Kevin G. Owens

Electrospray sample preparation for improved quantitation in matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

The goal of this work is the development of a rapid and objective matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) method for the quantitation of peptides and proteins in human plasma suitable for use in the Good Laboratory Practices (GLP) environment, where the analytical method, validation and pharmacokinetic parameters derived from concentration data will be scrutinized by global regulatory agencies. Electrospray deposition has traditionally been used to prepare thin, uniform samples for a number of techniques, including Cf-252 plasma desorption and secondary ion mass spectrometry. Here the electrospray process of sample application is used to reduce the segregation of analyte from matrix during the sample drying step. The small droplets formed during the electrospray process are found to significantly improve the homogeneity of the sample surface prepared. Experiments comparing the traditional air dried and electrosprayed methods of sample preparation show that the increase in sample homogeneity from electrosprayed samples decreases both the within-sample spot and between-sample spot variability, resulting in a decrease in percent coefficient of variation (%CV) for the recorded MALDI mass spectra. The increase in sample homogeneity permits a more objective use of MALDI-TOFMS as a quantitative analytical method and has led to the development of an assay for the determination of desamino-[8-D-arginine] vasopressin (DDAVP) using arginine vasopressin (AVP) as internal standard in human plasma. The range of quantitation observed (2.0-10 micrograms/mL) is of limited value for bioanalytical application; however, the analysis of neat standards shows lower quantitation limits are easily achieved.

p‐fluorotoluene. I. Methyl (CH3 and CD3) internal rotation in the S1 and S0 states

Supersonic jet S1‐S0 spectroscopy (resonance‐enhanced multiphoton ionization, fluorescence excitation, and dispersed single vibronic level fluorescence) has been used to determine the S1 and S0 internal rotation energy level structure of p‐fluorotoluene with a CD3 methyl rotor as well as to extend observations of the CH3 rotor structure. The observed rotor energy levels 2≤m≤8 for both species in both states are fit by a simple sixfold hindered rotor Hamiltonian for which the rotor inertial constants B and the internal rotation potential energy barriers V6 are evaluated. V6 may be obtained independently from B by observations of ΔE3, the observed splitting of the 3a‘1 and 3a‘2 rotor levels. Numerical solution of the wave equation shows that the perturbation theory relationship V6=−2ΔE3 holds well for any reasonable B value. Correspondingly, the B constant may be obtained from other level energies without appreciable sensitivity to (reasonably) assumed barrier heights. Earlier microwave and S1‐S0 fluorescen...

Investigations of matrix-assisted laser desorption/ionization sample preparation by time-of-flight secondary ion mass spectrometry

Matrix-assisted laser desorption/ionization (MALDI) and time-of-flight secondary ion mass spectrometry (ToF-SIMS) analyses are compared to gain insight into some of the details of sample preparation for MALDI analysis of synthetic polymers. ToF-SIMS imaging of MALDI samples shows segregation of the cationization agent from the matrix crystals. The amount of observed segregation can be controlled by the sample preparation technique. Electrospray sample deposition minimizes segregation. Comparing ToF-SIMS and MALDI mass spectra from the same samples confirms that ToF-SIMS is significantly more surface sensitive than MALDI. This comparison shows that segregation of the oligomers of a polymer sample can occur during MALDI sample preparation. Our data indicate that MALDI is not as sensitive to those species dominating the sample surface as to species better incorporated into the matrix crystals. Finally, we show that matrix-enhanced SIMS can be an effective tool to analyze synthetic polymers, although the sample preparation conditions may be different than those optimized for MALDI.

Silver cluster interferences in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry of nonpolar polymers

Potential difficulties associated with background silver salt clusters during matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) of nonpolar polymers are reported. Silver salt cluster ions were observed from m/z 1500 to 7000 when acidic, polar matrices, such as 2,5-dihydroxybenzoic acid (DHB), all-trans-retinoic acid (RTA) or 2-(4-hydroxyphenylazo)benzoic acid (HABA), were used for the analysis of nonpolar polymers. These background signals could be greatly reduced or eliminated by the use of nonpolar matrices such as anthracene or pyrene. Representative examples of these background interferences are demonstrated during the analysis of low molecular weight nonpolar polymers including polybutadiene and polystyrene. Nonpolar polymers analyzed with acidic, polar matrices (e.g., RTA) and silver cationization reagents can yield lower quality mass spectral results when interferences due to silver clusters are present. Replacing the polar matrices with nonpolar matrices or the silver salts with copper salts substantially improved the quality of the analytical results. In addition, it was found that silver contamination cannot be completely removed from standard stainless steel sample plates, although the presence of silver contamination was greatly reduced after thorough cleaning of the sample plate with aluminum oxide grit. Carry-over silver may cationize polymer samples and complicate the interpretation of data obtained using nonpolar matrices in the absence of added cationization reagents.

