Development and validation of a quantitative assay for the determination of tamoxifen and its five main phase I metabolites in human serum using liquid chromatography coupled with tandem mass spectrometry

A sensitive bioanalytical assay for the quantitative determination of tamoxifen and five of its phase I metabolites (N-desmethyltamoxifen, N-desmethyl-4-hydroxytamoxifen, N-desmethyl-4'-hydroxytamoxifen, 4-hydroxytamoxifen and 4'-hydroxytamoxifen) in serum is described. The method has been fully validated at ranges covering steady-state serum concentrations in patients receiving therapeutic dosages of tamoxifen. The bioanalytical assay is based on reversed phase liquid chromatography coupled with tandem mass spectrometry in the positive ion mode using multiple reaction monitoring for drug (-metabolite) quantification. The sample pretreatment consists of protein precipitation with acetonitrile using only 50 μL of serum. In the past, numerous assays have been developed by other groups for the quantification of tamoxifen and its phase I metabolites. However, the number of metabolites included in these studies is very limited and only very few of these assays have been fully validated. A liquid chromatography tandem mass spectrometry assay for the quantification of tamoxifen and four phase I metabolites in human serum that was previously developed by our group is now explicitly improved and described herein. Time of analysis has been reduced by 50% and sensitivity was increased by a reduction of the lower limit of quantification from 1.0 to 0.2 ng/mL for 4-hydroxytamoxifen and 4'-hydroxytamoxifen. Additionally, two phase I metabolites that have never been quantified in human serum hitherto, namely 4'-hydroxytamoxifen and N-desmethyl-4'-hydroxytamoxifen, were included in this assay. Validation results demonstrate an accurate and precise quantification of tamoxifen, N-desmethyltamoxifen, N-desmethyl-4-hydroxytamoxifen, N-desmethyl-4'-hydroxytamoxifen, 4-hydroxytamoxifen and 4'-hydroxytamoxifen in human serum. The applicability of the assay was demonstrated and it is now successfully used to support clinical studies in which patient-specific dose optimization is performed based on serum concentrations of tamoxifen metabolites.     

Analytical methods to determine phytoestrogenic compounds

The analytical methods for the determination of phytoestrogenic compounds in edible plants, plant products and biological matrices are reviewed. The detection, qualitative and quantitative methods based on different chromatographic separations of gas chromatography (GC), high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) coupled with various detections by ultraviolet absorption (UV), electrochemical detection (ED), fluorescence detection, mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR), as well as non-chromatographic immunoassay are each extensively examined and compared. An overview on phytoestrogen chemistry, bioactivities and health effects, plant precursors, metabolism and sample preparation is also presented.     

Isolation and Characterization of Human Intestinal Bacteria Capable of Transforming the Dietary Carcinogen 2-Amino-1-Methyl-6-Phenylimidazo[4,5-b]Pyridine

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a carcinogenic heterocyclic aromatic amine formed in meat products during cooking. Although the formation of hazardous PhIP metabolites by mammalian enzymes has been extensively reported, research on the putative involvement of the human intestinal microbiota in PhIP metabolism remains scarce. In this study, the in vitro conversion of PhIP into its microbial derivate, 7-hydroxy-5-methyl-3-phenyl-6,7,8,9-tetrahydropyrido[3′,2′:4,5]imidazo[1,2-a]pyrimidin-5-ium chloride (PhIP-M1), by fecal samples from 18 human volunteers was investigated. High-performance liquid chromatography analysis showed that all human fecal samples transformed PhIP but with efficiencies ranging from 1.8 to 96% after 72 h of incubation. Two PhIP-transforming strains, PhIP-M1-a and PhIP-M1-b, were isolated from human feces and identified by fluorescent amplified fragment length polymorphism and pheS sequence analyses as Enterococcus faecium strains. Some strains from culture collections belonging to the species E. durans, E. avium, E. faecium, and Lactobacillus reuteri were also able to perform this transformation. Yeast extract, special peptone, and meat extract supported PhIP transformation by the enriched E. faecium strains, while tryptone, monomeric sugars, starch, and cellulose did not. Glycerol was identified as a fecal matrix constituent required for PhIP transformation. Abiotic synthesis of PhIP-M1 and quantification of the glycerol metabolite 3-hydroxypropionaldehyde (3-HPA) confirmed that the anaerobic fermentation of glycerol via 3-HPA is the critical bacterial transformation process responsible for the formation of PhIP-M1. Whether it is a detoxification is still a matter of debate, since PhIP-M1 has been shown to be cytotoxic toward Caco-2 cells but is not mutagenic in the Ames assay.     

