Identification of 4-hydroxyandrost-4-ene-3,17-dione metabolites in prostatic cancer patients by liquid chromatography-mass spectrometry.

Liquid chromatography with thermospray mass spectrometry has proved to be an invaluable technique for the study of metabolic degradation of xenobiotics in complex biological fluids. This paper describes the detection of 4-hydroxyandrost-4-ene-3,17-dione and its metabolites in urinary extracts from prostatic cancer patients. Several metabolites were detected including 4 beta,5 alpha-dihydroxyandrostan-3,17-dione, 3,17-dihydroxyandrostan-4-ones and 3 alpha-hydroxy-5 beta-androstan-4,17-dione.     

Characterization of forsythoside A metabolites in rats by a combination of UHPLC‐LTQ‐Orbitrap mass spectrometer with multiple data processing techniques

Forsythoside A (FTA), the main active constituent isolated from Fructus Forsythiae, has various biological functions including anti‐oxidant, anti‐viral and anti‐microbial activities. However, while research on FTA has been mainly focused on the treatment of diseases on a material basis, FTA metabolites in vivo have not been comprehensively evaluated. Here, a rapid and sensitive method using a UHPLC‐LTQ‐Orbitrap mass spectrometer with multiple data processing techniques including high‐resolution extracted ion chromatograms, multiple mass defect filters and diagnostic product ions was developed for the screening and identification of FTA metabolites in rats. As the result, a total of 43 metabolites were identified in biological samples including 42 metabolites in urine, 22 metabolites in plasma and 15 metabolites in feces. These results demonstrated that FTA underwent a series of in vivo metabolic reactions including methylation, dimethylation, sulfation, glucuronidation, diglucuronidation, cysteine conjugation and their composite reactions. The research enhanced our understanding of FTA metabolism and built a foundation for further toxicity and safety studies.     

Comparative bioanalytical study of3H-deramciclane in dog plasma, using a gas chromatography-nitrogen-selective detection (GC-NPD), a new GC-radiochemical detection (GC-RD) and a liquid scintillation method

Summary A new, highly sensitive and selective gas chromatography method, using radiochemical detection (GC-DR) was developed for the selective determination of³H-labelled deramciclane and its N-desmethyl metabolite in dog plasma. Inter-day accuracy and precision, as well as system suitability of the GC-RD method was investigated during the method validation. The calibration curve was proved to be linear (r=0.9986) in a wide concentration range (13–1000 ngeqv mL⁻¹) The lower limit of quantitation (LLOQ) was 13.7 ngeqv mL⁻¹, and the limit of the detection (LOD) was 1 ngeqv mL⁻¹. Using this new GC-RD method, plasma levels of³H-labelled deramciclane and its metabolite were determined in dogs, after the administration of a single 10 mg kg⁻¹ oral dose. Pharmacokinetic curves and the calculated pharmacokinetic parameters were compared to those obtained using a previously elaborated gas chromatography-nitrogen selective detection method (GC-NPD) and to those obtained by measuring the plasma level of total radioactivity (liquid scintillation counting, LSC). Pharmacokinetic curves and the calculated pharmacokinetic parameters obtained with the two different gas chromatography detection methods (NPD and RD) showed good correlation. Comparison of these results to those acquired by total radioactivity measurement demonstrated that deramciclane was intensively metabolised. Moreover, the biological half-life (t ₁ ^2/β ) of the unknown metabolites proved to be more than a magnitude longer than the half-life of the parent compound or that of N-desmethyl metabolite. Presented at: Balaton Symposium on High-Performance Separation Methods, Siófok, Hungary, September 3–5, 1997.     

