Nonlinear rotational spectroscopy reveals many-body interactions in water molecules.

Because of their central importance in chemistry and biology, water molecules have been the subject of decades of intense spectroscopic investigations. Rotational spectroscopy of water vapor has yielded detailed information about the structure and dynamics of isolated water molecules, as well as water dimers and clusters. Nonlinear rotational spectroscopy in the terahertz regime has been developed recently to investigate the rotational dynamics of linear and symmetric-top molecules whose rotational energy levels are regularly spaced. However, it has not been applied to water or other lower-symmetry molecules with irregularly spaced levels. We report the use of recently developed two-dimensional (2D) terahertz rotational spectroscopy to observe high-order rotational coherences and correlations between rotational transitions that were previously unobservable. The results include two-quantum (2Q) peaks at frequencies that are shifted slightly from the sums of distinct rotational transitions on two different molecules. These results directly reveal the presence of previously unseen metastable water complexes with lifetimes of 100 ps or longer. Several such peaks observed at distinct 2Q frequencies indicate that the complexes have multiple preferred bimolecular geometries. Our results demonstrate the sensitivity of rotational correlations measured in 2D terahertz spectroscopy to molecular interactions and complexation in the gas phase.

Long-range model of vibrational autoionization in core-nonpenetrating Rydberg states of NO.

In high orbital angular momentum (ℓ ≥ 3) Rydberg states, the centrifugal barrier hinders the close approach of the Rydberg electron to the ion-core. As a result, these core-nonpenetrating Rydberg states can be well described by a simplified model in which the Rydberg electron is only weakly perturbed by the long-range electric properties (i.e., multipole moments and polarizabilities) of the ion-core. We have used a long-range model to describe the vibrational autoionization dynamics of high-ℓ Rydberg states of nitric oxide (NO). In particular, our model explains the extensive angular momentum exchange between the ion-core and the Rydberg electron that had been previously observed in vibrational autoionization of f (ℓ = 3) Rydberg states. These results shed light on a long-standing mechanistic question around these previous observations and support a direct, vibrational mechanism of autoionization over an indirect, predissociation-mediated mechanism. In addition, our model correctly predicts newly measured total decay rates of g (ℓ = 4) Rydberg states because for ℓ ≥ 4, the non-radiative decay is dominated by autoionization rather than predissociation. We examine the predicted NO+ ion rotational state distributions generated by vibrational autoionization of g states and discuss applications of our model to achieve quantum state selection in the production of molecular ions.

Analysis of vibrational autoionization of CaF Rydberg states.

We report calculations of vibrational autoionization rates of CaF Rydberg states, based on the results of a global multi-channel quantum defect theory (MQDT) fit. Our goal is to use intuitive physical models to interpret and extend the results from the MQDT calculations and, in particular, to characterize the physical mechanisms for the interaction between the Rydberg electron and the ion-core. The calculations indicate that, among the six strongly l-mixed core-penetrating (CP) Rydberg series of CaF, the n.36 p^Π Rydberg series has the fastest Δv = 1 vibrational autoionization rate, which is at least four times larger than that for the other CP Rydberg series, in agreement with experimental results. We first demonstrate that the rotational level dependence of the vibrational autoionization rate of the n.36 p^Π series is satisfactorily explained by l-uncoupling interactions, which differ for the positive and negative Kronig symmetry levels. Next, we interpret the relative vibrational autoionization rates of all six CP Rydberg series in the context of a valence-precursor (VP) model. The VP model is a consequence of Mulliken's rule, which states that the innermost lobe of the Rydberg wavefunction remains invariant in both the nodal position and shape for members of the same Rydberg series. The electronic properties of the six VP states, which are the terminus states (lowest-n) of each of the six CP Rydberg series, are further characterized in terms of a ligand-field model, providing insight into the intimate relationship between the Rydberg electron density in the ion-core region and the vibrational autoionization rate.

Electric potential invariants and ions-in-molecules effective potentials for molecular Rydberg states.

