Mass-resolving Power Research Articles

Design and commissioning of the ISOL beamline at the RAON facility

The Rare isotope Accelerator complex for ON-line experiment (RAON) located at the Daejeon site of the Institute for Basic Science (IBS) is the first Radioactive Isotope (RI) beam facility in Korea. The RAON facility employs an in-flight method using RI beams produced using an Isotope Separator On-Line (ISOL) method to explore unstable nuclei in uncharted regions. A high-power ISOL facility is critical for providing the required high-quality, intense RI beams. The design, construction, and installation of the ISOL system have been completed, and the beam transport line commissioning with stable isotope beams has been conducted. As a result, all requirements, such as the transmission and the mass-resolving power, for the beamlines were satisfied, and the beams from the Target Ion Source (TIS) were transported to the end of the ISOL beamline, which is the entrance to the superconducting linear accelerator system. The ISOL beamline is now ready for the transport of RI beams.

Generalized Kendrick analysis for improved visualization of atmospheric mass spectral data

Abstract. Mass spectrometry is an important analytical technique within the field of atmospheric chemistry. Owing to advances in instrumentation, particularly with regards to mass-resolving power and instrument response factors (sensitivities), hundreds of different mass-to-charge (m/z) signals are routinely measured. This large number of detected ions creates challenges for data visualization. Furthermore, assignment of chemical formulas to these ions is time consuming and increases in difficulty at the higher m/z ranges. Here, we describe generalized Kendrick analysis (GKA) to facilitate the visualization and peak identification processes for typical atmospheric organic (and to some extent inorganic) compounds. GKA is closely related to resolution-enhanced Kendrick mass defect analysis (REKMD), which introduces a tunable integer into the Kendrick equation that effectively contracts or expands the mass scale. A characteristic of all Kendrick analysis methods is that these changes maintain the horizontal alignment of ion series related by integer multiples of the chosen base unit. Compared to traditional Kendrick analysis, GKA and REKMD use a tunable parameter (“scaling factor”) to alter the mass defect spacing between different homologue ion series. As a result, the entire mass defect range (−0.5 to 0.5) is more effectively used simplifying data visualization and facilitating chemical formula assignment. We describe the mechanism of this transformation and discuss base unit and scaling factor selections appropriate for compounds typically found in atmospheric measurements. We present an open-source graphical user interface (GUI) for calculating and visualizing GKA results within the Igor Pro environment.

Correlating laser energy with compositional and atomic-level information of oxides in atom probe tomography

Atom probe tomography (APT) is a 3D analysis technique that offers unique chemical accuracy and sensitivity with sub-nanometer spatial resolution. There is an increasing interest in the application of APT to complex oxides materials, giving new insight into the relation between local variations in chemical composition and emergent physical properties. However, in contrast to the field of metallurgy, where APT is routinely applied to study materials at the atomic level, complex oxides and their specific field evaporation mechanisms are much less explored. Here, we perform APT measurements on the hexagonal manganite ErMnO3 and systematically study the effect of different experimental parameters on the measured composition and structure. We demonstrate that both the mass resolving power (MRP) and compositional accuracy can be improved by increasing the charge-state ratio (CSR) working at low laser energy (< 5 pJ) for a given fixed detection rate. Furthermore, we observe a substantial preferential retention of Er atoms, which is suppressed at higher CSRs. We explain our findings based on fundamental field evaporation concepts, expanding the knowledge about the impact of key experimental parameters and the field evaporation process in complex oxides in general.

Achieving high mass resolution with the NanoSIMS 50 while preserving signal transmission from submicron probe impact areas

Elemental and isotopic local analysis using secondary ion emission induced by the impact of a submicron ion probe (SIMS) requires: (i) efficient collection of ejected ions (sensitivity) (ii) high mass resolving power (MRP) to discard interfering species (selectivity). Matching the extracted secondary beam with the acceptance of the spectrometer is then an essential issue. The NanoSIMS 50 fits those requirements fairly well. Nonetheless, a thoughtful study of its mass spectrometer has led to higher performance and suggested further improvements.12C2D and 12C2H2 with similar peak amplitudes were separated, MRP ≅ 16 800, while preserving half of the maximum signal. MRP of about 9 000, with flat top peaks and a few percent valley between 16OD and 17OH, were obtained while maintaining about 2/3 of maximum signals. Such complete separation should allow D/H ratio measurements via 16OD/16OH with much better sensitivity than that obtained with monoatomic species.