Ultra-Performance Liquid Chromatography Coupled with Quadrupole Time-of-Flight Mass Spectrometry for Rapid Determination of the Metabolites of Baicalin Produced by Human Intestinal Bacteria

Baicalin, mainly isolated from Scutllaria baicalensis, has been reported to possess a wide range of biological activities. However, the information about the metabolic route and metabolites of baicalin was limited to the role of the human intestinal bacterial mixture. In this paper, four strains of bacteria including Bacteroides sp. 33 and 56, and Veillonella sp. 23 and 71 were isolated from human intestinal bacterial mixture and studied for their abilities to convert baicalin to different metabolites. A highly sensitive and specific ultraperformance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) method combined with mass defect filtering (MDF) provides high throughput capabilities for drug metabolism study. The chromatographic separation was performed on a 1.7 µm particle size C 18 column using gradient elution system. The components in the extract were identified and confirmed according to the mass spectrometric fragmentation mechanisms, MS/MS fragment ions and relevant literature by means of electrospray ionization mass spectrometry in negative ion mode. With this method, a total of 4 metabolites were identified based on MS and MS/MS data. The results indicated that hydrogenation, methylation, and deglycosylation were the major metabolic pathways of baicalin in vitro. The present study provides important information about the metabolism of baicalin which will be helpful for fully understanding the impact of the intestinal bacteria on this active component. Furthermore, this work demonstrated the potential of the ultraperformance liquid chromatography/quadrupole time-of-flight mass spectrometry approach for a rapid, simple, reliable, and automated identification of metabolites of natural products.     

Metabolite profiling of l‐isocorypalmine in rat urine,plasma, and feces after oral administration using high performance liquid chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry

l ‐Isocorypalmine, an active alkaloid compound isolated from Rhizoma Corydalis yanhusuo, has been reported to possess biological activity for treating cocaine use disorder. A high‐performance liquid chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry method was established for identification of the metabolites of l ‐isocorypalmine in urine, plasma and feces samples of rats after a single intragastric gavage of l ‐isocorypalmine at a dose of 15 mg/kg. As a result, a total of 21 metabolites (six phase ? metabolites and fifteen phase II metabolites) were detected and tentatively identified by mass spectrometry and fragment ions from tandem mass spectrometry spectra. All metabolites were present in the urine samples, nine metabolites were found in the plasma samples and three metabolites were found in the feces samples. Results indicated that metabolic pathways of l ‐isocorypalmine included oxidation, dehydrogenation, demethylation, sulfate conjugation, and glucuronide conjugation. In addition, glucuronidation was the major metabolic reaction. Results of this investigation could provide significant experimental basis for efficacy, safety and action mechanism of l ‐isocorypalmine, which will be advantageous to new drug development for treating cocaine addiction.     

Application of microcolumn liquid chromatography-continuous-flow fast atom bombardment mass spectrometry in environmental studies of sulfonylurea herbicides.

The use of 0.25-mm I.D. packed capillary liquid chromatography columns coupled with continuous-flow fast atom bombardment (FAB) mass spectrometry has proven to be a very valuable technique, especially for the identification of unknown sulfonylurea herbicide metabolites. Several new and unusual heterocycle ring-opened metabolites and hydrolysis products were identified, and metabolic pathways were proposed. Typical column flow-rates are 1-2 microliters/min, which allows direct coupling with no sample splitting. This is important in our metabolite identification work, since we are usually sample-limited. Techniques for increasing injection volume to allow analyses of dilute solutions and the use of polymeric packing for separation of polar metabolites are discussed. The FAB mass spectra usually provide unequivocal molecular weights and structurally useful fragments ions, which often allows structure assignments on exceedingly small quantities of isolated metabolites.     

Determination of methandrostenolone and its metabolites in equine plasma and urine by coupled-column liquid chromatography with ultraviolet detection and confirmation by tandem mass spectrometry

