TOF Mass Spectrometer Research Articles

Influence of a synthetic analogue of the active center of GM-CSF – peptide ZP2 on the growth and biofilm formation of gram-negative bacteria – causes of surgical infections

The aim is to analyze the nature of the effect of the synthetic analogue of the active center of GM- CSF – peptide ZP2 on the growth and biofilm formation (BPO) of gram–negative bacteria – pathogens of surgical infections. The study used 18 clinical isolates of gram-negative bacteria of different species (Klebsiella pneumoniae, Citrobacter freundii, Stenotrophamonas maltophilia) isolated from purulent wounds in patients with surgical pathology and showing pronounced resistance to many antibiotics used in clinical practice. The isolation of pure cultures of microorganisms was carried out by conventional methods; species were assessed by direct protein profiling using a MALDI TOF mass spectrometer. To study the effect of ZP2 peptide on bacterial growth and BPO, isolates were co-cultured with a solution of ZP2 peptide in meat-peptone broth (MPB) at a concentration of 5 micrograms/ml at 37°C for 24-48 hours. The effect of the ZP2 peptide was studied both on emerging biofilms and on those formed, according to the degree of binding of crystalline violet in sterile 96-well polystyrene plates. Next, Growth inhibition indices and coefficients of BPO of microorganisms were determined. It was found that the synthetic analogue of the active center of granulocyte-macrophage colony stimulating factor (GM-CSF) – peptide ZP2 inhibited the growth of the studied bacterial strains, reducing the biomass of experimental cultures during the development of bacterial populations. At the same time, the species-specific effect of the ZP2 peptide on the ability of surgical strains of the studied bacteria to form biofilms has been shown. It was experimentally established that all the studied surgical strains of microorganisms were capable of biofilm formation, and the maximum severity of the trait was characteristic of S. maltophilia isolates. The synthetic analogue of the active center of granulocyte-macrophage colony stimulating factor (GM-CSF) – peptide ZP2 caused a decrease in the ability to form biofilms in isolates S. maltophilia and destroyed already formed biofilms in all studied species of microorganisms. The experimental data obtained expand the range of potential clinical applications of the synthetic analogue of the active center of granulocyte-macrophage colony stimulating factor (GM-CSF) – peptide ZP2, and its use in new medicines can be effective in combating antibiotic-resistant pathogens of surgical infections.

Imaging Mass Spectrometry of Isotopically Resolved Intact Proteins on a Trapped Ion-Mobility Quadrupole Time-of-Flight Mass Spectrometer.

In this work, we demonstrate rapid, high spatial, and high spectral resolution imaging of intact proteins by matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) on a hybrid quadrupole-reflectron time-of-flight (qTOF) mass spectrometer equipped with trapped ion mobility spectrometry (TIMS). Historically, untargeted MALDI IMS of proteins has been performed on TOF mass spectrometers. While advances in TOF instrumentation have enabled rapid, high spatial resolution IMS of intact proteins, TOF mass spectrometers generate relatively low-resolution mass spectra with limited mass accuracy. Conversely, the implementation of MALDI sources on high-resolving power Fourier transform (FT) mass spectrometers has allowed IMS experiments to be conducted with high spectral resolution with the caveat of increasingly long data acquisition times. As illustrated here, qTOF mass spectrometers enable protein imaging with the combined advantages of TOF and FT mass spectrometers. Protein isotope distributions were resolved for both a protein standard mixture and proteins detected from a whole-body mouse pup tissue section. Rapid (∼10 pixels/s) 10 μm lateral spatial resolution IMS was performed on a rat brain tissue section while maintaining isotopic spectral resolution. Lastly, proof-of-concept MALDI-TIMS data was acquired from a protein mixture to demonstrate the ability to differentiate charge states by ion mobility. These experiments highlight the advantages of qTOF and timsTOF platforms for resolving and interpreting complex protein spectra generated from tissue by IMS.

