In-source Collision-induced Dissociation Research Articles

Development of a LC-QTOF-MS based dilute-and-shoot approach for the botanical discrimination of honeys

Honeys of particular botanical origins can be associated with premium market prices, a trait which also makes them susceptible to fraud. Currently available authenticity testing methods for botanical classification of honeys are either time-consuming or only target a few “known” types of markers. Simple and effective methods are therefore needed to monitor and guarantee the authenticity of honey. In this study, a ‘dilute-and-shoot’ approach using liquid chromatography (LC) coupled to quadrupole time-of-flight-mass spectrometry (QTOF-MS) was applied to the non-targeted fingerprinting of honeys of different floral origin (buckwheat, clover and blueberry). This work investigated for the first time the impact of different instrumental conditions such as the column type, the mobile phase composition, the chromatographic gradient, and the MS fragmentor voltage (in-source collision-induced dissociation) on the botanical classification of honeys as well as the data quality. Results indicated that the data sets obtained for the various LC-QTOF-MS conditions tested were all suitable to discriminate the three honeys of different floral origin regardless of the mathematical model applied (random forest, partial least squares-discriminant analysis, soft independent modelling by class analogy and linear discriminant analysis). The present study investigated different LC-QTOF-MS conditions in a “dilute and shoot” method for honey analysis, in order to establish a relatively fast, simple and reliable analytical method to record the chemical fingerprints of honey. This approach is suitable for marker discovery and will be used for the future development of advanced predictive models for honey botanical origin.

Two-Dimensional Tandem Mass Spectrometry for Biopolymer Structural Analysis.

Biopolymer analysis, including proteomics and glycomics, relies heavily on the use of mass spectrometry for structural elucidation, including sequence determination. Novel methods to improve sample workup, instrument performance, and data analysis continue to be developed to address shortcomings associated with sample preparation, analysis time, data quality, and data interpretation. Here, we present a new method that couples in-source collision-induced dissociation (IS-CID) with two-dimensional tandem mass spectrometry (2D MS/MS) as a way to simplify proteomics and glycomics workflows while also providing additional insight into analyte structures over traditional MS/MS experiments. Specifically, IS-CID is employed as a gas-phase digestion method, i.e., to break down intact full-length polysaccharide or peptide ions prior to mass analysis. The resulting mixtures of oligomeric ions are analyzed by 2D-MS/MS, a technique that allows association of product ions with their precursor ions without isolation of the latter. A novel data analysis strategy is introduced to leverage the second dimension of 2D MS/MS spectra, in which stairstep patterns, representing outputs of a molecule's MSn scans, are extracted for structural interconnectivity information on the oligomer. The results demonstrate the potential applicability of 2D MS/MS strategies to the modern omics workflow and structural analysis of various classes of biopolymers.

Two‐Dimensional Tandem Mass Spectrometry for Biopolymer Structural Analysis

AbstractBiopolymer analysis, including proteomics and glycomics, relies heavily on the use of mass spectrometry for structural elucidation, including sequence determination. Novel methods to improve sample workup, instrument performance, and data analysis continue to be developed to address shortcomings associated with sample preparation, analysis time, data quality, and data interpretation. Here, we present a new method that couples in‐source collision‐induced dissociation (IS‐CID) with two‐dimensional tandem mass spectrometry (2D MS/MS) as a way to simplify proteomics and glycomics workflows while also providing additional insight into analyte structures over traditional MS/MS experiments. Specifically, IS‐CID is employed as a gas‐phase digestion method, i.e., to break down intact full‐length polysaccharide or peptide ions prior to mass analysis. The resulting mixtures of oligomeric ions are analyzed by 2D‐MS/MS, a technique that allows association of product ions with their precursor ions without isolation of the latter. A novel data analysis strategy is introduced to leverage the second dimension of 2D MS/MS spectra, in which stairstep patterns, representing outputs of a molecule's MSn scans, are extracted for structural interconnectivity information on the oligomer. The results demonstrate the potential applicability of 2D MS/MS strategies to the modern omics workflow and structural analysis of various classes of biopolymers.

Rapid Chemical Screening of Microplastics and Nanoplastics by Thermal Desorption and Pyrolysis Mass Spectrometry with Unsupervised Fuzzy Clustering.