Monitoring steroid use requires an understanding of the metabolism in the species in question and development of sensitive methods for screening of the steroid or its metabolites in urine. Qualitative information for confirmation of methandrostenolone and identification of its metabolites was primarily obtained by coupled-column high-performance liquid chromatography-tandem mass spectrometry. The steroids and a sulphuric acid conjugate were isolated and identified by their daughter ion mass spectra in the urine of both man and the horse following administration of methandrostenolone. Spontaneous hydrolysis of methandrostenolone sulphate gave 17-epimethandrostenolone and several dehydration products. This reaction had a half-life of 16 min in equine urine at 27 degrees C. Mono- and dihydroxylated metabolites were also identified. Several screening methods were evaluated for detection and confirmation of methandrostenolone use including thin-layer chromatography and high-performance liquid chromatography. Coupled-column liquid chromatography was used for automated clean-up of analytes difficult to isolate by manual methods. The recovery of methandrostenolone was 101 +/- 3.3% (mean +/- S.D.) at 6.5 ng/ml and both methandrostenolone and 17-epimethandrostenolone were quantified in urine by ultraviolet detection up to six days after a 250-mg intramuscular dose to a horse. The utility of on-line tandem mass spectrometry for confirmation of suspected metabolites is also shown.     

Characterization of in Vitro Metabolism of Loganin by Human Intestinal Microflora Using Ultra-High Performance Liquid Chromatography–Quadrupole Time-of-Flight Mass Spectrometry

Human intestinal microbiota comprise a complex biological system with considerable metabolic activity. Various studies have focused on the bioconversion of flavonoids. However, in addition to flavonoids, bioactive components such as iridoids also exist in many natural and traditional Chinese medicines. Little is known about the interactions of the iridoids with bacteria. Loganin, one of the main effective iridoids in the valuable traditional Chinese herbal medicine Cornus officinalis, exhibits various pharmacological activities and biological effects. Human intestinal bacteria were isolated and the conversion capability of loganin was investigated. The metabolites were determined by ultra-high performance liquid chromatography–quadrupole time-of-flight mass spectrometry. Loganin was metabolized to hydrogenated and hydroxylated loganin sulfate, acetylated loganin, loganetin, methylated loganetin, hydrogenated, and hydroxylated loganetin. The metabolic routes and metabolites of loganin were reported for the first time. These metabolites may influence the biological activities of loganin in vivo. Thus, this study provides fundamental information about a traditional Chinese medicine.     

Identifying and overcoming bioanalytical challenges associated with chlorine-containing dehydrogenation metabolites

Liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS) is a widely utilized analytical tool for quantifying small molecules in complex biological matrices. In certain situations the mass-selection capabilities of the tandem mass spectrometer may be insufficient to discriminate between the analyte of interest and its metabolites, particularly those metabolites that are isobaric with the analyte. One scenario by which isobaric interference may occur is the metabolism of a chlorine- or bromine-containing small molecule to a metabolite with the concomitant loss of 2 Da. This report describes the detection and characterization of two distinct dehydrogenation [M-2] metabolites during LC/MS/MS quantification of a chlorinated small molecule in rat plasma samples derived from a toxicokinetic study. The potential isotope-related impact of these metabolites on quantification of the parent compound was assessed. Several alternate precursor ion and product ion combinations were evaluated and shown to minimize the quantitative impact of the interfering metabolites without having to rely on their stringent chromatographic resolution from the parent compound. These results indicate that when quantifying chlorine- or bromine-containing small molecules from in vivo samples or in vitro metabolic incubations: (1) efforts to detect potential dehydrogenation metabolites should be undertaken and (2) if such metabolites are detected, the judicious choice of alternate multiple-reaction monitoring (MRM) transitions can limit their impact on quantification of the parent molecule without the need for robust chromatographic resolution.     

Atrazine and its Metabolites as Indicators of Stream-aquifer Interaction in Kansas,USA

Abstract

A survey of atrazine and its metabolites in Kansas ground water indicated that ground-water quality was impacted by stream-aquifer interaction between rivers in the Kansas River basin and their adjacent alluvial aquifers. Atrazine was detected in 19 of the 78 samples. The most common metabolite, deethylatrazine, was detected in 25 samples, 18 of which also had atrazine. The deethylatrazine/atrazine ratio (DAR) of < 1.0 indicates rapid movement of agricultural chemicals to ground water. In this study, 12 of 18 samples had DAR values < 1.0, suggesting rapid recharge to the aquifers. Hydroxyatrazine is seldom detected in ground water. In this study hydroxyatrazine was detected primarily in wells sited in alluvium of rivers. These rivers contain atrazine in varying concentrations. Results of the study suggest that stream-aquifer interaction is a process contributing to the presence of both atrazine and its metabolites in ground water in these areas.     

