Particle interactions of fluticasone propionate and salmeterol xinafoate detected with single particle aerosol mass spectrometry (SPAMS).

Particle co-associations between the active pharmaceutical ingredients fluticasone propionate and salmeterol xinafoate were examined in dry powder inhaled (DPI) and metered dose inhaled (MDI) combination products. Single Particle Aerosol Mass Spectrometry was used to investigate the particle interactions in Advair Diskus® (500/50 mcg) and Seretide® (125/25 mcg). A simple rules tree was used to identify each compound, either alone or co-associated at the level of the individual particle, using unique marker peaks in the mass spectra for the identification of each drug. High levels of drug particle co-association (fluticasone-salmeterol) were observed in the aerosols emitted from Advair Diskus® and Seretide®. The majority of the detected salmeterol particles were found to be in co-association with fluticasone in both tested devices. Another significant finding was that rather coarse fluticasone particles (in DPI) and fine salmeterol particles (both MDI and DPI) were forming the particle co-associations.

Autonomous, broad-spectrum detection of hazardous aerosols in seconds.

Actual or surrogate chemical, biological, radiological, nuclear, and explosive materials and illicit drug precursors can be rapidly detected and identified when in aerosol form by a Single-Particle Aerosol Mass Spectrometry (SPAMS) system. This entails not only the sampling of such particles but also the physical analysis and subsequent data analysis leading to a highly reliable alarm state. SPAMS hardware is briefly reviewed. SPAMS software algorithms are discussed in greater detail. A laboratory experiment involving actual threat and surrogate releases mixed with ambient background aerosols demonstrates broad-spectrum detection within seconds. Data from a field test at the San Francisco International Airport demonstrate extended field operation with an ultralow false alarm rate. Together these data sets demonstrate a significant and important advance in rapid aerosol threat detection.

Single-particle aerosol mass spectrometry for the detection and identification of chemical warfare agent simulants.

Single-particle aerosol mass spectrometry (SPAMS) was used for the real-time detection of liquid nerve agent simulants. A total of 1000 dual-polarity time-of-flight mass spectra were obtained for micrometer-sized single particles each of dimethyl methyl phosphonate, diethyl ethyl phosphonate, diethyl phosphoramidate, and diethyl phthalate using laser fluences between 0.58 and 7.83 nJ/microm2, and mass spectral variation with laser fluence was studied. The mass spectra obtained allowed identification of single particles of the chemical warfare agent (CWA) simulants at each laser fluence used although lower laser fluences allowed more facile identification. SPAMS is presented as a promising real-time detection system for the presence of CWAs.

Online aerosol mass spectrometry of single micrometer-sized particles containing poly(ethylene glycol).

The analysis of poly(ethylene glycol) (PEG)-containing particles by online single particle aerosol mass spectrometers equipped with laser desorption/ionization (LDI) is reported. We demonstrate that PEG-containing particles are useful in the development of aerosol mass spectrometers because of their ease of preparation, low cost, and inherently recognizable mass spectra. Solutions containing millimolar quantities of PEGs were nebulized and, after drying, the resultant micrometer-sized PEG-containing particles were sampled. LDI (266 nm) of particles containing NaCl and PEG molecules of average molecular weight<500 Da generated mass spectra reminiscent of mass spectra of PEG collected by other mass spectrometer platforms including the characteristic distribution of positive ions (Na+ adducts) separated by the 44 m/z units of the ethylene oxide units separating each degree of polymerization. PEGs of average molecular weight>500 Da were detected from particles that also contained the tripeptide tyrosine-tyrosine-tyrosine or 2,5-dihydroxybenzoic acid, which were added to nebulized solutions to act as matrices to assist LDI using pulsed 266 nm and 355 nm lasers, respectively. Experiments were performed on two aerosol mass spectrometers, one reflectron and one linear, that each utilize two time-of-flight mass analyzers to detect positive and negative ions created from a single particle. PEG-containing particles are currently being employed in the optimization of our bioaerosol mass spectrometers for the application of measurements of complex biological samples, including human effluents, and we recommend that the same strategies will be of great utility to the development of any online aerosol LDI mass spectrometer platform.

Identification of high explosives using single-particle aerosol mass spectrometry.

The application of single-particle aerosol mass spectrometry (SPAMS) to the real-time detection of micrometer-sized single particles of high explosives is described. Dual-polarity time-of-flight mass spectra from 1000 single particles each of 2,4,6-trinitrotoluene (TNT), 1,3,5-trinitro-1,3,5-triazinane (RDX), and pentaerythritol tetranitrate (PETN), as well as those of complex explosives, Composition B, Semtex 1A, and Semtex 1H, were obtained over a range of desorption/ionization laser fluences between 0.50 and 8.01 nJ/microm2. Mass spectral variability with laser fluence for each explosive is discussed. The ability of the SPAMS system to identify explosive components in a single complex explosive particle ( approximately 1 pg) without the need for consumables is demonstrated.