Selective deuteration as a tool for resolving autoxidation mechanisms in α-pinene ozonolysis

Abstract. Highly oxygenated organic molecules (HOMs) from α-pinene ozonolysis have been shown to be significant contributors to secondary organic aerosol (SOA), yet our mechanistic understanding of how the peroxy-radical-driven autoxidation leads to their formation in this system is still limited. The involved isomerisation reactions such as H-atom abstractions followed by O2 additions can take place on sub-second timescales in short-lived intermediates, making the process challenging to study. Similarly, while the end-products and sometimes radical intermediates can be observed using mass spectrometry, their structures remain elusive. Therefore, we propose a method utilising selective deuterations for unveiling the mechanisms of autoxidation, where the HOM products can be used to infer which C atoms have taken part in the isomerisation reactions. This relies on the fact that if a C−D bond is broken due to an abstraction by a peroxy group forming a −OOD hydroperoxide, the D atom will become labile and able to be exchanged with a hydrogen atom in water vapour (H2O), effectively leading to loss of the D atom from the molecule. In this study, we test the applicability of this method using three differently deuterated versions of α-pinene with the newly developed chemical ionisation Orbitrap (CI-Orbitrap) mass spectrometer to inspect the oxidation products. The high mass-resolving power of the Orbitrap is critical, as it allows the unambiguous separation of molecules with a D atom (mD=2.0141) from those with two H atoms (mH2=2.0157). We found that the method worked well, and we could deduce that two of the three tested compounds had lost D atoms during oxidation, suggesting that those deuterated positions were actively involved in the autoxidation process. Surprisingly, the deuterations were not observed to decrease HOM molar yields, as would have been expected due to kinetic isotope effects. This may be an indication that the relevant H (or D) abstractions were fast enough that no competing pathways were of relevance despite slower abstraction rates of the D atom. We show that selective deuteration can be a very useful method for studying autoxidation on a molecular level and likely is not limited to the system of α-pinene ozonolysis tested here.

First application of the phase-imaging ion-cyclotron resonance technique at TRIGA-Trap

The phase-imaging ion cyclotron resonance technique (PI-ICR) has been implemented at TRIGA-Trap together with a newly built five-pole cylindrical trap. In PI-ICR the total phase of trapped ions is measured by projecting the ion motion onto a position-sensitive delay-line micro-channel plate detector. The systematic uncertainties have been investigated and first mass measurements on stable Pb isotopes have been performed with PI-ICR. The new technique offers higher mass-resolving power, allows checking for the presence of contaminant ion species, and it proved useful in tuning the harmonicity of the trapping potential as well as in aligning the trap symmetry axis with respect to the magnetic field axis by visualizing the radial ion motion. This is a non-scanning technique where every detected ion contributes equally, therefore it is more sensitive than the previously used time-of-flight ion-cyclotron-resonance (ToF-ICR) technique, which is based on the scanning of the sideband-frequency of trapped ions and recording their time of flight after ejection. It will enable us to carry out high-precision mass measurements in the actinide region with uncertainties on the ppb level.

Label-free Chemical Characterization of Polarized Immune Cells in vitro and Host Response to Implanted Bio-instructive Polymers in vivo Using 3D OrbiSIMS.

The Three-dimensional OrbiTrap Secondary Ion Mass Spectrometry (3D OrbiSIMS) is a secondary ion mass spectrometry instrument, a combination of a Time of Flight (ToF) instrument with an Orbitrap analyzer. The 3D OrbiSIMS technique is a powerful tool for metabolic profiling in biological samples. This can be achieved at subcellular spatial resolution, high sensitivity, and high mass-resolving power coupled with MS/MS analysis. Characterizing the metabolic signature of macrophage subsets within tissue sections offers great potential to understand the response of the human immune system to implanted biomaterials. Here, we describe a protocol for direct analysis of individual cells after in vitro differentiation of naïve monocytes into M1 and M2 phenotypes using cytokines. As a first step in vivo, we investigate explanted silicon catheter sections as a medical device in a rodent model of foreign body response. Protocols are presented to allow the host response to different immune instructive materials to be compared. The first demonstration of this capability illustrates the great potential of direct cell and tissue section analysis for in situ metabolite profiling to probe functional phenotypes using molecular signatures. Details of the in vitro cell approach, materials, sample preparation, and explant handling are presented, in addition to the data acquisition approaches and the data analysis pipelines required to achieve useful interpretation of these complex spectra. This method is useful for in situ characterization of both in vitro single cells and ex vivo tissue sections. This will aid the understanding of the immune response to medical implants by informing the design of immune-instructive biomaterials with positive interactions. It can also be used to investigate a broad range of other clinically relevant therapeutics and immune dysregulations. Graphical overview.