Quantitative and Qualitative Analyses of Mass Spectra of OEL Materials by Artificial Neural Network and Interface Evaluation: Results from a VAMAS Interlaboratory Study.

Quantitative analysis of binary mixtures of tris(2-phenylpyridinato)iridium(III) (Ir(ppy)3) and tris(8-hydroxyquinolinato)aluminum (Alq3) by using an artificial neural network (ANN) system to mass spectra was attempted based on the results of a VAMAS (Versailles Project on Advanced Materials and Standards) interlaboratory study (TW2 A31) to evaluate matrix-effect correction and to investigate interface determination. Monolayers of binary mixtures having different Ir(ppy)3 ratios (0, 0.25, 0.50, 0.75, and 1.00), and the multilayers containing these mixtures and pure samples were measured using time-of-flight secondary ion mass spectrometry (ToF-SIMS) with different primary ion beams, OrbiSIMS (SIMS with both Orbitrap and ToF mass spectrometers), laser desorption ionization (LDI), desorption/ionization induced by neutral clusters (DINeC), and X-ray photoelectron spectroscopy (XPS). The mass spectra were analyzed using a simple ANN with one hidden layer. The Ir(ppy)3 ratios of the unknown samples and the interfaces of the multilayers were predicted using the simple ANN system, even though the mass spectra of binary mixtures exhibited matrix effects. The Ir(ppy)3 ratios at the interfaces indicated by the simple ANN were consistent with the XPS results and the ToF-SIMS depth profiles. The simple ANN system not only provided quantitative information on unknown samples, but also indicated important mass peaks related to each molecule in the samples without a priori information. The important mass peaks indicated by the simple ANN depended on the ionization process. The simple ANN results of the spectra sets obtained by a softer ionization method, such as LDI and DINeC, suggested large ions such as trimers. From the first step of the investigation to build an ANN model for evaluating mixture samples influenced by matrix effects, it was indicated that the simple ANN method is useful for obtaining candidate mass peaks for identification and for assuming mixture conditions that are helpful for further analysis.

Evaluation of the proteomic profile in patients with celiac disease and malabsorption syndrome

The malabsorption syndrome includes many clinical units that lead to chronic diarrhea and malnutrition. In celiac disease and small intestine bacterial overgrowth making the diagnosis is not always straightforward, and the diagnosis often requires combining blood antibody tests, intestinal biopsies, and specific genetic testing. The study aimed to assess patients' protein/peptide profiles with celiac disease (n=31), small intestinal bacterial overgrowth (n=28), and the control group (n=30). Attempts have also been made to identify potential new diagnostic markers. After appropriate preparation, a proteomic analysis of peptides was performed using the MALDI–TOF mass spectrometer. Identified proteins were fibrinogen alpha-chain, kininogen-1, mucin 3A, complement C3, complement C4A, and inter-alpha-trypsin inhibitor heavy chain H4. Performed analyses indicated some proteins that could be potentially involved in the presence of the disease in question and proved that proteomic profiling might serve as a powerful diagnostic tool. Due to the difficulties in celiac disease and small intestine bacterial overgrowth diagnosis, there is a clear need for further investigation of the biological role of proteins potentially involved in the course of the disease.

Upcycling Polytetrahydrofuran to Polyester

Upcycling Polytetrahydrofuran to Polyester

Controllable Polymerization of N -Substituted β-Alanine N -Thiocarboxyanhydrides for Convenient Synthesis of Functional Poly(β-peptoid)s

Controllable Polymerization of <i>N</i> -Substituted β-Alanine <i>N</i> -Thiocarboxyanhydrides for Convenient Synthesis of Functional Poly(β-peptoid)s

Dissociation of Biomolecules by an Intense Low-Energy Electron Beam in a High Sensitivity Time-of-Flight Mass Spectrometer