The dependence of multipole moments and polarizabilities on external fields appears in many applications including biomolecular molecular mechanics, optical non-linearity, nanomaterial calculations, and the perturbation of spectroscopic signatures in atomic clocks. Over a wide range of distances, distributed multipole and polarizability potentials can be applied to obtain the variation of atom-centered atoms-in-molecules electric properties like bonding-quenched polarizability. For cylindrically symmetric charge distributions, we examine single-center and atom-centered effective polarization potentials in a non-relativistic approximation for Rydberg states. For ions, the multipole expansion is strongly origin-dependent, but we note that origin-independent invariants can be defined. The several families of invariants correspond to optimized representations differing by origin and number of terms. Among them, a representation at the center of dipole polarizability optimizes the accuracy of the potential with terms through 1/r4. We formulate the single-center expansion in terms of polarization-modified effective multipole moments, defining a form related to the source-multipole expansion of Brink and Satchler. Atom-centered potentials are an origin independent alternative but are limited both by the properties allowed at each center and by the neglected effects like bond polarizability and charge flow. To enable comparisons between single-center effective potentials in Cartesian or spherical form and two-center effective potentials with differing levels of mutual induction between atomic centers, we give analytical expressions for the bond-length and origin-dependence of multipole and polarizability terms projected in the multipole and polarizability expansion of Buckingham. The atom-centered potentials can then be used with experimental data and ab initio calculations to estimate atoms-in-molecules properties. Some results are given for BaF+ and HF showing the utility and limitations of the approach. More detailed results on X 1Σ+ CaF+ are published separately.

A differential mobility spectrometry/mass spectrometry platform for the rapid detection and quantitation of DNA adduct dG-ABP.

RATIONALE: There is continued interest in exploring new analytical technologies for the detection and quantitation of DNA adducts, biomarkers which provide direct evidence of exposure and genetic damage in cells. With the goal of reducing clean-up steps and improving sample throughput, a Differential Mobility Spectrometry/Mass Spectrometry (DMS/MS) platform has been introduced for adduct analysis. METHODS: A DMS/MS platform has been utilized for the analysis of dG-ABP, the deoxyguanosine adduct of the bladder carcinogen 4-aminobiphenyl (4-ABP). After optimization of the DMS parameters, each sample was analyzed in just 30 s following a simple protein precipitation step of the digested DNA. RESULTS: A detection limit of one modification in 10^6 nucleosides has been achieved using only 2 µg of DNA. A brief comparison (quantitative and qualitative) with liquid chromatography/mass spectrometry is also presented highlighting the advantages of using the DMS/MS method as a high-throughput platform. CONCLUSIONS: The data presented demonstrate the successful application of a DMS/MS/MS platform for the rapid quantitation of DNA adducts using, as a model analyte, the deoxyguanosine adduct of the bladder carcinogen 4-aminobiphenyl.

Chirped-pulse millimeter-wave spectroscopy: spectrum, dynamics, and manipulation of Rydberg-Rydberg transitions.

We apply the chirped-pulse millimeter-wave (CPmmW) technique to transitions between Rydberg states in calcium atoms. The unique feature of Rydberg-Rydberg transitions is that they have enormous electric dipole transition moments (~5 kiloDebye at n* ~ 40, where n* is the effective principal quantum number), so they interact strongly with the mm-wave radiation. After polarization by a mm-wave pulse in the 70-84 GHz frequency region, the excited transitions re-radiate free induction decay (FID) at their resonant frequencies, and the FID is heterodyne-detected by the CPmmW spectrometer. Data collection and averaging are performed in the time domain. The spectral resolution is ~100 kHz. Because of the large transition dipole moments, the available mm-wave power is sufficient to polarize the entire bandwidth of the spectrometer (12 GHz) in each pulse, and high-resolution survey spectra may be collected. Both absorptive and emissive transitions are observed, and they are distinguished by the phase of their FID relative to that of the excitation pulse. With the combination of the large transition dipole moments and direct monitoring of transitions, we observe dynamics, such as transient nutations from the interference of the excitation pulse with the polarization that it induces in the sample. Since the waveform produced by the mm-wave source may be precisely controlled, we can populate states with high angular momentum by a sequence of pulses while recording the results of these manipulations in the time domain. We also probe the superradiant decay of the Rydberg sample using photon echoes. The application of the CPmmW technique to transitions between Rydberg states of molecules is discussed.

