Screening and identification of GSH-trapped reactive metabolites using hybrid triple quadruple linear ion trap mass spectrometry

The present study describes a new analytical approach for the detection and characterization of GSH-trapped reactive metabolites using multiple reaction monitoring (MRM) as the survey scan to trigger the acquisition of enhanced product ion (EPI) spectra on a triple quadrupole linear ion mass spectrometer. The MRM scan step was carried out following up to 114 MRM transitions from the protonated molecules of potential GSH adducts to their product ions derived from a neutral loss of 129 or 307 Da. MRM transition protocols were constructed on the basis of common bioactivation reactions predicted to occur in human liver microsomes (HLM). The effectiveness and reliability of the approach were evaluated using acetaminophen, diclofenac, and carbamazepine as model compounds. The total ion chromatograms of the MRM for the HLM incubations with these compounds and GSH clearly displayed a number of GSH adducts, including acetaminophen-GSH adducts and carbamazepine-GSH adducts that were not previously observed in HLM incubations. In addition, clomipramine and mefenamic acid that have the frame structures susceptible to P450-mediated bioactivation were investigated. As a result, the MRM-EPI analysis revealed multiple GSH adducts of clomipramine and mefenamic acid in HLM incubations possibly mediated by epoxide and/or quinone imine intermediates. Compared with the neutral loss (NL) and precursor ion (PI) scanning analysis, the MRM-based approach provided superior sensitivity and selectivity for GSH adducts. It also enabled the sensitive acquisition of EPI spectra with rich fragmentation in the same LC/MS run, which were useful for the rapid structure elucidation of GSH adducts and the elimination of false positives. The MRM-EPI experiment can be employed for high throughput screening of reactive metabolites and should be especially applicable to compounds of the same chemotype. Also, it can be applied in conjunction with the PI or NL scan as a comprehensive method for the analysis of reactive metabolites in a drug discovery setting.     

Recent development in liquid chromatography/mass spectrometry and emerging technologies for metabolite identification

Metabolism studies play a pivotal role in drug discovery and development since the active metabolites is critical to toxicological profile, efficacy and designing new drug candidates. From the instrumentation standpoint, liquid chromatography/mass spectrometry (LC/MS) has secured a central analytical technique for metabolite identification with the continuous developments and improvements in LC and MS technologies. Recently, a wide range of experimental strategies and post acquisition data processing and mining modes have emerged driven by the need to identify and characterize metabolites at ever increasing sensitivity and in ever more complex samples. In this article, the classical and practical mass spectrometry-based techniques, such as low resolution MS (quadruple, ion trap, linear ion trap, etc), high resolution MS (time-of-flight, hybrid time-of-flight instruments, Qrbitrap, Fourier transform ion cyclotron resonance MS, etc) and corresponding post acquisition data processing and mining modes (precursor ion filtering, neutral loss filtering, mass defect filter, isotope-pattern-filtering, etc) are described comprehensively. In addition, this review is also devote to discuss several novel MS technologies (ambient ionization techniques, ion mobility MS, imaging MS, LC/MNR/MS, etc) that hold additional promise for the advancement of metabolism studies.     

The use of mass spectrometry in the study of chemically-reactive drug metabolites. Application of MS/MS and LC/MS to the analysis of glutathione- and related S-linked conjugates of N-methylformamide

The S-(N-methylcarbamoyl) derivatives of glutathione, cysteine and N-acetylcysteine, the S-linked conjugates derived from a reactive metabolite of N-methylformamide (NMF), were studied in mice dosed with an equimolar mixture of NMF and deuterium-labelled NMF. Following preparation of N-benzyloxycarbonyl derivatives in aqueous media, the title conjugates were isolated, purified as their methyl esters and subjected to analysis by fast atom bombardment mass spectrometry (FAB/MS), fast atom bombardment tandem mass spectrometry (FAB/MS/MS) or thermospray liquid chromatography/mass spectrometry (TSP LC/MS). Characteristic isotope clusters in the FAB or TSP mass spectra facilitated recognition of drug metabolites, while constant neutral loss (89 u) and daughter ion scanning tandem mass spectrometry (MS/MS) experiments provided unique structural information on the conjugates of interest. It is concluded that the combined use of stable isotopes, aqueousphase derivatization and contemporary mass spectrometric techniques represents a powerful approach for the analysis of glutathione adducts and related S-linked conjugates of chemically-reactive drug metabolites.     

