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.     

Triterpenoid saponins profiling by adducts-targeted neutral loss triggered enhanced resolution and product ion scanning using triple quadrupole linear ion trap mass spectrometry

Triterpenoid saponins (TSs) are a unique class of high molecular weight glycosides and have been frequently used in cosmetic and phytotherapy industry. There is a great need to comprehensively profile these plant metabolites for studying their functions. In the present study, a novel adducts targeted neutral loss (NL), triggered enhanced resolution (ER) and enhanced product ion (EPI) scanning approach were described for TSs profiling using a triple quadrupole linear ion trap mass spectrometry. This approach circumvented the disadvantages of poor glycosidic bond cleavage of TSs by monitoring the NH₃ (NL17) and HCOOH (NL46) loss of their abundant ammonium and formate adducts, respectively. The sugar-loss independent NL scanning served as a sensitive survey scan and triggered information-dependent ER and EPI scans to increase peak assignment confidence. NL17 was superior to NL46 for TSs characterization due to the better fragmentation of ammonium adducts than formate adducts. For those TSs undetectable by NL17, precursor ion (PI) scan for sapogenin fragments could be used to screen out non-adducted TSs. The NL/PI-ER-EPI approach was applied for TSs profiling in Astragali Radix, a famous medicinal and nutritional plant widely used in Asian countries and United States. In total, 136 TSs were detected while previous research using high resolution mass spectrometry based full scan only detected 22 TSs in this herb.     

Rapid screening and characterization of drug metabolites using a new quadrupole-linear ion trap mass spectrometer

The application of a new hybrid RF/DC quadrupole-linear ion trap mass spectrometer to support drug metabolism and pharmacokinetic studies is described. The instrument is based on a quadrupole ion path and is capable of conventional tandem mass spectrometry (MS/MS) as well as several high-sensitivity ion trap MS scans using the final quadrupole as a linear ion trap. Several pharmaceutical compounds, including trocade, remikiren and tolcapone, were used to evaluate the capabilities of the system with positive and negative turbo ionspray, using either information-dependent data acquisition (IDA) or targeted analysis for the screening, identification and quantification of metabolites. Owing to the MS/MS in-space configuration, quadrupole-like CID spectra with ion trap sensitivity can be obtained without the classical low mass cutoff of 3D ion traps. The system also has MS(3) capability which allows fragmentation cascades to be followed. The combination of constant neutral loss or precursor ion scan with the enhanced product ion scan was found to be very selective for identifying metabolites at the picogram level in very complex matrices. Owing to the very high cycle time and, depending on the mass range, up to eight different MS experiments could be performed simultaneously without compromising chromatographic performance. Targeted product ion analysis was found to be complementary to IDA, in particular for very low concentrations. Comparable sensitivity was found in enhanced product ion scan and selected reaction monitoring modes. The instrument is particularly suitable for both qualitative and quantitative analysis.     

Hybrid triple quadrupole-linear ion trap mass spectrometry in fragmentation mechanism studies: application to structure elucidation of buspirone and one of its metabolites

The use of a hybrid triple quadrupole-linear ion trap (QqQ(LIT)) mass spectrometer system for a comprehensive study of fragmentation mechanisms is described. The anxiolytic drug, buspirone, was chosen as a model compound for this study. With the advent of a QqQ(LIT) instrument, both the traditional quadrupole and the new linear ion trap scans (LIT) could be performed in a single LC run. In the past, a sample had to be run on two different instruments, namely, a triple quadrupole instrument (QqQ) and a 3D ion trap (3D IT) to obtain similar information. With the new QqQ(LIT) technology, collision-induced dissociation (CID) occur in a quadrupole collision cell, q2, and fragment ions are trapped and analyzed in Q3 operated in LIT mode. In this work, high-sensitivity product ion spectra of buspirone were obtained from the one-stage 'Enhanced Product Ion' scan (EPI) with rich product ions and no low mass cut-off. Furthermore, detailed fragmentation pathways were elucidated by further dissociation of each of the fragment ions in the EPI spectrum using MS(3) mode in the same run. The MS(3) scan was performed by incorporating CID in q2, and trapping, cooling, isolation, and resonance-excitation in Q3 when operating in LIT mode. This approach allowed unambiguous assignment of all fragment ions quickly with fewer experiments and easier interpretation than the previous approach. The overall sensitivity for obtaining complete fragment ion data was significantly improved for QqQ(LIT) as compared with that of QqQ and 3D IT mass spectrometers. This is beneficial for structure determination of unknown trace components. The method allowed structure determination of metabolites of buspirone in rat microsomes at 1 microM concentration, which was a 10-fold lower concentration than was needed for QqQ or 3D IT instruments. The QqQ(LIT) instrument provided a simple, rapid, sensitive and powerful approach for structure elucidation of trace components.     