Signatures of Reaction Mechanisms Encoded in the Vibrational Population Distribution of Small-Molecule Products: Photodissociation of Symmetric-Triazine Using 266 nm Radiation.

We utilize rotationally resolved Chirped-Pulse Fourier Transform millimeter-wave spectroscopy to study photodissociation dynamics of 1,3,5-Triazine (symmetric-Triazine) to form 3 HCN molecules. The state-specific vibrational population distribution (VPD) of the photofragments contains mechanistic details of the reaction. This photodissociation is performed using 266 nm radiation transverse to a seeded supersonic jet. The vibrational cooling inefficiency in the jet preserves the VPD of the photofragments, while rotational cooling enhances the signal of low-J pure-rotational transitions. The multiplexed nature of the spectrometer enables simultaneous sampling of several "vibrational satellites" of the J = 1 ← 0 transition of HCN. Excited state populations along the HCN bend (v2) and CN stretch (v3) modes are observed, which show ≥3.2% vibrational excitation of the photofragments. Observation of an at least bimodal VPD, along the even-v states of v2, implies an asymmetric partitioning of vibrational energy among the HCN photofragments. This suggests a sequential dissociation mechanism of symmetric-Triazine initiated by 266 nm radiation.

Chirped-pulse millimeter-wave spectroscopy of Rydberg-Rydberg transitions.

Transitions between Rydberg states of Ca atoms, in a pulsed, supersonic atomic beam, are directly detected by chirped-pulse millimeter-wave spectroscopy. Broadband, high-resolution spectra with accurate relative intensities are recorded instantly. Free induction decay (FID) of atoms, polarized by the chirped pulse, at their Rydberg-Rydberg transition frequencies, is heterodyne detected, averaged in the time domain, and Fourier transformed into the frequency domain. Millimeter-wave transient nutations are observed, and the possibility of FID evolving to superradiance is discussed.

A quantum defect model for the s, p, d, and f Rydberg series of CaF.

We present an improved quantum defect theory model for the "s," "p," "d," and "f" Rydberg series of CaF. The model, which is the result of an exhaustive fit of high-resolution spectroscopic data, parameterizes the electronic structure of the ten ("s"Σ, "p"Σ, "p"Π, "d"Σ, "d"Π, "d"Δ, "f"Σ, "f"Π, "f"Δ, and "f"Φ) Rydberg series of CaF in terms of a set of twenty µ(ll('))(Λ) quantum defect matrix elements and their dependence on both internuclear separation and on the binding energy of the outer electron. Over 1000 rovibronic Rydberg levels belonging to 131 observed electronic states of CaF with n∗ ≥ 5 are included in the fit. The correctness and physical validity of the fit model are assured both by our intuition-guided combinatorial fit strategy and by comparison with R-matrix calculations based on a one-electron effective potential. The power of this quantum defect model lies in its ability to account for the rovibronic energy level structure and nearly all dynamical processes, including structure and dynamics outside of the range of the current observations. Its completeness places CaF at a level of spectroscopic characterization similar to NO and H(2).

Chemical effects in the separation process of a differential mobility/mass spectrometer system.