The transport and chemical identification of microplastics and nanoplastics (MNPs) are critical to the concerns over plastic accumulation in the environment. Chemically and physically transient MNP species present unique challenges for isolation and analysis due to many factors such as their size, color, surface properties, morphology, and potential for chemical change. These factors contribute to the eventual environmental and toxicological impact of MNPs. As analytical methods and instrumentation continue to be developed for this application, analytical test materials will play an important role. Here, a direct mass spectrometry screening method was developed to rapidly characterize manufactured and weathered MNPs, complementing lengthy pyrolysis-gas chromatography-mass spectrometry analysis. The chromatography-free measurements took advantage of Kendrick mass defect analysis, in-source collision-induced dissociation, and advancements in machine learning approaches for the data analysis of complex mass spectra. In this study, we applied Gaussian mixture models and fuzzy c-means clustering for the unsupervised analysis of MNP sample spectra, incorporating clustering stability and information criterion measurements to determine latent dimensionality. These models provided insight into the composition of mixed and weathered MNP samples. The multiparametric data acquisition and machine learning approach presented improved confidence in polymer identification and differentiation.

Differential Mobility Spectrometry-Tandem Mass Spectrometry with Multiple Ion Monitoring Coupled with in Source-Collision Induced Dissociation: A New Strategy for the Quantitative Analysis of Pharmaceutical Polymer Excipients in Rat Plasma.

Polylactic acids (PLAs) are synthetic polymers composed of repeating lactic acid subunits. For their good biocompatibility, PLAs have been approved and widely applied as pharmaceutical excipients and scaffold materials. Liquid chromatography-tandem mass spectrometry is a powerful analytical tool not only for pharmaceutical ingredients but also for pharmaceutical excipients. However, the characterization of PLAs presents particular problems for mass spectrometry techniques. In addition to their high molecular weights and wide polydispersity, multiple charging and various adductions are intrinsic features of electrospray ionization. In the present study, a strategy combining of differential mobility spectrometry (DMS), multiple ion monitoring (MIM) and in-source collision-induced dissociation (in source-CID) has been developed and applied to the characterization and quantitation of PLAs in rat plasma. First, PLAs will be fragmented into characteristic fragment ions under high declustering potential in the ionization source. The specific fragment ions are then screened twice by quadrupoles to ensure a high signal intensity and low interference for mass spectrometry detection. Subsequently, DMS technique has been applied to further reduce the background noise. The appropriately chosen surrogate specific precursor ions could be utilized for the qualitative and quantitative analysis of PLAs, which provided results with the advantages of low endogenous interference, sufficient sensitivity and selectivity for bioassay. The linearity of the method was evaluated over the concentration range 3-100 μg/mL (r2 = 0.996) for PLA 20,000. The LC-DMS-MIM coupled with in source-CID strategy may contribute to the pharmaceutical studies of PLAs and the possible prospects of other pharmaceutical excipients.

High throughput LC/ESI-MS/MS method for simultaneous analysis of 20 oral molecular-targeted anticancer drugs and the active metabolite of sunitinib in human plasma

Many types of oral molecular-targeted anticancer drugs are clinically used in cancer genomic medicine. Combinations of multiple molecular-targeted anticancer drugs are also being investigated, expecting to prolong the survival of patients with cancer. Therapeutic drug monitoring of oral molecular-targeted drugs is important to ensure efficacy and safety. A liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) has been used for simultaneous determination of these drugs in human plasma. However, the sensitivity of mass spectrometers and differences in the therapeutic range of drugs have rendered the development of simultaneous LC/ESI-MS/MS methods difficult. In this study, a simultaneous quantitative method for 20 oral molecular-targeted anticancer drugs and the active metabolite of sunitinib was developed based on the results of linear range shifts of the calibration curves using four ion abundance adjustment techniques (collision energy defects, in-source collision-induced dissociation, secondary product ion selected reaction monitoring, and isotopologue selected reaction monitoring). The saturation of the detector for the seven analytes was resolved by incorporating optimal ion abundance adjustment techniques. Furthermore, the reproducibility of this method was confirmed in validation tests, and plasma from patients was measured by this method to demonstrate its usefulness in actual clinical practice. This analytical method is expected to make a substantial contribution to the promotion of personalized medicine in the future.

