Quantitative-qualitative data acquisition using a benchtop orbitrap mass spectrometer

Current approaches to discovery-stage drug metabolism studies (pharmacokinetics, microsomal stability, etc. ) typically use triple-quadrupole-based approaches for quantitative analysis. This necessitates the optimization of parameters such as Q1 and Q3 m/z values, collision energy, and interface voltages. These studies detect only the specified compound and information about other components, such as metabolites, is lost. The ability to perform full-scan acquisition for quantitative analysis would eliminate the need for compound optimization while enabling the detection of metabolites and other non-drug-related endogenous components. Such an instrument would have to provide sensitivity, selectivity, dynamic range, and scan speed suitable for discovery-stage quantitative studies. In this study, a prototype benchtop Orbitrap-based mass analyzer was used to collect both quantitative and qualitative data from human microsomal incubation samples as well as rat plasma from pharmacokinetic studies. Instrumental parameters such as scan speed, resolution, and mass accuracy are discussed in relation to the requirements for a quantitative-qualitative workflow. The ability to perform highly selective quantitative analysis while simultaneously characterizing metabolites from both in vitro and in vivo studies is discussed.     

Introduction of HPLC/orbitrap mass spectrometry as screening method for doping control

A new doping control screening method has been developed, for the analysis of doping agents in human urine, using HPLC/orbitrap with in-source collision-induced dissociation and atmospheric pressure chemical ionization. The developed method allows the detection of 29 compounds, including agents with antiestrogenic activity, beta(2) agonists, exogenous anabolic steroids, and other anabolic agents. The mass accuracy of this method is better at 2 ppm using an external reference. The detection limit for all compounds tested was better than 100 pg/ml. The recoveries of most analytes were above 70%. The measured median repeatability values for doping agents included in the method at concentrations of 1 and 10 ng/ml were 21 and 17%, respectively. The relative standard deviation (RSD) of the intraday precision (n = 6) ranged from RSD = 16-22%, whereas the interday precision (n = 18), ranged from RSD = 17-26%, depending on the solute concentration investigated.     

Dynamic range of mass accuracy in LTQ orbitrap hybrid mass spectrometer

Using a novel orbitrap mass spectrometer, the authors investigate the dynamic range over which accurate masses can be determined (extent of mass accuracy) for short duration experiments typical for LC/MS. A linear ion trap is used to selectively fill an intermediate ion storage device (C-trap) with ions of interest, following which the ensemble of ions is injected into an orbitrap mass analyzer and analyzed using image current detection and fast Fourier transformation. Using this technique, it is possible to generate ion populations with intraspectrum intensity ranges up to 10(4). All measurements (including ion accumulation and image current detection) were performed in less than 1 s at a resolving power of 30,000. It was shown that 5-ppm mass accuracy of the orbitrap mass analyzer is reached with >95% probability at a dynamic range of more than 5000, which is at least an order of magnitude higher than typical values for time-of-flight instruments. Due to the high resolving power of the orbitrap, accurate mass of an ion could be determined when the signal was reliably distinguished from noise (S/Np-p)>2...3).     

Suitability of an orbitrap mass spectrometer for the screening of pesticide residues in extracts of fruits and vegetables

For about 500 pesticides, the sensitivity of a benchtop high-resolution mass spectrometer using the Orbitrap for mass separation was compared to that of a widely used (low-resolution) tandem mass spectrometer. Both instruments were coupled to LC and used electrospray ionization. The selectivity of the Orbitrap in the full-scan acquisition mode without fragmentation was evaluated at a resolution of 100 000 full width at half maximum for all pesticides detectable with sufficient sensitivity. For this purpose, quasimolecular ions were extracted within 5 ppm windows from total ion chromatograms of two types of extracts of cucumber, lemon, wheat flour, raisin, and tea. In each of the obtained reconstructed ion chromatograms (individual chromatograms for 500 pesticides, each pesticide in 10 different extracts) the sum of signals not arising from the analyte was used to get a measure on selectivity. In addition, the target analyte list was checked for ions of similar mass. The influence of matrix on the ability to detect low concentrations of fortified pesticides was also studied, with the help of spiked extracts. This part of the survey tested whether analyte peaks were lost because of insufficient mass resolution or an early closing C-Trap (used to control the ion current into the Orbitrap). Finally, the stability of the ion ratio [M+H]+/[M+Na]+ was tested, which may be helpful to confirm the identity of an analyte.     