Enhanced Detection and Characterization of Glutathione-Trapped Reactive Metabolites by Pseudo-MS(3) Transition Using a Linear Ion Trap Mass Spectrometer

We present a simple and label-free approach to characterize glutathione (GSH)-trapped reactive metabolites from a single LC-MS analysis employing a linear ion trap mass spectrometer. The GSH specific fragment anion m/z 272 was first generated from the nonselective in-source fragmentation of intact conjugates. GSH conjugates were then detected by selected reaction monitoring (SRM) of the anion pair m/z 272 → 179 or 210. The resultant SRM peaks represented putative GSH conjugates which were then further characterized from their MS and MS(2) data acquired in both positive and negative ion modes. The method is demonstrated with test compounds that are all known to form GSH conjugates.     

The use of mass spectrometry in the study of chemically-reactive drug metabolises. Application of MS/MS and LC/MS to the analysis of glutathione- and related S-linked conjugates of N-methylformamide

The S-(N-methylcarbamoyl) derivatives of glutathione, cysteine and N-acetylcysteine, the S-linked conjugates derived from a reactive metabolite of N-methylformamide (NMF), were studied in mice dosed with an equimolar mixture of NMF and deuterium-labelled NMF. Following preparation of N-benzyloxycarbonyl derivatives in aqueous media, the title conjugates were isolated, purified as their methyl esters and subjected to analysis by fast atom bombardment mass spectrometry (FAB/MS), fast atom bombardment tandem mass spectrometry (FAB/MS/MS) or thermospray liquid chromatography/mass spectrometry (TSP LC/MS). Characteristic isotope clusters in the FAB or TSP mass spectra facilitated recognition of drug metabolites, while constant neutral loss (89 u) and daughter ion scanning tandem mass spectrometry (MS/MS) experiments provided unique structural information on the conjugates of interest. It is concluded that the combined use of stable isotopes, aqueousphase derivatization and contemporary mass spectrometric techniques represents a powerful approach for the analysis of glutathione adducts and related S-linked conjugates of chemically-reactive drug metabolites.     

Use of isotopic signatures for mass spectral detection of protein adduction by chemically reactive metabolites of bromobenzene: studies with model proteins

The cytotoxicity of many small organic compounds often apparently derives from their metabolic activation and covalent binding to cellular proteins. It is therefore of considerable interest to be able to determine, for a given protoxin, which metabolites modify which proteins at which sites. Our laboratory has identified more than 45 target proteins for bromobenzene metabolites in liver by peptide mass mapping after two-dimensional electrophoresis. Through all of this work, we have never observed a bromine-containing peptide. We therefore generated model adducted proteins by carbodiimide coupling of Nalpha-acetyl-Ntau-(p-bromophenyl)-L-histidine (1) and Nalpha-acetyl-Nepsilon-(p-bromophenyl)-L-lysine (2) to bovine pancreatic ribonuclease A. For the adducts, RNase-(1)n and RNase-(2)n, mass spectrometry indicated that n = 0-2 and 0-6, respectively. RNase-(2)n was submitted to in-gel and in-solution digestion with trypsin, and the digests were analyzed by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) and liquid chromatgraphy electrospray ionization MS (LC/ESI-MS) and tandem MS (MS/MS). Sequence coverages observed ranged from 67% with only three modified lysines observed using in-gel digestion and MALDI-TOF analysis, to 100% coverage with all 10 lysines observed in both modified and unmodified form using in-solution digestion and LC/ESI-MS. In the mass spectra of all modified peptides up to 2000 Da, the bromine isotope pattern was obvious by visual inspection; for peptides up to 3600 Da, the isotopic signature could be recognized by visual comparison to simulated spectra. The presence of Br-containing adducts was confirmed by MS/MS analysis of selected peptides. The selection of peaks for MS/MS analysis was significantly facilitated by visual recognition of the bromine isotope pattern, even at signal-to-noise ratios of 10 (or lower in favorable cases). These results indicate that stable isotope labeling may have considerable potential for detecting and locating protein adducts of reactive metabolites.     

