Screening strategy for the rapid detection of in vitro generated glutathione conjugates using high‐performance liquid chromatography and low‐resolution mass spectrometry in combination with LightSight® software for data processing

The knowledge of drug metabolism in the early phases of the drug discovery process is vital for minimising compound failure at later stages. As chemically reactive metabolites may cause adverse drug reactions, it is generally accepted that avoiding formation of reactive metabolites increases the chances of success of a molecule. In order to generate this important information, a screening strategy for the rapid detection of in vitro generated reactive metabolites trapped by glutathione has been developed. The bioassay incorporated the use of native glutathione and its close analogue the glutathione ethyl ester. The generic conditions for detecting glutathione conjugates that undergo constant neutral loss of 129 Da were optimised using a glutathione‐based test mix of four compounds. The final liquid chromatography/tandem mass spectrometry constant neutral loss method used low‐resolution settings and a scanning window of 200 amu. Data mining was rapidly and efficiently performed using LightSight® software. Unambiguous identification of the glutathione conjugates was significantly facilitated by the analytical characteristics of the conjugate pairs formed with glutathione and glutathione ethyl ester, i.e. by chromatographic retention time and mass differences. The reliability and robustness of the screening strategy was tested using a number of compounds known to form reactive metabolites. Overall, the developed screening strategy provided comprehensive and reliable identification of glutathione conjugates and is well suited for rapid routine detection of trapped reactive metabolites. This new approach allowed the identification of a previously unreported diclofenac glutathione conjugate. Copyright © 2009 John Wiley & Sons, Ltd.     

Identification of new minor metabolites of penicillin G in human serum by multiple-stage tandem mass spectrometry

Liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) were applied to characterize drug metabolites. Although these two methods have overcome the identification and structural characterization of metabolites analysis, they remain time‐consuming processes. In this study, a novel multiple‐stage tandem mass spectrometric method (MSⁿ) was evaluated for identification and characterization of new minor metabolism profiling of penicillin G, one of the β‐lactam antibiotics, in human serum. Seven minor metabolites including five phase I metabolites and two phase II metabolites of penicillin G were identified by using data‐dependent LC/MSⁿ screening in one chromatographic run. The accuracy masses of seven identified metabolites of penicillin G were also confirmed by mass spectral calibration software (MassWorks?). The proposed data‐dependent LC/MSⁿ method is a powerful tool to provide large amounts of the necessary structural information to characterize minor metabolite in metabolism profiling. Copyright © 2010 John Wiley & Sons, Ltd.     

Method for rapid metabolite profiling of drug candidates in fresh hepatocytes using liquid chromatography coupled with a hybrid quadrupole linear ion trap

Rapid information on metabolic profiling is required to evaluate the structural liabilities of drug candidates in early drug discovery. In this study, a sensitive and rapid semi-quantitative method was developed to simultaneously monitor the drug candidate and metabolites as well as collect tandem mass (MS/MS) spectra for subsequent metabolite identification. The simultaneous semi-quantitation and identification of metabolites in fresh hepatocytes is achieved using high-performance liquid chromatography (HPLC) coupled with a hybrid quadrupole linear ion trap. The survey experiment consists of monitoring multiple-reaction monitoring (MRM) transitions for the internal standard, the parent, and 48 MRM transitions designed to cover the most common phase I and II biotransformations. An information-dependent acquisition (IDA) method was employed to trigger product ion scans above the MRM signal threshold. Three biotransformations of a lead compound have been identified through enhanced product ion scans and the respective MRM transitions of those metabolites were selected for semi-quantitation. Parent disappearance and formation of the metabolites as a function of incubation time in five different species were monitored by their respective MRM responses. The method provides the necessary sensitivity to detect minor metabolites in a relevant therapeutic concentration range. Enzymatic turnover of the parent and the metabolites in different species are revealed based on the different initial concentrations of the parent. This methodology integrates the parent disappearance, metabolite identification, and the formation of the metabolites along the time course using a single rapid LC/MS/MS analysis. This method can be used as a complementary tool to the conventional method of metabolic profiling. It provides a rapid and sensitive initial profile of the metabolism of potential structural series at the lead selection stage. The method can also be incorporated into the overall metabolite profiling scheme to evaluate the drug candidates in drug discovery.     