In differential mobility spectrometry (also referred to as high-field asymmetric waveform ion mobility spectrometry), ions are separated on the basis of the difference in their mobility under high and low electric fields. The addition of polar modifiers to the gas transporting the ions through a differential mobility spectrometer enhances the formation of clusters in a field-dependent way and thus amplifies the high- and low-field mobility difference, resulting in increased peak capacity and separation power. Observations of the increase in mobility field dependence are consistent with a cluster formation model, also referred to as the dynamic cluster-decluster model. The uniqueness of chemical interactions that occur between an ion and cluster-forming neutrals increases the selectivity of the separation, and the depression of low-field mobility relative to high-field mobility increases the compensation voltage and peak capacity. The effect of a polar modifier on the peak capacity across a broad range of chemicals has been investigated. We discuss the theoretical underpinnings which explain the observed effects. In contrast to the result with a polar modifier, we find that using mixtures of inert gases as the transport gas improves the resolution by reducing the peak width but has very little effect on the peak capacity or selectivity. The inert gas helium does not cluster and thus does not reduce low-field mobility relative to high-field mobility. The observed changes in the differential mobility alpha parameter exhibited by different classes of compounds when the transport gas contains a polar modifier or has a significant fraction of inert gas can be explained on the basis of the physical mechanisms involved in the separation processes.

Planar differential mobility spectrometer as a pre-filter for atmospheric pressure ionization mass spectrometry.

Ion filters based on planar DMS can be integrated with the inlet configuration of most mass spectrometers, and are able to enhance the quality of mass analysis and quantitative accuracy by reducing chemical noise, and by pre-separating ions of similar mass. This paper is the first in a series of three papers describing the optimization of DMS / MS instrumentation. In this paper the important physical parameters of a planar DMS-MS interface including analyzer geometry, analyzer coupling to a mass spectrometer, and transport gas flow control are considered. The goal is to optimize ion transmission and transport efficiency, provide optimal and adjustable resolution, and produce stable operation under conditions of high sample contamination. We discuss the principles of DMS separations and highlight the theoretical underpinnings. The main differences between planar and cylindrical geometries are presented, including a discussion of the advantages and disadvantages of RF ion focusing. In addition, we present a description of optimization of the frequency and amplitude of the DMS fields for resolution and ion transmission, and a discussion of the influence and importance of ion residence time in DMS. We have constructed a mass spectrometer interface for planar geometries that takes advantage of atmospheric pressure gas dynamic principles, rather than ion focusing, to minimize ion losses from diffusion in the analyzer and to maximize total ion transport into the mass spectrometer. A variety of experimental results has been obtained that illustrate the performance of this type of interface, including tests of resistance to high contamination levels, and the separation of stereoisomers. In a subsequent publication the control of the chemical interactions that drive the separation process of a DMS / MS system will be considered. In a third publication we describe novel electronics designed to provide the high voltages asymmetric waveform fields (SV) required for these devices as well as the effects of different waveforms.

Detection of Radiation-Exposure Biomarkers by Differential Mobility Prefiltered Mass Spectrometry (DMS-MS).

Technology to enable rapid screening for radiation exposure has been identified as an important need, and, as a part of a NIH / NIAD effort in this direction, metabolomic biomarkers for radiation exposure have been identified in a recent series of papers. To reduce the time necessary to detect and measure these biomarkers, differential mobility spectrometry - mass spectrometry (DMS-MS) systems have been developed and tested. Differential mobility ion filters preselect specific ions and also suppress chemical noise created in typical atmospheric-pressure ionization sources (ESI, MALDI, and others). Differential-mobility-based ion selection is based on the field dependence of ion mobility, which, in turn, depends on ion characteristics that include conformation, charge distribution, molecular polarizability, and other properties, and on the transport gas composition which can be modified to enhance resolution. DMS-MS is able to resolve small-molecule biomarkers from nearly-isobaric interferences, and suppresses chemical noise generated in the ion source and in the mass spectrometer, improving selectivity and quantitative accuracy. Our planar DMS design is rapid, operating in a few milliseconds, and analyzes ions before fragmentation. Depending on MS inlet conditions, DMS-selected ions can be dissociated in the MS inlet expansion, before mass analysis, providing a capability similar to MS/MS with simpler instrumentation. This report presents selected DMS-MS experimental results, including resolution of complex test mixtures of isobaric compounds, separation of charge states, separation of isobaric biomarkers (citrate and isocitrate), and separation of nearly-isobaric biomarker anions in direct analysis of a bio-fluid sample from the radiation-treated group of a mouse-model study. These uses of DMS combined with moderate resolution MS instrumentation indicate the feasibility of field-deployable instrumentation for biomarker evaluation.

