Quantitation of hepcidin in serum using ultra‐high‐pressure liquid chromatography and a linear ion trap mass spectrometer

Hepcidin is a peptide hormone that functions as a key regulator of mammalian iron metabolism. Biological levels are increased in end‐stage renal disease and during inflammation but suppressed in hemochromatosis. Thus hepcidin levels have diagnostic importance. This study describes the development of an analytical method for the quantitative determination of the concentration of hepcidin in clinical samples. The fragmentation of hepcidin was investigated using triple quadrupole and linear ion trap mass spectrometers. A standard quantity of a stable isotopically labelled hepcidin internal standard was added to serum samples. Extraction was performed by protein precipitation and weak cation‐exchange magnetic nanoparticles. Chromatography was carried out on sub 2 µm particle stationary phase, using ultra‐high‐pressure liquid chromatography and a linear ion trap for quantitation. The lower limit of quantitation was 0.4 nmol/L with less than 20% accuracy and precision. The mean hepcidin concentration in sera for controls was 4.6 ± 2.7 nmol/L, in patients with sickle cell disease, 7.0 ± 8.9 nmol/L; in patients with end‐stage renal disease, 30.5 ± 15.7 nmol/L; and patients with penetrant hereditary hemochromatosis, 1.4 ± 0.8 nmol/L. Copyright © 2010 John Wiley & Sons, Ltd.     

Accuracy of relative isotopic abundance and mass measurements in a single‐stage orbitrap mass spectrometer

Orbitrap technology offers a combination of different technical specifications which have not yet been achieved by other high‐resolution mass spectrometry instrumentation. This refers to the combination of sensitivity, dynamic range, mass accuracy, resolution and speed. The high stability of the mass axis and the general ease of use made the orbitrap instrumentation attractive for routine laboratories. However, there are circumstances where significantly deviating relative isotopic abundance (RIA) and shifting accurate masses can be observed. RIA becomes biased at low ion counts. Furthermore, two adjacent, only partially resolved near‐isobaric ions are detected with a deviating RIA. The presence of a very intensive mass peak does not only induce Fourier transformation related artefacts (side‐lobes) but can cause mass shifts of small adjacent near‐isobaric mass peaks. These effects are not as drastic as known for Fourier transform ion cyclotron resonance instruments. Still, users trying to identify or quantify trace level compounds should be aware about such limitations in order to avoid possible pitfalls. Copyright © 2012 John Wiley & Sons, Ltd.     

Use of benchtop exactive high resolution and high mass accuracy orbitrap mass spectrometer for screening in horse doping control

Liquid chromatography-mass spectrometry (LC-MS) has been widely used in doping control laboratories over the last two decades. Currently, simple quadrupole, triple quadrupole and ion trap are the most commonly employed analyzers in toxicological analysis. Nevertheless, the main lack of these technologies is the restricted number of target compounds simultaneously screened without loss of sensitivity. In this article we present an innovative screening approach routinely applied in the French horse doping control laboratory based on high resolution (50000) and high mass accuracy (<5 ppm) in full scan MS mode for more than 235 target analytes screened from an initial volume of 5 mL of urine. The sample preparation was classically founded on solid phase extraction by means of reverse phase C18 cartridges. LC-MS analyses were carried out on a Shimadzu binary HPLC pumps linked to a C18 Sunfire column associated with the high resolution exactive benchtop orbitrap mass spectrometer. This screening was performed alternatively in positive-negative ionization mode during the same run. Thus, the identification of compounds of interest was made using their exact mass in positive-negative ionization mode at their expected retention time. All data obtained were processed by ToxID software (ThermoFisherScientific) which is able to identify a molecule by theoretical mass and retention time. In order to illustrate this innovative technology applied in our laboratory, sample preparation, validation data performed on 20 target compounds from 16 different horse urine samples, chromatograms and spectra will be discussed in this paper.     

High-throughput direct-infusion ion trap mass spectrometry: a new method for metabolomics

A fast method was developed to directly infuse raw plant extracts into a linear ion trap mass spectrometer, using the ion trap to isolate and fragment as many ions as possible from the extract. The full mass spectra can be analysed by multivariate statistics to determine discriminating ions, and the fragmentation data allows rapid classification or identification of these ions. The methodology was used to screen a wide range of strains of endophytic fungi in perennial ryegrass seeds for differences in metabolic profiles. The results show that this newly developed methodology is able to determine discriminating ions that can be present in very low concentrations. It also yielded sufficient fragmentation data to classify or identify the discriminating ions.     

Investigation of space charge effects and ion trapping capacity on direct introduction ultra‐high‐resolution mass spectrometry workflows for metabolomics

