Phosphopeptide quantitation using amine-reactive isobaric tagging reagents and tandem mass spectrometry: application to proteins isolated by gel electrophoresis

Polyacrylamide gel electrophoresis is widely used for protein separation and it is frequently the final step in protein purification in biochemistry and proteomics. Using a commercially available amine-reactive isobaric tagging reagent (iTRAQ) and mass spectrometry we obtained reproducible, quantitative data from peptides derived by tryptic in-gel digestion of proteins and phosphoproteins. The protocol combines optimized reaction conditions, miniaturized peptide handling techniques and tandem mass spectrometry to quantify low- to sub-picomole amounts of (phospho)proteins that were isolated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Immobilized metal affinity chromatography (FeIII-IMAC) was efficient for removal of excess reagents and for enrichment of derivatized phosphopeptides prior to matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis. Phosphopeptide abundance was determined by liquid chromatography/tandem mass (LC/MS/MS) using either MALDI time-of-flight/time-of-flight (TOF/TOF) MS/MS or electrospray ionization quadrupole time-of-flight (ESI-QTOF) MS/MS instruments. Chemically labeled isobaric phosphopeptides, differing only by the position of the phosphate group, were distinguished and characterized by LC/MS/MS based on their LC elution profile and distinct MS/MS spectra. We expect this quantitative mass spectrometry method to be suitable for systematic, comparative analysis of molecular variants of proteins isolated by gel electrophoresis.     

Application of gas and liquid chromatography coupled to time‐of‐flight mass spectrometry in pesticides: Multiresidue analysis

Analysis of pesticide residues in water and food matrices is an active research area closely related to food safety and environmental issues. In this aspect mass spectrometry (MS) coupled to gas chromatography (GC) and liquid chromatography (LC) has been increasingly used in the analysis of pesticide residues in water and food. The increasing interest in application of high‐resolution mass spectrometry with time‐of‐flight (TOF) and hybrid triple quadrupole TOF in pesticide analysis is due to its capability of performing both targeted and nontargeted analysis. This article discusses an overview of the application of GC‐TOF‐MS and LC‐TOF‐MS in water and food matrices.     

High-resolution liquid chromatography/electrospray ionization time-of-flight mass spectrometry combined with liquid chromatography/electrospray ionization tandem mass spectrometry to identify polyphenols from grape antioxidant dietary fiber

Grape antioxidant dietary fiber (GADF) is a dietary supplement that combines the benefits of both fiber and antioxidants that help prevent cancer and cardiovascular diseases. The antioxidant polyphenolic components in GADF probably help prevent cancer in the digestive tract, where they are bioavailable. Mass spectrometry coupled to liquid chromatography is a powerful tool for the analysis of complex plant derivatives such as GADF. We use a combination of MS techniques, namely liquid chromatography/electrospray ionization time-of-flight mass spectrometry (LC/ESI-TOF-MS) and liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) on a triple quadrupole, for the identification of the polyphenolic constituents of the soluble fraction of GADF. First, we separated the mixture into four fractions which were tested for phenolic constituents using the TOF system in the full scan mode. The high sensitivity and resolution of the TOF detector over the triple quadrupole facilitate the preliminary characterization of the fractions. Then we used LC/ESI-MS/MS to identify the individual phenols through MS/MS experiments (product ion scan, neutral loss scan, precursor ion scan). Finally, most of the identities were unequivocally confirmed by accurate mass measurements on the TOF spectrometer. LC/ESI-TOF-MS combined with MS/MS correctly identifies the bioactive polyphenolic components from the soluble fraction of GADF. High-resolution TOF-MS is particularly useful for identifying the structure of compounds with the same LC/ESI-MS/MS fragmentation patterns.     