Novel approach to performing metabolite identification in drug metabolism

A novel online method is developed, using liquid chromatography (LC)-accurate radioisotope counting dynamic-flow (ARC) coupled with a radioactivity detector and mass spectrometer, for metabolite identification in drug discovery and development. This method offers the advantages of improved sensitivity for detecting radiolabeled drugs as well as streamlining the process of identifying and characterizing metabolites. For the purposes of evaluating this method, in vitro human liver microsomal incubations with [(14)C]dextromethorphan are conducted. Online separation and identification of [(14)C]dextromethorphan metabolites are achieved without intensive sample preparation, concentration, or fraction collection. Mass spectrometric analysis identified and characterized the metabolites of dextromethorphan formed by N - and O -dealkylation, correlated well with previously published results. Chromatographic peaks for [(14)C]dextromethorphan and its metabolites are collected online, then infused for extended periods of time at a flow rate of 10 microL/min while maintaining the column pressure. The continuous analytical signal input allowed acquisition of a higher order of multistage fragmentation for both major and minor metabolites. The multistage MS fragmentation pattern obtained for the metabolites allowed defining the sites of metabolism for dextromethorphan. Further evaluations of this method are also conducted using a [(14)C]compound A to check the linearity and sensitivity of the dynamic-flow method. The R(2) value is 0.996 for the dynamic-flow method between 50 and 600 disintegrations per minute (dpm); the limit of detection for LC-ARC is 20 dpm, which is approximately 10 times more sensitive than conventional continuous-flow radioactivity detection techniques. The overall results suggest that the combination of LC-ARC with radioactivity detection and mass spectrometry has great potential as a powerful tool for enhancing the sensitivity of radioisotope measurement in metabolite identification studies during drug discovery and development.     

A high-throughput liquid chromatography/tandem mass spectrometry method for screening glutathione conjugates using exact mass neutral loss acquisition

Chemically reactive metabolites may cause hepatotoxicity and as a result liver failure or other adverse side reactions. Therefore, this is a vital topic of interest because early reactive metabolite screening may prevent compound failure at a later stage. In order to address this issue, a screening assay has been developed to detect the formation of reactive metabolites by using glutathione as a trapping reagent, which will allow us to search for phase I metabolites and also glutathiones during in vitro metabolite screening using liquid chromatography/tandem mass spectrometry (LC/MS/MS) with exact mass. Glutathione conjugations when fragmented by the mass spectrometer give a common loss corresponding to the pyroglutamic acid moiety, which can be monitored. Until recently, this work has been carried out with triple quadrupole technology using nominal mass. The advantage of the hybrid quadrupole time-of-flight mass spectrometer is the selectivity and sensitivity that can be achieved. Exact neutral loss detection is achieved via sequential low- and high-energy MS acquisitions. After detection of the loss of the pyroglutamic acid moiety, using a window of +/-20 mDa on the high-energy scan, MS/MS is carried out on the parent mass of interest to confirm the common neutral loss.     

Improved detection of reactive metabolites with a bromine‐containing glutathione analog using mass defect and isotope pattern matching