We report the progress on an electron-activated dissociation (EAD) device coupled to a quadrupole TOF mass spectrometer (QqTOF MS) developed in our group. This device features a new electron beam optics design allowing up to 100 times stronger electron currents in the reaction cell. The electron beam current reached the space-charge limit of 0.5 μA at near-zero electron kinetic energies. These advances enable fast and efficient dissociation of various analytes ranging from singly charged small molecules to multiply protonated proteins. Tunable electron energy provides access to different fragmentation regimes: ECD, hot ECD, and electron-impact excitation of ions from organics (EIEIO). The efficiency of the device was tested on a wide range of precursor charge states. The EAD device was installed in a QqTOF MS employing a novel trap-and-release strategy facilitating spatial mass focusing of ions at the center of the TOF accelerator. This technique increased the sensitivity 6-10 times and allows for the first time comprehensive structural lipidomics on an LC time scale. The system was evaluated for other compound classes such as intact proteins and glycopeptides. Application of hot ECD for the analysis of glycopeptides resulted in rich fragmentation with predominantly peptide backbone fragments; however, glycan fragments attributed to the ECD process were also observed. A standard small protein ubiquitin (8.6 kDa) was sequenced with 90% cleavage coverage at spectrum accumulation times of 100 ms and 98% at 800 ms. Comparable cleavage coverage for a medium-size protein (carbonic anhydrase: 29 kDa) could be achieved, albeit with longer accumulation times.

Fluorescent Visualization of Nucleolar G-Quadruplex RNA and Dynamics of Cytoplasm and Intranuclear Viscosity

The nucleolus, the locus of ribosome biogenesis, was found to be the predominant intracellular target of a new fluorescent probe, V-P1. In solution, the probe demonstrated both a selectivity to RNA...

MALDI‐TOF IP‐MS quantification of plasma amyloid peptides in Alzheimer’s disease

AbstractBackgroundThe use of biomarkers which reflect Alzheimer’s disease (AD) improved the stratification of patients and the ability to propose personalized care. Their detection using either cerebrospinal fluid analysis (CSF), which is invasive, or using PET imaging, which is costly, is not adapted to large‐scale use. In 2018, Nakamura et al. using MALDI‐TOF IP‐MS generated a new composite biomarker with the amyloid‐ß precursor protein (APP)669–711/Aß42 and Aß40/Aß42 ratios with an AUC above 0.94 to identify subjects with PETAß+. Our primary objective here was to validate the use of this approach to identify AD and predicts its apparence in MCI and pre‐symptomatic patients.MethodThe blood‐based Aß markers were measured with an innovative MALDI‐TOF IP‐MS approach adapted from Nakamura et al., 2018. Briefly, 250 μl of plasma was diluted and spiked with 10 pM of stable‐isotope‐labelled (SIL) Aβ1–38 peptide. Aβ‐related peptides were immunoprecipitated, washed, and eluted with with 70% acetonitrile / 5 mM HCl to a 900‐μm μFocus MALDI plateTM (Hudson Surface Technology) which was pre‐spotted with a mix of α‐cyano‐4‐hydroxycinnamic acid (CHCA) and methanediphosphonic acid (MDPNA). Mass spectra were acquired using a MALDI‐linear TOF mass spectrometer (AXIMA Assurance, Shimadzu/KRATOS) equipped with a 337‐nm nitrogen laser in the positive ion mode. References AD cohorts with CSF biomarkers and longitudinal follow‐up were used.ResultsQuality controls (QC) plasma pools with Low (LQC), Medium (MQC), and High (HQC) (APP)669–711, Aß42 and Aß40 levels were generated to perform the analytical validation. Intra‐day variability were for LQC, MQC and HQC ≈19% for raw MS intensities and ≈6% after internal normalization. Inter‐day variability were ≈16% after normalization. Dilution integrity indicated linear results between 100% and 50% of the initial plasma volume. Three cycles of freeze/thaw test showed a good stability for the 3 target peptides. A cohort of &gt;200 subject’s plasma samples from the memory consultation of the CMRR of Montpellier with Alzheimer’s disease, Mild Cognitive Impairment and other degenerative diseases are being analyzed to demonstrate the clinical interest of this measurement.ConclusionMALDI‐TOF IP‐MS quantification of plasma amyloid peptides reached satisfactory analytical performance for clinical use. Its relevance for Alzheimer disease will be illustrated.