High- and low-resolution mass spectrometry coupled to liquid chromatography as confirmatory methods of anabolic residues in crude meat and infant foods

Within the European Union the use of anabolic steroids for promoting growth and improving meat-to-fat ratio in food-producing animals has been banned since 1988. For the unequivocal identification of hormone residues in a complex matrix such as meat we have developed a rapid, specific and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method, in combination with a simple extraction procedure based on the matrix solid-phase dispersion (MSPD). The performances of a triple quadrupole (QqQ) and a quadrupole/time-of-flight (QqTOF) were compared: the QqQ mass spectrometer was found to be more sensitive for almost all studied analytes, but the selectivity was superior using the QqTOF system; the full-scan spectra (acquired without losing sensitivity), mass accuracy and resolution of the hybrid instrument enabled a more probatory analyte identification than that obtained selecting two multiple-reaction monitoring (MRM) transitions with a QqQ. Average recoveries ranged from 80 to 100%, and the detection capabilities (CCbetas) were less than 1.06 ppb with the QqQ instrument and less than 5.20 ppb with the QqTOF instrument for the bovine meat, which proved to be the most complex matrix.     

Determination of heterocyclic amines by liquid chromatography-quadrupole time-of-flight mass spectrometry

This paper discusses the applicability of coupling liquid chromatography (LC) to mass spectrometry (MS) using a time-of-flight (TOF) mass analyzer for the analysis of heterocyclic amines (HAs). Accurate mass measurement (<2 mDa) with both MS-MS and in-source CID MS-MS was carried out to confirm the elemental composition of some fragments previously reported. Some isobaric assignments (fragments containing N versus CH2 and NH3 versus CD2H) were distinguished by taking advantage of the resolution provided by the TOF mass analyzer. On the other hand, the LC-MS analysis of HAs in MS acquisition mode was also performed. Quality parameters of the method were established. The linearity range extended over three orders of magnitude, limits of detection were in the pg level and good short-term precision values (R.S.D., 1.2-8.0%) were obtained. The LC-ESI-TOF method was applied to the determination of HAs in a lyophilized meat extract and the results obtained were comparable to those given by MS-MS with triple quadrupole and ion trap instruments.     

High mass accuracy in-source collision-induced dissociation tandem mass spectrometry and multi-step mass spectrometry as complementary tools for fragmentation studies of quaternary ammonium herbicides

Fragmentation studies using both an ion-trap mass analyzer and a hybrid quadrupole time-of-flight (Q-TOF) mass spectrometer were performed in order to establish the fragmentation pathways of organic molecules. A general strategy combining MSn data (n = 1-4) in an ion-trap analyzer with tandem mass spectrometry and in-source collision-induced dissociation tandem mass spectrometry (CID MS/MS) in a Q-TOF instrument was applied. The MSn data were used to propose a tentative fragmentation pathway following genealogical relationships. When several assignments were possible, MS/MS and in-source CID MS/MS (Q-TOF) allowed the elemental compositions of the fragments to be confirmed. Quaternary ammonium herbicides (quats) were used as test compounds and their fragmentation pathways were established. The elemental composition of the fragments was confirmed using the TOF analyzer with relative errors <0.0023 Da. Some fragments previously reported in the literature were reassigned taking advantage of the high mass resolution and accuracy of the Q-TOF instrument, which made it possible to solve losses where nitrogen was involved.     

Quadrupole time-of-flight versus triple-quadrupole mass spectrometry for the determination of non-steroidal antiinflammatory drugs in surface water by liquid chromatography/tandem mass spectrometry

A triple-quadrupole instrument and a hybrid quadrupole/time-of-flight (TOF) mass spectrometer were compared for the determination of pharmaceutical compounds in water samples. The drugs investigated were the analgesics Ibuprofen, Fenoprofen, Ketoprofen, Naproxen, and Diclofenac. The recently introduced Q2-pulsing function, which enhances the transmission of fragment ions of a selected m/z window from the collision cell into the TOF analyzer, improved the sensitivity of product ion scans on the quadrupole/TOF instrument. The selectivity is much better on quadrupole/TOF systems than on triple quadrupoles because the high resolving power of the reflectron-TOF mass analyzer permits high-accuracy fragment ion selection. This minimizes interferences from environmental matrices and allows acquisition of full spectra for selected analytes with better signal-to-noise characteristics than comparable spectra obtained with a scanned quadrupole. The qualitative information obtained (mass accuracy, resolution and full-scan spectra) by hybrid quadrupole/TOF mass spectrometry allows a more certain identification of analytes in environmental matrices at trace levels. Sample enrichment of water samples was achieved by a solid-phase extraction procedure. Average recoveries for loading 1 L of samples varied from 88 to 110%, and the quantification limits were less than 1.2 ng/L for the triple-quadrupole instrument (in MRM mode) and less than 3 ng/L for the quadrupole/TOF instrument.     