Methods for the Analysis of Hydrocarbon Polymers by Matrix‐assisted Laser Desorption/Ionization Time‐of‐flight Mass Spectrometry

With the addition of silver acetoacetonate to matrices of trans-3-indoleacrylic acid or 1,4-di-(2-(5-phenyloxazolyl))benzene, methods have been developed for the analysis of hydrocarbon polymers by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Silver-cationized molecular ions were produced for polymers of styrene, butadiene and isoprene up to mass 125 000 Da. For lower-mass styrene polymers, the resolved oligomer molecular ions provide information concerning the end group. Average molecular weights determined by MALDI-TOF are in good agreement with values from gel-permeation chromatography.

Application of correlation analysis techniques to mass spectral data

Abstract The power of mass spectrometry as an analytical technique lies in the ability of the analyst to deduce the molecular structure of the compound under study from the data collected. Correlation analysis techniques (from information theory) can be employed to enhance the quantitative information present in the spectrum, as well as aid the analyst in the interpretation of the mass spectral data.

Updating Evidence for Cationization of Polymers in the Gas Phase during Matrix-Assisted Laser Desorption/Ionization

Matrix-assisted laser desorption/ionization (MALDI) techniques have been developed to determine the chemical structure of a variety of industrial polymers. Despite the enormous popularity and power of MALDI, the details of the cationization mechanisms of the process are currently rather poorly understood. The MALDI cationization of polymer analytes was investigated previously by Hoberg and co-workers.1 They used layered samples to explore the role of gas-phase cationization in MALDI of polymers. This paper seeks to extend the work initiated by Hoberg and co-workers and update the results of the earlier work. The new experiments take advantage of a MALDI instrument with delayed extraction and show that separation in the initial acceleration region of the mass spectrometer is not a key component of cationization. Investigations of tri-layer samples by time-of-flight secondary ion mass spectrometry (ToF-SIMS) also show that there is considerable interaction among the layers. These interactions, specifically the solubility of the salts in the solvents, account for the presence of the bottom layer cation in the mass spectra. The absence of the top layer cation in the mass spectra is due to the inability of the polymer oligomers to penetrate that layer during desorption.

Intensity-dependent laser ionization experiments involving the 1b1u state of benzene

Abstract Benzene is two-step photoionized with ArF laser radiation and the resulting mass- and photoelectron spectra obtained versus laser intensity. In agreement with previous work, benzene ionizes very inefficiently at this wavelength. Attempts to probe the rapid non-radiative relaxation of its 1B1u excited state by monitoring the intensity dependence of the photoelectron spectrum are impeded by ionization of neutral atomic carbon fragments.

Development of a Combined Standard Additions/Internal Standards Method to Quantify Residual PEG in Ethoxylated Surfactants by MALDI TOFMS

Measuring the residual polyethylene glycol (PEG) in polyethylene oxide (PEO)-based surfactants is important to fully understanding the performance of these materials. Traditional methods of quantitating PEG in PEO-based surfactants can be time-consuming and struggle with low amounts or overlapping molecular mass distributions. This paper describes a matrix-assisted laser desorption/ionization (MALDI) mass spectrometry method developed to quantitate residual PEG in a series of ethoxylated surfactants. The technique addresses the difficulties faced in doing quantitative MALDI experiments by utilizing both internal standard and standard additions protocols. The method produces excellent straight line standard addition plots, and the quantitative results are verified using both a constructed standard and an independent traditional chromatographic separation.

Atmospheric pressure matrix-assisted laser desorption/ionisation ion trap mass spectrometry of synthetic polymers: a comparison with vacuum matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry

Atmospheric pressure matrix-assisted laser desorption/ionisation quadrupole ion trap (AP-MALDI/QIT) mass spectrometry has been investigated for the analysis of polyethylene glycol (PEG 1500) and a hyperbranched polymer (polyglycidol) in the presence of alkali–metal salts. Mass spectra of PEG 1500 obtained at atmospheric pressure showed dimetallated matrix/analyte adducts, in addition to the expected alkali–metal/PEG ions, for all matrix/alkali–metal salt combinations. The relative intensities of the desorbed ions were dependent on the matrix, the alkali–metal salt added to aid cationisation and the ion trap interface conditions [capillary temperature, in-source collisionally-induced dissociation (CID)]. These data indicate that the adducts are rapidly stabilised by collisional cooling enabling them to be transferred into the ion trap. Experiments using identical sample preparation conditions were carried out on a vacuum MALDI time-of-flight (ToF) mass spectrometer. In all cases, vacuum MALDI-ToF spectra showed only alkali–metal/PEG ions and no matrix/analyte adducts. The tandem mass spectrometry (MS/MS) capability of the ion trap has been demonstrated for a lithiated polyglycol yielding a rich fragment-ion spectrum. Analysis of the hyperbranched polymer polyglycidol by AP-MALDI/QIT reveals the characteristic ion series for these polymers as also observed under vacuum MALDI-ToF conditions.