Phase-enhanced selective ion ejection in an orbitrap mass spectrometer

The mass resolution achieved in selective ion isolation using resonance excitation is usually limited by the frequency resolution of the ac waveform and by unintended off-resonance excitation. A new method of phase-enhanced selective ion ejection based on broadband dipolar excitation and ion ejection applicable to the Orbitrap is described and shown to allow an isolation resolution of 28,400. The method is calculated to be able to provide a mass resolution for ion ejection of up to 100,000.     

Evaluating a direct swabbing method for screening pesticides on fruit and vegetable surfaces using direct analysis in real time (DART) coupled to an Exactive benchtop orbitrap mass spectrometer

Rapid screening of pesticides present on the surfaces of fruits and vegetables has been facilitated by using a Direct Analysis in Real Time (DART(?)) open air surface desorption ionization source coupled to an Exactive(?) high-resolution accurate mass benchtop orbitrap mass spectrometer. The use of cotton and polyester cleaning swabs to collect and retain pesticides for subsequent open air desorption ionization is demonstrated by sampling the surface of various produce to which solutions of pesticides have been applied at levels 10 and 100 times below the tolerance levels established by the United States Environmental Protection Agency (US EPA). Samples analyzed include cherry tomatoes, oranges, peaches and carrots each chosen for their surface characteristics which include: smooth, pitted, fuzzy, and rough respectively. Results from the direct analysis of fungicides on store-bought oranges are also described. In all cases, the swabs were introduced directly into the heated ionizing gas of the DART source resulting in production of protonated pesticide molecules within seconds of sampling. Operation of the orbitrap mass spectrometer at 25,000 full-width half maximum resolution was sufficient to generate high-quality accurate mass data. Stable external mass calibration eliminated the need for addition of standards typically required for mass calibration, thus allowing multiple analyses to be completed without instrument recalibration.     

The development of a high resolution mass spectrometer: A reminiscence

This article is a review of the events leading to the development of the double-focusing tandem mass spectrometer system, which is sometimes called the Johnson-Nier geometry. An essential feature of the geometry is that it eliminates second-order angular aberration, enhancing the sensitivity, without a loss in resolution. Flexibility in the choice of ion source and collector designs is assured because both are outside of regions of electric and magnetic fields. The vacuum housing permits high-temperature baking, ensuring ultrahigh vacuum conditions. Although introduced initially for the purpose of determining precise atomic masses, the design has found its greatest application in studies of structure of heavy molecules, making use of the high resolution to identify fragments. In many cases the composition of a molecule, or fragment, can be deduced from its exact mass by utilizing the known atomic masses of likely constituent atoms.     

A Quadrupole mass spectrometer for resolution of low mass isotopes

The qualitative and quantitative identification of low mass isotopes in the mass range 1–6 u poses certain difficulties when attempting to achieve the required resolution with an instrument suitable for deployment within a process environment. Certain adjacent species present in the process sample (HT and D₂) require a resolution greater than 930 to achieve an accurate measurement. We demonstrate here through simulation techniques that this level of performance required is unachievable using commercially available instruments. Using previously reported simulation techniques, this article demonstrates how the required performance for resolving the low mass isotopes can be achieved by a quadrupole mass spectrometer (QMS), which incorporates a quadrupole mass filter (QMF) constructed from hyperbolic electrodes and operated in zone 3 of the Mathieu stability diagram.     

Sensitive determination of prohibited drugs in dried blood spots (DBS) for doping controls by means of a benchtop quadrupole/Orbitrap mass spectrometer