Negative ion tandem mass spectrometry for the detection of glutathione conjugates

Characterization of S-linked conjugates of the endogenous tripeptide glutathione (gamma-glutamyl-cysteinylglycine, GSH) represents a valuable indirect approach for the identification of chemically reactive, electrophilic intermediates formed during the metabolism of both foreign compounds and endogenous substances. In most cases, GSH adducts generated in vitro or excreted in the bile of animals are detected by the use of liquid chromatography-tandem mass spectrometry (LC-MS/MS), employing survey scans based on characteristic fragmentations of this class of conjugates. However, a limitation of current LC-MS/MS approaches, which typically employ electrospray ionization with analysis of positive ions, is that no single survey scan exhibits broad utility in the detection of unknown GSH adducts, since different structural classes of conjugate (aromatic, benzylic, aliphatic, thioester, etc.) behave differently upon collision-induced dissociation (CID) of the respective [M + H]+ parent ions. In the present study, we evaluated MS/MS in the negative ion mode as an alternative approach and report herein that the spectra obtained by CID of the [M - H]- ions of a number of representative GSH adducts, as well as GSH itself, are dominated by fragments originating from the glutathionyl moiety of the tripeptide. In particular, the anion at m/z 272, corresponding nominally to deprotonated gamma-glutamyl-dehydroalanyl-glycine, was abundant in the negative ion spectra of free GSH and all GSH conjugates examined, suggesting that scanning for precursors of this ion may provide a generally applicable technique for the detection of adducts of unknown structure. The utility of this novel detection strategy was demonstrated in a series of in vitro and in vivo experiments where compounds known to undergo metabolic activation were examined for their propensity to form conjugates with GSH. In all cases, scanning for precursors of m/z 272 in the negative ion mode revealed the presence of the expected adducts and in some instances revealed additional conjugates that had not been reported previously. Positive ion MS/MS, on the other hand, was more useful than the corresponding negative ion scans in providing information on the molecular structure of GSH conjugates.     

Identification of a novel glutathione adduct of diclofenac, 4'-hydroxy-2'-glutathion-deschloro-diclofenac, upon incubation with human liver microsomes.

Clinical use of the nonsteroidal anti-inflammatory drug diclofenac (DF) is associated with an incidence of idiosyncratic hepatoxicity. The formation of reactive metabolites of DF in vivo has been proposed to be responsible for such toxicity. One type of reactive metabolite, a benzoquinone imine of DF formed through oxidation by cytochromes P450, can be trapped by glutathione in vitro in liver microsomes to form glutathione (GS) adducts. Three GS adducts from DF were reported in the literature, namely, 5-hydroxy (OH)-4-glutathione-DF, 4'-OH-3'-glutathione-DF and 5-OH-6-glutathione-DF, and they all have the same molecular weight of 616. Recently, we developed a sensitive and high throughput method for the detection of GS adducts from liver microsome incubation. This method uses a constant neutral loss scan of m/z 129, a "structure-characteristic" fragment for GS adduct, on an automated chip-based nanoelectrospray (Advion NanoMate 100) attached to a tandem mass spectrometer (Sciex API 3000). The analysis of GS adducts from human liver microsome incubation with DF by the NanoMate 100-API 3000 method unambiguously revealed a new adduct ion with m/z 583 (MH+), in addition to the known adduct peak with m/z 617 (MH+). This new adduct was further confirmed to be 4'-OH-2'-glutathion-deschloro-diclofenac by liquid chromatography (LC) tandem mass spectrometry (MS), LC/MS-NMR, and comparison to a synthetic standard.     

2,7-Disubstituted-pyrrolotriazine kinase inhibitors with an unusually high degree of reactive metabolite formation

There are numerous published studies establishing a link between reactive metabolite formation and toxicity of various drugs. Although the correlation between idiosyncratic reactions and reactive metabolite formation is not 1:1, the association between the two is such that many pharmaceutical companies now monitor for reactive metabolites as a standard part of drug candidate testing and selection. The most common method involves in vitro human microsomal incubations in the presence of a thiol trapping agent, such as glutathione (GSH), followed by LC/MS analysis. In this study, we describe several 2,7-disubstituted-pyrrolotriazine analogues that are extremely potent reactive metabolite precursors. Utilizing a UPLC/UV/MS method, unprecedented levels of GSH adducts were measured that are 5-10 times higher than previously reported for high reactive metabolite-forming compounds such as clozapine and troglitazone.     