Evaluation of automated single mass spectrometry to tandem mass spectrometry function switching for comprehensive drug profiling analysis using a quadrupole time-of-flight mass spectrometer

A liquid chromatographic mass spectrometric strategy for systematic toxicological analysis (STA) is presented using the automatic 'on-the-fly' single mass spectrometry mode to tandem mass spectrometry mode (MS to MS/MS) switching abilities of a quadrupole time-of-flight (Q-TOF) instrument. During the chromatographic run, the quadrupole is initially set to transmit all masses until (an) ion(s) reaches a certain set threshold. Thereupon, the quadrupole automatically switches to the MS/MS mode, selecting the ion(s), which are subsequently fragmented in the high-efficiency hexapole collision cell, thus generating product ions that are further mass analyzed by the TOF. By limiting the TOF spectral accumulation time in the MS/MS mode to a statistically acceptable minimum, the quadrupole almost instantly switches back to the MS mode. Qualitative information, comprising the complementary MS ([M + H](+) ion mass) and MS/MS (informative product ion profile) data, as well as quantitative information obtained by integration of the MS extracted ion chromatogram(s), can be obtained in one single acquisition. Optimization of the automatic switching parameters, such as threshold, TOF spectral accumulation time, detection window and collision energy, was carried out by injection of a mix of 17 common drugs which were not necessarily baseline separated in the chromatographic system used. Indeed, the complete separation of the drugs is not deemed necessary since up to 8 different ions can 'simultaneously' be selected for MS/MS if they reach the preset criteria. In addition, the quantitative performance of the method was defined. In a second phase, the developed method was field-tested. To that end, the resulting data from extracts of urine samples were compared with and found to be in close concordance with those obtained by a standard toxicological analysis. This innovative approach clearly holds the potential for a substantial advance in the introduction of LC/MS in STA.     

Addressing the analytical throughput challenges in ADME screening using rapid ultra-performance liquid chromatography/tandem mass spectrometry methodologies

High-throughput ADME screening for compound drug development properties has become an essential part of the modern drug discovery process, allowing more informed decisions to be made on the best compounds to take forward in the discovery/development process. This however is a time-consuming process requiring multiple tests to be performed, demanding a significant amount of liquid chromatography/mass spectrometry (LC/MS) instrument time. This article focuses on the use of sub-2 microm porous particle LC coupled to tandem quadrupole MS/MS mass spectrometry for the rapid screening of ADME properties. Using this approach analysis times from 30 s to 1 min were achievable allowing analysis times to be cut by 80%. The use of the small particles coupled to high flow rates allowed for sufficient resolution, even with very short analysis time, to resolve the analytes of interest from similar compounds that would interfere with the assay. The use of dedicated, intelligent, software packages allowed for the user-free generation of MS/MS conditions and the processing of the data.     

Full-scan accurate mass selectivity of ultra-performance liquid chromatography combined with time-of-flight and orbitrap mass spectrometry in hormone and veterinary drug residue analysis