Experience of using MALDI TOF minisequencing for studing TEM beta-lactamases in K. pneumoniae strains circulating in pediatric hospitals

Study of the nature of K. pneumoniae antibiotic resistance and analysis of the structure of extended-spectrum β-lactamases (ESBL) are one of the most important aspects of modern clinical microbiology and epidemiology. The issue of studying the gene context of these enzymes in order to search for point mutations that lead to ESBL formation is urgent. Research objective : to analyze and search for SNPs in nucleotide sequences of genes encoding TEM β-lactamases in K. pneumoniae strains using MALDI TOF minisequencing. Materials and methods: bla TEM genes of 36 K. pneumoniae strains isolated during episodes of epidemic trouble in pediatric hospital settings were studied. The strains were identified using an Autoflex MALDI TOF mass spectrometer. DNA isolation was performed using a DNA-express kit, bla TEM genes were detected by PCR. The minisequencing reaction for SNP detection was performed using oligonucleotide probes described in the academic literature. The spectral data were taken using an Autoflex MALDI TOF mass spectrometer and FlexControl software in the manual mode. Results. In the course of the work it was found that SNPs in the bla TEM gene sequence at positions 104, 240 and 238 and those ones leading to the substitution of glutamic acid by lysine – Glu104Lys, Glu240Lys, as well as glycine substitution by serine – Gly238Ser were absent in all studied strains. In 67% strains, a mutation was found that led to alanine substitution by threonine at position 237 (Ala237Thr), which led to a change in the proteins substrate specificity and to formation of ESBLs resistant to inhibitors. Conclusions. In 67% strains, single nucleotide substitutions were found, leading to a change in the enzyme substrate specificity and to formation of ESBLs. This confirms the need to monitor the frequency of significant mutations in antibiotic resistance genes in hospital strains in order to ensure epidemic forecasting.

RMSIproc: an R package for mass spectrometry imaging data processing.

Mass spectrometry imaging (MSI) can reveal biochemical information directly from a tissue section. MSI generates a large quantity of complex spectral data which is still challenging to translate into relevant biochemical information. Here, we present rMSIproc, an open-source R package that implements a full data processing workflow for MSI experiments performed using TOF or FT-based mass spectrometers. The package provides a novel strategy for spectral alignment and recalibration, which allows to process multiple datasets simultaneously. This enables to perform a confident statistical analysis with multiple datasets from one or several experiments. rMSIproc is designed to work with files larger than the computer memory capacity and the algorithms are implemented using a multi-threading strategy. rMSIproc is a powerful tool able to take full advantage of modern computer systems to completely develop the whole MSI potential. rMSIproc is freely available at https://github.com/prafols/rMSIproc. pere.rafols@urv.cat. Supplementary data are available at Bioinformatics online.

Subcellular Chemical Imaging: New Avenues in Cell Biology.

To better understand the physiology and acclimation capability of the cell, one of the great challenges of the future is to access the interior of a cell and unveil its chemical landscape (composition and distribution of elements and molecules). Chemical imaging has greatly improved in sensitivity and spatial resolution to visualize and quantify nutrients, metabolites, toxic elements, and drugs in single cells at the subcellular level. This review aims to present the current potential of these emerging imaging technologies and to guide biologists towards a strategy for interrogating biological processes at the nanoscale. We also describe various solutions to combine multiple imaging techniques in a correlative way and provide perspectives and future directions for integrative subcellular imaging across different disciplines.