In the present study, a new type of mass spectrometer combining a quadrupole mass filter, a higher collision dissociation (HCD) cell and an Orbitrap detector, was evaluated for the analysis of dried blood spots (DBS) in doping controls. DBS analysis is characterized by the necessity to detect prohibited compounds in sub-nanogram-per-milliliter levels with high identification capacity. After extraction of DBS with an organic solvent and liquid chromatographic separation (using a regular C18-RP-analytical UHPLC-column) of target analytes, mass spectrometry is performed with a high-resolution full scan in positive and negative mode by means of electrospray ionisation. Single-product ion mass spectra are acquired using the data-dependent analysis mode (employing an inclusion list) for previously selected precursors of known prohibited compounds with fixed retention time ranges. Besides, a sensitive screening in a targeted approach, non-targeted analysis for retrospective data evaluation is thus possible. The chosen experimental design enables the determination of various drugs from different classes with one generic sample preparation which is shown for 26 selected model compounds (Δ⁹-tetrahydrocannabinol (THC), tetrahydrocannabinol-9-carboxylic acid (THC-COOH), methylhexaneamine, methylphenidate, cocaine, nikethamide, 3,4-methylenedioxyamphetamine, N-methyl-3,4-methylenedioxyamphetamine, strychnine, mesocarb, salbutamol, formoterol, clenbuterol, metandienone, stanozolol, bisoprolol, propranolol, metoprolol, anastrazole, clomiphene, exemestane, dexamethasone, budesonide, selective androgen receptor modulator (SARM) S4 (andarine), SARM S1, hydrochlorothiazide). Generally, only qualitative result interpretation was focussed upon, but for target analytes with deuterium-labelled internal standards (salbutamol, clenbuterol, cocaine, dexamethasone, THC-COOH and THC) quantitative analysis was also possible. Especially the most challenging analytes, THC and its carboxy metabolite, were detected in DBS at relevant concentrations (<0.5 ng/mL) using targeted HCD experiments. The method was validated for the parameters: specificity, linearity (0–20 ng/mL), precision (<25%), recovery (mean 60%), limit of detection/quantification, ion suppression, stability and accuracy (80–120%). Six isotope-labelled analogues used as internal standards facilitate a quantitative result interpretation which is of utmost importance especially for in-competition drug sports testing.     

Analytical performance of a triple quadrupole mass spectrometer compared to a high resolution mass spectrometer for the analysis of polybrominated diphenyl ethers in fish

Polybrominated diphenyl ethers (PBDEs) are a class of flame retardants used globally in many consumer products and industrial applications. Traditionally, gas chromatography–high resolution mass spectrometry (GC–HR-MS) is the method of choice for analysis of PBDEs in environmental samples because it offers high sensitivity and selectivity, resulting in less interferences. However, the specificity offered by gas chromatography-triple quadrupole tandem mass spectrometry (GC–QQQ-MS/MS), operated in selected reaction monitoring mode, provides a more affordable alternative to GC–HR-MS for the analysis of PBDEs in complex environmental samples. In this study, an analytical method was developed for the analysis of 41 PBDE congeners in fish using GC–QQQ-MS/MS. Results from the analysis of three fish species [lake trout (Salvelinus namaycush), yellow perch (Perca flavescens), and round goby (Neogobius melanostomus)] using GC–QQQ-MS/MS were compared with those obtained by GC–HR-MS. These species were selected because they represent varying levels of lipid-rich matrix and contaminant loads. Instrumental limits of detection for the GC–QQQ-MS/MS ranged from 0.04 pg to 41 pg, whereas those for the GC–HR-MS ranged from 5 pg to 85 pg. The PBDE values obtained from these two methods were highly correlated, R² values >0.7, for all three fish species, supporting the suitability of GC–QQQ-MS/MS for analysis of PBDEs in fish with varying fat content.     

High resolution orbitrap mass spectrometry in comparison with tandem mass spectrometry for confirmation of anabolic steroids in meat

A prominent trend which has been observed in recent years in the analysis of veterinary drugs and growth-promoting agents is the shift from target-oriented procedures, mainly based on liquid chromatography coupled to triple-quadrupole mass spectrometry (LC-QqQ-MS), towards accurate mass full scan MS (such as time of flight (ToF) and Fourier Transform (FT) Orbitrap MS). In this study the applicability of high resolution single-stage-Orbitrap-MS for confirmatory analysis of growth-promoting agents in meat was compared to that of a QqQ-MS. Validation according to CD 2002/657/EC demonstrated that steroid analysis based on Orbitrap MS, operating at a resolution of 50,000 FWHM, is indeed capable to compete with QqQ-MS in terms of selectivity/specificity, while providing excellent linearity (for most compounds >0.99) but somewhat inferior sensitivity. Indeed, CC_αs reached from 0.04–0.88 μg kg⁻¹ for the 34 anabolic steroids upon MS/MS detection, while upon Orbitrap MS detection a range of 0.07–2.50 μg kg⁻¹ was observed. Using QqQ-MS adequate precision was obtained since relative standard deviations, associated with the repeatability and intra-laboratory reproducibility, were below 20%. In the case of Orbitrap MS, for some compounds (i.e. some estrogens) this threshold was exceeded and thus poor precision was observed, which is possibly caused by the lack in sensitivity. Overall, it may be concluded that Orbitrap-MS offers an adequate performance in terms of linearity and precision but lacks in sensitivity for some of the compounds.     