Determination of 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP) and its metabolite 2-hydroxyamino-PhIP by liquid chromatography/electrospray ionization-ion trap mass spectrometry.

2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is the most abundant heterocyclic aromatic amine found in cooked meat. It is metabolically activated by the human cytochrome P450 enzymes to form the carcinogenic metabolite N2-OH-PhIP. PhIP has been found to induce tumors in rats and is a suspected human carcinogen. In the present work, we have developed and validated a liquid chromatography-electrospray ionization/ion trap mass spectrometry (LC-ESI/ITMS) method for the determination of PhIP and N2-OH-PhIP. PhIP was incubated with microsomes prepared from the human liver; the PhIP and N2-OH-PhIP formed were isolated from the biomatrices by solid-phase extraction using C18 cartridges, with recoveries greater than 86%. Subsequently, the products were separated on a microbore reversed-phase C18 liquid chromatograph coupled to an ESI-ITMS. The ESI interface and the ITMS were tuned for various parameters, and data acquisition was performed in selective ion monitoring mode. The detection limit of PhIP and N2-OH-PhIP was 1 and 10 pg, respectively. The method is highly sensitive and selective, has simple sample preparation protocols, and should be applicable to the study of the metabolic activation of PhIP in various human tissues.     

Structure elucidation of metabolites of gambogic acid in vivo in rat bile by high-performance liquid chromatography-mass spectrometry and high-performance liquid chromatography-nuclear magnetic resonance

Gambogic acid (GBA), the main component of Gamboge, possesses significant anti-tumour activity. Due to its structural complexity, little is known about GBA metabolism. Here, we investigate the metabolism of GBA in vivo in rat bile. Identification of the metabolites formed was elucidated using high-performance liquid chromatography (HPLC) with UV-vis detection, HPLC/ion trap electrospray ionization-mass spectrometry, as well as HPLC/nuclear magnetic resonance. Four main metabolites were determined. Two phase I metabolites, 10-hydroxygambogic acid and 9,10-epoxygambogic acid, were oxides on the 9,10-olefinic bond of GBA. The others phase II metabolites, were 9,10-epoxygambogic acid-30-O-glucuronide and 10-hydroxylgambogic acid-30-O-glucuronide.     

Analytical strategies for the direct mass spectrometric analysis of steroid and corticosteroid phase II metabolites

The use of steroid hormones as growth promoters remains illegal in Europe. A classical approach used to control their utilization consists to measure the parent drug in target biological matrices. However, this strategy may fail when the parent drug is submitted to extensive metabolism reactions. For urine and tissue samples, chemical or enzymatic hydrolysis is usually applied in order to deconjugate glucuronide and sulfate phase II metabolites. But this treatment lead to the loss of information such as nature and relative proportions of the different conjugated forms, which can be useful, for example, to discriminate an endogenous production from an exogenous administration for natural hormones, or for other clinical or biochemical specific applications. For these purposes, direct measurement of conjugated metabolites using liquid chromatography-tandem mass spectrometry may represent a solution of choice. In this context, the mass spectrometric behavior of 14 steroid and corticosteroid phase II metabolites after electrospray ionization was investigated. Their fragmentation pathways in tandem mass spectrometry revealed some specificities within the different group of conjugates. A specific acquisition program (MRM mode) was developed for the unambiguous identification of the studied reference compounds. A more generic method (Parent Scan mode) was also developed for fishing approaches consisting to monitor several fragment ions typical of each conjugate class. A reverse phase HPLC procedure was also proposed for efficient retention and separation of the studied compounds. Finally, a protocol based on quaternary amine SPE was developed, permitting the separation of free, glucuronide, and sulfate fractions. Preliminary results on biological samples demonstrated the suitability of this analytical strategy for direct measurement of dexamethasone glucuronide and sulfate residues in bovine urine.     

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.     