Phenyl Sulfate Derivatives: New Thermometer Ions for Characterization of Internal Energy of Negative Ions Produced by Electrospray Ionization.

Although positive thermometer ions are widely used for evaluating the internal energy distribution of gas-phase ions, negative thermometer ions have not yet been proposed. In this study, phenyl sulfate derivatives were tested as thermometer ions to characterize the internal energy distribution of ions produced by electrospray ionization (ESI) in the negative mode because the activation of phenyl sulfate preferentially undergoes SO3 loss, providing a phenolate anion. The dissociation threshold energies for the phenyl sulfate derivatives were determined using quantum chemistry calculations at the CCSD(T)/6-311++G(2df,p)//M06-2X-D3/6-311++G(d,p) level of theory. The values for the appearance energies of the fragment ions of the phenyl sulfate derivatives depend on the dissociation time scale in the experiment; therefore, the dissociation rate constants of the corresponding ions were estimated using the Rice-Ramsperger-Kassel-Marcus theory. The phenyl sulfate derivatives were used as thermometer ions to determine the internal energy distribution of negative ions activated by the in-source collision-induced dissociation (CID) and higher-energy collisional dissociation. Both mean and full width at half-maximum values increased with increasing ion collision energy. In the in-source CID experiments, the internal energy distributions obtained by phenyl sulfate derivatives are similar to that when all voltages are mirrored, and the traditional benzylpyridinium thermometer ions are used. The reported method will aid in determining the optimum voltage for ESI mass spectrometry and the subsequent tandem mass spectrometry of acidic analyte molecules.

Integrating Effect-Directed Analysis and Chemically Indicative Mass Spectral Fragmentation to Screen for Toxic Organophosphorus Compounds.

Analytical chemists are often challenged to screen for bioactive compounds in complex matrices, sometimes without a priori knowledge of the exact compound of interest. Therefore, "flagging" techniques, highlighting common characteristics of bioactive compounds, are highly sought after. In this work, we demonstrate a double flagging method, where unknown organophosphorus acetylcholinesterase inhibitors are "flagged" out of a complex matrix by the presence of organophosphorus-indicative ions as well as their acetylcholinesterase inhibition. This is accomplished by flagging the LC chromatographic retention time of phosphorus-indicative ions using accurate mass high-energy in-source CID products, and the retention time of acetylcholinesterase inhibiting compounds using a parallel microfractionation-based bioassay. We successfully apply this method to screen VX, VM, and RVX nerve agents as well as methomyl, a carbamate pesticide, out of soil and whole blood samples at low μM to sub-μM concentrations. This methodology can be easily extended to diverse chemical families and biological activities of interest.

A feasibility study of direct analysis in real time-mass spectrometry for screening organic gunshot residues from various substrates.

This study reports the use of direct analysis in real time-mass spectrometry (DART-MS) for the detection of organic gunshot residues (OGSR) in a variety of matrices of interest for forensics, customs, and homeland security. Detection limits ranged from (0.075 to 12) ng, with intra- and inter-day reproducibility below 0.0012% CV. The collection of mass spectra at multiple in-source collision-induced dissociation (is-CID) voltages produced distinctive mass spectral signatures with varying levels of fragmentation and allowed differentiation of isomers. To test method performance, a collection of 330 authentic specimens from various substrates were analyzed - (1) neat smokeless powders, (2) spent cartridge cases, (3) burnt particles removed from clothing via carbon stubs or (4) with tweezers, and hand samples from (5) non-shooters, and (6) shooters. A subset of hand specimens (n = 80) was further analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) for confirmation and comparison. Seven types of ammunition from five manufacturers and two calibers were monitored for OGSR profiles with similar compositions observed for paired sets (e.g., unburnt smokeless powder and the respective residues on spent cartridges, clothing, and hands). No false positives were observed across all datasets. A 100% true positive rate (TPR) was observed for all substrates except the shooters' hands. Depending on the ammunition type and classification criteria, the shooters' hands exhibited a TPR ranging from 19% to 73%. The results show that DART-MS is feasible and versatile for fast screening of OGSR across various substrates but may benefit from alternative approaches to improve detection at trace levels.

