Rapid screening and characterization of drug metabolites using a multiple ion monitoring-dependent MS/MS acquisition method on a hybrid triple quadrupole-linear ion trap mass spectrometer

A novel LC/MS/MS method that uses multiple ion monitoring (MIM) as a survey scan to trigger the acquisition of enhanced product ions (EPI) on a hybrid quadrupole-linear ion trap mass spectrometer (Q TRAP) was developed for drug metabolite identification. In the MIM experiment, multiple predicted metabolite ions were monitored in both Q1 and Q3. The collision energy in Q2 was set to a low value to minimize fragmentation. Results from analyzing ritonavir metabolites in rat hepatocytes demonstrate that MIM-EPI was capable of targeting a larger number of metabolites regardless of their fragmentation and retained sensitivity and duty cycle similar to multiple reaction monitoring (MRM)-EPI. MIM-based scanning methods were shown to be particularly useful in several applications. First, MIM-EPI enabled the sensitive detection and MS/MS acquisition of up to 100 predicted metabolites. Second, MIM-MRM-EPI was better than MRM-EPI in the analysis of metabolites that undergo either predictable or unpredictable fragmentation pathways. Finally, a combination of MIM-EPI and full-scan MS (EMS), as an alternative to EMS-EPI, was well suited for routine in vitro metabolite profiling. Overall, MIM-EPI significantly enhanced the metabolite identification capability of the hybrid triple quadrupole-linear ion trap LC/MS.     

Isobaric metabolite interferences and the requirement for close examination of raw data in addition to stringent chromatographic separations in liquid chromatography/tandem mass spectrometric analysis of drugs in biological matrix

In addition to matrix effects, common interferences observed in liquid chromatography/tandem mass spectrometry (LC/MS/MS) analyses can be caused by the response of drug-related metabolites to the multiple reaction monitoring (MRM) channel of a given drug, as a result of in-source reactions or decomposition of either phase I or II metabolites. However, it has been largely ignored that, for some drugs, metabolism can lead to the formation of isobaric or isomeric metabolites that exhibit the same MRM transitions as parent drugs. The present study describes two examples demonstrating that interference caused by isobaric or isomeric metabolites is a practical issue in analyzing biological samples by LC/MS/MS. In the first case, two sequential metabolic reactions, demethylation followed by oxidation of a primary alcohol moiety to a carboxylic acid, produced an isobaric metabolite that exhibits a MRM transition identical to the parent drug. Because the drug compound was rapidly metabolized in rats and completely disappeared in plasma samples, the isobaric metabolite appeared as a single peak in the total ion current (TIC) trace and could easily be quantified as the drug since it was eluted at a retention time very close to that of the drug in a 12-min LC run. In the second example, metabolism via the ring-opening of a substituted isoxazole moiety led to the formation of an isomeric product that showed an almost identical collision-induced dissociation (CID) MS spectrum as the original drug. Because two components were co-eluted, the isomeric product could be mistakenly quantified and reported by data processing software as the parent drug if the TIC trace was not carefully inspected. Nowadays, all LC/MS data are processed by computer software in a highly automated fashion, and some analysts may spend much less time to visually examine raw TIC traces than they used to do. Two examples described in this article remind us that quality data require both adequate chromatographic separations and close examination of raw data in LC/MS/MS analyses of drugs in biological matrix.     