The applicability of ultra-performance liquid chromatography (UPLC) combined with full-scan accurate mass time-of-flight (TOF) and Orbitrap mass spectrometry (MS) to the analysis of hormone and veterinary drug residues was evaluated. Extracts from blank bovine hair were fortified with 14 steroid esters. UPLC-Orbitrap MS performed at a resolving power of 60,000 (FWHM) enabled the detection and accurate mass measurement (<3 ppm error) of all 14 steroid esters at low ng/g concentration level, despite the complex matrix background. A 5 ppm mass tolerance window proved to be essential to generate highly selective reconstructed ion chromatograms (RICs) having reduced background from the hair matrix. UPLC-Orbitrap MS at a lower resolving power of 7500 and UPLC-TOFMS at mass resolving power 10,000 failed both to detect all of the steroid esters in hair extracts owing to the inability to mass resolve analyte ions from co-eluting isobaric matrix compounds. In a second application, animal feed extracts were fortified with coccidiostats drugs at levels ranging from 240 to 1900 ng/g. UPLC-Orbitrap MS conducted at a resolving power of 7500 and 60,000 and UPLC-TOFMS detected all of the analytes at the lowest investigated level. Thanks to the higher analyte-to-matrix background ratio, the utilization of very narrow mass tolerance windows in the RIC was not required. This study demonstrates that even when the targeted sample preparation from conventional LC-MS/MS is applied to UPLC with full-scan accurate mass MS, false compliant (false negative) results can be obtained when the mass resolving power of the MS is insufficient to separate analyte ions from isobaric co-eluting sample matrix ions. The current trend towards more generic and less selective sample preparation is expected to aggravate this issue further.     

Surface swabbing technique for the rapid screening for pesticides using ambient pressure desorption ionization with high-resolution mass spectrometry

A rapid screening method for pesticides has been developed to promote more efficient processing of produce entering the United States. Foam swabs were used to recover a multiclass mixture of 132 pesticides from the surfaces of grapes, apples, and oranges. The swabs were analyzed using direct analysis in real time (DART) ionization coupled with a high‐resolution Exactive Orbitrap? mass spectrometer. By using a DART helium temperature gradient from 100–350°C over 3 min, a minimal separation of analytes based on volatility differences was achieved. This, combined with the Exactive's mass resolution of 100 000, allowed the chromatographic step, along with the typical compositing and extraction steps associated with gas chromatography/mass spectrometry (GC/MS) or liquid chromatography/mass spectrometry (LC/MS) approaches, to be eliminated. Detection of 86% of the analytes present was consistently achieved at levels of 2 ng/g (per each apple or orange) and 10 ng/g (per grape). A resolution study was conducted with four pairs of isobaric compounds analyzed at a mass resolution of 100 000. Baseline separation was achieved with analyte ions differing in mass by 25 ppm and analyte ions with a mass difference of 10 ppm were partially resolved. In addition, field samples that had undergone traditional sample preparation using QuEChERS (quick, easy, cheap, rugged, and safe) were analyzed using both LC/MS and DART‐MS and the results from the two techniques were found to be comparable in terms of identification of the pesticides present. The use of swabs greatly increased sample throughput by reducing sample preparation and analysis time. Published in 2010 by John Wiley & Sons, Ltd.     

Selenium speciation by HPLC with tandem mass spectrometric detection

An HPLC/MS-MS method was developed for the analysis of selenium species. Tandem mass spectrometry (MS-MS) was chosen as a detector to provide structural and molecular information allowing the identification of species, which are not commercially available as standards. A new separation method for selenium species was developed, based on porous graphitic carbon (PGC) as the stationary phase. The applicability of the optimized HPLC/MS-MS system was demonstrated by the analysis of a mixture containing Se-methyl-selenocysteine, selenomethionine, selenocystine, selenoethionine and selenocystamine. All peaks were baseline-resolved and eluted within 16 min. Positive ionization led to higher intensities than negative ionization. Signal suppression tests showed that electrospray ionization (ESI) is a more effective ionization method than atmospheric pressure chemical ionization (APCI) for selenium species in a matrix containing pentafluoropropionic acid, heptafluorobutyric acid or ammonium formate. Comparative experiments with a triple quadrupole mass spectrometer (Quattro LC) and a time-of-flight instrument (Q-Tof-2) showed a 20 fold higher mass resolution of the latter mass spectrometer (m/Am= 5000) and significantly lower intensities for analyte signals as well as background noise compared to the triple quadrupole instrument. MS-MS spectra of the investigated selenium species including characteristic fragmentation patterns are presented.     