Energy selective time of flight spectrometer to study low energy dissociation channels of PAHs

SynopsisDissociation channels induced by sequential UV multiphoton absorption of vapour phase fluorene cation is investigated using energy selective ToF mass spectrometer. Neutral fluorene molecule is ionized by resonantly enhanced multiphoton absorption and dissociated in the same photon field. The main goal of this experiment is to determine the rate constants of statistical dissociation channels of PAH cations for a wide range of internal energies.

Universal Affinity Capture Liquid Chromatography-Mass Spectrometry Assay for Evaluation of Biotransformation of Site-Specific Antibody Drug Conjugates in Preclinical Studies.

Antibody drug conjugates (ADCs) can undergo in vivo biotransformation (e.g., payload metabolism, deconjugation) leading to reduced or complete loss of activity. The location/site of conjugation of payload-linker can have an effect on ADC stability and hence needs to be carefully optimized. Affinity capture LC-MS of intact ADCs or ADC subfragments has been extensively used to evaluate ADC biotransformation. However, the current methods have certain limitations such as the requirement of specific capture reagents, limited mass resolution of low mass change metabolites, low sensitivity, and use of capillary or nanoflow LC-MS. To address these challenges, we developed a generic affinity capture LC-MS assay that can be utilized to evaluate the biotransformation of any site-specific ADC independent of antibody type and site of conjugation (Fab and Fc) in preclinical studies. The method involves a combination of some or all of these steps: (1) "mono capture" or "dual capture" of ADCs from serum with streptavidin magnetic beads coated with a generic biotinylated antihuman capture reagent, (2) "on-bead" digestion with IdeS and/or PNGase F, and (3) reduction of interchain disulfide bonds to generate ∼25 kDa ADC subfragments, which are finally analyzed by LC-HRMS on a TOF mass spectrometer. The advantages of this method are that it can be performed using commercially available generic reagents and requires sample preparation time of less than 7 h. Furthermore, by reducing the size of intact ADC (∼150 kDa) to subfragments (∼25 kDa), the identification of conjugated payload and its metabolites can be achieved with excellent sensitivity and resolution (hydrolysis and other small mass change metabolites). This method was successfully applied to evaluate the in vitro and in vivo biotransformation of ADCs conjugated at different sites (LC, HC-Fab, and HC-Fc) with various classes of payload-linkers.

Der Alleskönner der Messtechnik

SummaryThe all‐rounder of measurement technology – Monitoring of vacuum propertiesThe Novion is an all‐rounder of vacuum measurement technology. The newly developed sensor is based on a novel ion source and an integrated TOF mass spectrometer. The new vacuum measuring instrument is versatile: precise total pressure measurement over a wide range and simultaneously determination of helium partial pressure and residual gas analysis. By using the patented ion source, the instrument also achieves a high dynamic range without the use of a cost‐intensive electron multiplier, down to the UHV range. The Novion comes with a compact design, easy maintenance and an intuitive operation. The article covers the fields of application of this multi‐method sensor and explains its functionality in detail.

Mass spectrometry transanal minimally invasive surgery (MS-TAMIS) to promote organ preservation in rectal cancer