Use of a quadrupole linear ion trap mass spectrometer in metabolite identification and bioanalysis

A new type of quadrupole linear ion trap mass spectrometer, Q TRAP trade mark LC/MS/MS system (Q TRAP trade mark ), was evaluated for its performance in two studies: firstly, the in vitro metabolism of gemfibrozil in human liver microsomes, and, secondly, the quantification of propranolol in rat plasma. With the built-in information-dependent-acquisition (IDA) software, the instrument utilizes full scan MS in the ion trap mode and/or constant neutral loss scans as survey scans to trigger product ion scan (MS(2)) and MS(3) experiments to obtain structural information of drug metabolites 'on-the-fly'. Using this approach, five metabolites of gemfibrozil were detected in a single injection. This instrument combines some of the unique features of a triple quadrupole mass spectrometer, such as constant neutral loss scan, precursor ion scan and multiple reaction monitoring (MRM), together with the capability of a three-dimensional ion trap. Therefore, it becomes a powerful instrument for metabolite identification. The fast duty cycle in the ion trap mode allows the use of full product ion scan for quantification. For the quantification of propranolol, both MRM mode and full product ion scan in the ion trap mode were employed. Similar sensitivity, reproducibility and linearity values were established using these two approaches. The use of the product ion scan mode for quantification provided a convenient tool in selecting transitions for improving selectivity during the method development stage.     

General unknown screening procedure for the characterization of human drug metabolites in forensic toxicology: Applications and constraints

LC coupled to single (LC–MS) and tandem (LC–MS/MS) mass spectrometry is recognized as the most powerful analytical tools for metabolic studies in drug discovery. In this article, we describe five cases illustrating the utility of screening xenobiotic metabolites in routine analysis of forensic samples using LC–MS/MS. Analyses were performed using a previously published LC–MS/MS general unknown screening (GUS) procedure developed using a hybrid linear IT–tandem mass spectrometer. In each of the cases presented, the presence of metabolites of xenobiotics was suspected after analyzing urine samples. In two cases, the parent drug was also detected and the metabolites were merely useful to confirm drug intake, but in three other cases, metabolite detection was of actual forensic interest. The presented results indicate that: (i) the GUS procedure developed is useful to detect a large variety of drug metabolites, which would have been hardly detected using targeted methods in the context of clinical or forensic toxicology; (ii) metabolite structure can generally be inferred from their “enhanced” product ion scan spectra; and (iii) structure confirmation can be achieved through in vitro metabolic experiments or through the analysis of urine samples from individuals taking the parent drug.     

An introduction to hybrid ion trap/time‐of‐flight mass spectrometry coupled with liquid chromatography applied to drug metabolism studies

Metabolism studies play an important role at various stages of drug discovery and development. Liquid chromatography combined with mass spectrometry (LC/MS) has become a most powerful and widely used analytical tool for identifying drug metabolites. The suitability of different types of mass spectrometers for metabolite profiling differs widely, and therefore, the data quality and reliability of the results also depend on which instrumentation is used. As one of the latest LC/MS instrumentation designs, hybrid ion trap/time‐of‐flight MS coupled with LC (LC‐IT‐TOF‐MS) has successfully integrated ease of operation, compatibility with LC flow rates and data‐dependent MSⁿ with high mass accuracy and mass resolving power. The MSⁿ and accurate mass capabilities are routinely utilized to rapidly confirm the identification of expected metabolites or to elucidate the structures of uncommon or unexpected metabolites. These features make the LC‐IT‐TOF‐MS a very powerful analytical tool for metabolite identification. This paper begins with a brief introduction to some basic principles and main properties of a hybrid IT‐TOF instrument. Then, a general workflow for metabolite profiling using LC‐IT‐TOF‐MS, starting from sample collection and preparation to final identification of the metabolite structures, is discussed in detail. The data extraction and mining techniques to find and confirm metabolites are discussed and illustrated with some examples. This paper is directed to readers with no prior experience with LC‐IT‐TOF‐MS and will provide a broad understanding of the development and utility of this instrument for drug metabolism studies. Copyright © 2012 John Wiley & Sons, Ltd.     