Novel identification of a sulfur-centered, radical-derived 5,5-dimethyl-1-pyrroline N-oxide nitrone adduct formed from the oxidation of DTT by LC/ELISA, LC/electrospray ionization-MS, and LC/tandem MS

The detection of highly reactive free radicals generated in biological systems by an ESR spin-trapping technique is always difficult and limited due to the short lifetimes of ESR active spin-trapping radical adducts and poor structural information provided by ESR spectra. In this investigation, we have for the first time employed anti-5,5-dimethyl-1-pyrroline N-oxide (DMPO) polyclonal antiserum that specifically recognizes stable, ESR silent end products of DMPO radical adducts and combined HPLC with ELISA, electrospray ionization mass spectrometry (ESI-MS), and tandem mass spectrometry (MS/MS) to separate and characterize DMPO nitrone adducts derived from free radical metabolites. When mircoperoxidase-11 (MP-11) reacted with DTT in the presence of DMPO with or without H2O2, we detected radical-derived DMPO nitrone adducts by ELISA. Similar results were obtained when MP-11 was replaced by hemin. To identify the DMPO nitrone adducts formed in both reaction systems, LC separation was carried out, and the fractions eluted from the LC column were collected and analyzed by ELISA. In both reaction mixtures, we found that only one peak with the same retention time showed a strong positive ELISA signal, suggesting that this peak was from radical-derived DMPO nitrone adducts and that both systems produced the same free radical metabolites. Using online LC/ESI-MS, LC/MS/MS, and (1)H NMR, we demonstrated that the DMPO nitrone adducts formed are from the DMPO adducts of the sulfur-centered radical of DTT. The successful application of LC/ELISA, LC/MS, and LC/MS/MS in this study makes it possible to separate and identify the stable DMPO nitrone adducts derived from free radical metabolites generated in biological systems.     

Identification and characterization of the glutathione and N-acetylcysteine conjugates of (E)-2-propyl-2,4-pentadienoic acid, a toxic metabolite of valproic acid, in rats and humans.

The severe hepatotoxicity of valproic acid (VPA) is believed to be mediated through reactive metabolites. The formation of glutathione (GSH) and N-acetylcysteine (NAC) adducts of reactive intermediates derived from VPA and two of its metabolites, 2-propyl-4-pentenoic acid (4-ene-) and 2-propyl-2,4-pentadienoic acid [(E)-2,4-diene VPA], was investigated in the rat. Rats were dosed ip with 100 mg/kg of VPA, 4-ene-, or 2,4-diene-VPA, and methylated bile and urine extracts were analyzed by LC/MS/MS and GC/MS, respectively. The GSH conjugate of (E)-2,4-diene VPA was detected in the bile of rats treated with 4-ene- and (E)-2,4-diene VPA. The NAC conjugate was a major urinary metabolite of rats given (E)-2,4-diene VPA and was a prominent urinary metabolite of those animals given 4-ene VPA. The NAC conjugate was also found to be a metabolite of VPA in patients. Both the GSH and NAC adducts were chemically synthesized and their structures established to be 5-(glutathion-S-yl)3-ene VPA and 5-(N-acetylcystein-S-yl)3-ene VPA by NMR and mass spectrometry. In contrast to the very slow reaction of the free acid of (E)-2,4-diene VPA with GSH, the methyl ester reacted rapidly with GSH to yield the adduct. In vivo it appears the diene forms an intermediate with enhanced electrophilic reactivity to GSH as indicated by the facile reaction of the diene with GSH in vivo [about 40% of the (E)-2,4-diene VPA administered to rats was excreted as the NAC conjugate in 24 hr]. The characterization of the GSH and NAC (in humans and rats) conjugates of (E)-2,4-diene VPA suggests that VPA is metabolized to a chemically reactive intermediate that may contribute to the hepatotoxicity of the drug.     

Cyanide trapping of iminium ion reactive intermediates followed by detection and structure identification using liquid chromatography-tandem mass spectrometry (LC-MS/MS)

Secondary and tertiary alicyclic amines are widely found in pharmaceuticals and environmental compounds. The formation of iminium ions as reactive intermediates in the metabolic activation of alicyclic amines has previously been investigated in radiometric assays where radiolabeled cyanide is typically employed. In this paper, we report a relatively high throughput LC-MS/MS method for the detection of the nonradiolabeled cyanide adduct formed in rat or human liver microsomal incubations via constant neutral loss scan followed by structural characterization using product ion scan on a triple quadrupole mass spectrometer. A total of 14 alicyclic amine compounds were investigated with the cyanide trapping LC-MS/MS screen and also with the glutathione (GSH) trapping screen, a well-established and commonly employed technique for reactive metabolite screening. Our results are found to be in general agreement with the previous metabolism reports for these compounds, demonstrating the effectiveness, speed, and simplicity of the cyanide trapping LC-MS/MS method to study the iminium ion intermediates from alicyclic amines and its complementarities to GSH trapping method for reactive metabolite screenings.     