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.     

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.     

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.     

High-performance liquid chromatography-tandem mass spectrometry with a new quadrupole/linear ion trap instrument

The use of a new hybrid quadrupole/linear ion trap known as the Q TRAP offers unique benefits as a LC-MS-MS detector for both small and large molecule analyses. The instrument combines the capabilities of a triple quadrupole mass spectrometer and ion trap technology on a single platform. Product ion scans are conducted in a hybrid fashion with the fragmentation step accomplished via acceleration into the collision cell followed by trapping and mass analysis in the Q3 linear ion trap. This results in triple quadrupole fragmentation patterns with no inherent low molecular mass cutoff. In-trap fragmentation is also possible in order to provide triple MS (MS3) capabilities. There are also several scan modes that are not possible on conventional instruments that enable identification of analytes within complex biological matrixes for subsequent high sensitivity product ion scans. This report will describe the new hybrid instrument and the principles of operation, and also provide examples of the unique scan modes and capabilities of the Q TRAP for LC-MS-MS detection in metabolism identification.     

Screening and characterization of reactive metabolites using glutathione ethyl ester in combination with Q-trap mass spectrometry

The present study describes a new analytical approach for the detection and characterization of chemically reactive metabolites using glutathione ethyl ester (GSH-EE) as the trapping agent in combination with hybrid triple quadrupole linear ion trap mass spectrometry. Polarity switching was applied between a negative precursor ion (PI) survey scan and the positive enhanced product ion (EPI) scan. The negative PI scan step was carried out monitoring the anion at m/z 300, corresponding to deprotonated gamma-glutamyl-dehydroalanyl-glycine ethyl ester originating from the GSH-EE moiety. Samples resulting from incubations in the presence of GSH-EE were cleaned and concentrated by solid-phase extraction, followed by the PI-EPI analysis. Unambiguous identification of GSH-EE-trapped reactive metabolites was greatly facilitated by the unique survey scan of the anion at m/z 300, which achieved less background interference, in particular, from endogenous glutathione adducts present in human liver microsomes. Further structural characterization was achieved by analyzing positive MS(2) spectra that featured rich fragments without mass cutoff and were acquired in the same liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The effectiveness and reliability of this approach was evaluated using a number of model compounds in human liver microsomal incubations, including acetaminophen, amodiaquine, carbamazepine, 4-ethylphenol, imipramine and ticlopidine. In addition, iminoquinone reactive metabolites of mianserin were trapped and characterized for the first time using this method. Compared to neutral loss (NL) scanning assays using GSH as the trapping agent, the results have demonstrated superior selectivity, sensitivity, and reliability of this current approach.     

Complete profiling and characterization of in vitro nefazodone metabolites using two different tandem mass spectrometric platforms

This paper describes the complete profiling and characterization of in vitro metabolites of the antidepressant agent nefazodone (NEF) generated by human liver microsome (HLM). Two new metabolic pathways (biotransformation) for NEF have been discovered by the characterization of three new metabolites, including two new metabolites (M24, M25) formed due to the N-dealkylation reaction that occurred between the triazolone and propyl units, and one new metabolite (M26) formed due to the O-dearylation reaction that occurred on the phenoxyethyl unit. These metabolites were initially detected by a 4000 Q-Trap instrument and then confirmed by exact mass measurement using an LTQ-Orbitrap. Both instruments proved to be capable of providing complete in vitro metabolite information in a single liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis, although each had its advantages and disadvantages. One noticeable disadvantage of the 4000 Q-Trap was the reduced quality of isotopic pattern in the enhanced mass scan (EMS) spectrum when it was used as survey scan to trigger multiple dependent product ion scans. The problem was especially exacerbated for minor metabolites with low signal intensity. On the other hand, the LTQ-Orbitrap maintained excellent isotopic pattern when used as a full scan survey scan. Twenty-six metabolites were detected and identified. The formation of these new metabolites was also confirmed by analyzing duplicate incubations at different time points.     