Development of electrostatic-induced charge detector for multiturn time-of-flight mass spectrometer.

We developed an autocorrelation function to resolve the overtaking problem in a multiturn time-of-flight mass spectrometer (TOF-MS). The function analyzes the characteristic period for one lap of each ion packet and derives a mass spectrum from a signal pulse train composed of multiturn ion packets. To detect the ion pulse train, a new nondestructive ion detector was developed and installed in the multiturn orbit of MULTUM-S II. This detector is composed of an electrostatically induced charge detector, a preamplifier, and a digitizer. The electrostatic noises are smaller than the single-ion signals owing to the accumulation of the multiturn TOF spectrum. The conventional ion detector of TOF-MS is operated after collecting the signal pulse train. The multiturn TOF spectrum was convolved with an autocorrelation function to derive the mass spectrum. The convolved mass spectrum performed a mass resolving power (MRP) of 28,200 at m/z 69 and mass accuracy of 28 ppm for the perfluorotributylamine (PFTBA) gas sample.

Recent Advances in Single-Cell Metabolomics Based on Mass Spectrometry

Recent Advances in Single-Cell Metabolomics Based on Mass Spectrometry

FT-ICR Mass Spectrometry Imaging at Extreme Mass Resolving Power Using a Dynamically Harmonized ICR Cell with 1ω or 2ω Detection.

MALDI mass spectrometry imaging (MALDI MSI) is a powerful analytical method for achieving 2D localization of compounds from thin sections of typically but not exclusively biological samples. The dynamically harmonized ICR cell (ParaCell) was recently introduced to achieve extreme spectral resolution capable of providing the isotopic fine structure of ions detected in complex samples. The latest improvement in the ICR technology also includes 2ω detection, which significantly reduces the transient time while preserving the nominal mass resolving power of the ICR cell. High-resolution MS images acquired on FT-ICR instruments equipped with 7T and 9.4T superconducting magnets and the dynamically harmonized ICR cell operating at suboptimal parameters suffered severely from the pixel-to-pixel shifting of m/z peaks due to space-charge effects. The resulting profile average mass spectra have depreciated mass measurement accuracy and mass resolving power under the instrument specifications that affect the confidence level of the identified ions. Here, we propose an analytical workflow based on the monitoring of the total ion current to restrain the pixel-to-pixel m/z shift. Adjustment of the laser parameters is proposed to maintain high spectral resolution and mass accuracy measurement within the instrument specifications during MSI analyses. The optimized method has been successfully employed in replicates to perform high-quality MALDI MS images at resolving power (FWHM) above 1,000,000 in the lipid mass range across the whole image for superconducting magnets of 7T and 9.4T using 1 and 2ω detection. Our data also compare favorably with MALDI MSI experiments performed on higher-magnetic-field superconducting magnets, including the 21T MALDI FT-ICR prototype instrument of the NHMFL group at Tallahassee, Florida.

First Offline Results from the S3 Low-Energy Branch

We present the first results obtained from the S3 Low-Energy Branch, the gas cell setup at SPIRAL2-GANIL, which will be installed behind the S3 spectrometer for atomic and nuclear spectroscopy studies of exotic nuclei. The installation is currently being commissioned offline, with the aim to establish optimum conditions for the operation of the radio frequency quadrupole ion guides, mass separation and ion bunching, providing high-efficiency and low-energy spatial spread for the isotopes of interest. Transmission and mass-resolving power measurements are presented for the different components of the S3-LEB setup. In addition, a single-longitudinal-mode, injection-locked, pumped pulsed-titanium–sapphire laser system has been recently implemented and is used for the first proof-of-principle measurements in an offline laser laboratory. Laser spectroscopy measurements of erbium, which is the commissioning case of the S3 spectrometer, are presented using the 4f126s23H6→4f12(3H)6s6p optical transition.