BackgroundTransanal minimally invasive surgery (TAMIS) is deployed for organ preservation in early rectal cancer and significant rectal polyps. Rapid evaporative ionisation mass spectrometry (REIMS) provides biochemical tissue analysis, which could be applied intraoperatively to give real-time tissue feedback to the surgeon and decrease the risk of an involved margin. However, the accuracy and feasibility of this approach have not been established.MethodsIn this prospective observational study, patients undergoing resection of rectal adenomas or carcinomas were recruited. An electrosurgical handpiece analysed tissues ex vivo using diathermy, with the aerosol aspirated into a Xevo G2-S ToF mass spectrometer. The relative abundance of lipids underwent predictive statistical modelling and leave-one-patient-out cross-validation. The outcomes of interest were the ability of REIMS to differentiate normal, adenomatous and cancerous tissue, or any disease subtype from normal. REIMS was coupled with TAMIS for in vivo sampling, assessing the accuracy of tissue recognition and distinguishing bowel wall layers.ResultsForty-seven patients were included, yielding 266 spectra (121 normal, 109 tumour and 36 adenoma). REIMS differentiates normal, adenomatous and cancerous rectal tissues with 86.8% accuracy, and normal and adenomatous tissue with 92.4% accuracy and 91.4% accuracy when differentiating disease from normal. We have performed the first five in-man mass spectrometry augmented TAMIS (MS-TAMIS). In real time, MS-TAMIS can differentiate rectal mucosa and submucosa based on their relative abundance of triglycerides and glycerophospholipids. The ex vivo accuracy distinguishing diseased and normal tissues is maintained in vivo at 90%, with negative predictive value of 95%. The system identified a deep and lateral involved tumour margin during TAMIS.ConclusionsREIMS distinguishes rectal tissue types based on underlying lipid biology, and this can be translated in vivo by coupling it to TAMIS. There is a role for this technology in improving the efficacy of resection of early rectal cancers.

Comprehensive lipidomics of mouse plasma using class-specific surrogate calibrants and SWATH acquisition for large-scale lipid quantification in untargeted analysis

Lipidomics has gained rising attention in recent years. Several strategies for lipidomic profiling have been developed, with targeted analysis of selected lipid species, typically utilized for lipid quantification by low-resolution triple quadrupole MS/MS, and untargeted analysis by high-resolution MS instruments, focusing on hypothesis generation for prognostic, diagnostic and/or disease-relevant biomarker discovery. The latter methodologies generally yield relative quantification data with limited inter-assay comparability. In this work we aimed to combine untargeted analysis and absolute quantification to enhance data quality and to obtain independent results for optimum comparability to previous studies or database entries. For the lipidomic analysis of mouse plasma, RP-UHPLC hyphenated to a high-resolution quadrupole TOF mass spectrometer in comprehensive data-independent SWATH acquisition mode was employed. This way, quantifiable data on the MS and the MS/MS level were recorded, which increases assay specificity and quantitative performance. Due to the lack of an appropriate blank matrix for untargeted lipidomics, we herein established a sophisticated strategy for lipid class-specific calibration with stable isotope labeled standards (surrogate calibrants). LLOQs were in the range between 10 and 50 ng mL−1 for LPC, LPE, PI, PS, PG, SM, PC, PE, DAG) or 100–700 ng mL−1 (MAG, TAG), except for cholesterol and CE (1–20 μg mL−1). Acceptable values for accuracy and precision well below ±15% bias were reached for the majority of surrogate calibrants. However, to achieve sufficient accuracy for target lipids, response factors to corresponding surrogate calibrants are required. An approach to estimate response factors via a standard reference material (NIST SRM 1950) was therefore conducted. Furthermore, a useful workflow for post-acquisition re-calibration, involving response factor determination and iteratively built libraries, is suggested. In comparison to single-point calibration, the presented surrogate calibrant method was shown to yield results with improved accuracy that are largely in accordance with standard addition. Quantitative results of real samples (high-fat diet vs control diet) were then compared to two previously published dietary mouse plasma studies that provided absolute lipid levels and showed similar trends.

Gallium selenide clusters generated via laser desorption ionisation quadrupole ion trap time-of-flight mass spectrometry.