Drug bioactivation leading to the formation of reactive species capable of covalent binding to proteins represents an important cause of drug‐induced toxicity. Reactive metabolite detection using in vitro microsomal incubations is a crucial step in assessing potential toxicity of pharmaceutical compounds. The most common method for screening the formation of these unstable, electrophilic species is by trapping them with glutathione (GSH) followed by liquid chromatography/mass spectrometry (LC/MS) analysis. The present work describes the use of a brominated analog of glutathione, N‐(2‐bromocarbobenzyloxy)‐GSH (GSH‐Br), for the in vitro screening of reactive metabolites by LC/MS. This novel trapping agent was tested with four drug compounds known to form reactive metabolites, acetaminophen, fipexide, trimethoprim and clozapine. In vitro rat microsomal incubations were performed with GSH and GSH‐Br for each drug with subsequent analysis by liquid chromatography/high‐resolution mass spectrometry on an electrospray time‐of‐flight (ESI‐TOF) instrument. A generic LC/MS method was used for data acquisition, followed by drug‐specific processing of accurate mass data based on mass defect filtering and isotope pattern matching. GSH and GSH‐Br incubations were compared to control samples using differential analysis (Mass Profiler) software to identify adducts formed via the formation of reactive metabolites. In all four cases, GSH‐Br yielded improved results, with a decreased false positive rate, increased sensitivity and new adducts being identified in contrast to GSH alone. The combination of using this novel trapping agent with powerful processing routines for filtering accurate mass data and differential analysis represents a very reliable method for the identification of reactive metabolites formed in microsomal incubations. Copyright © 2010 John Wiley & Sons, Ltd.     

Simultaneous Screening of Glutathione and Cyanide Adducts Using Precursor Ion and Neutral Loss Scans-Dependent Product Ion Spectral Acquisition and Data Mining Tools

Drugs can be metabolically activated to soft and hard electrophiles, which are readily trapped by glutathione (GSH) and cyanide (CN), respectively. These adducts are often detected and structurally characterized using separate tandem mass spectrometry methods. We describe a new method for simultaneous screening of GSH and CN adducts using precursor ion (PI) and neutral loss (NL) scans-dependent product ion spectral acquisition and data mining tools on an triple quadrupole linear ion trap mass spectrometry. GSH, potassium cyanide, and their stable isotope labeled analogues were incubated with liver microsomes and a test compound. Negative PI scan of m/z 272 for detection of GSH adducts and positive NL scans of 27 and 29 Da for detection of CN adducts were conducted as survey scans to trigger acquisition of enhanced resolution (ER) spectrum and subsequent enhanced product ion (EPI) spectrum. Post-acquisition data mining of EPI data set using NL filters of 129 and 27 Da was then performed to reveal the GSH adducts and CN adducts, respectively. Isotope patterns and EPI spectra of the detected adducts were utilized for identification of their molecular weights and structures. The effectiveness of this method was evaluated by analyzing reactive metabolites of nefazodone formed from rat liver microsomes. In addition to known GSH- and CN-trapped reactive metabolites, several new CN adducts of nefazodone were identified. The results suggested that current approach is highly effective in the analysis of both soft and hard reactive metabolites and can be used as a high-throughput method in drug discovery.     

Single-step analysis of low abundance phosphoamino acids via on-line sample preconcentration with chemical derivatization by capillary electrophoresis

New strategies for rapid, sensitive and high-throughput analysis of low abundance metabolites in biological samples are required for future metabolomic research. In this report, a direct method for sub-micromolar analyses of phosphoamino acids was developed using on-line sample preconcentration with 9-fluorenylmethyloxycarbonyl chloride (FMOC) derivatization by capillary electrophoresis (CE) and UV detection. Analyte focusing by dynamic pH junction and FMOC labeling efficiency were influenced by several experimental factors including buffer pH, ionic strength, sample injection length and FMOC concentration. About a 200-fold enhancement in concentration sensitivity was achieved under optimal conditions relative to conventional off-line derivatization, as reflected by a detection limit (S/N approximately 3) of 0.1 microM. In-capillary sample preconcentration with chemical labeling by CE offers a unique single-step analytical platform for high-throughput screening of low abundance metabolites without intrinsic chromophores.     