Gas chromatography/miniaturized time-of-flight mass spectrometry technique for high-throughput quantitative on-site field analysis

Previously, we reviewed several mass spectrometers designed for field analysis, also known as harsh mass spectrometers. The majority of these instruments are focused on the instrument footprint size and weight, which compromise the performance, for instance, in terms of mass-resolving power. Our recent work employed a shoebox-size (mini-tower personal computer size) high-resolving-power mass analyzer equipped with a robust gas chromatograph in the firm field, which successfully monitored the soil-gas compositional changes on-the-fly in real time. We evaluated the system performance for mass accuracy, self-validation testing for analyte peaks, and quantitative precision through long-term continuous data acquisitions of 1429 replicates of model gas samples for 48 h. The observed mass drift was 4 mDa, which was compensated within 0.45 mDa using a novel algorithm without the addition of mass reference materials. In addition, the optimized algorithm for high-resolution ion chromatogram extraction for this application was evaluated. Results indicated that the proposed method limited the chromatographic peak area variance. The coefficient of variance (CV) for determining CO2 amounts for the 1429 sample runs was 8.6%. Furthermore, the CO2 average retention time was 48.06 ± 0.29 s (CV = 0.61%), average number of theoretical plates was 3500, and asymmetry factor was 1.09.

FT-ICR mass spectrometry: Superconducting magnet, external ion source, ion-molecule reactions, and ion-ion traps.

The world of Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometry has witnessed, especially in the last 30 years significant advances in many fields of science, such as electronics, magnets, new ICR cell designs, developed ICR event sequences, modern external ionization sources, and linear ion beam guides, as well as modern vacuum technology. In this review, a brief account is given focusing especially on the studies performed in Wanczek's group and ICR research laboratory at the University of Bremen. An FT-ICR mass spectrometer has been developed with a high magnetic field superconducting magnet, operating at 4.7 T. At this magnetic field, a trapping time of 13.5 h was obtained with 30% efficiency. For the tetrachloromethane molecular ion, m/z 166, a mass-resolving power m/Δm = 1.5 × 106 was measured at a pressure of 2 × 10-8 Torr. The transition from magnet sweep to frequency sweep and the application of Fourier-transform has greatly enhanced the ICR technology. External ion sources were invented and differential pumping schemes were developed for enabling ultrahigh vacuum condition for ICR detection, while guiding ions at relatively higher pressures, during their flight to the ICR cell. With the external ion source, a time-of-flight ICR tandem instrument is built. A method to measure the ion flight time and to trap the ions in the ICR cell is described. Many ICR cell characteristics such as z-axis ion ejection and coupling of radial and axial ion motions in a superposed homogeneous magnetic and inhomogeneous trapping electric field were extensively studied. Gas-phase ion-molecule reactions of several reactive inorganic compounds with a focus on phosphorous and sulfur as well as silicon chemistry were also studied in great detail. The gas-phase ion chemistry of several trifluoromethyl-reagents such as trifluoromethyltrimethylsilane and tris(trifluoromethyl)phosphine were also investigated in ICR. Dual polarities multisegmented ICR cells were invented and deeply characterized. Sophisticated ICR pulse event programs were developed to enable long-range ion-ion interactions between simultaneously trapped positive and negative ions.

Fourier-transform ion cyclotron resonance mass spectrometry for characterizing proteoforms.