Control of chemical effects in the separation process of a differential mobility mass spectrometer system.

Differential mobility spectrometry (DMS) separates ions on the basis of the difference in their migration rates under high versus low electric fields. Several models describing the physical nature of this field mobility dependence have been proposed but emerging as a dominant effect is the clusterization model sometimes referred to as the dynamic cluster-decluster model. DMS resolution and peak capacity is strongly influenced by the addition of modifiers which results in the formation and dissociation of clusters. This process increases selectivity due to the unique chemical interactions that occur between an ion and neutral gas-phase molecules. It is thus imperative to bring the parameters influencing the chemical interactions under control and find ways to exploit them in order to improve the analytical utility of the device. In this paper, we describe three important areas that need consideration in order to stabilize and capitalize on the chemical processes that dominate a DMS separation. The first involves means of controlling the dynamic equilibrium of the clustering reactions with high concentrations of specific reagents. The second area involves a means to deal with the unwanted heterogeneous cluster ion populations emitted from the electrospray ionization process that degrade resolution and sensitivity. The third involves fine control of parameters that affect the fundamental collision processes, temperature and pressure.

The Use of DMS-MS for the Quantitative Analysis of Acylcarnitines.

Differential mobility spectrometry (DMS) is capable of separating molecules based on their size and shape. When coupled to mass spectrometry (MS), DMS reduces chemical background and enhances signal-to-noise (S/N) ratio. Flow injection analysis (FIA) is a technique used to introduce samples into the source of the DMS-MS platform. Here we describe the application of FIA-DMS-MS/MS for the analysis of urinary acylcarnitine species. More than 20 acylcarnitine species can be detected and quantified during a single FIA-DMS-MS/MS acquisition.

Quantitation of Cyclosporin A in Cell Culture Media by Differential Mobility Mass Spectrometry (DMS-MS/MS).

Cell permeability is an important factor in determining the bioavailability of therapeutics that is usually measured by cell culture testing. The concentration of pharmaceutical in a medium such as Hank's Balanced Salt Solution with HEPES organic buffer (HBSS-HEPES) is measured at a series of time points, making simplicity and high throughput of the analytical method important characteristics. We report an electrospray differential mobility spectrometry mass spectrometry method (nanoESI-DMS-MS) for the rapid determination of cyclosporin A (CsA, cyclosporine) concentration in such a buffer. DMS technology provides gas phase atmospheric pressure ion filtration for small-molecule bioanalytical methods that suppresses interfering ions and reduces chemical noise, without the use of chromatography. This allows simplified sample preparation, fast calibration curve development, and shortened analysis times. It has also been noted that the DMS prefilter can reduce contamination of the mass spectrometer by salts, thereby extending mass spectrometer system uptime.In the application described here, DMS-MS/MS is applied to cyclosporine A (CsA) in cell medium. Sample preparation is limited to dilution with an ammonium acetate-methanol-water mobile phase and the addition of CsA-d4 internal standard. The isotope ratio data are obtained in DMS-MS MRM mode observing NH3 loss from the ammonium adduct of the two species. A calibration curve with high linearity (R2 = 0.998) is rapidly obtained with nearly zero intercept, while it was found that a liquid chromatography LC-MS method required a preliminary SPE step to obtain a linear calibration curve. The time for data acquisition in the DMS-MS MRM method with flow injection (FIA) or infusion introduction at ESI flow of 400 nL/min is typically 30 s leading to a cycle time of less than 1 min.

Quantitation of Urinary Acylcarnitines by DMS-MS/MS Uncovers the Effects of Total Body Irradiation in Cancer Patients.