Mass defect profiles of biological matrices and the general applicability of mass defect filtering for metabolite detection

Recent examples have demonstrated that the high-resolution liquid chromatography/mass spectrometry (LC/MS)-based mass defect filtering (MDF) technique was effective in selectively detecting drug metabolites regardless of their molecular weights or fragmentation patterns. The main objective of the current study was to evaluate the general applicability of MDF for drug metabolite detection in typical biological matrices. Mass defect profiles of commonly used biological matrices including plasma, urine, bile, and feces were obtained using an LTQ FT mass spectrometer and were compared with those of 115 commonly prescribed drugs. The mass defect profiles were presented as two-dimensional Y-X plots with the determined mass defects of components on the y-axis versus the corresponding m/z values on the x-axis. The mass defect profiles of the matrices appeared to be similar for each type of matrix across species, yet marked differences were apparent between matrices of a given species. The mass defect profiles of components in plasma, bile, and feces showed significant separation from most of the 115 drugs. The mass defect profiles of urine did not show such clean separation from that of the 115 drugs. The results suggest that MDF has a broad applicability for selective detection of drug metabolites in plasma, bile and feces although the selectivity for detecting urinary drug metabolites is not as good as in the other matrices. In addition, the mass defect profiles of the biological matrices allow for prediction of the effectiveness of MDF for certain applications, and for designing specific MDF windows for selective detection of drug metabolites.     

Metabolism of 14C-miloxacin in rats--metabolites in urine, bile and feces (author's transl)

Isolation and characterization of metabolites of miloxacin, a new antimicrobial agent, were undertaken with rats. 14C-Miloxacin was orally administered to Sprague-Dawley rats at a dose of 50 mg/kg, and urine, bile and feces were collected. The metabolites extracted from the biological samples were isolated by column and thin-layer chromatographies. Characterization of the isolated metabolites was carried out by comparison with the authentic materials in various physicochemical analyses. Eight metabolites together with intact miloxacin were identified; containing the metabolites of N-demethoxy (M-1), catechol (M-3) and 6-methoxy (M-2 and M-4) types and their conjugates with glucuronic acid.     

Strategy of using microsome-based metabolite production to facilitate the identification of endogenous metabolites by liquid chromatography mass spectrometry

One of the challenges in metabolomic profiling of complex biological samples is to identify new and unknown compounds. Typically, standards are used to help identify metabolites, yet standards cannot be purchased or readily synthesized for many unknowns. In this work we present a strategy of using human liver microsomes (HLM) to metabolize known endogenous human metabolites (substrates), producing potentially new metabolites that have yet to be documented. The metabolites produced by HLM can be tentatively identified based on the associated substrate structure, known metabolic processes, tandem mass spectrometry (MS/MS) fragmentation patterns and, if necessary, accurate mass measurements. Once identified, these metabolites can be used as references for identification of the same compounds in complex biological samples. As a proof of principle, a total of 9 metabolites have been identified from individual HLM incubations using 5 different substrates. Each metabolite was used as a standard. In the analysis of human urine sample by liquid chromatography MS/MS, four spectral matches were found from the 9 microsome-produced metabolite standards. Two of them have previously been documented as endogenous human metabolites, the third is an isomer of a microsome-metabolite and the fourth compound has not been previously reported and is also an isomer of a microsome-metabolite. This work illustrates the feasibility of using microsome-based metabolism to produce metabolites of endogenous human metabolites that can be used to facilitate the identification of unknowns in biological samples. Future work on improving the performance of this strategy is also discussed.     