Chemopreventive efficacy of geraniol against 7,12-dimethylbenz[a]anthracene-induced hamster buccal pouch carcinogenesis

The status of lipid peroxidation, antioxidants, and detoxification enzymes were used as biochemical end points to assess the chemopreventive potential of geraniol, a monoterpene, in 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch carcinogenesis. Topical application of 0.5% DMBA in liquid paraffin, three times a week, for 14 weeks developed well-differentiated squamous cell carcinoma in the buccal pouch of golden Syrian hamsters. Although 100% tumor formation was noticed in hamsters treated with DMBA alone, intragastric administration of geraniol, at a dose of 250 mg/kg body weight (b.w.) to DMBA-treated hamster completely prevented the formation of oral tumors. Furthermore, geraniol significantly reduced lipid peroxidation by-products and improved the status of enzymatic and non-enzymatic antioxidants as well as modulated the status of phase I and phase II detoxification enzymes, favoring the excretion of carcinogenic metabolite, during DMBA-induced oral carcinogenesis. The present study concludes that the chemopreventive potential of geraniol relies on its anti-lipid peroxidative and antioxidant function as well as modulatory effects on phase I and II detoxification enzymes to excrete the carcinogenic metabolite, during DMBA-induced hamster buccal pouch carcinogenesis.     

Simultaneous determination of benzo[a]pyrene and eight of its metabolites in Fundulus heteroclitus bile using ultra-performance liquid chromatography with mass spectrometry

A sensitive and fast method was developed to quantitate the carcinogenic polycyclic aromatic hydrocarbon benzo[a]pyrene (BaP) and eight of its oxidized metabolites by ultra-performance liquid chromatography (UPLC) coupling with mass spectrometry (MS). The UPLC method, using an acetonitrile:water gradient as a mobile phase, provided baseline separation of the BaP metabolites including three BaP diones. Linearity of detection was in the range of 0.2-5.0ng/microL, and limits of detection (LOD) were lower than 0.01ng/microL for BaP and all of the metabolites except BaP tetrol. In order to test this method in environmentally relevant samples, we exposed the small fish Fundulus heteroclitus to BaP and quantitated biliary BaP metabolites. Extraction recovery of all compounds varied from 65.4+/-21.3% to 92.4+/-3.0%. In exposed fish bile, the BaP diones, BaP-7,8-dihydrodiol, and 3-hydroxy BaP metabolites predominated, existing mainly as glucuronic acid conjugates. This UPLC-MS method will be useful for further defining the roles of cytochrome P450s with both in vivo and in vitro models in the understanding of the mechanisms of metabolic activation and detoxification of BaP.     

Chemopreventive efficacy of geraniol against 7,12-dimethylbenz[a]anthracene-induced hamster buccal pouch carcinogenesis

Abstract

The status of lipid peroxidation, antioxidants, and detoxification enzymes were used as biochemical end points to assess the chemopreventive potential of geraniol, a monoterpene, in 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch carcinogenesis. Topical application of 0.5% DMBA in liquid paraffin, three times a week, for 14 weeks developed well-differentiated squamous cell carcinoma in the buccal pouch of golden Syrian hamsters. Although 100% tumor formation was noticed in hamsters treated with DMBA alone, intragastric administration of geraniol, at a dose of 250 mg/kg body weight (b.w.) to DMBA-treated hamster completely prevented the formation of oral tumors. Furthermore, geraniol significantly reduced lipid peroxidation by-products and improved the status of enzymatic and non-enzymatic antioxidants as well as modulated the status of phase I and phase II detoxification enzymes, favoring the excretion of carcinogenic metabolite, during DMBA-induced oral carcinogenesis. The present study concludes that the chemopreventive potential of geraniol relies on its anti-lipid peroxidative and antioxidant function as well as modulatory effects on phase I and II detoxification enzymes to excrete the carcinogenic metabolite, during DMBA-induced hamster buccal pouch carcinogenesis.     

Glucuronidation and sulfation of 7-hydroxycoumarin in liver matrices from human, dog, monkey, rat, and mouse

Summary Uridine 5′-diphospho-N-acetylgalactosamine glycosyltransferases (UGTs) and sulfotransferases (SULTs) are 2 phase II enzymes that are actively involved in detoxification processes as well as in drug metabolism. Compared with cytochrome P450 enzymes, the role of UGTs and SULTs in drug metabolism has received little attention. Liver microsomes, S9 fractions, and cryopreserved hepatocytes from human, dog, cynomolgus monkey, mouse, and rat were used as matrices in the study. Single compound, 7-hydroxycoumarin (7-HC), along with necessary cofactors was dosed into the matrices and incubated at 37° C; formation of two metabolites, 7-HC-glucuronide and 7-HC-sulfate, was determined with liquid chromatography with tandem mass spectrometry. Within the same species, the UGTs activities in microsomes and S9 fractions were comparable. In addition, UGTs activities in cryopreserved hepatocytes were lower than in the other matrices. Also, the SULTs activities were much higher in S9 fractions than in cryopreserved hepatocytes and microsomes. Species differences on UGTs and SULTs activities were also observed. The results indicated that S9 fractions, microsomes, and cryopreserved hepatocytes might be useful for UGTs metabolism study, whereas S9 fractions appear to be the most appropriate matrix for both UGTs and SULTs metabolism. Species differences with respect to phase II metabolism also need to be taken into consideration when selecting an in vitro system to evaluate various aspects of drug metabolism.     