Rapid screening and characterization of drug metabolites using multiple ion monitoring dependent product ion scan and postacquisition data mining on a hybrid triple quadrupole‐linear ion trap mass spectrometer

Multiple ion monitoring (MIM)‐dependent acquisition with a triple quadrupole‐linear ion trap mass spectrometer (Q‐trap) was previously developed for drug metabolite profiling. In the analysis, multiple predicted metabolite ions are monitored in both Q1 and Q3 regardless of their fragmentations. The collision energy in Q2 is set to a low value to minimize fragmentation. Once an expected metabolite is detected by MIM, enhanced product ion (EPI) spectral acquisition of the metabolite is triggered. To analyze in vitro metabolites, MIM‐EPI retains the sensitivity and selectivity similar to that of multiple reaction monitoring (MRM)‐EPI in the analysis of in vitro metabolites. Here we present an improved approach utilizing MIM‐EPI for data acquisition and multiple data mining techniques for detection of metabolite ions and recovery of their MS/MS spectra. The postacquisition data processing tools included extracted ion chromatographic analysis, product ion filtering and neutral loss filtering. The effectiveness of this approach was evaluated by analyzing oxidative metabolites of indinavir and glutathione (GSH) conjugates of clozapine and 4‐ethylphenol in liver microsome incubations. Results showed that the MIM‐EPI‐based data mining approach allowed for comprehensive detection of metabolites based on predicted protonated molecules, product ions or neutral losses without predetermination of the parent drug MS/MS spectra. Additionally, it enabled metabolite detection and MS/MS acquisition in a single injection. This approach is potentially useful in high‐throughout screening of metabolic soft spots and reactive metabolites at the drug discovery stage. Copyright © 2009 John Wiley & Sons, Ltd.     

In vivo metabolite detection and identification in drug discovery via LC-MS/MS with data-dependent scanning and postacquisition data mining

An important aspect in drug discovery is the early structural identification of the metabolites of potential new drugs. This gives information on the metabolically labile points in the molecules under investigation, suggesting structural modifications to improve their metabolic stability, and allowing an early safety assessment via the identification of metabolic activation products. From an analytical point of view, metabolite identification still remains a challenging task, especially for in vivo samples, in which they occur at trace levels together with high amounts of endogenous compounds. Here we describe a method, based on LC-ion trap tandem MS, for the rapid in vivo metabolite identification. It is based on the automatic, data-dependent acquisition of multiple product ion MS/MS scans, followed by a postacquisition search, within the entire MS/MS data set obtained, for specific neutral losses or marker ions in the tandem mass spectra of parent molecule and putative metabolites. One advantage of the method is speed, since it requires minimum sample preparation and all the necessary data can be obtained in one chromatographic run. In addition, it is highly sensitive and selective, allowing detection of trace metabolites even in the presence of a complex matrix. As an example of application, we present the studies of the in vivo metabolism of the compound MEN 15916 (1). The method allowed identification of monohydroxy ([M + H](+) = m/z 655), dihydroxy ([M + H](+) = m/z 671), and trihydroxy ([M + H](+) = m/z 687) metabolites, as well as some unexpected biotransformation products such as a carboxylic acid ([M + H](+) = m/z 669), a N-dealkylated metabolite ([M + H](+) = m/z 541), and its hydroxy-analog ([M + H](+) = m/z 557).     

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.     

'All-in-one' analysis for metabolite identification using liquid chromatography/hybrid quadrupole time-of-flight mass spectrometry with collision energy switching

The removal of bottlenecks in discovery stage metabolite identification studies is an ongoing challenge for the pharmaceutical industry. We describe the use of an 'All-in-One' approach to metabolite characterization that leverages the fast scanning and high mass accuracy of hybrid quadrupole time-of-flight mass spectrometry (QqToFMS) instruments. Full-scan MS and MS/MS data is acquired using collision energy switching without the preselection, either manually or in a data-dependent manner, of precursor ions. The acquisition of 'clean' MS/MS data is assisted by the use of ultrahigh-performance chromatography. Data acquired using this method can then be mined post-acquisition in a number of ways. These include using narrow window extracted ion chromatograms (nwXICs) for expected biotransformations, XICs for the product ions of the parent compound and/or expected modification of these product ions, and neutral loss chromatograms. This approach has the potential to be truly comprehensive for the determination of in vitro biotransformations in a drug discovery environment.     