Assessing the effectiveness of the NIST DART-MS Forensics Database and Data Interpretation Tool for designer drug screening with alternative instrumentation

The increasing prevalence of designer drugs has contributed towards the adoption of rapid screening techniques, such as direct analysis in real time-mass spectrometry (DART-MS). Recently, the National Institute of Standards and Technology (NIST) developed a DART-MS Forensics Database and Data Interpretation Tool (DIT) to assist practitioners with the interpretation of seized drug mixtures. However, this resource was only designed for DART ionization coupled with AccuTOF high-resolution mass spectrometers. This study assesses the effectiveness of the NIST DART-MS Forensics Database and DIT for mixture interpretation using other soft ionization sources, such as electrospray ionization (ESI), and coupling DART to an Agilent 6530 quadrupole time-of-flight (Q-TOF) mass spectrometer. As the seized drug community continues to shift towards the use of in-source collision-induced dissociation (IS-CID) with single-stage mass spectrometers coupled with soft ionization sources, it is imperative to understand the similarity of the generated IS-CID fragment ion spectra and the effectiveness of the DIT for seized drug screening using instrumentation other than the DART-AccuTOF.Pearson product-moment correlations (PPMCs) revealed a high degree of spectral similarity between the ESI-IS-CID and DART-IS-CID fragment ion spectra collected across a range of activation conditions. The 95% confidence interval for the average highest PPMC coefficient across the 12 designer drugs in this study was 0.9295 ± 0.0031. More importantly, nearly identical mixture interpretation results were obtained when the ESI-IS-CID and DART-IS-CID fragment ion spectra were analyzed with the DIT. In all cases, the DIT was able to identify a potential library match with at least two match types between the low and high IS-CID spectra for all compounds present in each mixture. Demonstrating the potential applicability of the NIST DART-MS Forensics Database and DIT for the screening of seized drug mixtures using instrumentation other than the DART-AccuTOF provides support for the expanded applicability of this freely available resource.

How Soft Is Secondary Electrospray Ionization?

Secondary electrospray ionization (SESI) mass spectrometry (MS) is a direct infusion technique often used for untargeted metabolomics, e.g., for online breath analysis. SESI is thought to be a soft ionization method, which is important to avoid interference from in-source fragments and to simplify compound annotation. In this work, benzylammonium ions, formed from volatile benzylamines, with known bond dissociation enthalpies were used as thermometer ions to investigate the internal energy distribution of ions that are produced by SESI. It is shown that SESI is softer than electrospray ionization (ESI), and therefore, SESI indeed qualifies as a soft ionization technique. However, we also found that the standard MS instrument settings used in the SESI community are relatively harsh. Proper soft tuning of the instrument is essential to fully benefit from the softness that SESI can provide. Moreover, there is evidence from in-source collision-induced dissociation (CID) experiments that analytes can be solvated in SESI under soft conditions, which supports a recently proposed SESI mechanism referred to as ligand switching.

Fragmentation of Protonated Histamine and Histidine by Electrospray Ionization In-Source Collision-Induced Dissociation

Electrospray ionization (ESI) generally produces intact gas-phase ions without extensive fragmentation; however, for histamine and histidine, ESI provides fragment ions through in-source collision-induced dissociation (CID). In this study, we investigated the fragmentation of these compounds both experimentally and using density functional theory calculations. We found that histamine undergoes protonation with subsequent NH3 loss by ESI in-source CID. The corresponding fragmentation mainly produces bicyclo and spiro compounds. In contrast, the ESI in-source CID of protonated histidine preferentially results in H2O loss rather than NH3 loss. However, the corresponding fragment ion is not observed in the ESI mass spectrum of histidine, because it undergoes further CO loss within 100 ps. Consequently, protonated histidine produces a fragment ion arising from a 46 Da loss, which corresponds to the masses of H2O and CO, by ESI in-source CID. The fragment ion yields of histamine and histidine produced by ESI in-source CID are then estimated from the dissociation rate constant and internal energy of the analyte ion, respectively. The dissociation rate constant and internal energy of the analyte ion were determined by double-hybrid density functional theory calculations and the survival yield method using benzylpyridinium thermometer ions, respectively. Because intense fragment ion signals are present in the ESI mass spectrum, the analysis of the fragment ions produced by ESI in-source CID facilitates the identification of metabolites originating from aromatic amino acids, such as histamine.