Application of ion trap technology to liquid chromatography/mass spectrometry quantitation of large peptides

Triple quadrupole mass spectrometers are generally considered the instrument of choice for quantitative analysis. However, for the analysis of large peptides we have encountered some cases where, as the data presented here would indicate, ion trap mass spectrometers may be a good alternative. In general, specificity and sensitivity in bioanalytical liquid chromatography/mass spectrometry (LC/MS) assays are achieved via tandem MS (MS/MS) utilizing collision-induced dissociation (CID) while monitoring unique precursor to product ion transitions (i.e. selected reaction monitoring, SRM). Due to the difference in CID processes, triple quadrupoles and ion traps often generate significantly different fragmentation spectra of product ion species and intensities. The large peptidic analytes investigated here generated fewer fragments with higher relative abundance on the ion trap as compared to those generated on the triple quadrupole, resulting in lower limits of detection on the ion trap.     

A reliable analytical approach based on gas chromatography coupled to triple quadrupole and time‐of‐flight mass analyzers for the determination and confirmation of polycyclic aromatic hydrocarbons in complex matrices from aquaculture activities

The potential of gas chromatography coupled to tandem mass spectrometry (GC/MS/MS) with a triple quadrupole analyzer (QqQ) has been investigated for the quantification and reliable identification of sixteen polycyclic aromatic hydrocarbons (PAHs) from the EPA priority list in animal and vegetable samples from aquaculture activities, whose fat content ranged from 5 to 100%. Matrices analyzed included fish fillet, fish feed, fish oil and linseed oil. Combining optimized saponification and solid‐phase extraction led to high efficiency in the elimination of interfering compounds, mainly fat, from the extracts. The developed procedure minimized the presence of these interfering compounds in the extracts and provided satisfactory recoveries of PAHs. The excellent sensitivity and selectivity of GC/(QqQ)MS/MS in selected reaction monitoring (SRM) allowed to reach limits of detection at pg/g levels. Two SRM transitions were acquired for each analyte to ensure reliable identification of compounds detected in samples. Confirmation of positive findings was performed by GC coupled to high‐resolution time‐of‐flight mass spectrometry (GC/TOFMS). The accurate mass information provided by GC/TOFMS in full acquisition mode together with its high mass resolution makes it a powerful analytical tool for the unequivocal confirmation of PAHs in the matrices tested. The method developed was applied to the analysis of real‐world samples of each matrix studied with the result of detecting and confirming the majority of analytes at the µg/kg level by both QqQ and TOF mass spectrometers. Copyright © 2009 John Wiley & Sons, Ltd.     

Use of high‐resolution mass spectrometry to investigate a metabolite interference during liquid chromatography/tandem mass spectrometric quantification of a small molecule in toxicokinetic study samples

During routine liquid chromatography/tandem mass spectrometric (LC/MS/MS) bioanalysis of a small molecule analyte in rat serum samples from a toxicokinetic study, an unexpected interfering peak was observed in the extracted ion chromatogram of the internal standard. No interfering peaks were observed in the extracted ion chromatogram of the analyte. The dose‐dependent peak area response and peak area response versus time profiles of the interfering peak suggested that it might have been related to a metabolite of the dosed compound. Further investigation using high‐resolution mass spectrometry led to unequivocal identification of the interfering peak as an N‐desmethyl metabolite of the parent analyte. High‐resolution mass spectrometry (HRMS) was also used to demonstrate that the interfering response of the metabolite in the multiple reaction monitoring (MRM) channel of the internal standard was due to an isobaric relationship between the ¹³C‐isotope of the metabolite and the internal standard (i.e., common precursor ion mass), coupled with a metabolite product ion with identical mass to the product ion used in the MRM transition of the internal standard. These results emphasize (1) the need to carefully evaluate internal standard candidates with regard to potential interferences from metabolites during LC/MS/MS method development, validation and bioanalysis of small molecule analytes in biological matrices; (2) the value of HRMS as a tool to investigate unexpected interferences encountered during LC/MS/MS analysis of small molecules in biological matrices; and (3) the potential for interference regardless of choice of IS and therefore the importance of conducting assay robustness on incurred in vitro or in vivo study samples. Copyright © 2010 John Wiley & Sons, Ltd.     

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.     

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.     