Fast-atom bombardment and thermospray mass spectrometry for the characterization of two glucuronide metabolites of methapyrilene

Two conjugated metabolites of methapyrilene hydrochloride isolated from mouse-hepatocytes were examined by mass spectrometry using fast-atom bombardment (FAB) and thermospray ionization. The major metabolite, methapyrilene glucuronide, was identified based on a prominent peak due to the protonated molecule as well as the loss of the dimethylamine and sugar moieties. Identification of the second metabolite was complicated by large signals associated with the biological sample matrix. The complementary nature of the fragmentation observed in the mass spectra using FAB and thermospray ionization allowed this metabolite to be identified as the desmethylmethapyrilene glucuronide. The fragmentation observed using FAB ionization was not greatly affected by the presence of the glucuronide moiety. While loss of the sugar moiety indicated a glucuronide, additional fragmentation confirmed the presence of the underlying ethylenediamine substructure which is characteristic of this class of antihistamines.     

Emerixanthone E,a new xanthone derivative from deep sea fungus Emericella sp SCSIO 05240

The marine fungus Emericella sp was isolated from the deep sea sediments. The fungus was identified by its morphology and ITS region. A new emerixanthone E (1) together with four (2–5) known emodin derivatives were isolated from the metabolites of the fungus Emericella SCSIO05240. The structures were elucidated on the basis of NMR spectroscopic analysis and mass spectrometry. The biological properties of those compounds (1–5) were explored for antimicrobial, antifungal and antitumor activity.     

A new strategy for the discovery of epimedium metabolites using high-performance liquid chromatography with high resolution mass spectrometry

In this paper, a new strategy of drug metabolite discovery and identification was established using high-performance liquid chromatography with high resolution mass spectrometry (HPLC–HRMS) and a mass spectral trees similarity filter (MTSF) technique. The MTSF technique was developed as a means to rapidly discover comprehensive metabolites from multiple active components in a complicated biological matrix. Using full-scan mass spectra as the stem and data-dependent subsequent stage mass spectra to form branches, the HRMS and multiple-stage mass spectrometric data from detected compounds were converted to mass spectral trees data. Potential metabolites were discovered based on the similarity between their mass spectral trees and that known compounds or metabolites in a mass spectra trees library. The threshold value for match similarity scores was set at above 200, allowing approximately 80% of interference to be filtered out. A total of 115 metabolites of five flavonoid monomers (epimedin A, epimedin B, epimedin C, icariin, and baohuoside I) and herbal extract of epimedium were discovered and identified in rats via this new strategy. As a result, a metabolic profile for epimedium was obtained and a metabolic pathway was proposed. In addition, comparing to the widely used neutral loss filter (NLF), product ion filter (PIF), and mass defect filter (MDF) techniques, the MTSF technique was shown superior efficiency and selectivity for discovering and identifying metabolites in traditional Chinese medicine (TCM).     

Isolation from rat urine and human liver microsomes and identification by electrospray and nanospray tandem mass spectrometry of new malagashanine metabolites

Malagashanine has been isolated from indigenous madagascan Strychnos myrtoides alkaloids used traditionally to treat malaria. This alkaloid was found to enhance the action of chloroquine against chloroquine-resistant strains of Plasmodium falciparum when combined with classical antimalarial drugs (chloroquine, quinine). The present study was carried out in order to investigate by electrospray mass and tandem mass spectrometry and NMR spectroscopy the structure of two new metabolites isolated from rat urine and human liver microsomes. We were able to demonstrate the presence of two new metabolites of malagashanine corresponding to a malagashanine N-demethylated metabolite and to the oxidation of malagashanine in the alpha-position of the N-methyl group to produce a carbinolamine function. The latter metabolite may be subject to ring and open-chain tautomerism effects and dimeric species were detected in the electrospray mass spectrum.     