Proteoforms contribute functional diversity to the proteome and aberrant proteoforms levels have been implicated in biological dysfunction and disease. Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR MS), with its ultrahigh mass-resolving power, mass accuracy, and versatile tandem MS capabilities, has empowered top-down, middle-down, and native MS-based approaches for characterizing proteoforms and their complexes in biological systems. Herein, we review the features which make FT-ICR MS uniquely suited for measuring proteoform mass with ultrahigh resolution and mass accuracy; obtaining in-depth proteoform sequence coverage with expansive tandem MS capabilities; and unambiguously identifying and localizing post-translational and noncovalent modifications. We highlight examples from our body of work in which we have quantified and comprehensively characterized proteoforms from cardiac and skeletal muscle to better understand conditions such as chronic heart failure, acute myocardial infarction, and sarcopenia. Structural characterization of monoclonal antibodies and their proteoforms by FT-ICR MS and emerging applications, such as native top-down FT-ICR MS and high-throughput top-down FT-ICR MS-based proteomics at 21 T, are also covered. Historically, the information gleaned from FT-ICR MS analyses have helped provide biological insights. We predict FT-ICR MS will continue to enable the study of proteoforms of increasing size from increasingly complex endogenous mixtures and facilitate the benchmarking of sensitive and specific assays for clinical diagnostics. © 2020 John Wiley & Sons Ltd. Mass Spec Rev.

New Method for Improving LC/Time-of-Flight Mass Spectrometry Detection Limits Using Simultaneous Ion Counting and Waveform Averaging.

Simultaneous ion counting and waveform averaging implemented on a field-programmable gate array compiled with a high-speed digitizer was applied to ultraperformance liquid chromatography-time-of-flight mass spectrometric analysis of sulfa drugs. Ion counting was carried out by a "Peak Detection" (PKD) function that works together with signal averaging (AVG). Sulfadimidine (SDD) and sulfadimethoxine (SDMX) were measured in human serum (HS) model sample matrix. By using simultaneous PKD and AVG acquisition, we observed a unified calibration curve for more than 3 orders of magnitude of sample amounts (0.010-100.0 pmol). The ion count rate for the "practical" sample amounts, such as less than 1 pmol, was below 30%, which is suitable for PKD-based ion counting for quantitative accuracy and excellent peak identification performance. Samples containing 200 fmol or less could not be identified from the AVG waveform. Adding HS treated with acetonitrile severely suppressed the SDMX ion to less than one-half (58.1%). However, a linear response was observed for chromatographic peak area for analytes calculated from PKD waveforms. Also, the mass-resolving power calculated from the peak on the PKD waveform was 24% better than the corresponding AVG waveform, which also improves performance for analyte identification.

Isobar-free neon isotope measurements of flux-fused potential reference minerals on a Helix-MC-Plus10K mass spectrometer

This work presents new analytical techniques for extraction and analysis of neon from a suite of different mineral phases, including quartz, pyroxene, hematite, apatite, zircon, topaz, and fluorite. Neon was quantitatively extracted at 1100 °C from all of these minerals using an in-vacuum lithium borate-flux fusion technique. Evolved neon was purified using a cryogenic method capable of separating Ne from He present in abundances ~8 orders of magnitude higher, typical of samples carrying nucleogenic/radiogenic noble gases. The purified neon was measured on a Helix-MC-Plus10K mass spectrometer that permits isobar-free measurement of all three neon isotopes. When operated at its highest mass resolving power (MRP) of ~10,300, the shoulder representing solely 22Ne on the low mass-side of the 22Ne-CO2+2 doublet is wide enough to permit measurement of isobar free 22Ne. Operating in this mode comes with the penalty of a 50% reduction in neon sensitivity. Coupled with a mathematical isobar-stripping method, this approach excludes 99.5% of the CO2+2 while still collecting >99% of the 22Ne beam. Routine edge-centering on the dynamic CO2+2 peak prior to introduction of a sample permits rapid and robust relocation of the desired measure point in the mass spectrum.Cosmogenic 21Ne and 22Ne concentrations obtained using these methods on the Cronus-A quartz and Cronus-P pyroxene international reference materials are in excellent agreement with previous work or expectations. Similarly, the concentration of nucleogenic 21Ne and 22Ne in Durango apatite and the CIT hematite standard agree well with previous work. Durango apatite has notable heterogeneity in neon concentrations, consistent with previous observations of heterogeneous He, U and Th concentrations in this apatite. Nucleogenic neon concentrations are also presented for previously unstudied minerals including a Sri Lanka zircon (SLC), a topaz from the Imperial Topaz mine in Brazil (ITP1), and a fluorite (W-90) from New Hampshire. Taken together this set of potential reference minerals and the associated dataset provide a starting point for intercalibration among multiple mineral phases carrying 21Ne and 22Ne of cosmogenic or nucleogenic origin.

