in the presence of hamster liver 10000g supernatant produces several metabolites that are more polar than the parent compound. Two of these metabolites are identical with synthetic all-trans-4-hydroxyretinoic acid and all-trans-4-oxoretinoic acid both in ultraviolet absorption and mass spectral characteristics and in migration rates on two different reverse-phase high-pressure liquid chromatographic systems. The metabolites produced in a cell-free liver incubation reaction also migrate on a high-pressure liquid chromatography column together with metabolites isolated from a tracheal organ culture system. Both the metabolites and the synthetic standards show less biological activity than the parent all-trans-retinoic acid in a tracheal organ culture assay.     

Gas chromatographic–mass spectrometry and gas chromatographic–Fourier transform infrared spectroscopy assay for the simultaneous identification of fentanyl metabolites

Fentanyl, a synthetic opioid, undergoes important biotransformation to several metabolites. A gas chromatographic–mass spectrometric assay was applied for the simultaneous analysis of fentanyl and its major metabolites in biological samples. The identification of different metabolites was performed by gas chromatography–mass spectrometry (electronic impact and chemical ionisation modes) and gas chromatography–Fourier transform infrared spectroscopy. In the present study, rat and human microsomes incubation mixtures and human urines were analysed. In vitro formation of already known fentanyl metabolites was confirmed. The presence of metabolites not previously detected in human urine is described.     

Quantitative determination of histamine metabolites by capillary gas chromatography

A method using capillary gas chromatography is described for the determination of histamine and eight of its basic and acid metabolites in a single biological sample of serum, urine, or gastric juice. Ion-exchange chromatography and extraction with organic solvents are used for isolation and purification, and gas chromatography for identification and quantitation. The heptafluorobutyryl derivatives of histamine and some basic metabolites are compatible with nitrogen-phosphorus and electron capture detection modes and offer an excellent sensitivity (detection limit 0.1 pmol with electron capture). The acid metabolites are quantitated after esterification. The linearity range, the sensitivity, a partial study of reproducibility and typical chromatograms show that the method is adaptable to a variety of applications.     

Identification of the 11,12-dihydro-11,12-dihydroxybenzo(a)pyrene as a major metabolite produced by the green alga, Selenastrum capricornutum

Benzo(a)pyrene metabolites were isolated after incubation of [14C]-benzo(a)pyrene with the green alga, Selenastrum capricornutum. A significant amount of radioactivity chromatographed in the dihydrodiol region which did not coelute with any of the previously identified dihydrodiol metabolites isolated from this system. Following characterization by mass spectrometry, fluorescence spectroscopy, and high pressure liquid chromatography, this metabolite was identified as the cis-11,12-dihydro-11,12-dihydroxybenzo(a)pyrene. This metabolite has not been identified previously as a metabolite formed in a plant system.     

Process for the integrated extraction, identification and quantification of metabolites, proteins and RNA to reveal their co-regulation in biochemical networks

A novel extraction protocol is described with which metabolites, proteins and RNA are sequentially extracted from the same sample, thereby providing a convenient procedure for the analysis of replicates as well as exploiting the inherent biological variation of independent samples for multivariate data analysis. A detection of 652 metabolites, 297 proteins and clear RNA bands in a single Arabidopsis thaliana leaf sample was validated by analysis with gas chromatography coupled to a time of flight mass spectrometer for metabolites, two-dimensional liquid chromatography coupled to mass spectrometry for proteins, and Northern blot analysis for RNA. A subset of the most abundant proteins and metabolites from replicate analysis of different Arabidopsis accessions was merged to form an integrative dataset allowing both classification of different genotypes and the unbiased analysis of the hierarchical organization of proteins and metabolites within a real biochemical network.     

Formation of 5,16-androstadien-3 beta-ol from pregnenolone in human testicular microsomes

A microsomal fraction of testicular tissue from a patient with prostatic carcinoma was incubated with [4-14C]pregnenolone in the presence of an NADPH-generating system for different periods of time. The metabolites were separated by Sephadex LH-20 column chromatography and then identified by thin-layer chromatography, radio-gas chromatography, and crystallization studies. Pregnenolone was converted to a major metabolite, 5-androstene-3 beta,17 beta-diol via 17-hydroxypregnenolone and then dehydroepiandrosterone. Another major metabolite was 5,16-androstadien-3 beta-ol, which increased with the time of incubation and accumulated in the incubation medium. After 120 min of incubation, 34.6% of the precursor was converted to 5-androstene-3 beta,17 beta-diol and 15.1% to 5,16-androstadien-3 beta-ol. In addition to the above-mentioned steroids, 16 alpha-hydroxypregnenolone, 5-pregnene-3 beta,20 alpha-diol, and 5-androstene-3 beta,17 alpha-diol were identified as minor metabolites of pregnenolone. From these results it was concluded that human testicular microsomes possess enzymic activities for the synthesis of 5,16-androstadien-3 beta-ol, as well as androgens from pregnenolone.     

A revision of the metabolic disposition of amantadine

Amantadine is one of the most commonly used drugs for the control of tremor in Parkinson's disease. Additionally, it has an antiviral action in the prevention of type A influenza. It has been previously reported that amantadine is nearly completely eliminated in the urine. No metabolites have been detected. Surprisingly, in a case of amantadine overdose, several metabolites could be identified by gas chromatography/mas spectrometry. This finding prompted us to re-investigate the metabolism of amantadine under a therapeutic dosing regimen. The bulk of the dose was eliminated unchanged. However, eight metabolites could be identified. Besides N-acetylation which is the major metabolic pathway, several rather unusual metabolic pathways were observed: N-methylation, formation of Schiff bases and N-formiates. No metabolites with a hydroxylated adamantane ring system could be detected.     

Chromatographic and capillary electrophoretic methods for the analysis of nicotinic acid and its metabolites

Methods for the assay of nicotinic acid (NiAc) and its metabolites in biological fluids using high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) are reviewed. Most of the references cited in this review concern HPLC methods. A few CE methods that have been recently reported are also included. As these compounds are relatively polar and have a wide range of physico-chemical properties, the sample pre-treatment or clean-up process prior to analysis is included. Most HPLC methods using an isocratic elution system allow determination of a single or few metabolites, but gradient HPLC methods enable simultaneous determination of five to eight compounds. Simultaneous determination of NiAc including many metabolites in a single run can be achieved by CE. We also discuss the pharmacokinetics of NiAc and some of its metabolites.     

High-performance liquid chromatographic analysis of 2-aminofluorene and its metabolites in monolayer cultures of rat hepatocytes

A high-pressure liquid chromatography method was developed to separate 2-aminofluorene and several ring-hydroxylated metabolites. The acetylated derivatives of these compounds and N-hydroxy-2-acetylaminofluorene were also separated simultaneously. HPLC analyses were performed using a Dupont Zorbax C-8 HPLC column and a solvent mixture of 0.02 M acetic acid and isopropanol. Isopropanol concentrations were increased from 27 to 100% over 45 min using a concave gradient system. Desferal mesylate was added to the aqueous component to improve the resolution of several hydroxylated arylamine metabolites. The method was used to quantitate the metabolites of 2-aminofluorene in monolayer cultures of rat hepatocytes.