Analysis of β‐blockers in groundwater using large‐volume injection coupled‐column reversed‐phase liquid chromatography with fluorescence detection and liquid chromatography time‐of‐flight mass spectrometry

Atenolol, nadolol, metoprolol, bisoprolol and betaxolol were simultaneously determined in groundwater samples by large‐volume injection coupled‐column reversed‐phase liquid chromatography with fluorescence detection (LVI‐LC‐LC‐FD) and liquid chromatography‐time‐of‐flight mass spectrometry (LC‐TOF‐MS). The LVI‐LC‐LC‐FD method combines analyte isolation, preconcentration and determination into a single step. Significant reductions in costs for sample pre‐treatment (solvent and solid phases for clean up) and method development times are also achieved. Using LC‐TOF‐MS, accurate mass measurements within 3 ppm error were obtained for all of the β‐blockers studied. Empirical formula information can be obtained by this method, allowing the unequivocal identification of the target compounds in the samples. To increase the sensitivity, a solid‐phase extraction step with Oasis MCX cartridge was carried out yielding recoveries of 79–114% (n=5) with RSD 2–7% for the LC‐TOF‐MS method. SPE gives a high purification of β‐blockers compared with the existing methods. A 100% methanol wash was allowed for these compounds with no loss of analytes. Limit of quantification was 1–7 ng/L for LVI‐LC‐LC‐FD and 0.25–5 ng/L for LC‐TOF‐MS. As a result of selective extraction and effective removal of coextractives, no matrix effect was observed in LVI‐LC‐LC‐FD and LC‐TOF‐MS analyses. The methods were applied to detect and quantify β‐blockers in groundwater samples of Almería (Spain).     

Tandem mass spectrometric accurate mass performance of time-of-flight and Fourier transform ion cyclotron resonance mass spectrometry: a case study with pyridine derivatives

The interpretation of mass spectra is a key process during compound identification, and the combination of tandem mass spectrometry (MS/MS) with high-accuracy mass measurements may deliver crucial information on the identity of a compound. Obtaining accurate mass data of fragment ions in MS/MS reveals the particular problem of mass calibration when a lockmass, which is frequently used to obtain accurate masses in MS, is absent. An alternative technique is to recalibrate the MS/MS spectrum using a reference MS/MS spectrum acquired under the same conditions. We have tested and validated this approach using a hybrid quadrupole/orthogonal acceleration reflectron-type time-of-flight (TOF) mass spectrometer. The results were compared with those obtained under similar conditions on a Fourier transform ion cyclotron resonance (FT-ICR) instrument. We found that the mass accuracy observed with such an "external" recalibration on the TOF instrument in MS/MS is identical to what can be obtained on a similar instrument operating in one-dimensional MS mode using the lockmass technique. However, mass accuracy in both cases is one order of magnitude inferior to that obtained using FTMS, and also inferior to that observed using sector field MS when operated at comparable resolution. Nevertheless, for small (<200 Da) molecules, this mass accuracy was still sufficient to have the "true" elemental composition identified as the first hit in about 70% of all cases. It was possible to elucidate the fragmentation mechanism of eight azaheterocycles containing a pyridine moiety, where the accurate mass data from the TOF instrument allowed distinction between two alternative fragmentation pathways.     

Simultaneous determination of the urinary metabolites of benzene, toluene, xylene and styrene using high-performance liquid chromatography/hybrid quadrupole time-of-flight mass spectrometry

A simple and rapid method using reversed-phase liquid chromatography/tandem mass spectrometry (LC/MS/MS) for the simultaneous determination of the urinary metabolites of benzene, toluene, xylene and styrene in human urine specimens and standard solutions is described. A hybrid quadrupole/time-of-flight (QqTOF) mass spectrometer was compared for the determination of metabolite of aromatic solvents in urine samples. The metabolites selected were: trans,trans-muconic acid, hippuric acid, o-, m- and p-methylhippuric acid and phenylglyoxylic acid. The compounds were well separated from each other on narrow-bore 1-mm i.d. reversed-phase LC C-18 columns. Average recoveries for loading 100 microL of urine samples varied from 88-110% and the quantification limits were less than 30 ng/mL for each analyte (3 ng/mL for trans,trans-muconic acid). The qualitative information obtained (mass accuracy, resolution and full-scan spectra) with the QqTOF mass spectrometer allows a secure identification of analytes in biological matrices.