Acylcarnitines have been identified in human and animal metabolomic-profiling studies as urinary markers of radiation exposure, a result which is consistent with their cytoprotective effects and roles in energy metabolism. In the present work, a rapid method for quantitation of the more abundant acylcarnitines in human urine is developed using a valuable set of samples from cancer patients who received total body irradiation (TBI) at Memorial Sloan Kettering Cancer Center. The method uses solid-phase extraction (SPE) processing followed by differential mobility spectrometry (DMS with ethyl acetate modifier) tandem mass spectrometry (ESI-DMS-MS/MS) with deuterated internal standards. The analyzed human urine samples were collected from 38 individual patients at three time points over 24 h during and after the course of radiation treatment, a design allowing each patient to act as their own control and creatinine normalization. Creatinine-normalized concentrations for nine urinary acylcarnitine (acyl-CN) species are reported. Six acyl-CN species were reduced at the 6 h point. Acetylcarnitine (C2:0-CN) and valerylcarnitine (C5:0-CN) showed recovery at 24 h, but none of the other acyl-CN species showed recovery at that point. Levels of three acyl-CN species were not significantly altered by radiation. This rapid quantitative method for clinical samples covers the short- and medium-chain acylcarnitines and has the flexibility to be expanded to cover additional radiation-linked metabolites. The human data presented here indicates the utility of the current approach as a rapid, quantitative technique with potential applications by the medical community, by space research laboratories concerned with radiation exposure, and by disaster response groups.

The Stark effect in Rydberg states of a highly polar diatomic molecule: CaF.

The Stark effect in molecular Rydberg states is qualitatively different from the Stark effect in atomic Rydberg states because of the anisotropy of the ion core and the existence of rotational and vibrational degrees of freedom. These uniquely molecular features cause the electric-field-induced decoupling of the Rydberg electron from the body frame to proceed in several stages in a molecule. Because the transition dipole moment among the same-n* Rydberg states is much larger than the permanent dipole moment of the ion core, the decoupling of the Rydberg electron from the ion core proceeds gradually. In the first stage, analyzed in detail in this paper, l and N are mixed by the external electric field, while N+ is conserved. In the further stages, as the external electric field increases, N+, n*, and v+ are expected to undergo mixing. We have characterized these stages in n*=13, v+=1 states of CaF. The large permanent dipole moment of CaF+ makes CaF qualitatively different from the other molecules in which the Stark effect in Rydberg states has been described (H2, Na2, Li2, NO, and H3) and makes it an ideal testbed for documenting the competition between the external and CaF+ dipole electric fields. We use the weak-field Stark effect to gain access to the lowest-N rotational levels of f, g, and h states and to assign their actual or nominal N+ quantum number. Lowest-N rotational levels provide information needed to disentangle the short-range and long-range interactions between the Rydberg electron and the ion core. We diagonalize an effective Hamiltonian matrix to determine the l-characters of the 3 < or = l < or = 5 core-nonpenetrating 2Sigma+ states and to characterize their mixing with the core-penetrating states. We conclude that the mixing of the l=4, N-N+=-4(g(-4)) state with lower-l 2Sigma+ states is stronger than documented in our previous multichannel quantum defect theory and long-range fits to zero-field spectra.

Differential Mobility Spectrometry-Mass Spectrometry (DMS-MS) in Radiation Biodosimetry: Rapid and High-Throughput Quantitation of Multiple Radiation Biomarkers in Nonhuman Primate Urine.