Quantitative analysis of cocaine and its metabolites in whole blood and urine by high-performance liquid chromatography coupled with tandem mass spectrometry

AIM: In forensic toxicology it is important to have specific and sensitive analysis for quantification of illicit drugs in biological matrices. This paper describes a quantitative method for determination of cocaine and its major metabolites (ecgonine methyl ester, benzoylecgonine, norcocaine and ethylene cocaine) in whole blood and urine by liquid chromatography coupled with tandem mass spectrometry LC/MS/MS. METHOD: The sample pre-treatment (0.20 g) consisted of acid precipitation, followed by centrifugation and solid phase extraction of supernatant using mixed mode sorbent columns (SPEC MP1 Ansys Diag. Inc.). Chromatographic separation was performed at 30 degrees C on a reverse phase Zorbax C18 column with a gradient system consisting of formic acid, water and acetonitrile. The analysis was performed by positive electrospray ionisation with a triple quadropole mass spectrometer operating in multiple reaction monitoring (MRM) mode. Two MRM transitions of each analyte were established and identification criteria were set up based on the retention time and the ion ratio. The quantification was performed using deuterated internal analytes of cocaine, benzoylecgonine and ecgonine methyl ester. The calibration curves of extracted standards were linear over a working range of 0.001-2.00 mg/kg whole blood for all analytes. The limit of quantification was 0.008 mg/kg; the interday precision (measured by relative standard deviation-%RSD) was less than 10% and the accuracy (BIAS) less than 12% for all analytes in whole blood. Urine samples were estimated semi-quantitatively at a cut-off level of 0.15 mg/kg with an interday precision of 15%. CONCLUSION: A liquid chromatography mass spectrometric (LC/MS/MS) method has been developed for confirmation and quantification of cocaine and its metabolites (ecgonine methyl ester, benzoylecgonine, norcocaine and ethylene cocaine) in whole blood and semi-quantitative in urine. The method is specific and sensitive and offers thereby an excellent alternative to other methods such as GC-MS that involves derivatisation.     

Separation and mass spectrometry in microbial metabolomics

Measurements of low molecular weight metabolites have been increasingly incorporated in the characterization of cellular physiology, qualitative studies in functional genomics, and stress response determination. The application of cutting edge analytical technologies to the measurement of metabolites and the changes in metabolite concentrations under defined conditions have helped illuminate the effects of perturbations in pathways of interest, such as the tricarboxylic acid cycle, as well as unbiased characterizations of microbial stress responses as a whole. Owing to the complexity of microbial metabolite extracts and the large number of metabolites therein, advanced and high-throughput separation techniques in gas chromatography, liquid chromatography, and capillary electrophoresis have been coupled to mass spectrometry - usually high-resolution mass spectrometry, but not exclusively - to make these measurements.     

Granger causality in integrated GC–MS and LC–MS metabolomics data reveals the interface of primary and secondary metabolism

Metabolomics has emerged as a key technique of modern life sciences in recent years. Two major techniques for metabolomics in the last 10 years are gas chromatography coupled to mass spectrometry (GC–MS) and liquid chromatography coupled to mass spectrometry (LC–MS). Each platform has a specific performance detecting subsets of metabolites. GC–MS in combination with derivatisation has a preference for small polar metabolites covering primary metabolism. In contrast, reversed phase LC–MS covers large hydrophobic metabolites predominant in secondary metabolism. Here, we present an integrative metabolomics platform providing a mean to reveal the interaction of primary and secondary metabolism in plants and other organisms. The strategy combines GC–MS and LC–MS analysis of the same sample, a novel alignment tool MetMAX and a statistical toolbox COVAIN for data integration and linkage of Granger Causality with metabolic modelling. For metabolic modelling we have implemented the combined GC–LC–MS metabolomics data covariance matrix and a stoichiometric matrix of the underlying biochemical reaction network. The changes in biochemical regulation are expressed as differential Jacobian matrices. Applying the Granger causality, a subset of secondary metabolites was detected with significant correlations to primary metabolites such as sugars and amino acids. These metabolic subsets were compiled into a stoichiometric matrix N. Using N the inverse calculation of a differential Jacobian J from metabolomics data was possible. Key points of regulation at the interface of primary and secondary metabolism were identified.     

