Two key bottlenecks in pharmaceutical bioanalysis are sample cleanup and chromatographic separations. Although multiple approaches have been developed in the past decade to either shorten or multiplex these steps, they remain the rate limiting steps as ADME (Absorption, Distribution, Metabolism, and Excretion) property screening is being routinely incorporated into the drug discovery process. In this work, a novel system incorporating an automated Direct Analysis in Real Time (DART) ionization source coupled with a triple-quadrupole mass spectrometer has been developed and evaluated for quantitative bioanalysis. This system has the capability of directly analyzing samples from their biological matrixes and therefore potentially eliminating the need for sample cleanup and chromatographic separations. A LEAP Technologies autosampler was customized to perform the automated sample introduction into the DART beam with high precision, which significantly improved the reproducibility of the method. Additional pumping was applied to the atmospheric pressure inlet on the mass spectrometer to compensate for the increased vacuum load because of the use of high-flow helium by the DART. This resulted in an improvement of detection sensitivity by a factor of 10 to 100 times. Matrix effects for a diversified class of compounds were evaluated directly from untreated raw plasma and were found to range from approximately 0.05 to 0.7. Precision and accuracy were also tested for multiple test compounds over a dynamic range of four orders of magnitude. The system has been used to analyze biological samples from both in vivo pharmacokinetic studies and in vitro microsomal/S9 stability studies, and the results generated were similar to those obtained with conventional LC/MS/MS methods. Overall, this new automated DART-triple quadrupole mass spectrometer system has demonstrated significant potential for high-throughput bioanalysis.

Direct analysis in real time (DART) is a novel ionization technique that provides for the rapid ionization of small molecules under ambient conditions. In this study, several commercially available drugs as well as actual compounds from drug discovery research were examined by LC/UV/ESI-MS and DART interfaced to a quadrupole mass spectrometer. For most compounds, the molecular ions observed by ESI-MS were observed by DART/MS. DART/MS was also studied as a means to quickly monitor synthetic organic reactions and to obtain nearly instantaneous molecular weight confirmations of final products in drug discovery. For simple, synthetic organic transformations, the trends in the intensities of the mass spectral signals for the reactant and product obtained by DART/MS scaled closely with those of the diode array or the total ion chromatogram obtained by LC/UV/ESI-MS. In summary, DART is a new tool that complements electrospray ionization for the rapid ionization and subsequent mass spectral analysis of compounds in drug discovery.

Nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) are the two most commonly used analytical tools in metabolomics, and their complementary nature makes the combination particularly attractive. A combined analytical approach can improve the potential for providing reliable methods to detect metabolic profile alterations in biofluids or tissues caused by disease, toxicity, etc. In this paper, 1 H NMR spectroscopy and direct analysis in real time (DART)-MS were used for the metabolomics analysis of serum samples from breast cancer patients and healthy controls. Principal component analysis (PCA) of the NMR data showed that the first principal component (PC1) scores could be used to separate cancer from normal samples. However, no such obvious clustering could be observed in the PCA score plot of DART-MS data, even though DART-MS can provide a rich and informative metabolic profile. Using a modified multivariate statistical approach, the DART-MS data were then reevaluated by orthogonal signal correction (OSC) pretreated partial least squares (PLS), in which the Y matrix in the regression was set to the PC1 score values from the NMR data analysis. This approach, and a similar one using the first latent variable from PLS-DA of the NMR data resulted in a significant improvement of the separation between the disease samples and normals, and a metabolic profile related to breast cancer could be extracted from DART-MS. The new approach allows the disease classification to be expressed on a continuum as opposed to a binary scale and thus better represents the disease and healthy classifications. An improved metabolic profile obtained by combining MS and NMR by this approach may be useful to achieve more accurate disease detection and gain more insight regarding disease mechanisms and biology.

Over the last decade, direct analysis in real time (DART) has emerged as a viable method for fast, easy, and reliable "ambient ionization" for forensic analysis. The ability of DART to generate ions from chemicals that might be present at the scene of a criminal activity, whether they are in the gas, liquid, or solid phase, with limited sample preparation has made the technology a useful analytical tool in numerous forensic applications. This review paper summarizes many of those applications, ranging from the analysis of trace evidence to security applications, with a focus on providing the forensic scientist with a resource for developing their own applications. The most common uses for DART in forensics are in studying seized drugs, drugs of abuse and their metabolites, bulk and detonated explosives, toxic chemicals, chemical warfare agents, inks and dyes, and commercial plant and animal products that have been adulterated for economic gain. This review is meant to complement recent reviews that have described the fundamentals of the ionization mechanism and the general use of DART. We describe a wide range of forensic applications beyond the field of analyzing drugs of abuse, which dominates the literature, including common experimental and data analysis methods. © 2016 Wiley Periodicals, Inc. Mass Spec Rev 37:171-187, 2018.

Accurate mass measurements were obtained for a number of compounds with masses In the 500-16 000 u range analyzed with a double-focusing magnetic-sector mass spectrometer equipped with an electrospray Ionization (ESI) source. Mass accuracies In the low-ppm range were obtained for positive and negative Ions by using either magnetic field scans or acderating voltage scan methods. These mass measurements were sufficiently accurate to permit the determination of elemental compositions for smaler molecules, and they provide confidence In the mass assignments for larger biomolecules such as hen egg-white lysozyme and equine myoglobin. The utility of poly(ethylene oxide) and polypropylene oxide) as posltlve-km reference standards and poly(ethylene oxide), poly(propylene oxide), and poly (propylene oxide) sulfates as negative-ton reference standards Is demonstrated, and factors affecting accurate mass measurements for large molecules are discussed. The ESI mass spectrum of the lysozyme [M + 9Hf+ species measured at a resolution of 10 000 permits the determination of the masses of Individual Isotopes to within 0.15 u (12 ppm) of the theoretical value, and a peak assigned to water toss Is observed.