Gallium selenide thin films important for electronics and phase-change materials are prepared via pulsed laser deposition (PLD); however, there are no studies concerning the analysis of gallium selenide clusters formed in the gas phase. Laser desorption ionisation (LDI) combined with time-of-flight mass spectrometry (TOF-MS) has great potential to generate charged Gam Sen clusters, to analyse them and thus to develop new materials. LDI of Ga-Se mixtures using a pulsed laser (337 nm nitrogen) was used to generate gallium-selenide clusters. Mass spectra were recorded (in positive and negative ion mode) on a TOF mass spectrometer equipped with a quadrupole ion trap and reflectron mass analyser. Ga-Se mixtures were found to be suitable for laser ablation synthesis (LAS) of gallium selenide clusters, although their composition was strongly dependent on the laser energy. The effect of laser energy on the stoichiometry of the generated clusters was established. In total, over 100 gallium selenide Gam Sen clusters were generated and identified from Ga-Se mixtures. LDI of Ga2 Se3 crystals showed almost the same clusters up to m/z 1000 with lower intensities, whereas no clusters from Ga2 Se3 were observed above m/z 1000. A family of over 100 gallium selenide clusters, generated and identified for the first time, shows rich and complex chemistry. Some of the clusters represent new compounds that have the potential to be used in the development of advanced materials.

Simultaneous Determination of 15 Flavonoids in Scutellaria barbata-Hedyotis diffusa Herb Pair by HPLC Q-TOF MS.

Background: The Scutellaria barbata-Hedyotis diffusa herb pair has appeared in classical prescriptions and has usually been used for the treatment of cancer and inflammatory conditions. Objective: For quality control of this herb pair, a rapid and reliable HPLC-quadrupole-time-of-flight(Q-TOF)-MS method was used for the simultaneous determination of alpinetin, apigenin-7-O-β-D-glucopyranoside, scutellarein, apigenin, quercetin-3-O-β-D-glucopyranoside, wogonoside, quercetin, amentoflavone, wogonin, chrysin, luteolin, rutin, naringenin, baicalein, and baicalin in the S. barbata-H. diffusa herb pair. Methods: LC-MS analysis was performed on a Shimadzu HPLC 20ADXR LC system in-line with an AB-SCIEX 5600 Triple TOF mass spectrometer. Results: The calibration curves showed good linearity in a relatively wide concentration range (r >0.996). The LOD and LOQ were less than 20 and 68 ng/mL, respectively. The relative standard deviation (RSD) values of intraday and inter-day precision were <3.31% for all the analytes. The recoveries of the method were in the range of 94.6-106.0%, with RSD <4.72%. The RSD values of peak areas in the stability study were not more than 4.28% for all analytes within 2 days. These results revealed that the method exhibited good sensitivity, precision, accuracy, and stability. Conclusions: This method could be applied to the quantitative determination of 15 flavonoids in the S. barbata - H. diffusa herb pair. Highlights: (1) The established method of HPLC-Q-TOF-MS is simple, fast, sensitive, and reliable. (2) 15 Flavonoids in the S. barbata-H. diffusa herb pair were simultaneous determined.

Two-dimensional liquid chromatography-mass spectrometry for the characterization of modified oligonucleotide impurities

A 2D-LC system coupled with a TOF mass spectrometer has been evaluated for its capabilities to provide enhanced characterization of oligonucleotide impurities. To address loop limitations in the total volume (40 μL) sampled across 1D peaks, a column trap was incorporated between the 1D and 2D columns. The main advantages of the column trap include reduction of the total number of sequential 2D runs required to fully sample broad 1D peaks, and most importantly, reduction of the error in quantitative determination of the components in broad 1D peaks by avoiding the numerical stitching of data from several 2D runs. Comprehensive RP x IP provided orthogonal separation despite its lower 1D resolution. In contrast, IP x IP did not provide orthogonal separation. RP x IP using the direct on-line extended heart-cutting system with the column trap showed additional benefits, in the elimination of off-line fractionation and sample handling errors and was successfully applied in a pH stability study of a crude oligonucleotide. SAX x IP successfully separated the isobaric “n+16” doublet of the “n + O” and “[n + S-O]” impurity species, a feat not currently possible by mass spectrometry alone or 1D-LC, demonstrating the importance of the added capabilities of the 2D-LC approach.