Assessment of the similarity between in-source collision-induced dissociation (IS-CID) fragment ion spectra and tandem mass spectrometry (MS/MS) product ion spectra for seized drug identifications

The use of in-source collision-induced dissociation (IS-CID) with single-stage mass spectrometers has grown in popularity within the field of seized drug analysis as a pseudo-tandem mass spectrometry (MS/MS) analysis to gain structural information about unknown compounds. However, there is currently no statistical assessment of the similarity and thus, the comparability, of IS-CID fragment ion spectra and MS/MS product ions spectra for representative novel psychoactive substances (NPS), such as synthetic cannabinoids, synthetic cathinones, and fentanyl analogs. This study investigates the use of spectral comparisons, breakdown curves, and Pearson product-moment correlations (PPMCs) to assess the similarity between IS-CID fragment ion spectra and MS/MS product ion spectra both visually and statistically. In addition, this study also examines the repeatability of IS-CID fragment ion spectra relative to MS/MS product ion spectra to assess the short-term repeatability of IS-CID fragment ion spectra. The 95% confidence interval average PPMC for the highest pairwise comparison across this study was 0.9294 +/- 0.0106. The optimum comparison range between IS-CID fragment ion spectra and MS/MS product ion spectra was identified between a fragmentor voltage of 150–280 V and a collision energy of 0–22 eV. Similar repeatability of IS-CID fragment ion spectra (15.21%) and MS/MS product ion spectra (14.72%) was also observed. Demonstrating the achievable spectral similarity between IS-CID fragment ion spectra and MS/MS product ion spectra for representative NPS, as well as highlighting the potential deleterious effects of high activation conditions on spectral comparisons, will help inform analysts’ decisions regarding seized drug identifications based on IS-CID fragment ion spectra.

Systematic Identification of Bioactive Compositions in Leaves of Morus Cultivars Using UHPLC-ESI-QTOF-MS/MS and Comprehensive Screening of High-Quality Resources

Morus spp. leaves (MSLs) show various beneficial effects in the treatment of metabolic-related diseases, which have created a growing interest in MSL development as dietary supplements and functional foods. The illustration of chemical compositions and screening of high-quality MSL resources are therefore necessary for further application. This study developed a new UHPLC-ESI-QTOF-MS/MS strategy of in-source collision-induced dissociation (IS-CID) and target collision-cell CID (TCC-CID) to quickly capture analogues with consistent skeleton, and combined global natural product social molecular networking (GNPS) to efficiently annotate bioactive phytochemicals in MSLs. For the results, 49 bioactive ingredients, including quercetin-type flavonoids, kaempferol-type flavonoids, chlorogenic acid isomers, 1-deoxynojirimycin, γ-aminobutyric acid, amino acids, and unsaturated fatty acids, were systematically identified in MSLs for the first time. Quantification for the typical components was simultaneously carried out in MSLs of 90 Morus resources collected from different locations. Partial least squares discriminant analysis (PLS-DA) indicated that quercetin-3-O-(6″-O-malonyl)-glucoside, rutin, kaempferol-3-O-(6″-O-malonyl)-glucoside, kaempferol-3-O-rutinoside, and chlorogenic acid showed high variable importance in the project (VIP > 1) that were significant constituents for the differences between MSL species. Then, high-quality MSLs were comprehensively screened in multiple Morus cultivars based on the criteria importance through intercriteria correlation (CRITIC) method. This study presented an efficient strategy to annotate bioactive compounds, revealed the difference of bioactive components in MSLs, and provided important information for the high-value production of Morus cultivars in food and supplement fields.

Cutting-edge LC-MS/MS applications in clinical mass spectrometry: Focusing on analysis of drugs and metabolites.

In recent years, liquid chromatography with tandem mass spectrometry (LC-MS/MS) has become a fundamental technology in clinical practice. In Japan, the LC-MS/MS system is used in many large hospitals. It has become popular among pharmacists and laboratory technicians. LC-MS/MS has some advantages in terms of accuracy, speed, and comprehensiveness compared to conventional automated chemical testing equipment. However, LC-MS/MS is by no means a universal method, and it is necessary to understand its characteristics before using it. In the field of therapeutic drug monitoring (TDM), there is an issue with linearity in comprehensive measurement; however, ion-abundance adjustment methods, such as in-source collision-induced dissociation, have been proposed as a solution to this problem. The development of a biomarker analysis includes search, identification, and quantification, and it is necessary to select an appropriate mass spectrometric method for each step. In this paper, we review cutting-edge technologies that can expand the performance of LC-MS/MS in the clinical field and consider current issues and future prospects.

