Electrospray Ionization Source Research Articles

All Ion Fragmentation Analysis Enhances the Untargeted Profiling of Glucosinolates in Brassica Microgreens by Liquid Chromatography and High-Resolution Mass Spectrometry.

An analytical approach based on reversed-phase liquid chromatography coupled to electrospray ionization Fourier-transform mass spectrometry in negative ion mode (RPLC-ESI-(-)-FTMS) was developed for the untargeted characterization of glucosinolates (GSL) in the polar extracts of four Brassica microgreen crops, namely, garden cress, rapeseed, kale, and broccoli raab. Specifically, the all ion fragmentation (AIF) operation mode enabled by a quadrupole-Orbitrap mass spectrometer, i.e., the systematic fragmentation of all ions generated in the electrospray source, followed by the acquisition of an FTMS spectrum, was exploited. First, the best qualifying product ions for GSL were recognized from higher-energy collisional dissociation (HCD)-FTMS2 spectra of representative standard GSL. Extracted ion chromatograms (EIC) were subsequently obtained for those ions from RPLC-ESI(-)-AIF-FTMS data referred to microgreen extracts, by plotting the intensity of their signals as a function of retention time. The alignment of peaks detected in the EIC traces was finally exploited for the recognition of peaks potentially related to GSL, with the EIC obtained for the sulfate radical anion [SO4]•- (exact m/z 95.9523) providing the highest selectivity. Each putative GSL was subsequently characterized by HCD-FTMS2 analyses and by collisionally induced dissociation (CID) multistage MSn (n = 2, 3) acquisitions based on a linear ion trap mass spectrometer. As a result, up to 27 different GSLs were identified in the four Brassica microgreens. The general method described in this work appears as a promising approach for the study of GSL, known and novel, in plant extracts.

Development and validation of a sensitive LC-MS/MS method for determination of intracellular concentration of fluconazole in Candida albicans.

Systemic candidiasis is the fourth leading cause of healthcare-associated infections worldwide. The combination therapy based on existing antifungal agents is well-established to overcome drug resistance and restore antifungal efficacy against drug-resistant strains. In this study, a simple and sensitive liquid chromatography with tandem mass spectrometry (LC-MS/MS) method was developed to quantify the intracellular fluconazole (FLC) content in the opportunistic human fungal pathogen Candida albicans. The cell lysates were prepared by lysing C. albicans cells with Precellys homogenizers and FLC was extracted with methylene chloride. The entire extraction approach was simple, precise and reliable. The extracts were separated on a Zorbax SB-C18 column using a mobile phase of acetonitrile (solvent A) and deionized water plus 0.1% formic acid. FLC and ketoconazole (KCZ, internal standard) were monitored in positive mode using electrospray ionization source. The multiple reaction monitoring transitions (precursor to product) were monitored for FLC m/z 307.1 → 238.2 and for the internal standard KCZ m/z 531.2 → 489.1. The linear for this method were in the range from 5.0 to 1000.0 ng/mL. The precision and accuracy of the samples were relative standard deviations (RSD) < 1.0% for intra-day and RSD < 0.51% for inter-day. The overall recovery of FLC from samples was higher than 77.61%. Furthermore, this method was successfully applied and validated in 36 clinical isolated strains. Taken together, we established a highly accurate, efficient, and reproducible method for quantifying the intracellular content of FLC in C. albicans.

Rapid Online Oxidation of Proteins and Peptides via Electrospray-Accelerated Ozonation.

Mass spectrometry-based analyses of protein conformation continue to grow in utilization due their speed, low sample requirements, and applicability to most protein systems. These techniques typically rely on chemical derivatization of proteins and as with all label-based analyses must ensure the integrity of the protein conformation throughout the duration of the labeling reaction. Hydroxyl radical footprinting of proteins and the recently developed fast fluoroalkylation of proteins attempt to bypass this consideration via rapid reactions that occur on time scales faster than protein folding, but they often require microfluidic setups or electromagnetic radiation sources. In this work, we demonstrate that ozonation of proteins and peptides, which normally occurs in the second to minute time scales, can be accelerated to the submillisecond to millisecond time scale with an electrospray ionization source. This rapid ozonation results in selective labeling of tryptophan and methionine residues. When applied to cytochrome C and carbonic anhydrase, this labeling technique is sensitive to solution conditions and correlates with solution-phase analyses of conformation. While significant work is still needed to characterize this fast chemical labeling strategy, it requires no complicated sample handling, electromagnetic radiation sources, or microfluidic systems outside of the electrospray source and may represent a facile alternative to other rapid labeling technologies that are utilized today.