Accurate mass filtering of ion chromatograms for metabolite identification using a unit mass resolution liquid chromatography/mass spectrometry system

Acceleration of liquid chromatography/mass spectrometric (LC/MS) analysis for metabolite identification critically relies on effective data processing since the rate of data acquisition is much faster than the rate of data mining. The rapid and accurate identification of metabolite peaks from complex LC/MS data is a key component to speeding up the process. Current approaches routinely use selected ion chromatograms that can suffer severely from matrix effects. This paper describes a new method to automatically extract and filter metabolite-related information from LC/MS data obtained at unit mass resolution in the presence of complex biological matrices. This approach is illustrated by LC/MS analysis of the metabolites of verapamil from a rat microsome incubation spiked with biological matrix (bile). MS data were acquired in profile mode on a unit mass resolution triple-quadrupole instrument, externally calibrated using a unique procedure that corrects for both mass axis and mass spectral peak shape to facilitate metabolite identification with high mass accuracy. Through the double-filtering effects of accurate mass and isotope profile, conventional extracted ion chromatograms corresponding to the parent drug (verapamil at m/z 455), demethylated verapamil (m/z 441), and dealkylated verapamil (m/z 291), that contained substantial false-positive peaks, were simplified into chromatograms that are substantially free from matrix interferences. These filtered chromatograms approach what would have been obtained by using a radioactivity detector to detect radio-labeled metabolites of interest.     

High-throughput, accurate mass liquid chromatography/tandem mass spectrometry on a quadrupole time-of-flight system as a 'first-line' approach for metabolite identification studies

Throughput for drug metabolite identification studies has been increased significantly by the combined use of accurate mass liquid chromatography/tandem mass spectrometry (LC/MS/MS) data on a quadrupole time-of-flight (QTOF) system and targeted data analysis procedures. Employed in concert, these tools have led to the implementation of a semi-automated high-throughput metabolite identification strategy that has been incorporated successfully into lead optimization efforts in drug discovery. The availability of elemental composition data on precursor and all fragment ions in each spectrum has greatly enhanced confidence in ion structure assignments, while computer-based algorithms for defining sites of biotransformation based upon mass shifts of diagnostic fragment ions have facilitated identification of positions of metabolic transformation in drug candidates. Adoption of this technology as the 'first-line' approach for in vitro metabolite profiling has resulted in the analysis of as many as 21 new chemical entities on one day from diverse structural classes and therapeutic programs.     

A novel approach to perform metabolite screening during the quantitative LC-MS/MS analyses of in vitro metabolic stability samples using a hybrid triple-quadrupole linear ion trap mass spectrometer

In vitro metabolic stability experiments using microsomes or other liver preparations are important components in the discovery and lead-optimization stages of compound selection in the pharmaceutical industry. Currently, liquid chromatography-tandem mass spectrometric (LC-MS/MS) support of in vitro metabolic stability studies primarily involves the monitoring of disappearance of parent compounds, using selected reaction monitoring (SRM) on triple-quadrupole instruments. If moderate to high turnover is observed, separate metabolite identification experiments are then conducted to characterize the biotransformation products. In this paper, we present a novel method to simultaneously perform metabolite screening in addition to the quantitative stability measurements, both within the same chromatographic run. This is accomplished by combining SRM and SRM-triggered, information-dependent acquisition (IDA) of MS/MS spectra on a hybrid triple-quadrupole linear ion trap (QqQLIT) mass spectrometer. Microsomal stability experiments using model compounds, bufuralol, propranolol, imipramine, midazolam, verapamil and diclofenac, were used to demonstrate the applicability of our approach. This SRM + SRM-IDA approach generated metabolic stability results similar to those obtained by conventional SRM-only approach. In addition, MS/MS spectra from potential metabolites were obtained with the enhanced product ion (EPI) scan function of LIT during the same injection. These spectra were correlated to the spectra of parent compounds to confirm the postulated structures. The time-concentration profiles of identified metabolites were also estimated from the acquired data. This approach has been successfully used to support discovery programs.     