Sensitivity improvement in 19F NMR-based screening experiments: theoretical considerations and experimental applications

NMR-based binding and functional screening performed with FAXS (fluorine chemical shift anisotropy and exchange for screening) and 3-FABS (three fluorine atoms for biochemical screening) represents a potential alternative approach to high-throughput screening for the identification of novel potential drug candidates. The major limitation of this method in its current status is its intrinsic low sensitivity that limits the number of tested compounds. One approach for overcoming this problem is the use of a cryogenically cooled (19)F probe that reduces the thermal noise in the receiver circuitry. Sensitivity improvement in the two screening techniques achieved with the novel cryogenic (19)F probe technology permits an increased throughput, detection of weaker binders and inhibitors (relevant in a fragment-based lead discovery program), detection of slow binders, and reduction in protein and substrate consumption. These aspects are analyzed with theoretical simulations and experimental quantitative performance evaluation. Application of 3-FABS combined with the cryogenic (19)F probe technology to rapid screening at very low enzyme concentrations and the current detection limits reached with this approach are also presented.     

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.     

High-Throughput Screening of Drugs of Abuse in Urine by Supported Liquid–Liquid Extraction and UHPLC Coupled to Tandem MS

A qualitative method, involving supported liquid–liquid extraction (SLE) and ultra high pressure liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS–MS), was developed for the rapid tentative identification of various drugs of abuse in urine. In this study, 28 drugs and metabolites were covered by the screening procedure. Before analysis, urine samples were extracted by SLE and good extraction recoveries were obtained for most investigated compounds. The UHPLC strategy was then selected for the rapid separation of amphetamines, cocaine, opiates and related compounds in urine. Using columns packed with sub-2 µm particles, analysis time was reduced down to 2 min, while maintaining acceptable performance. Finally, the detection was by tandem MS operating in the single reaction monitoring (SRM) mode. The most intense transition was selected for the different drugs and SRM dwell times set at 5 ms, to maintain sufficient data points across the narrow UHPLC peaks. The tentative identification of the drugs of interest, including amphetamines, opiates and cocaine, was based on both, retention times and mass spectrometry information. With the proposed method, limits of detection were estimated at about 1 ng mL^?1 and the applicability was assessed by successfully analyzing several samples of drug abusers. Finally, this study demonstrates the potential of UHPLC coupled to tandem MS for the rapid screening of drugs of abuse in urine.     

Enzyme-catalyzed amperometric oxidation of neurotransmitters in chip-capillary electrophoresis

The determination of biogenic monoamines by enzyme-catalyzed oxidation after electrophoretical separation on a microfluidic chip decreases their detection limits significantly. An amperometric system with a chemically amplified response for neurotransmitters and their metabolites is presented. The principle is the rapid cyclic oxidation of the analyte on the amperometric detector in the presence of the redoxactive enzyme glucose oxidase in the capillary electrophoresis buffer. With this approach, detection limits in the range of 10(-7)-10(-8) M could be reached. Because of the good linearity between the current response and the concentration of catecholamines and their metabolites at concentrations up to 300 microM, this method is attractive for the analytical detection at low concentration levels such as in biological fluids.     

Parallel determination of multiple protein metabolite interactions using cell extract, protein microarrays and mass spectrometric detection

Analysis of interactive networks between proteins and other molecular constituents is of paramount importance to delineate complex cellular processes. In order to facilitate this process, new technologies that allow rapid, high-throughput parallel screening, as well as identification of constituents, are necessary. A particularly powerful combination in this regard could be the use of multiprotein microarrays coupled with mass spectrometry (MS). In the initial step of the method development we applied MS to single-protein microarrays. We demonstrated that even a simplified version of the method allows rapid parallel label-free assay of specific protein interactions with multiple metabolites derived from complex artificial and natural mixtures. The microarrays fabricated by the electrospray deposition technique and cross-linked in glutaraldehyde vapor were brought into contact with droplets of solution containing either a natural extract of baker's yeast cells or an artificial cocktail of metabolites. After washing, the microarrays were placed into 75% methanol to denature proteins and release specifically bound metabolites. The eluates were then analyzed by electrospray ionization mass spectrometry (ESI-MS) to simultaneously detect all the metabolites bound. Such a procedure applied to ten different proteins demonstrated that 50-400 ng of cross-linked protein is enough to obtain ion intensities from metabolites that are well distinguishable above noise. The compatibility of microplates and different microarray designs with MS detection is discussed.     