Nontargeted High-Resolution Mass Spectrometric Workflow for the Detection of Butyrylcholinesterase-Derived Adducts with Organophosphorus Toxicants and Structural Characterization of Their Phosphyl Moiety after In-Source Fragmentation.

Organophosphorus (OP) nerve agents were used for chemical warfare, assassination, and attempted murder of individuals. Therefore, forensic methods are required to identify known and unknown incorporated OP poisons. Serum is tested for the presence of covalent reaction products (adducts) of the toxicant with, e.g., butyrylcholinesterase (BChE) typically by targeted analysis, thus only detecting known OP adducts. We herein present a nontargeted two-step mass spectrometry (MS)-based workflow taking advantage of a high-resolution (HR) Orbitrap mass spectrometer and its option for in-source collision-induced dissociation (IS-CID) highly valuable for the detection of unknown agents. BChE adducts are extracted by immunomagnetic separation and proteolyzed with pepsin yielding a phosphylated nonapeptide (NP) biomarker NP(OP). In step 1, the sample is separated by micro liquid chromatography (μLC) detecting the NP(OP) by nontargeted HR MS followed by data-dependent tandem-MS (ddMS2). Extracted ion chromatograms of diagnostic product ions at m/z 778.33661, 673.29402, and 602.25690 reveal the accurate mass of the NP(OP) precursor ion as well as the elemental composition of the adducted phosphyl moiety. Considering this information, a second μLC run is performed (step 2) for nonselective IS-CID of NP(OP) yielding the cleaved charged phosphyl moiety. This fragment ion is immediately subjected to targeted CID in parallel reaction monitoring (PRM). The accurate mass of its product ions allows the determination of their elemental composition and thus supports its structural elucidation. The described workflow was exemplarily applied to NP(OP) of three Tamelin esters and VX providing highly appropriate abilities for the detection of adducts even of unknown OP poisons like Novichok agents.

Fragmentation efficiency of phenethylamines in electrospray ionization source estimated by theoretical chemistry calculation.

Small molecules with polar functional groups, including substituted phenethylamines, are commonly analyzed by liquid chromatography-mass spectrometry (LC-MS) with electrospray ionization (ESI). Analyte molecules are mostly detected in protonated and cation-adducted forms through positive-ion electrospray ionization-mass spectrometry (ESI-MS). However, the ESI of substituted phenethylamines commonly provides an intense signal of fragment ions by ESI in-source collision-induced dissociation (IS-CID), which hinders the unambiguous identification of phenethylamines. This phenomenon was approximated as a unimolecular dissociation model, and the dissociation efficiency was evaluated by various quantum chemistry calculations to determine the ESI IS-CID efficiency. The calculated results were consistent with the experimental data, when the dissociation threshold energy of phenethylamines was calculated using the post-Hartree-Fock (post-HF) method, CCSD(t)/cc-pVTZ//MP2(full)/6-311++G(d,p). In contrast to post-HF methods, the utilization of density functional theory calculations with a suitable functional is recognized as an accurate and competitive low-cost approach. In particular, ωB97-XD, M06-2X-D3, and recently developed Minnesota functionals, such as M11, MN12-SX, and MN15, provided reliable results, as in the case of the post-HF method. The results obtained by the recently developed double hybrid functionals, DSD-PEBP86-D3(BJ), PBE0-DH, and PBE-QIDH, were also reliable. The consideration of ESI IS-CID can facilitate the identification of analyte molecules because most phenethylamines, except for N-methylated analogs, provide an intense signal in the ESI mass spectrum.