Strategies for ascertaining the interference of phase II metabolites co‐eluting with parent compounds using LC–MS/MS

LC‐MS/MS is currently the most selective and efficient tool for the quantitative analysis of drugs and metabolites in the pharmaceutical industry and in clinical assays. However, phase II metabolites sometimes negatively affect the selectivity and efficiency of the LC‐MS/MS method, especially for the metabolites that possess similar physicochemical characteristics and generate the same precursor ions as their parent compounds due to the in‐source collision‐induced dissociation during the ionization process. This paper proposes some strategies for examining co‐eluting metabolites existing in real samples, and further assuring whether these metabolites could affect the selectivity and accuracy of the analytical methods. Strategies using precursor‐ion scans and product‐ion scans were applied in this study. An example drug, namely, caffeic acid phenethyl ester, which can generate many endogenous phase II metabolites, was selected to conduct this work. These metabolites, generated during the in vivo metabolic processes, can be in‐source‐dissociated to the precursor ions of their parent compounds. If these metabolites are not separated from their parent compounds, the quantification of the target analytes (parent compounds) would be influenced. Some metabolites were eluted closely to caffeic acid phenethyl ester on LC columns, although long columns and relatively long elution programs were used. The strategies can be utilized in quantitative methodologies that apply LC‐MS/MS to assure the performance of selectivity, thus enhancing the reliability of the experimental data.     

Semi‐quantitative and structural metabolic phenotyping by direct infusion ion trap mass spectrometry and its application in genetical metabolomics

The identification of quantitative trait loci (QTL) for plant metabolites requires the quantitation of these metabolites across a large range of progeny. We developed a rapid metabolic profiling method using both untargeted and targeted direct infusion tandem mass spectrometry (DIMSMS) with a linear ion trap mass spectrometer yielding sufficient precision and accuracy for the quantification of a large number of metabolites in a high‐throughput environment. The untargeted DIMSMS method uses top‐down data‐dependent fragmentation yielding MS² and MS³ spectra. We have developed software tools to assess the structural homogeneity of the MS² and MS³ spectra hence their utility for phenotyping and genetical metabolomics. In addition we used a targeted DIMS(MS) method for rapid quantitation of specific compounds. This method was compared with targeted LC/MS/MS methods for these compounds. The DIMSMS methods showed sufficient precision and accuracy for QTL discovery. We phenotyped 200 individual Lolium perenne genotypes from a mapping population harvested in two consecutive years. Computational and statistical analyses identified 246 nominal m/z bins with sufficient precision and homogeneity for QTL discovery. Comparison of the data for specific metabolites obtained by DIMSMS with the results from targeted LC/MS/MS analysis showed that quantitation by this metabolic profiling method is reasonably accurate. Of the top 100 MS¹ bins, 22 ions gave one or more reproducible QTL across the 2 years. Copyright © 2009 John Wiley & Sons, Ltd.     

Characterization of metabolites of Celecoxib in rabbits by liquid chromatography/tandem mass spectrometry