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.     

Development of a multi-target screening analysis for 301 drugs using a QTrap liquid chromatography/tandem mass spectrometry system and automated library searching

A new multi-target screening (MTS) procedure for drugs in blood and urine for toxicological analysis has been developed using a hybrid triple-quadrupole linear ion trap mass spectrometer (QTrap) for the fast detection and identification of 301 forensically important drugs, e.g. tranquilizers (benzodiazepines), hypnotics, drugs of abuse (opiates, cocaine, amphetamines, cannabinoids), antidepressants, neuroleptics, and some cardiac drugs, in one single liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis. Samples were extracted either with liquid-liquid extraction or solid-phase extraction. A multiple reaction monitoring (MRM) as survey scan and an enhanced product ion (EPI) scan as dependent scan were performed in an information-dependent acquisition (IDA) experiment. Finally, drug identification was carried out by library search with a newly developed MS/MS library based on EPI spectra at three different collision energies in positive mode. The advantage of this newly developed method is the possibility to detect and identify 301 drugs in one single LC/MS/MS run.     

Triple quadrupole linear ion trap mass spectrometer for the analysis of small molecules and macromolecules

Recently, linear ion traps (LITs) have been combined with quadrupole (Q), time-of-flight (TOF) and Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS). LITs can be used either as ion accumulation devices or as commercially available, stand-alone mass spectrometers with MSn capabilities. The combination of triple quadrupole MS with LIT technology in the form of an instrument of configuration QqLIT, using axial ejection, is particularly interesting, because this instrument retains the classical triple quadrupole scan functions such as selected reaction monitoring (SRM), product ion (PI), neutral loss (NL) and precursor ion (PC) while also providing access to sensitive ion trap experiments. For small molecules, quantitative and qualitative analysis can be performed using the same instrument. In addition, for peptide analysis, the enhanced multiply charged (EMC) scan allows an increase in selectivity, while the time-delayed fragmentation (TDF) scan provides additional structural information. Various methods of operating the hybrid instrument are described for the case of the commercial Q TRAP (AB/MDS Sciex) and applications to drug metabolism analysis, quantitative confirmatory analysis, peptides analysis and automated nanoelectrospray (ESI-chip-MS) analysis are discussed.     

Product ion scanning using a Q-q-Q linear ion trap (Q TRAP) mass spectrometer

The use of a Q-q-Q(linear ion trap) instrument to obtain product ion spectra is described. The instrument is based on the ion path of a triple quadrupole mass spectrometer with Q3 operable as either a conventional RF/DC quadrupole mass filter or a linear ion trap mass spectrometer with axial ion ejection. This unique ion optical arrangement allows de-coupling of precursor ion isolation and fragmentation from the ion trap itself. The result is a high sensitivity tandem mass spectrometer with triple quadrupole fragmentation patterns and no inherent low mass cut-off. The use of the entrance RF-only section of the instrument as accumulation ion trap while the linear ion trap mass spectrometer is scanning enhances duty cycles and results in increased sensitivities by as much as a factor of 20. The instrument is also capable of all of the triple quadrupole scans including multiple-reaction monitoring (MRM) as well as precursor and constant neutral loss scanning. The high product ion scanning sensitivity allows the recording of useful product ion spectra near the MRM limit of quantitation.     