New finding of nalbuphine metabolites in men: NMR spectroscopy and UPLC–MS/MS spectrometry assays in a pilot human study

A safe and efficient semi-synthetic narcotic nalbuphine (NAL) which was broadly applied in analgesic therapy has long been considered to eliminate from human body via phase II conjugation. However, up to the present, neither the complete metabolic pathways nor the identified metabolites of NAL have been clarified in documented reports. In this study, four novel metabolites were discovered by incubating NAL with human liver microsomes. These metabolites were later quantified in blood samples from human volunteers treated with NAL. An accurate and precise new method for simultaneously determining NAL and its metabolites was also established. Their chemical structures were elucidated on the basis of one- and two-dimensional NMR spectroscopic analyses including ¹H–¹H correlation spectroscopy, nuclear overhauser enhancement spectroscopy, heteronuclear single-quantum correlation, and heteronuclear multiple bond correlation, and further confirmed by mass spectrometry. The analytical method was validated and applied successfully to a pilot human study with ultra-high performance liquid chromatography–tandem mass spectrometry employed with positive ion electrospray ionization via multiple reaction monitoring mode. This is the first report on the qualitative and quantitative analysis of NAL coupled with its two hydroxylated (3′-hydroxynalbuphine and 4′-hydroxynalbuphine) and two conjugated metabolites (nalbuphine-3-β-d-glucuronide and nalbuphine-6-β-d-glucuronide). The present method offers a rapid and simple way of performing pharmacokinetic studies of NAL, and assists in elucidating its metabolic pathway in humans.     

Qualitative Profiling and Quantification of Neonicotinoid Metabolites in Human Urine by Liquid Chromatography Coupled with Mass Spectrometry

Neonicotinoid pesticides have been widely applied for the production of fruits and vegetables, and occasionally detected in conventionally grown produce. Thus oral exposure to neonicotinoid pesticides may exist in the general population; however, neonicotinoid metabolites in human body fluids have not been investigated comprehensively. The purpose of this study is the qualitative profiling and quantitative analysis of neonicotinoid metabolites in the human spot urine by liquid chromatography coupled with mass spectrometry (LC/MS). Human urine samples were collected from three patients suspected of subacute exposure to neonicotinoid pesticides. A qualitative profiling of urinary metabolites was performed using liquid chromatography/time-of-flight mass spectrometry (LC/TOFMS) with a database of nominal molecular weights of 57 known metabolites of three neonicotinoid pesticides (acetamiprid, Imidacloprid, and clothianidin), as well as the parent compounds. Then a quantitative analysis of selected urinary metabolites was performed using liquid chromatography/tandem mass spectrometry (LC/MS/MS) with a standard pesticide and metabolite, which were detected by the qualitative profiling. The result of qualitative profiling showed that seven metabolites, i.e. an acetamiprid metabolite, N-desmethyl-acetamiprid; three Imidacloprid metabolites, 5-hydroxy-Imidacloprid, 4,5-dihydroxy-imidacloprid, 4,5-dehydro-Imidacloprid; a common metabolite of acetamiprid and Imidacloprid, N-(6-chloronicotinoyl)-glycine; and two clothianidin metabolites, N-desmethyl-clothianidin, N-(2-(methylsulfanyl)thiazole-5-carboxyl)-glycine, as well as acetamiprid, were detected in the urine of three cases. The result of the quantitative analysis showed N-desmethyl-acetamiprid was determined in the urine of one case, which had been collected on the first visit, at a concentration of 3.2 ng/mL. This is the first report on the qualitative and quantitative detection of N-desmethyl-acetamiprid in the human urine. The results suggest that the one case with detection of N-desmethyl-acetamiprid was exposed to acetamiprid through the consumption of contaminated foods. Urinary N-desmethyl-acetamiprid, as well as 5-hydroxy-Imidacloprid and N-desmethyl-clothianidin, may be a good biomarker for neonicotinoid exposure in humans and warrants further investigation.     