Screening botanical extracts for quinoid metabolites.

Botanical dietary supplements represent a significant share of the growing market for alternative medicine in the USA, where current regulations do not require assessment of their safety. To help ensure the safety of such products, an in vitro assay using pulsed ultrafiltration and LC-MS-MS has been developed to screen botanical extracts for the formation of electrophilic and potentially toxic quinoid species upon bioactivation by hepatic cytochromes P450. Rat liver microsomes were trapped in a flow-through chamber by an ultrafiltration membrane, and samples containing botanical extracts, GSH and NADP(H), were flow-injected into the chamber. Botanical compounds that were metabolized to reactive intermediates formed stable GSH adducts mimicking a common in vivo detoxification pathway. If present in the ultrafiltrate, GSH conjugates were detected using LC-MS-MS with precursor ion scanning followed by additional characterization using product ion scanning and comparison to standard compounds. As expected, no GSH adducts of reactive metabolites were found in extracts of Trifolium pratense L. (red clover), which are under investigation as botanical dietary supplements for the management of menopause. However, extracts of Sassafras albidum (Nutt.) Nees (sassafras), Symphytum officinale L. (comfrey), and Rosmarinus officinalis L. (rosemary), all of which are known to contain compounds that are either carcinogenic or toxic to mammals, produced GSH adducts during this screening assay. Several compounds that formed GSH conjugates including novel metabolites of rosmarinic acid were identified using database searching and additional LC-MS-MS studies. This assay should be useful as a preliminary toxicity screen during the development of botanical dietary supplements. A positive test suggests that additional toxicological studies are warranted before human consumption of a botanical product.     

Formation of deoxyguanosine cross-links from calf thymus DNA treated with acrolein and 4-hydroxy-2-nonenal

Acrolein (AC) and 4-hydroxy-2-nonenal (HNE) are endogenous bis-electrophiles that arise from the oxidation of polyunsaturated fatty acids. AC is also found in high concentrations in cigarette smoke and automobile exhaust. These reactive α,β-unsaturated aldehyde (enal) covalently modify nucleic acids, to form exocyclic adducts, where the three-carbon hydroxypropano unit bridges the N1 and N(2) positions of deoxyguanosine (dG). The bifunctional nature of these enals allows them to undergo reaction with a second nucleophilic group and form DNA cross-links. These cross-linked enal adducts are likely to contribute to the genotoxic effects of both AC and HNE. We have developed a sensitive mass spectrometric method to detect cross-linked adducts of these enals in calf thymus DNA (CT DNA) treated with AC or HNE. The AC and HNE cross-linked adducts were measured by the stable isotope dilution method, employing a linear quadrupole ion trap mass spectrometer and consecutive reaction monitoring at the MS(3) or MS(4) scan stage. The lower limit of quantification of the cross-linked adducts is ～1 adduct per 10(8) DNA bases, when 50 μg of DNA is assayed. The cross-linked adducts occur at levels that are ～1-2% of the levels of the monomeric 1,N(2)-dG adducts in CT DNA treated with either enal.     

P450-mediated metabolism of 1-[3-(aminomethyl)phenyl]-N-[3-fluoro-2'-(methylsulfonyl)- [1,1'-biphenyl]-4-yl]-3-(trifluoromethyl)-1H-pyrazole- 5-carboxamide (DPC 423) and its analogues to aldoximes. Characterization of glutathione conjugates of postulated intermediates derived from aldoximes.