银离子高效液相色谱-质谱法分析血清中甘油三酯类化合物的组成

针对甘油三酯(TAG)类化合物的复杂性,建立了分析小鼠血清中TAG类化合物的方法。采用经典的氯仿-甲醇溶剂体系对血中的TAG类化合物进行提取。脂质提取物经Varian ChromSpher 5 Lipids柱分离,在0.75 mL/min的流速下以乙腈-正己烷(1:99, v/v)为流动相进行等度洗脱,采用大气压化学电离源正离子模式电离,质谱增强型全扫描、增强型子离子扫描和中性丢失扫描模式检测。根据银离子色谱对双键的保留规律以及质谱所给出的碎片离子信息,对血清中TAG类化合物进行了结构鉴定。结果表明采用该方法可以从小鼠血清中鉴定到66个TAG类化合物以及5个胆固醇酯。该方法简单,重现性好,可通用于其他样品中TAG类化合物的检测。     

Comparison of three types of mass spectrometers for HPLC/MS analysis of perfluoroalkylated substances and fluorotelomer alcohols

The performance of three different types of mass spectrometers (MS) coupled to high performance liquid chromatography (HPLC) was compared for trace analysis of perfluoroalkylated substances (PFAS) and fluorotelomer alcohols (FTOHs). Ion trap MS in the full scan and product ion MS2 mode, time-of-flight (TOF) high resolution MS and quadrupole MS in the selected ion mode as well as triple quadrupole tandem MS were tested. Electrospray ionisation in the negative ion mode [ESI-] was best suited for all instruments and compounds. PFAS could only be separated by a buffered mobile phase, but the presence of buffer suppressed the ionisation of FTOHs. Therefore, two independent chromatographic methods were developed for the two compound classes. Mass spectra and product ion spectra obtained by in-source and collision induced dissociation fragmentation are discussed including ion adduct formation. Product ion yields of PFAS were only in the range of 0.3 to 12%, independent from the applied MS instrument. Ion trap MS2 gave product ion yields of 20 to 62% for FTOHs, whereas only 4.1 to 5.8% were obtained by triple quadrupole tandem MS. Ion trap MS was best suited for qualitative analysis and structure elucidation of branched isomeric structures of PFAS. Providing typical detection limits of 5 ng injected in MS2 mode, it was not sensitive enough for selective trace amount quantification. TOF high resolution MS was the only technique combining high selectivity and excellent sensitivity for PFAS analysis (detection limits of 2 to 10 pg), but lacked the possibility of MS-MS. Triple quadrupole tandem MS was the method of choice for quantification of FTOHs with detection limits in the low pg range. It is also well suited for the determination of PFAS, though its detection limits of 10 to 100 pg in tandem MS mode are about one order of magnitude higher than for TOF MS.     

Automated neutral loss and data dependent energy resolved "pseudo MS3" for the targeted identification, characterization and quantitative analysis of methionine- containing peptides

A strategy involving the fixed-charge sulfonium ion derivatization, stable isotope labeling, capillary high- performance liquid chromatography and automated data dependent neutral loss scan mode tandem mass spectrometry (MS/MS) and "pseudo multiple mass spectrometry (MS(3))" product ion scans in a triple quadrupole mass spectrometer has been developed for the "targeted" gas-phase identification, characterization and quantitative analysis of low abundance methionine-containing peptides present within complex protein digests. Selective gas-phase "enrichment" and identification is performed via neutral loss scan mode MS/MS, by low energy collision-induced dissociation of the derivatized methionine side chain, resulting in the formation of a single characteristic product ion. Structural characterization of identified peptides is then achieved by automatically subjecting the characteristic neutral loss product ion to further dissociation by data dependent product ion scan mode pseudo MS(3) under higher collision energy conditions. Quantitative analysis is achieved by measurement of the abundances of characteristic product ions formed by sequential neutral loss scan mode MS/MS experiments from "light" ((12)C) and "heavy" ((13)C) stable isotope encoded fixed-charge derivatized peptides. In contrast to MS-based quantitative analysis strategies, the neutral loss scan mode MS/MS method employed here was able to achieve accurate quantification for individual peptides at levels as low as 100 fmol and at abundance ratios ranging from 0.1 to 10, present within a complex protein digest.     