High-throughput methods to assess radiation exposure are a priority due to concerns that include nuclear power accidents, the spread of nuclear weapon capability, and the risk of terrorist attacks. Metabolomics, the assessment of small molecules in an easily accessible sample, is the most recent method to be applied for the identification of biomarkers of the biological radiation response with a useful dose-response profile. Profiling for biomarker identification is frequently done using an LC-MS platform which has limited throughput due to the time-consuming nature of chromatography. We present here a chromatography-free simplified method for quantitative analysis of seven metabolites in urine with radiation dose-response using urine samples provided from the Pannkuk et al. (2015) study of long-term (7-day) radiation response in nonhuman primates (NHP). The stable isotope dilution (SID) analytical method consists of sample preparation by strong cation exchange-solid phase extraction (SCX-SPE) to remove interferences and concentrate the metabolites of interest, followed by differential mobility spectrometry (DMS) ion filtration to select the ion of interest and reduce chemical background, followed by mass spectrometry (overall SID-SPE-DMS-MS). Since no chromatography is used, calibration curves were prepared rapidly, in under 2 h (including SPE) for six simultaneously analyzed radiation biomarkers. The seventh, creatinine, was measured separately after 2500× dilution. Creatinine plays a dual role, measuring kidney glomerular filtration rate (GFR), and indicating kidney damage at high doses. The current quantitative method using SID-SPE-DMS-MS provides throughput which is 7.5 to 30 times higher than that of LC-MS and provides a path to pre-clinical radiation dose estimation. Graphical Abstract.

Differential mobility spectrometry (DMS) reveals the elevation of urinary acetylcarnitine in non-human primates (NHPs) exposed to radiation.

Acetylcarnitine has been identified as one of several urinary biomarkers indicative of radiation exposure in adult rhesus macaque monkeys (non-human primates, NHPs). Previous work has demonstrated an up-regulated dose-response profile in a balanced male/female NHP cohort. As a contribution toward the development of metabolomics-based radiation biodosimetry in human populations and other applications of acetylcarnitine screening, we have developed a quantitative, high-throughput method for the analysis of acetylcarnitine. We employed the Sciex SelexIon DMS-MS/MS QTRAP 5500 platform coupled to flow injection analysis (FIA), thereby allowing for fast analysis times of less than 0.5 minutes per injection with no chromatographic separation. Ethyl acetate is used as a DMS modifier to reduce matrix chemical background. We have measured NHP urinary acetylcarnitine from the male cohorts that were exposed to the following radiation levels: control, 2, 4, 6, 7, and 10 Gy. Biological variability, along with calibration accuracy of the FIA-DMS-MS/MS method, indicates LOQ of 20 µM, with observed biological levels on the order of 600 µM and control levels near 10 µM. There is an apparent onset of intensified response in the transition from 6 to 10 Gy. The results demonstrate that FIA-DMS-MS/MS is a rapid, quantitative technique that can be utilized for the analysis of urinary biomarker levels for radiation biodosimetry.

Rapid and High-Throughput Detection and Quantitation of Radiation Biomarkers in Human and Nonhuman Primates by Differential Mobility Spectrometry-Mass Spectrometry.

Radiation exposure is an important public health issue due to a range of accidental and intentional threats. Prompt and effective large-scale screening and appropriate use of medical countermeasures (MCM) to mitigate radiation injury requires rapid methods for determining the radiation dose. In a number of studies, metabolomics has identified small-molecule biomarkers responding to the radiation dose. Differential mobility spectrometry-mass spectrometry (DMS-MS) has been used for similar compounds for high-throughput small-molecule detection and quantitation. In this study, we show that DMS-MS can detect and quantify two radiation biomarkers, trimethyl-L-lysine (TML) and hypoxanthine. Hypoxanthine is a human and nonhuman primate (NHP) radiation biomarker and metabolic intermediate, whereas TML is a radiation biomarker in humans but not in NHP, which is involved in carnitine synthesis. They have been analyzed by DMS-MS from urine samples after a simple strong cation exchange-solid phase extraction (SCX-SPE). The dramatic suppression of background and chemical noise provided by DMS-MS results in an approximately 10-fold reduction in time, including sample pretreatment time, compared with liquid chromatography-mass spectrometry (LC-MS). DMS-MS quantitation accuracy has been verified by validation testing for each biomarker. Human samples are not yet available, but for hypoxanthine, selected NHP urine samples (pre- and 7-d-post 10 Gy exposure) were analyzed, resulting in a mean change in concentration essentially identical to that obtained by LC-MS (fold-change 2.76 versus 2.59). These results confirm the potential of DMS-MS for field or clinical first-level rapid screening for radiation exposure. Graphical Abstract ᅟ.