The in vivo and in vitro disposition of DPC 423, a highly potent, selective, and orally bioavailable inhibitor of blood coagulation factor Xa, has recently been described. Several metabolites, some of which were considered potentially reactive, were identified in rats. A novel GSH adduct, the structure of which was not determined conclusively, was isolated from bile of rats dosed with DPC 423. Herein, we describe the complete structural elucidation of this unique GSH conjugate employing LC/MS and high-field NMR. Similar GSH adducts of DPC 602, [13CD2]DPC 602, and SX 737, all structural analogues of DPC 423, were isolated, characterized spectroscopically, and shown to have identical mass fragmentation pathways. The structures of these conjugates were initially suspected to be either an amide with N-S bond or a nitrogen-oxygen juxtaposed amide with a C-S bond. Studies conducted with [13CD2]DPC 602 indicated an aldoxime structure. The concluding evidence came from HMBC NMR spectrum of the conjugate, which showed strong correlation of the cysteine methylene protons with the imino carbon. Further spectroscopic studies with chemically prepared GSH adduct from benzaldehyde oxime confirmed this pattern of correlation. In vivo and in vitro studies with the synthetic oxime intermediate from DPC 423 showed an adduct identical to the one isolated from the bile of rats dosed with DPC 423. This supported the intermediacy of an aldoxime as a precursor to the GSH adducts. It is postulated that the benzylamine moiety of DPC 423 (and its analogues) is oxidized to a hydroxylamine, which is subsequently converted to a nitroso intermediate. Subsequent rearrangement of the nitroso leads to an aldoxime which in turn is metabolized by P450 to a reactive intermediate. The formation of oxime from DPC 423 (and its analogues) was found to be mediated by rat CYP 3A1/2, which were also responsible for converting the oxime to the GSH trappable reactive intermediate. It is postulated that the aldoxime produces a radical or a nitrile oxide intermediate that reacts with GSH and hence produces this unusual GSH adduct. On the basis of synthetic analogy, it is more likely that the nitrile oxide resulting from two-electron oxidation of the aldoxime is the reactive intermediate. Intramolecular kinetic isotope effects were studied with [13CD2]DPC 602 to assess the importance of the metabolic cleavage of the aminomethyl carbon-hydrogen bond in forming this GSH adduct. The lack of isotope effect in forming the aldoxime from [13CD2]DPC 602 suggests its formation does not occur through the imine intermediate. Instead the data supports the postulated mechanism of hydroxylamine and nitroso intermediates as precursors to the aldoxime. However, the formation of the GSH adduct from [13CD2]DPC 602 did show a significant intramolecular kinetic isotope effect (kH/kD = 2.3) since a carbon-deuterium bond had to be broken on the aldoxime prior to the formation of the adduct. A stable nitrile oxide derived from DPC 602 was postulated as the reactive intermediate responsible for forming this unique GSH adduct.     

d-Isomer of gly-tyr-pro-cys-pro-his-pro peptide: A novel and sensitive in vitro trapping agent to detect reactive metabolites by electrospray mass spectrometry

This paper describes a d-peptide isomer-based trapping assay using an LC/MS ion-trap spectrometer with an electrospray ionization (ESI) source as the analytical tool to study bioactivation of xenobiotics. Reactive metabolites were generated from parent compounds in in vitro incubations with different sources of CYP enzymes. A short d-isomer of gly-tyr-pro-cys-pro-his-pro proved to be a sensitive trapping agent and resistant to proteases. This method was tested with 16 probe substances. Acetaminophen, 1-chloro 2,4-dinitrobenzene, clozapine, diclofenac, imipramine, menthofuran, propranolol, pulegone and ticlopidine all formed d-peptide adducts, which were analogous to the GSH adducts previously described in the literature. New adducts were identified with clopidogrel (-Cl + peptide), nicotine (-CH₃₊H + peptide), nimesulide (+peptide) and tolcapone (+peptide), i.e., no GSH adducts of those drugs have been described in the literature. No adducts were identified with ciprofloxacin, ketoconazole and verapamil. In the literature no GSH adducts have been described with ciprofloxacin and verapamil. d-Peptide-based trapping proved to be a reliable and reproducible method to identify bioactivated intermediates. d-Peptide is a new and convenient protein trapping agent for use in early phase screening of bioactivation of new chemical entities and evaluation of toxic properties of chemicals.     