Identification of NTBC metabolites in urine from patients with hereditary tyrosinemia type 1 using two different mass spectrometric platforms: triple stage quadrupole and LTQ‐Orbitrap

The objective of our work was to identify known and unknown metabolites of the drug NTBC (2‐(2‐nitro‐4‐trifluoromethylbenzoyl)‐1,3‐cyclohexanedione) in urine from patients during the treatment of hereditary tyrosinemia type 1 (HT‐1) disease, a severe inborn error of tyrosine metabolism. Two different mass spectrometric techniques, a triple stage quadrupole and an LTQ‐Orbitrap (Fourier transform mass spectrometry (FTMS)), were used for the identification and the structural elucidation of the detected metabolites. Initially, the mass spectrometric (MS) approach consisted of the precursor ion scan detection of the selected product ions, followed by the corresponding collision‐induced dissociation (CID) fragmentation analysis (MS²) for the targeted selected reaction monitoring (SRM) mode. Subsequently, accurate and high‐resolution full scan and MS/MS measurements were performed on the possible metabolites using the LTQ‐Orbitrap. Final confirmation of the identified metabolites was achieved by measuring commercially supplied or laboratory‐synthesized standards. Altogether six metabolites, including NTBC itself, were extracted, detected and identified. In addition, two new NTBC metabolites were unambiguously identified as amino acid conjugates, namely glycine‐NTBC and β‐alanine‐NTBC. These identifications were based on their characteristics of chromatographic retention times, protonated molecular ions, elemental compositions, product ions (using CID and higher‐energy C‐trap dissociation (HCD) techniques) and synthesized references. The applied MS strategy, based on two different MS platforms (LC/MS/MS and FTMS), allowed the rapid identification analysis of the drug metabolites from human extracts and could be used for pharmaceutical research and drug development. Copyright © 2010 John Wiley & Sons, Ltd.     

Application of a linear ion trap/orbitrap mass spectrometer in metabolite characterization studies: Examination of the human liver microsomal metabolism of the non-tricyclic anti-depressant nefazodone using data-dependent accurate mass measurements

We report herein, facile metabolite identification workflow on the anti-depressant nefazodone, which is derived from accurate mass measurements based on a single run/experimental analysis. A hybrid LTQ/orbitrap mass spectrometer was used to obtain accurate mass full scan MS and MS/MS in a data-dependent fashion to eliminate the reliance on a parent mass list. Initial screening utilized a high mass tolerance ( approximately 10 ppm) to filter the full scan MS data for previously reported nefazodone metabolites. The tight mass tolerance reduces or eliminates background chemical noise, dramatically increasing sensitivity for confirming or eliminating the presence of metabolites as well as isobaric forms. The full scan accurate mass analysis of suspected metabolites can be confirmed or refuted using three primary tools: (1) predictive chemical formula and corresponding mass error analysis, (2) rings-plus-double bonds, and (3) accurate mass product ion spectra of parent and suspected metabolites. Accurate mass characterization of the parent ion structure provided the basis for assessing structural assignment for metabolites. Metabolites were also characterized using parent product ion m/z values to filter all tandem mass spectra for identification of precursor ions yielding similar product ions. Identified metabolite parent masses were subjected to chemical formula calculator based on accurate mass as well as bond saturation. Further analysis of potential nefazodone metabolites was executed using accurate mass product ion spectra. Reported mass measurement errors for all full scan MS and MS/MS spectra was <3 ppm, regardless of relative ion abundance, which enabled the use of predictive software in determining product ion structure. The ability to conduct biotransformation profiling via tandem mass spectrometry coupled with accurate mass measurements, all in a single experimental run, is clearly one of the most attractive features of this methodology.     

The rapid identification of drug metabolites using capillary liquid chromatography coupled to an ion trap mass spectrometer

Capillary liquid chromatography (LC) using a 320 microns column and a flow rate of 10 microL/min has been coupled to an ion trap mass spectrometer using electrospray ionisation (ESI) to enable the rapid and effective identification of metabolites in urine, following oral administration of a novel human neutrophil elastase inhibitor, GW311616. Metabolites were identified from their mass (MS) spectra and tandem (MS/MS) mass spectra using minimal sample (1 microL of urine) and no sample pretreatment. Sensitivity assessment has shown that both molecular weight and structural information is obtainable on as little as 5 pg of compound, making the capillary LC/ion trap system as described an ideal analytical tool for the detection and characterisation of low level metabolites in biofluids (particularly when sample volume is limited). This level of detection was unattainable using a triple quadrupole mass spectrometer operating in full-scan mode, although 200 fg on column was detected using selected reaction monitoring target analysis.