Following the biochemical and morphological changes of Bacillus atrophaeus cells during the sporulation process using Bioaerosol Mass Spectrometry.

Bioaerosol Mass Spectrometry (BAMS), a real-time single cell analytical technique, was used to follow the biochemical and morphological changes within a group of Bacillus atrophaeus cells by measuring individual cells during the process of sporulation. A mutant of B. atrophaeus that lacks the ability to produce dipicolinic acid (DPA) was also analyzed. Single cell aerodynamic sizing was used to follow gross morphological changes, and chemical analysis of single cells by mass spectrometry was used to follow some biochemical changes of B. atrophaeus cells during endospore formation.

Desorption/ionization fluence thresholds and improved mass spectral consistency measured using a flattop laser profile in the bioaerosol mass spectrometry of single Bacillus endospores.

Bioaerosol mass spectrometry is being developed to analyze and identify biological aerosols in real time. Mass spectra of individual Bacillus endospores were measured with a bipolar aerosol time-of-flight mass spectrometer in which molecular desorption and ionization were produced using a single laser pulse from a Q-switched, frequency-quadrupled Nd:YAG laser that was modified to have an approximately flattop profile. The flattened laser profile allowed the minimum fluence required to desorb and ionize significant numbers of ions from single aerosol particles to be determined. For Bacillus spores, this threshold had a mean value of approximately 1 nJ/microm(2) (0.1 J/cm(2)). Thresholds for individual spores, however, could apparently deviate by 20% or more from the mean. Threshold distributions for clumps of MS2 bacteriophage and bovine serum albumin were subsequently determined. Finally, the flattened profile was observed to increase the reproducibility of single-spore mass spectra. This is consistent with the general conclusions of our earlier paper on the fluence dependence of single-spore mass spectra and is particularly significant because it is expected to enable more robust differentiation and identification of single bioaerosol particles.

Bioaerosol mass spectrometry for rapid detection of individual airborne Mycobacterium tuberculosis H37Ra particles.

Single-particle laser desorption/ionization time-of-flight mass spectrometry, in the form of bioaerosol mass spectrometry (BAMS), was evaluated as a rapid detector for individual airborne, micron-sized, Mycobacterium tuberculosis H37Ra particles, comprised of a single cell or a small number of clumped cells. The BAMS mass spectral signatures for aerosolized M. tuberculosis H37Ra particles were found to be distinct from M. smegmatis, Bacillus atrophaeus, and B. cereus particles, using a distinct biomarker. This is the first time a potentially unique biomarker was measured in M. tuberculosis H37Ra on a single-cell level. In addition, M. tuberculosis H37Ra and M. smegmatis were aerosolized into a bioaerosol chamber and were sampled and analyzed using BAMS, an aerodynamic particle sizer, a viable Anderson six-stage sampler, and filter cassette samplers that permitted direct counts of cells. In a background-free environment, BAMS was able to sample and detect M. tuberculosis H37Ra at airborne concentrations of >1 M. tuberculosis H37Ra-containing particles/liter of air in 20 min as determined by direct counts of filter cassette-sampled particles, and concentrations of >40 M. tuberculosis H37Ra CFU/liter of air in 1 min as determined by using viable Andersen six-stage samplers. This is a first step toward the development of a rapid, stand-alone airborne M. tuberculosis particle detector for the direct detection of M. tuberculosis bioaerosols generated by an infectious patient. Additional instrumental development is currently under way to make BAMS useful in realistic environmental and respiratory particle backgrounds expected in tuberculosis diagnostic scenarios.

Comprehensive assignment of mass spectral signatures from individual Bacillus atrophaeus spores in matrix-free laser desorption/ionization bioaerosol mass spectrometry.

We have fully characterized the mass spectral signatures of individual Bacillus atrophaeus spores obtained using matrix-free laser desorption/ionization bioaerosol mass spectrometry (BAMS). Mass spectra of spores grown in unlabeled, 13C-labeled, and 15N-labeled growth media were used to determine the number of carbon and nitrogen atoms associated with each mass peak observed in mass spectra from positive and negative ions. To determine the parent ion structure associated with fragment ion peaks, the fragmentation patterns of several chemical standards were independently determined. Our results confirm prior assignments of dipicolinic acid, amino acids, and calcium complex ions made in the spore mass spectra. The identities of several previously unidentified mass peaks, key to the recognition of Bacillus spores by BAMS, have also been revealed. Specifically, a set of fragment peaks in the negative polarity is shown to be consistent with the fragmentation pattern of purine nucleobase-containing compounds. The identity of m/z = +74, a marker peak that helps discriminate B. atrophaeus from Bacillus thuringiensis spores grown in rich media is [N1C4H12]+. A probable precursor molecule for the [N1C4H12]+ ion observed in spore spectra is trimethylglycine (+N(CH3)3CH2COOH), which produces a m/z = +74 peak when ionized in the presence of dipicolinic acid. A clear assignment of all the mass peaks in the spectra from bacterial spores, as presented in this work, establishes their relationship to the spore chemical composition and facilitates the evaluation of the robustness of "marker" peaks. This is especially relevant for peaks that have been used to discriminate Bacillus spore species, B. thuringiensis and B. atrophaeus, in our previous studies.

