Use of single particle aerosol mass spectrometry for the automated nondestructive identification of drugs in multicomponent samples.

In this work, single particle aerosol mass spectrometry (SPAMS) was used to identify the active drug ingredients in samples of multicomponent over-the-counter (OTC) drug tablets with minimal damage to the tablets. OTC drug tablets in various formulations were analyzed including single active ingredient tablets and multi-ingredient tablets. Using a sampling apparatus developed in-house, micrometer-sized particles were simultaneously dislodged from tablets and introduced to the SPAMS, where dual-polarity mass spectra were obtained from individual particles. Active ingredients were identified from the parent ions and fragment ions formed from each sample, and alarm files were developed for each active ingredient, allowing successful automated identification of each compound in a mixture. The alarm algorithm developed for SPAMS correctly identified all drug compounds in all single-ingredient and multi-ingredient tablets studied. A further study demonstrated the ability of this technique to identify the active ingredient in a single tablet analyzed in the presence of several other nonidentical tablets. In situ measurements were also made by sampling directly from a drug sample in its original bottle. A single tablet embedded in 11 identical tablets of different composition was detected in this manner. Overall, this work demonstrates the ability of the SPAMS technique to detect a target drug compound both in complex tablets, i.e., multidrug ingredient tablets, and complex sampling environments, i.e., multitablet sampling sources. The technique is practically nondestructive, leaving the characteristic shape, color, and imprint of a tablet intact for further analysis. Applications of this technique may include forensic and pharmaceutical analysis.

Reagentless detection of Mycobacteria tuberculosis H37Ra in respiratory effluents in minutes.

Two similar mycobacteria, Mycobacteria tuberculosis H37Ra and Mycobacteria smegmatis are rapidly detected and identified within samples containing a complex background of respiratory effluents using single-particle aerosol mass spectrometry (SPAMS). M. tuberculosis H37Ra (TBa), an avirulent strain, is used as a surrogate for virulent tuberculosis; M. smegmatis (MSm) is utilized as a near-neighbor confounder for TBa. Bovine lung surfactant and human exhaled breath condensate are used as first-order surrogates for infected human lung expirations from patients with pulmonary tuberculosis. This simulated background sputum is mixed with TBa or MSm and nebulized to produce conglomerate aerosol particles, single particles that contain a bacterium embedded within a background respiratory matrix. Mass spectra of single conglomerate particles exhibit ions associated with both respiratory effluents and mycobacteria. Spectral features distinguishing TBa from MSm in pure and conglomerate particles are shown. SPAMS pattern matching alarm algorithms are able to distinguish TBa-containing particles from background matrix and MSm for >50% of the test particles, which is sufficient to enable a high probability of detection and a low false alarm rate if an adequate number of such particles are present. These results indicate the potential usefulness of SPAMS for rapid, reagentless tuberculosis screening.

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.

Parameters contributing to efficient ion generation in aerosol MALDI mass spectrometry.

The Bioaerosol Mass Spectrometry (BAMS) system was developed for the real-time detection and identification of biological aerosols using laser desorption ionization. Greater differentiation of particle types is desired; consequently MALDI techniques are being investigated. The small sample size ( approximately 1 microm3), lack of substrate, and ability to simultaneously monitor both positive and negative ions provide a unique opportunity to gain new insight into the MALDI process. Several parameters known to influence MALDI molecular ion yield and formation are investigated here in the single particle phase. A comparative study of five matrices (2,6-dihydroxyacetophenone, 2,5-dihydroxybenzoic acid, alpha-cyano-4-hydroxycinnamic acid, ferulic acid, and sinapinic acid) with a single analyte (angiotensin I) is presented and reveals effects of matrix selection, matrix-to-analyte molar ratio, and aerosol particle diameter. The strongest analyte ion signal is found at a matrix-to-analyte molar ratio of 100:1. At this ratio, the matrices yielding the least and greatest analyte molecular ion formation are ferulic acid and alpha-cyano-4-hydroxycinnamic acid, respectively. Additionally, a significant positive correlation is found between aerodynamic particle diameter and analyte molecular ion yield for all matrices. SEM imaging of select aerosol particle types reveals interesting surface morphology and structure.

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.

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.

Improved sensitivity and mass range in time-of-flight bioaerosol mass spectrometry using an electrostatic ion guide.

Bioearosol mass spectrometry (BAMS) analyzes single particles in real time from ambient air, placing strict demands on instrument sensitivity. Modeling of the BAMS reflectron time of flight (TOF) with SIMION revealed design limitations associated with ion transmission and instrument sensitivity at higher masses. Design and implementation of a BAMS linear TOF with electrostatic ion guide and delayed extraction capabilities has greatly increased the sensitivity and mass range relative to the reflectron design. Initial experimental assessment of the new instrument design revealed improved sensitivity at high masses as illustrated when using standard particles of cytochrome C (m/z approximately 12,000), from which the compound's monomer, dimer (m/z approximately 24,000) and trimer (m/z approximately 36,000) were readily detected.

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.

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.