The metabolism of the anti-inflammatory drug Celecoxib in rabbits was characterized using liquid chromatography (LC)/tandem mass spectrometry (MS/MS) with precursor ion and constant neutral loss scans followed by product ion scans. After separation by on-line liquid chromatography, the crude urine samples and plasma and fecal extracts were analyzed with turbo-ionspray ionization in negative ion mode using a precursor ion scan of m/z 69 (CF(3)) and a neutral loss scan of 176 (dehydroglucuronic acid). The subsequent product ion scans of the [M - H] ions of these metabolites yielded the identification of three phase I and four phase II metabolites. The phase I metabolites had hydroxylations at the methyl group or on the phenyl ring of Celecoxib, and the subsequent oxidation product of the hydroxymethyl metabolite formed the carboxylic acid metabolite. The phase II metabolites included four positional isomers of acyl glucuronide conjugates of the carboxylic acid metabolite. These positional isomers were caused by the alkaline pH of the rabbit urine and were not found in rabbit plasma. The chemical structures of the metabolites were characterized by interpretation of their product ion spectra and comparison of their LC retention times and the product ion spectra with those of the authentic synthesized standards.     

Rapid metabolite identification with sub parts-per-million mass accuracy from biological matrices by direct infusion nanoelectrospray ionization after clean-up on a ZipTip and LTQ/Orbitrap mass spectrometry

Metabolite identification studies remain an integral part of pre-clinical and clinical drug development programs. Analysis of biological matrices, such as plasma, urine, feces and bile, pose challenges due to the large amounts of endogenous components that can mask a drug and its metabolites. Although direct infusion nanoelectrospray using capillaries has been used routinely for proteomic studies, metabolite identification has traditionally employed liquid chromatographic (LC) separation prior to analysis. A method is described here for rapid metabolite profiling in biological fluids that involves initial sample clean-up using pipette tips packed with reversed-phase material (i.e. ZipTips) to remove matrix components followed by direct infusion nanoelectrospray on an LTQ/Orbitrap mass spectrometer using a protonated polydimethylcyclosiloxane cluster ion for internal calibration. We re-examined samples collected from a prazosin metabolism study in the rat. Results are presented that demonstrate that sub parts-per-million accuracies can be achieved on molecular ions, facilitating identification of metabolites, and on product ions, facilitating structural assignments. The data also show that the high-resolution measurements (R = 100 000 at m/z 400) enable metabolites of interest to be resolved from endogenous components. The extended analysis times available with nanospray enables signal averaging for 1 min or more that is valuable when metabolites are present in low concentrations as encountered here in plasma and brain. Using this approach, the metabolic fate of a drug can be quickly obtained. A limitation of this approach is that metabolites that are structural isomers cannot be distinguished, although such information can be collected by LC/MS during follow-on experiments. Copyright (c) 2008 John Wiley & Sons, Ltd.     

A strategy for identification of drug metabolites from dried blood spots using triple-quadrupole/linear ion trap hybrid mass spectrometry

Discovery stage studies that address issues of absorption, distribution, metabolism and excretion (ADME) are vital for lead optimization resulting in new drug candidates. Often pharmacokinetics (PK) is assessed in these experiments without regard for the metabolism of the compound or the potential for metabolites to circulate in vivo. This work presents a strategy for drug level determination and detection of metabolites using dried blood spots for sample collection. Initially, metabolites are detected from microsomal incubations and characterized using tandem mass spectrometry. Data dependent enhanced MS and enhanced product ion (EMS-EPI) scanning with dynamic background subtraction was used on a hybrid quadruple linear ion trap mass spectrometer. On-the-fly background subtraction greatly improved the detection of metabolites. These data were used to build a multiple reaction monitoring (MRM) method for the parent and metabolites. MRM-EPI scanning was used to analyze the extracted dried blood spots from the PK study. Circulating metabolites were detected using MRM and their identities confirmed on the basis of fragment ion spectra collected simultaneously. The use of dried blood spots provides a means for re-analysis of PK samples for metabolite identification without the need for complex sample storage and preparation. Both parent compound and metabolite information can be collected in these studies, resulting in a savings of time and resources.     