Stable isotope labeling of entire Bacillus atrophaeus spores and vegetative cells using bioaerosol mass spectrometry.

Single vegetative cells and spores of Bacillus atrophaeus, formerly Bacillus subtilis var. niger, were analyzed using bioaerosol mass spectrometry. Key biomarkers were identified from organisms grown in 13C and 15N isotopically enriched media. Spore spectra contain peaks from dicipolinate and amino acids. The results indicate that compounds observed in the spectra correspond to material from the spore's core and not the exosporium. Standard compounds and mixtures were analyzed for comparison. The biomarkers for vegetative cells were clearly different from those of the spores, consisting mainly of phosphate clusters and amino acid fragments.

Toward understanding the ionization of biomarkers from micrometer particles by bio-aerosol mass spectrometry.

The appearance of informative signals in the mass spectra of laser-ablated bio-aerosol particles depends on the effective ionization probabilities (EIP) of individual components during the laser ionization process. This study investigates how bio-aerosol chemical composition governs the EIP values of specific components and the overall features of the spectra from the bio-aerosol mass spectrometry (BAMS). EIP values were determined for a series of amino acid, dipicolinic acid, and peptide aerosol particles to determine what chemical features aid in ionization. The spectra of individual amino acids and dipicolinic acid, as well as mixtures, were examined for extent of fragmentation and the presence of molecular ion dimers, which are indicative of ionization conditions. Standard mixtures yielded information with respect to the significance of secondary ion plume reactions on observed spectra. A greater understanding of how these parameters affect EIP and spectra characteristics of bio-aerosols will aid in the intelligent selection of viable future biomarkers for the identification of bio-terrorism agents.

Reagentless detection and classification of individual bioaerosol particles in seconds.

The rapid chemical analysis of individual cells is an analytical capability that will profoundly impact many fields including bioaerosol detection for biodefense and cellular diagnostics for clinical medicine. This article describes a mass spectrometry-based analytical technique for the real-time and reagentless characterization of individual airborne cells without sample preparation. We characterize the mass spectral signature of individual Bacillus spores and demonstrate the ability to distinguish two Bacillus spore species, B. thuringiensis and B.atrophaeus, from one another very accurately and from the other biological and nonbiological background materials tested with no false positives at a sensitivity of 92%. This example demonstrates that the chemical differences between these two Bacillus spore species are consistently and easily detected within single cells in seconds.

Laser power dependence of mass spectral signatures from individual bacterial spores in bioaerosol mass spectrometry.

Bioaerosol mass spectrometry is being developed to analyze and identify biological aerosols in real time. Characteristic mass spectra from individual bacterial endospores of Bacillus subtilis var. niger were obtained in a bipolar aerosol time-of-flight mass spectrometer using a pulsed 266-nm laser for molecular desorption and ionization. Spectra from single spores collected at an average fluence of approximately 0.1 J/cm2 frequently contain prominent peaks attributed to arginine, dipicolinic acid, and glutamic acid, but the shot-to-shot (spore-to-spore) variability in the data may make it difficult to consistently distinguish closely related Bacillus species with an automated routine. Fortunately, a study of the laser power dependence of the mass spectra reveals clear trends and a finite number of "spectral types" that span most of the variability. This, we will show, indicates that a significant fraction of the variability must be attributed to fluence variations in the profile of the laser beam.

A field-based approach for deterimining ATOFMS instrument sensitities to ammonium and nitrate.

Aerosol time-of-flight mass spectrometry (ATOFMS) instruments measure the size and chemical composition of individual particles in real-time. ATOFMS chemical composition measurements are difficult to quantify, largely because the instrument sensitivities to different chemical species in mixed ambient aerosols are unknown. In this paper, we develop a field-based approach for determining ATOFMS instrument sensitivities to ammonium and nitrate in size-segregated atmospheric aerosols, using tandem ATOFMS-impactor sampling. ATOFMS measurements are compared with collocated impactor measurements taken at Riverside, CA, in September 1996, August 1997, and October 1997. This is the first comparison of ion signal intensities from a single-particle instrument with quantitative measurements of atmospheric aerosol chemical composition. The comparison reveals that ATOFMS instrument sensitvities to both NH4+ and NO3- decline with increasing particle aerodynamic diameter over a 0.32-1.8 microm calibration range. The stability of this particle size dependence is tested overthe broad range of fine particle concentrations (PM1.8) = 17.6 +/- 2.0-127.8 +/- 1.8 microg m(-3)), ambient temperatures (23-35 degrees C), and relative humidity conditions (21-69%), encountered during the field experiments. This paper describes a potentially generalizable methodology for increasing the temporal and size resolution of atmospheric aerosol chemical composition measurements, using tandem ATOFMS-impactor sampling.