Rapid monitoring and sensitive determination of DDT and its metabolites in water sample using solid-phase extraction followed by ion mobility spectrometry

Dichlorodiphenyl trichloroethane (DDT) as an organochlorine compound has been globally used as a pesticide for controlling soil-dwelling insects and treating diseases such as malaria and typhus. The degradation products of DDT and its metabolites have also negative effects on the environment. The present study has investigated the determination of DDT and its metabolites in water sample using ion mobility spectrometry (IMS) as a rapid and sensitive detection technique. For this purpose, DDT and its metabolites were extracted using reverse phase solid-phase extraction (SPE) from water samples. The samples were then recovered by eluting with methanol and finally, quantified using the corona discharge IMS technique. Injection and oven temperatures and the effect of dopant were optimized as experimental parameters influencing both detection and determination efficiencies. Degradation of DDT in IMS drift tube was studied and reduced mobility values of DDT and its metabolites were calculated. The developed method was validated using water sample to obtain good results for the determination of DDT at low levels (1 ng ml^?1) while spiked recoveries were obtained to be between 95.0–96.7%. The proposed method based on IMS proved to be a simple, inexpensive, rapid and sensitive procedure for the fast monitoring and determination of DDT and its main metabolites in water sample.     

Rapid detection and characterization of reactive drug metabolites in vitro using several isotope‐labeled trapping agents and ultra‐performance liquid chromatography/time‐of‐flight mass spectrometry

Reactive metabolites are believed to be one of the main reasons for unexpected drug‐induced toxicity issues, by forming covalent adducts with cell proteins or DNA. Due to their high reactivity and short lifespan they are not directly detected by traditional analytical methods, but are most traditionally analyzed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) after chemical trapping with nucleophilic agents such as glutathione. Here, a simple but very efficient assay was built up for screening reactive drug metabolites, utilizing stable isotope labeled glutathione, potassium cyanide and semicarbazide as trapping agents and highly sensitive ultra‐performance liquid chromatography/time‐of‐flight mass spectrometry (UPLC/TOFMS) as an analytical tool. A group of twelve structurally different compounds was used as a test set, and a large number of trapped metabolites were detected for most of them, including many conjugates not reported previously. Glutathione‐trapped metabolites were detected for nine of the twelve test compounds, whereas cyanide‐trapped metabolites were found for eight and semicarbazide‐trapped for three test compounds. The high mass accuracy of TOFMS provided unambiguous identification of change in molecular formula by formation of a reactive metabolite. In addition, use of a mass defect filter was found to be a usable tool when mining the trapped conjugates from the acquired data. The approach was shown to provide superior detection sensitivity in comparison to traditional methods based on neutral loss or precursor ion scanning with a triple quadrupole mass spectrometer, and clearly more efficient detection and characterization of reactive drug metabolites with a simpler test setup. Copyright © 2009 John Wiley & Sons, Ltd.     

LC-ESI/MS/MS method for rapid screening and confirmation of 44 exogenous anabolic steroids in human urine

A sensitive and rapid method based on liquid chromatography-triple-quadrupole tandem mass spectrometry (LC-MS/MS) with electrospray ionization (ESI) has been developed and validated for the screening and confirmation of 44 exogenous anabolic steroids (29 parent steroids and 15 metabolites) in human urine. The method involves an enzymatic hydrolysis, liquid-liquid extraction, and detection by LC-MS/MS. A triple-quadrupole mass spectrometer was operated in positive ESI mode with selected reaction monitoring (SRM) mode for the screening and product ion scan mode for the confirmation. The protonated molecular ions were used as precursor ions for the SRM analysis and product ion scan. The intraday and interday precisions of the target analytes at concentrations of the minimum required performance levels for the screening were 2-14% and 2-15%, respectively. The limits of detection for the screening and confirmation method were 0.1-10 ng/mL and 0.2-10 ng/mL, respectively, for 44 steroids. This method was successfully applied to analysis of urine samples from suspected anabolic steroid abusers.     