液相色谱—电喷雾离子阱质谱法检测体液中士的宁、马钱子碱及其主要代谢物

目的：检测体液中士的宁、马钱子碱及其主要代谢物。方法：采用液相色谱－电喷雾离子阱质谱联用法,对送检样品进行了鉴定。结果：与士的宁、马钱子碱对照品的LC／MS^n分析结果比较,证明送检样品中含有大量士的宁、马钱子碱。并根据色谱和质谱行为,推测士的宁在人体内的主要代谢产物为葡萄粮苷酸结合物,马钱子碱的代谢途径为去甲基化。最低检测限为5ng。结论：本法快速、简捷,尤其适合于毒物检测等相关部门的分析检测用。     

Bioactivation of substituted thiophenes including α-chlorothiophene-containing compounds in human liver microsomes

The thiophene moiety has been recognized as a toxicophore because of the potential of oxidative bioactivation leading to electrophilic species. The introduction of bulky or electron-withdrawing groups at the α-carbon to the sulfur atom has the potential to reduce or eliminate bioactivation. In this article, we describe the bioactivation of a variety of substituted thiophenes. These compounds were incubated in NADPH-fortified human liver microsomes with or without the addition of reduced glutathione (GSH) as a trapping agent. The resulting GSH adducts were characterized by high performance liquid chromatography/high resolution mass spectrometry with the aid of a background subtraction methodology. Four of the five α-chlorothiophenes tested formed NADPH-dependent GSH adducts. Most adducts had masses consistent with the nominal substitution of chlorine by GSH. LC/MS/MS and proton NMR of the major GSH adduct of 1-(5-chlorothiophen-2-yl)ethanone (1a) confirmed that GSH displaced chlorine. To further explore the effect of different substitutions on the bioactivation potential, a series of 2-acetylthiophenes substituted at the C4 or C5 positions were tested in a quantitative thiol-trapping assay using dansyl glutathione. Substitutions at the C4 or C5 positions gave adduct levels that decreased in the following order: 4-H, 5-H (no substitution) > 4-Br ～ 4-Cl > 5-Cl > 5-CN > 4-CH(3) > 5-Br > 5-CH(3) (no adduct detected). In conclusion, bioactivation was detected in a series of substituted thiophenes. Although substitutions on the thiophene ring can reduce the formation of reactive metabolites, the degree of reduction is dependent on the substitution position and substituent.     

Tea polyphenol (-)-epigallocatechin-3-gallate: a new trapping agent of reactive dicarbonyl species

Previous studies have demonstrated that reactive dicarbonyl compounds [e.g., methylglyoxal (MGO) and glyoxal (GO)] irreversibly and progressively modify proteins over time and yield advanced glycation end products (AGEs), which are thought to contribute to the development of diabetes mellitus and its subsequent complications. Thus, decreasing the levels of MGO and GO will be an effective approach to reduce the formation of AGEs and the development of diabetic complications. In our studies to find nontoxic trapping agents of reactive dicarbonyl species from dietary sources, we found that (-)-epigallocatechin-3-gallate (EGCG), the major bioactive green tea polyphenol, could efficiently trap reactive dicarbonyl compounds (MGO or GO) to form mono- and di-MGO or GO adducts under physiological conditions (pH 7.4, 37 degrees C). The products formed from EGCG and MGO (or GO), combined at different ratios, were analyzed using LC/MS. We also developed a method to purify the two major mono-MGO adducts of EGCG without derivatization, and their structures were identified as stereoisomers of mono-MGO adducts of EGCG based on their 1D and 2D NMR spectra. Our LC/MS and NMR data showed that positions 6 and 8 of the EGCG A-ring were the major active sites for trapping reactive dicarbonyl compounds. We also found that EGCG lost its trapping efficacy under acidic conditions (pH相似文献   

A semi-automated method for the integrated evaluation of half-life and metabolic soft spots of discovery compounds

Background: An integrated method that provides rates of both parent disappearance and metabolite formation was developed. Results: Buspirone, mirtazapine and verapamil were used as model compounds in developing the method. Incubations were carried out on a robotic platform. Qualitative analysis of metabolites in 30 μM samples was conducted by data-dependent HPLC-MS/MS on a high-resolution instrument. Quantitative analysis of the parent compound and metabolites in 0.5 μM samples was conducted by full-scan MS(2) with product ion extraction using an ion trap mass spectrometer. Data generated for the compounds included half-life and intrinsic clearance of the parent molecule, characterization of metabolites and relative rates of metabolite formation. A correction factor was used to convert MS responses of metabolites in 0.5 μM samples to UV areas in order to compare relative metabolite concentrations. Conclusion: The approach allows for the investigation of a set of six compounds simultaneously, with a turnaround time of 1 week or less.