Identification of urinary metabolites of imperatorin with a single run on an LC/Triple TOF system based on multiple mass defect filter data acquisition and multiple data mining techniques

The detection of drug metabolites, especially for minor metabolites, continues to be a challenge because of the complexity of biological samples. Imperatorin (IMP) is an active natural furocoumarin component originating from many traditional Chinese herbal medicines and is expected to be pursued as a new vasorelaxant agent. In the present study, a generic and efficient approach was developed for the in vivo screening and identification of IMP metabolites using liquid chromatography-Triple TOF mass spectrometry. In this approach, a novel on-line data acquisition method mutiple mass defect filter (MMDF) combined with dynamic background subtraction was developed to trace all probable urinary metabolites of IMP. Comparing with the traditionally intensity-dependent data acquisition method, MMDF method could give the information of low-level metabolites masked by background noise and endogenous components. Thus, the minor metabolites in complex biological matrices could be detected. Then, the sensitive and specific multiple data-mining techniques extracted ion chromatography, mass defect filter, product ion filter, and neutral loss filter were used for the discovery of IMP metabolites. Based on the proposed strategy, 44 phase I and 7 phase II metabolites were identified in rat urine after oral administration of IMP. The results indicated that oxidization was the main metabolic pathway and that different oxidized substituent positions had a significant influence on the fragmentation of the metabolites. Two types of characteristic ions at m/z 203 and 219 can be observed in the MS/MS spectra. This is the first study of IMP metabolism in vivo. The interpretation of the MS/MS spectra of these metabolites and the proposed metabolite pathway provide essential data for further pharmacological studies of other linear-type furocoumarins.     

Identification of astilbin metabolites produced by human intestinal bacteria using UPLC‐Q‐TOF/MS

Astilbin, mainly isolated from a commonly used herbal medicine, Smilax glabra Roxb (SGR), exhibits a variety of pharmacological activities and biological effects. It is metabolized by intestinal bacteria after oral administration which leads to the variation of ethnopharmacological profile of this traditional medicine. However, little is known on the interactions of this active compound with intestinal bacteria, which would be very helpful in unravelling how SGR works. In this study, different pure bacteria from human feces were isolated and were used to investigate their conversion capability of astilbin. Ultra‐performance liquid chromatography/quadrupole‐time‐of‐flight mass spectrometry (UPLC‐Q‐TOF/MS) technique combined with Metabolynx^TM software was used to analyze astilbin and its metabolites. The parent compound and two metabolites (quercetin and eriodictyol) were detected in the isolated bacterial samples compared with blank samples. Quercetin was present in Enterococcus sp. 8B, 8–2 and 9–2 samples. Eriodictyol was only identified in Enterococcus sp. 8B sample. The metabolic routes and metabolites of astilbin produced by the different intestinal bacteria are reported for the first time. This will be useful for the investigation of the pharmacokinetic study of astilbin in vivo and the role of different intestinal bacteria in the metabolism of natural compounds. Copyright © 2014 John Wiley & Sons, Ltd.     

Identification of isomeric flavonoid glucuronides in urine and plasma by metal complexation and LC-ESI-MS/MS

Noncovalent complexes were used for structural determination and isomer differentiation of flavonoid glucuronides. Several flavonoid glucuronides including naringenin-7-O-glucuronide, synthesized here for the first time, were used as test compounds. Electrospray ionization quadrupole ion trap mass spectrometry with collision-induced dissociation (CID) was used to analyze complexes of the form [Co(II) (L-H) (Aux)]+ and [Co(II) (L-H) (Aux)2]+, in which L is the flavonoid glucuronide and Aux is a phenanthroline-based ligand. These complexes yielded characteristic fragmentation patterns that facilitated assignment of the substitution position of the glucuronides. The methods were adapted to liquid chromatography/tandem mass spectrometry (LC-MS/MS) with postcolumn cobalt complexation and were tested on extracts from biological fluids. The metabolites naringenin-7-O-glucuronide and naringenin-4'-O-glucuronide were detected in human urine following the consumption of grapefruit juice. Isomeric quercetin glucuronides were identified and differentiated after spiking rat plasma at the 1 microM level, proving that the new methods are effective at biologically relevant concentrations.