Multidimensional on-line sample preparation of verapamil and its metabolites by a molecularly imprinted polymer coupled to liquid chromatography-mass spectrometry

A new molecularly imprinted polymer (MIP) material was synthesized selective for verapamil and utilized for on-line metabolic screening of this common calcium antagonist in biological samples. Since some metabolites of verapamil have also shown pharmacological properties, a selective and sensitive sample preparation approach that provides a metabolic profile in biologically relevant samples is important. The MIP material was coupled on-line to a restricted access material (RAM) precolumn. The multidimensional nature of this set-up removed large matrix interferents such as proteins from the sample, while the selectivity of the MIP enabled further cleanup of the smaller analytes. The selectivity and extraction efficiency of the MIP for verapamil and its metabolites was evaluated in various biological matrices, such as cell cultures and urine. The experimental set-up with the developed method enabled the direct injection of biological samples for the selective isolation, preconcentration, identification and analysis of verapamil and its phase I metabolites by LC-MS(n). This multidimensional approach provided much qualitative information about the metabolic profile of verapamil in various biological matrices. An analytical method was developed for the quantification of verapamil and gallopamil in urine, plasma and cell culture. Acceptable linearity (R(2)=0.9996, 0.9982 and 0.9762) with an average injection repeatability (n=3) of 10, 25 and 15% R.S.D. was determined for urine, plasma and cell culture, respectively. This is the first application of the procedure for the selective metabolic screening of verapamil in biological samples.     

Development and validation of column-switching high-performance liquid chromatographic methods for the determination of a potent AII receptor antagonist, TCV-116, and its metabolites in human serum and urine

Column-switching HPLC methods have been developed and validated for the determination of a new antihypertensive prodrug, TCV-116 (I), and its metabolites, CV-11974 (II) and CV-15959 (III), in human serum and urine. Initial sample cleanup was achieved by extracting the analytes into an organic solvent. After chromatographing on an ODS column with a mobile phase consisting of acetonitrile and an acidic phosphate buffer, the zone of the analyte's retention was heart-cut onto a second ODS column with a mobile phase of acetonitrile and a phosphate buffer at a higher pH. Complete separation of the analytes and the endogenous peaks was accomplished by the two-dimensional chromatography. Good precision and linearity of the calibration standards, as well as the inter-day and intra-day precision and accuracy of quality control samples, were achieved. The limit of quantitation (LOQ), using 0.5 ml of serum, was 2 ng/ml for I, 0.8 ng/ml for II, and 0.5 ng/ml for III. The LOQ for urine sample was 10 ng/ml for II and III. Stability of the analytes during storage, extraction, and chromatography processes was established. The results illustrate the versatile application of column switching to method development of multiple analytes in various biological matrices. The methods have been successfully used for the analyses of I and its metabolites in thousands of clinical samples to provide pharmacokinetics data.     

Measurement of angiotensin metabolites in organ bath and cell culture experiments by liquid chromatography - electrospray ionization - mass spectrometry (LC-ESI-MS).

The metabolism of renin-angiotensin system (RAS) is more complicated than previously expected and understanding the biological phenomena regulated by variety of angiotensin metabolites requires their precise and possibly comprehensive quantitation. Physiological concentrations of angiotensins (Ang) in biological fluids are low, therefore their accurate measurements require very sensitive and specific analytical methods. In this study we developed an accurate and reproducible method of quantitation of angiotensin metabolites through coupling of liquid chromatography and electrospray ionization - mass spectrometry (LC-ESI-MS). With this method main angiotensin metabolites (Ang I, II, III, IV, 1-9, 1-7, 1-5) can be reliably measured in organ bath of rat tissues (aorta, renal artery, periaortal adipose tissue) and in medium of cultured endothelial cells (EA.hy926), exposed to Ang I for 15 minutes, in the absence or in the presence of angiotensin converting enzyme inhibitor, perindoprilat. Presented LC-ESI-MS method proved to be a quick and reliable solution to comprehensive analysis of angiotensin metabolism in biological samples.     

Microbiologic transformation of progesterone by Curvularia clavata Jain

Progesterone was transformed microbiologically by the fungal strain Curvularia clavata Jain. Progesterone (I) was added as substrate when the microorganism reached its exponential growth phase. Three substances were isolated after the fermentation: a non-steroidal substance, radicinin (II), which has been established to be a metabolic product of the fungus and acts as a phytotoxin, and two steroidal substances which resulted from fungal enzymatic action on the progesterone molecule. The structure of each microbial metabolite was elucidated by 1H-nuclear magnetic resonance, mass spectrometry, and infrared and UV analyses, and the yields were determined by high-pressure liquid chromatography. The progesterone metabolites were characterized as 7 alpha,14 alpha-dihydroxypregn-4-ene-3,20-dione (III) and 11 beta, 14 alpha-dihydroxypregn-4-ene-3,20-dione (IV). Evidence for the structure of these steroidal products came from derivatives resulting from acetylation and dehydration.