High‐resolution extracted ion chromatography,a new tool for metabolomics and lipidomics using a second‐generation orbitrap mass spectrometer

Most analytical methods in metabolomics are based on one of two strategies. The first strategy is aimed at specifically analysing a limited number of known metabolites or compound classes. Alternatively, an unbiased approach can be used for profiling as many features as possible in a given metabolome without prior knowledge of the identity of these features. Using high‐resolution mass spectrometry with instruments capable of measuring m/z ratios with sufficiently low mass measurement uncertainties and simultaneous high scan speeds, it is possible to combine these two strategies, allowing unbiased profiling of biological samples and targeted analysis of specific compounds at the same time without compromises. Such high mass accuracy and mass resolving power reduces the number of candidate metabolites occupying the same retention time and m/z ratio space to a minimum. In this study, we demonstrate how targeted analysis of phospholipids as well as unbiased profiling is achievable using a benchtop orbitrap instrument after high‐speed reversed‐phase chromatography. The ability to apply both strategies in one experiment is an important step forward in comprehensive analysis of the metabolome. Copyright © 2009 John Wiley & Sons, Ltd.     

Rephasing ion packets in the orbitrap mass analyzer to improve resolution and peak shape

A method is described to improve resolution and peak shape in the Orbitrap under certain experimental conditions. In these experiments, an asymmetric anharmonic axial potential was first produced in the Orbitrap by detuning the voltage on the compensator electrode, which results in broad and multiply split mass spectral peaks. An AC waveform applied to the outer electrode, 180° out of phase with ion axial motion and resonant with the frequency of ion axial motion, caused ions of a given m/z to be de-excited to the equator (z=0) and then immediately re-excited. This process, termed “rephasing,” leaves the ion packet with a narrower axial spatial extent and frequency distribution. For example, when the Orbitrap axial potential is thus anharmonically de-tuned, a resolution of 124,000 to 171,000 is obtained, a 2- to 3-fold improvement over the resolution of 40,000 to 60,000 without rephasing, at 10 ng/μL reserpine concentration. Such a rephasing capability may ultimately prove useful in implementing tandem mass spectrometry (MS/MS) in the Orbitrap, bringing the Orbitrap’s high mass accuracy and resolution to bear on both the precursor and product ions in the same MS/MS scan and making available the collision energy regime of the Orbitrap, ∼1500 eV.     

The direct coupling of high resolution glass open tubular columns to a mass spectrometer

Summary The coupling of high resolution open tubular glass columns to a mass spectrometer is achieved by avoiding any interfaces like seperators or splitting devices. In this way all the effluent without any loss is reaching the ion source and is available for mass spectrometric identification. It is shown that by this coupling the gas chromatographic resolution is fully retained and that the mass spectrometer proves to be a most sensitive detector. The usefullnes of a low cost computer for data aquisition and data reduction of the wealth of information inherent in a multi component chromatogram is demonstrated.

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Die direkte Kopplung von Glaskapillarsäulen mit höchster Trennleistung an ein Massenspektrometer

Zusammenrassung Es wird die Kopplung von hochauflösenden Glaskapillarsäulen mit einem Massenspektrometer beschrieben, wobei auf jede Art von Seperator oder Stromtrennung verzichtet wird. Es zeigt sich, daß die gaschromatografische Trennung voll erhalten bleibt, und daß sich das Massenspektrometer als ein äußerst empfindlicher Detektor erweist, da die gesamte eluierende Substanz zur massenspektrometrischen Indentifizierung zur Verfügung steht. Der Vorteil eines Kleincomputers für die Datenaufnahme und Datenreduktion bei der massenspektrometrischen Aufnahme eines komponentenreichen Gas-Chromatogramms wird demonstriert.