Ultra‐high‐resolution mass spectrometry, in the absence of chromatography, is finding its place for direct analyses of highly complex mixtures, such as those encountered during untargeted metabolomics screening. Advances, however, have been tempered by difficulties such as uneven signal suppression experienced during electrospray ionization. Moreover, ultra‐high‐resolution mass spectrometers that use Orbitrap and ICR analyzers both suffer from limited ion trapping capacities, owing principally to space‐charge effects. This study has evaluated and contrasted the above two types of Fourier transform mass spectrometers for their abilities to detect and identify by accurate mass measurement, small molecule metabolites present in complex mixtures. For these direct introduction studies, the Orbitrap Fusion showed a major advantage in terms of speed of analysis, enabling detection of 218 of 440 molecules (<2 ppm error, 500 000 resolution at m/z 200) present in a complex mixture in 5 min. This approach is the most viable for high‐throughput workflows, such as those used in investigations involving very large cohorts of metabolomics samples. From the same mixture, 183 unique molecules were observed by FT‐ICR in the broadband mode, but this number was raised to 235 when “selected ion monitoring‐stitching” (SIM‐stitching) was employed (<0.1 ppm error, 7 T magnet with dynamic harmonization cell, 1.8 million resolution at m/z 200, both cases). SIM‐stitching FT‐ICR thus offered the most complete detection, which may be of paramount importance in situations where it is essential to obtain the most complete metabolic profile possible. This added completeness, however, came at the cost of a more lengthy analysis time (120 min including manual treatment). Compared to the data presented here, future automation of processing, plus the use of absorption mode detection, segmented ion detection (stepwise detection of smaller width m/z sections), and higher magnetic field strengths, can substantially reduce FT‐ICR acquisition times.     

High-resolution accurate mass measurements of biomolecules using a new electrospray ionization ion cyclotron resonance mass spectrometer

A novel electrospray ionization/Fourier transform ion cyclotron resonance mass spectrometer based on a 7-T superconducting magnet was developed for high-resolution accurate mass measurements of large biomolecules. Ions formed at atmospheric pressure using electrospray ionization (ESI) were transmitted (through six differential pumping stages) to the trapped ion cell maintained below 10^?9 torr. The increased pumping speed attainable with cryopumping (> 10⁵ L/s) allowed brief pressure excursions to above 10^?4 torr, with greatly enhanced trapping efficiencies and subsequent short pumpdown times, facilitating high-resolution mass measurements. A set of electromechanical shutters were also used to minimize the effect of the directed molecular beam produced by the ES1 source and were open only during ion injection. Coupled with the use of the pulsed-valve gas inlet, the trapped ion cell was generally filled to the space charge limit within 100 ms. The use of 10–25 ms ion injection times allowed mass spectra to be obtained from 4 fmol of bovine insulin (M_r 5734) and ubiquitin (M_r 8565, with resolution sufficient to easily resolve the isotopic envelopes and determine the charge states. The microheterogeneity of the glycoprotein ribonuclease B was examined, giving a measured mass of 14,898.74 Da for the most abundant peak in the isotopic envelope of the normally glycosylated protein (i.e., with five mannose and two N-acetylglucosamine residues (an error of approximately 2 ppm) and an average error of approximately 1 ppm for the higher glycosylated and various H₃PO₄ adducted forms of the protein. Time-domain signals lasting in excess of 80 s were obtained for smaller proteins, producing, for example, a mass resolution of more than 700,000 for the 4⁺ charge state (m/z 1434) of insulin.     

Screening and confirmation criteria for hormone residue analysis using liquid chromatography accurate mass time-of-flight, Fourier transform ion cyclotron resonance and orbitrap mass spectrometry techniques

An emerging trend is recognised in hormone and veterinary drug residue analysis from liquid chromatography tandem mass spectrometry (LC/MS/MS) based screening and confirmation towards accurate mass alternatives such as LC coupled with time-of-flight (TOF), Fourier transform ion cyclotron resonance (FTICR) or Fourier transform orbitrap (FT Orbitrap) MS. In this study, mass resolution and accuracy are discussed for LC/MS screening and confirmation of targeted analytes and for the identification of unknowns using the anabolic steroid stanozolol and the designer beta-agonist "Clenbuterol-R" as model substances. It is shown theoretically and experimentally that mass accuracy criteria without proper mass resolution criteria yield false compliant (false negative) results, both in MS screening and MS/MS confirmation of stanozolol. On the other hand, previous medium resolution accurate mass TOFMS/MS data of the designer beta-agonist were fully confirmed by high resolution FT Orbitrap MS(n) experiments. A discussion is initiated through a proposal for additional criteria for the use of accurate mass LC/MS technologies, to be implemented in Commission Decision 2002/657/EC.     

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.     

Design of a new multi‐turn ion optical system ‘IRIS’ for a time‐of‐flight mass spectrometer

A new multi‐turn ion optical system ‘IRIS’ has been designed for use with a high‐performance time‐of‐flight (TOF) mass spectrometer, which satisfies the new design concepts of time focusing and phase space stability. It has an elliptical flight path composed of four toroidal electric sectors, with a flight path length for one lap of 0.974 m. Dimensions and voltages of sector electrodes have been optimized to satisfy theoretical requirements by simulations using surface charge method. Generally, multi‐turn instruments require an injection and ejection system to inject and eject ions. On the basis of this ion optical study, we have designed an injection and ejection ion optical system, which achieves time focusing for the total system. Furthermore, we have designed novel field‐adjusting electrodes (FAEs) for the perforated sectors in the injection and ejection systems, which accurately correct the electric potential around the perforated sector's hole. We have also used simulations to evaluate mass resolving power and ion transmissions for various lap numbers or flight path lengths. Through these we have confirmed that mass resolving powers of over 100 000 can be achieved with reasonable ion transmissions for a given set of initial conditions. Usually a multi‐turn TOF mass spectrometer with a closed optic axis has mass range limitations from overtaking ions. To solve this problem, a TOF segmentation method is proposed that identifies all peaks in a TOF spectrum, including those from overtaking ions. Copyright © 2008 John Wiley & Sons, Ltd.