Use of liquid chromatography/time-of-flight mass spectrometry and multivariate statistical analysis shows promise for the detection of drug metabolites in biological fluids

The process of metabolite identification is essential to the drug discovery and development process; this is usually achieved by liquid chromatography/tandem mass spectrometry (LC/MS/MS) or a combination of liquid chromatography/mass spectrometry (LC/MS) and nuclear magnetic resonance (NMR) spectroscopy. Metabolite identification is, however, a time-consuming process requiring an experienced skilled scientist. Multivariate statistical analysis has been used in the field of metabonomics to elucidate differences in endogenous biological profiling due to a toxic effect or a disease state. In this paper we show how a combination of liquid chromatography/time-of-flight mass spectrometry (LC/TOFMS) and multivariate statistical analysis can be used to detect drug metabolites in a biological fluid with no prior knowledge of the compound administered.     

Increase the accessibility and scale of targeted metabolomics: Construction of a human urinary metabolome-wide multiple reaction monitoring library using directly-coupled reversed-phase and hydrophilic interaction chromatography

Multiple reaction monitoring (MRM) is wildly employed to research drug absorption, distribution, metabolism, excretion and pharmacokinetics in pharmaceutical and clinical laboratories. Recently, scientists in these areas have shown great interest in utilization of metabolomics to evaluate drug efficacy and toxicity. MRM-based targeted metabolomics is intrinsically more sensitive and selective than MS based untargeted metabolomics in complex biological samples. MRM also minimizes data complexity for fast and focused analysis of core metabolites. Nevertheless, to mitigate the intrinsic targeted nature of MRM and promote it as a discovery toolbox for metabolomics, larger scale MRM assays providing more comprehensive biological information are highly desirable. Here, we employed data-dependent and data-independent strategies to perform extensive MS/MS mapping of human urinary metabolome with the assistance of a directly-coupled reversed-phase liquid chromatography and hydrophilic interaction chromatography (RPLC-HILIC) for simultaneous profiling of hydrophilic and hydrophobic metabolites. RPLC-HILIC enables to save time, limit sample consumption and facilitate data interpretation by removing data redundancy occurring between separate RPLC and HILIC methods. Major product ions in the raw MS/MS spectra were used to build a human urinary metabolome-wide MRM library which contains 749 refined MRM tags in negative ion mode with 198 of them being unambiguously or tentatively assigned for particular metabolites. The library relieves researchers from the most time-consuming setup of massive MRM transitions and making an important step toward large-scale targeted urinary metabolomics.     

Feasibility of different mass spectrometric techniques and programs for automated metabolite profiling of tramadol in human urine

The purpose of the study was to determine the advantages of different mass spectrometric instruments and commercially available metabolite identification programs for metabolite profiling. Metabolism of tramadol hydrochloride and the excretion of it and its metabolites into human urine were used as a test case because the metabolism of tramadol is extensive and well known. Accurate mass measurements were carried out with a quadrupole time-of-flight mass spectrometer (Q-TOF) equipped with a LockSpray dual-electrospray ionization source. A triple quadrupole mass spectrometer (QqQ) was applied for full scan, product ion scan, precursor ion scan and neutral loss scan measurements and an ion trap instrument for full scan and product ion measurements. The performance of two metabolite identification programs was tested. The results showed that metabolite programs are time-saving tools but not yet capable of fully automated metabolite profiling. Detection of non-expected metabolites, especially at low concentrations in a complex matrix, is still almost impossible. With low-resolution instruments urine samples proved to be challenging even in a search for expected metabolites. Many false-positive hits were obtained with the automated searching and manual evaluation of the resulting data was required. False positives were avoided by using the higher mass accuracy Q-TOF. Automated programs were useful for constructing product ion methods, but the time-consuming interpretation of mass spectra was done manually. High-quality MS/MS spectra acquired on the QqQ instrument were used for confirmation of the tramadol metabolites. Although the ion trap instrument is of undisputable benefit in MS(n), the low mass cutoff of the ion trap made the identification of tramadol metabolites difficult. Some previously unreported metabolites of tramadol were found in the tramadol urine sample, and their identification was based solely on LC/MS and LC/MS/MS measurements.