Pharmacokinetics and biodistribution of a novel anticancer thyrointegrin \u03b1v\u03b23 antagonist: triazole modified tetraiodothyroacetic acid conjugated to polyethylene glycol (P-bi-TAT)

We previously developed a triazole modified tetraiodothyroacetic acid (TAT) conjugated to a polyethylene glycol (PEG)-based thyrointegrin αvβ3 antagonist targeted compound, called P-bi-TAT. It exhibited potent anti-angiogenic and anticancer activities in vivo. The objective of the current study is to develop a quantitative bioanalytical method for P-bi-TAT using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and to elucidate pharmacokinetics (PK) and biodistribution of P-bi-TAT in animals. We used in-source collision-induced dissociation (CID) for ionization of P-bi-TAT in the positive mode, followed by multiple reaction monitoring (MRM) for quantification. P-bi-TAT was quantified using P-mono-TAT as an internal standard because of its similarity in structure and physicochemical properties to P-bi-TAT. The LOQ for P-bi-TAT was 30 ng/μL and the recovery efficiency was 76% with the developed method. Cmax and AUC results at different doses (1, 3, 10 mg/kg) in rats suggest that P-bi-TAT is dose-dependent within the range administered. Results for Cmax and AUC in monkeys at a low dose (25 mg/kg) were comparable to those in rats. Biodistribution of subcutaneously administered P-bi-TAT in the brain of rats ranged from 7.90 to 88.7 ng/g brain weight, and levels of P-bi-TAT in the brain were dose-dependent. The results suggest that P-bi-TAT is a potential candidate as a molecular-targeted anticancer therapeutic with blood-brain barrier permeability and acceptable PK parameters. Its accumulation in organs, toxicokinetic, and pharmacodynamics needs to be further investigated.Graphical

Study of Substituted Phenethylamine Fragmentation Induced by Electrospray Ionization Mass Spectrometry and Its Application for Highly Sensitive Analysis of Neurotransmitters in Biological Samples.

Although liquid chromatography-tandem mass spectrometry (LC-MS/MS) equipped with electrospray ionization (ESI) is widely employed for metabolite analysis, substituted phenethylamines commonly undergo fragmentation during ESI in-source collision-induced dissociation (CID). Unexpected fragmentation hampers not only unambiguous identification but also accurate metabolite quantification. ESI in-source CID induces N-Cα bond dissociation in substituted phenethylamines lacking a β-hydroxy group to produce fragment ions with a spiro[2.5]octadienylium motif. In contrast, phenethylamines with a β-hydroxy group generate substituted 2-phenylaziridium through ESI in-source CID-induced H2O loss. The fragment ion yield produced by ESI in-source CID can be estimated by the dissociation rate constant and internal energy of the analyte ion, determined by employing density functional theory calculations and the survival yield method using a thermometer ion, respectively. Fragmentation is strongly enhanced by the presence of an β-hydroxy group, whereas N-methylation suppresses fragmentation. In particular, octopamine and noradrenaline, which contain an β-hydroxy and primary amine groups, produce more intense fragment ion signals than protonated molecules. Regarding the quantitative analysis of phenethylamines present in the mouse brain, the noradrenaline fragment ion used as the precursor in multiple reaction monitoring (MRM) provided a higher signal-to-noise ratio in the resulting spectra than protonated noradrenaline. The present method allows for the quantitative analysis of substituted phenethylamines with high sensitivity.

A New Library-Search Algorithm for Mixture Analysis Using DART-MS.

Forensic analysis of seized drug evidence often involves determining whether the components of an unknown mixture are illicit compounds. One approach to this task is to screen the evidence using direct analysis in real time mass spectrometry (DART-MS) to make presumptive identifications. This manuscript introduces a new library-search algorithm that enhances presumptive identifications of mixture components using a series of in-source collision-induced dissociation mass spectra collected through DART-MS. The multistage search, titled the Inverted Library-Search Algorithm (ILSA), identifies potential components in a mixture by first searching the lowest fragmentation mass spectrum for target peaks, assuming these peaks are protonated molecules, and then scoring each target peak with possible library matches. As a proof of concept, the ILSA is demonstrated through several example searches of model seized drug mixtures of acetyl fentanyl, benzyl fentanyl, amphetamine, and methamphetamine searched against a small library of select compounds and the freely available NIST DART-MS Forensics Database. Discussion of the search results and several open areas of research to further extend the method are provided. This new approach for presumptive identification provides analysts with refined information about mixture components and will be of immediate importance in forensic analysis using DART-MS. A prototype implementation of the ILSA is available at https://github.com/asm3-nist/DART-MS-DST.