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Le couplage direct de colonnes capillaires à très haut pouvoir séparateur avec un spectromètre de masse

Résumé Le couplage de colonnes capillaires à haut pouvoir séparateur avec un spectromètre de masse est réalisé sans aucun intermédiaire tels que séparateurs ou diviseurs. De cette manière la totalité du composé élué atteint la source d'ions et est ainsi disponible pour l'identification par spectrométrie de masse. On montre que grâce à ce couplage la résolution du chromatogramme est conservée et que le spectromètre de masse devient un détecteur trés sensible. L'utilité d'un petit ordinateur pour l'acquisition et la réduction des données de l'ensemble des informations provenant d'un chromatogramme complexe est démontrée.

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Presented in part at the Analytikertagung in Basel (Switzerland) November 1970.     

Microderivatization of 4-[18O]hydroxyproline and quantitation with a benchtop mass spectrometer

Accurate estimation of in vivo turnover rates of collagen is complicated by amino acid reutilization. It was previously shown that the ideal, non-recycling tracer was [18O]hydroxyproline synthesized in vivo. The analytical method for measuring turnover rates with [18O]hydroxyproline must include analyte quantitation for pool size determination and isotope ratio measurement for determining levels of label incorporation. For ease of use and widest availability, a benchtop gas chromatograph-mass spectrometer in the electron-impact ionization mode was chosen. Here we present a versatile procedure for hydroxyproline derivatization that is well suited for routine, large-scale determination of analyte concentrations and relative levels of 18O incorporation.     

Optimizing signal and mass resolution for matrix-assisted laser desorption utilizing a linear time-of-flight mass spectrometer.

Results are presented for various instrumental configurations employed for matrix-assisted laser desorption mass spectrometry. Mass resolution is determined for a linear time-of-flight mass spectrometer for various lengths of the field-free region. A wire ion guide is utilized and is shown to improve ion transport efficiencies for longer field-free regions. It is also determined experimentally that a modest mass resolution increase is often obtained in configurations employing the wire ion guide when compared to the mass resolution obtained with the same geometry without the wire ion guide. Optimal applied potentials are determined for the wire ion guide. No mass dependence on the optimal applied potential (-100 V) for the wire ion guide is observed for samples of equine myoglobin (MW 16,951.5 Da) and a bacterial protease (MW 27,228.4 Da). The optimal applied voltage was also found to be identical (-100 V) for the singly through quadruply charged molecular ion species of rabbit gamma globulin (MW approximately 150,000 Da). It is shown that a 2 m flight tube with a wire ion guide provides better signal-to-noise mass spectra than a 1 m flight tube without the wire ion guide and can more than double the mass resolution obtainable. Utilization of a 4 m flight tube gives minimal mass resolution enhancement at the expense of signal-to-noise.     

Structural validation of saccharomicins by high resolution and high mass accuracy fourier transform-ion cyclotron resonance-mass spectrometry and infrared multiphoton dissociation tandem mass spectrometry

Exceptionally high mass resolving power and mass accuracy combined with tandem mass spectrometry (MSⁿ) capability make Fourier transform ion cyclotron resonance mass spectrometry a powerful tool for structure verification and determination of biological macromolecules. By means of local internal calibration and electron mass correction, mass accuracy better than ±0.5 ppm was achieved for two oligosaccharide antibiotics, Saccharomicins A and B, consistent with the proposed elemental compositions based upon NMR data. High resolution and high mass accuracy MS/MS data were obtained for both oligosaccharides by use of infrared multiphoton dissociation (IRMPD) with a 40 W continuous-wave CO₂ laser. The spectra were charge-state deconvolved by the “Z-score” algorithm to yield much simpler mass-only spectra. Sequences of 15 sugar residues could be confirmed from the charge state deconvolved accurate mass MS/MS spectra for Saccharomicins A and B, even without use of traditional prior permethylation. A fragment corresponding to an internal sugar loss rearrangement was observed by IRMPD and studied by collision activated dissociation MS⁴.     

A microcomputer system for mass spectrometer control and data acquisition

A microcomputer system has been designed for semi-automatic operation of a solid source mass spectrometer used for geochronological studies. It sequentially steps the magnetic field through pre-selected values, reads the digitized ion currents for a given time and temporarily stores the data which can be transferred to a paper tape or directly to a desk top calculator for further analysis. The unit is relatively inexpensive, made of readily available components and can be adapted to many laboratory automation tasks