Design of a multiple-reflection time-of-flight mass spectrometer for barium-tagging

The search for neutrinoless double beta decay requires increasingly advanced methods of background reduction. A bold approach to solving this problem, in experiments using Xe-136, is to extract and identify the daughter Ba-136 ion produced by double beta decay. Tagging events in this manner allows for a virtually background-free verification of double beta decay signals. Various approaches are being pursued by the nEXO collaboration to achieve Ba-tagging. A Multi-Reflection Time-of-Flight Mass Spectrometer (MR TOF) has been designed and optimized as one of the ion-identification methods, where it will investigate the ion-extraction efficiency, as well as provide further identification of the Ba isotope. The envisioned mode of operation allows the MR TOF to achieve a quickly adjustable mass-range and resolution, with simulations suggesting that a mass-resolving power of 140,000 is within reach. This work will discuss the MR TOF design and the methods employed to simulate and optimize it.

Perspectives on Liquid Chromatography-High-Resolution Mass Spectrometry for Pesticide Screening in Foods.

This perspective discusses the use of liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) for multiresidue analysis of pesticides in foods and agricultural commodities. HRMS has the important distinction and advantage of mass-resolving power and, therefore, requires different concepts, experiments, and guidance for screening, identification, and quantitation of pesticides in complex food matrices over triple quadrupole mass spectrometry. HRMS approaches for pesticide screening, including full-scan experiments in conjunction with tandem mass spectrometry (MS/MS) experiments, are described. This approach results in the generation of chromatographic retention times and high-resolution mass spectra with accurate mass measurements that can be used to create compound databases. New data processing tools can create an efficient and optimized screening approach that can speed the analysis and identification of compounds, reduce the need for chemical standards, and harmonize pesticide analytical procedures.

21 Tesla FT-ICR Mass Spectrometer for Ultrahigh-Resolution Analysis of Complex Organic Mixtures.

We describe complex organic mixture analysis by 21 tesla (T) Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). Ultrahigh mass-resolving power (m/Δm50% > 2 700 000 at m/z 400) and mass accuracy (80 ppb rms) enable resolution and confident identification of tens of thousands of unique elemental compositions. We demonstrate 2.2-fold higher mass-resolving power, 2.6-fold better mass measurement accuracy, and 1.3-fold more assigned molecular formulas compared to our custom-built, state-of-the-art 9.4 T FT-ICR mass spectrometer for petroleum and dissolved organic matter (DOM) analyses. Analysis of a heavy petroleum distillate exemplifies the need for ultrahigh-performance mass spectrometry (49 040 assigned molecular formulas for 21 T versus 29 012 for 9.4 T) and extends the identification of previously unresolved Oo, SsOo, and NOo classes. Mass selective ion accumulation (20 Thompson isolation) of an asphalt volcano sample yields 462 resolved mass spectral peaks at m/z 677 and reveals previously unresolved CcHhNnOoSs mass differences at high mass (m/z > 600). Similar performance gains are realized in the analysis of dissolved organic matter, where doubly charged Oo species are resolved from singly charged SOo species, which requires a mass-resolving power greater than 1 400 000 (at m/z 600). This direct comparison reveals the continued need for higher mass-resolving power and better mass accuracy for comprehensive molecular characterization of the most complex organic mixtures.

The 3D OrbiSIMS-label-free metabolic imaging with subcellular lateral resolution and high mass-resolving power.

We report the development of a 3D OrbiSIMS instrument for label-free biomedical imaging. It combines the high spatial resolution of secondary ion mass spectrometry (SIMS; under 200 nm for inorganic species and under 2 μm for biomolecules) with the high mass-resolving power of an Orbitrap (>240,000 at m/z 200). This allows exogenous and endogenous metabolites to be visualized in 3D with subcellular resolution. We imaged the distribution of neurotransmitters-gamma-aminobutyric acid, dopamine and serotonin-with high spectroscopic confidence in the mouse hippocampus. We also putatively annotated and mapped the subcellular localization of 29 sulfoglycosphingolipids and 45 glycerophospholipids, and we confirmed lipid identities with tandem mass spectrometry. We demonstrated single-cell metabolomic profiling using rat alveolar macrophage cells incubated with different concentrations of the drug amiodarone, and we observed that the upregulation of phospholipid species and cholesterol is correlated with the accumulation of amiodarone.