Rapid screening for cyclo-dopa and diketopiperazine alkaloids in crude extracts of Portulaca oleracea L. using liquid chromatography/tandem mass spectrometry

A simple and rapid qualitative liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed and validated for screening cyclo-dopa and diketopiperazine alkaloids in crude extracts of Portulaca oleracea L. at sub-ppm levels. An electrospray ionization orbitrap mass spectrometer, which provides accurate full scan MS and MS/MS data, was used in this study. After simple extraction with ethanol and purification by AB-8 resin, the extracts were subjected to LC/MS/MS analysis. A high mass tolerance (10 ppm) was used in the initial screening to filter the full scan MS data. The cyclo-dopa and diketopiperazine alkaloid standards gave limits of detection (LODs) at or below 5 ng/mL. The results also indicated that the method had an acceptable precision for day-to-day use in the identification of compounds. The alkaloids could be identified based on their MS/MS data, elemental compositions, and retention behavior. This system was used to assay trace amounts of cyclo-dopa and diketopiperazine alkaloids in crude extracts of Portulaca oleracea L., leading to the identification of 5/2 confirmed/unconfirmed cyclo-dopa and 7/6 confirmed/unconfirmed diketopiperazine alkaloids, respectively. The screening method considerably reduces the time and cost involved in the identification of cyclo-dopa and diketopiperazine alkaloids in Portulaca oleracea L., as well as being a simple and convenient approach to the identification of other structural families of natural products.     

Rapid detection of Staphylococcus aureus by a combination of monoclonal antibody-coated latex and capillary electrophoresis

The rapid detection of pathogenic bacteria is extremely important in biotechnology and clinical diagnosis. CE has been utilized in the field of bacterial analysis for many years, but to some extent, simultaneous separation and identification of certain microbes from complex samples by CE coupled with UV detector is still a challenge. In this paper, we propose a new strategy for rapid separation and identification of Staphylococcus aureus (S. aureus) in bacterial mixtures by means of specific mAb-coated latex coupled with CZE. An appropriate set of conditions that selectively isolated S. aureus from the microorganisms Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae were established. S. aureus could be differentiated from the others by unique peaks in the electropherograms. The validity was also confirmed by LIF with antibodies specific to both the latex and the microbial cells. The LOD is as low as 9.0 x 10(5) colony forming unit/mL. We have also utilized this technology to identify S. aureus in a stool sample coming from a healthy volunteer spiked successfully with S. aureus. This CZE-UV technique can be applied to rapid diagnosis of enteritis caused by S. aureus or other bacterial control-related fields needing rapid identification of target pathogens from microbial mixtures. In theory, this method is suitable for the detection of any bacterium as long as corresponding bacterium-specific antibody-coated latex is available.     

Fungal metabolite screening: database of 474 mycotoxins and fungal metabolites for dereplication by standardised liquid chromatography-UV-mass spectrometry methodology

A standardised LC-UV-MS micro-scale method for screening of fungal metabolites and mycotoxins in culture extracts is presented. The paper includes data for detection and dereplication of > 400 fungal metabolites to facilitate detection and identification when standards are not available. The data also shows the types of components that can be analysed by positive electrospray (ESI+) mass spectrometry (MS) along with common fragments and adducts of these, as well as giving suggestions on whether UV or ESI+-MS methods should be used. Examples of dereplication of penitrems and macro-cyclic ichothecenes, and detection of several novel compounds are shown. This was done by UV spectroscopy combined with accurate mass determination of adduct and fragment ions obtained by high-resolution orthogonal time-of-flight MS.