Method optimization of the simultaneous detection of B12 congeners leading to the detection of a novel isomer of hydroxycobalamin in seawater.

More than half of surveyed microalgae and over 90% of harmful algae have an obligate requirement for vitamin B12 , but methods for directly measuring dissolved B12 in seawater are scarce due to low concentrations and rapid light-induced hydrolysis. We present a method to detect and measure the four main congeners of vitamin B12 dissolved in seawater. The method includes solid-phase extraction, separation by ultrahigh-performance liquid chromatography and detection by triple-quadrupole tandem mass spectrometry utilizing an electrospray ion source. This method was applied to coastal field samples collected in the German Bay, Baltic Sea and the Danish Limfjord system. The total dissolved B12 pool ranged between 0.5 and 2.1pM. Under ambient conditions methyl-B12 and adenosyl-B12 were nearly fully hydrolyzed to hydroxy-B12 in less than 1 h. Hydroxy-B12 and a novel, corresponding isomer were the main forms of B12 found at all field sites. This isomer eluted well after the OH-B12 peak and was also detected in commercially available OH-B12 . Both compounds showed very high similarity in their collision-induced dissociation spectra. The high instability of the biologically active forms of Me-B12 and Ado-B12 towards hydrolysis was shown, highlighting the importance of reducing the duration of the extraction protocol. In addition, the vitamin B12 pool in the study area was mostly comprised of a previously undescribed isomer of OH-B12 . Further studies into the structure of this isomer and its bioavailability are needed.

Next-Generation Infrared Matrix-Assisted Laser Desorption Electrospray Ionization Source for Mass Spectrometry Imaging and High-Throughput Screening.

Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) is a hybrid, ambient ionization source that combines the advantages of electrospray ionization and matrix-assisted laser desorption/ionization, making it a versatile tool for both high-throughput screening (HTS) and mass spectrometry imaging (MSI) studies. To expand the capabilities of the IR-MALDESI source, an entirely new architecture was designed to overcome the key limitations of the previous source. This next-generation (NextGen) IR-MALDESI source features a vertically mounted IR-laser, a planar translation stage with computerized sample height control, an aluminum enclosure, and a novel mass spectrometer interface plate. The NextGen IR-MALDESI source has improved user-friendliness, improved overall versatility, and can be coupled to numerous Orbitrap mass spectrometers to accommodate more research laboratories. In this work, we highlight the benefits of the NextGen IR-MALDESI source as an improved platform for MSI and direct analysis. We also optimize the NextGen MALDESI source component geometries to increase target ion abundances over a wide m/z range. Finally, documentation is provided for each NextGen IR-MALDESI part so that it can be replicated and incorporated into any lab space.

Oral fluid as a new investigative matrix for the determination of organic gunshot residue exposure

In recent years, increased use of ammunition without lead and heavy metals was observed, leading to a growing interest in the detection of organic gunshot residues (OGSR) as evidence of firearms related crimes. The wide range of compounds belonging to the OGSR class hinders their mass spectrometric detection as different ionization techniques may be needed to obtain good results for all compounds.The purpose of this work was the development of a reliable analytical method by means of UHPLC-HRMS for the determination in oral fluid (OF) of the most common explosives and the most used stabilizers, arising from fire discharge and post-deflagration residues.For this purpose, SPE was used for OF clean-up before UHPLC-HRMS analysis. All target analytes were chromatographically separated by means of a Polar-C18 column. A chlorinated compound was added to the mobile phases in order to promote the formation of chloride adduct ions in the electrospray ion source operating in polarity switching to allow the best conditions for each analyte. The detection was conducted by means of a high-resolution mass spectrometer equipped with Orbitrap technology working in data dependent acquisition mode, in order to detect both the precursor ions and/or the most intense fragments for stabilizers.To verify its potential, the method was tested on real samples: a shooting session was performed in an open shooting range; the shooters fired from 2 to 20 rounds with a 9x21 caliber, thereafter OF was sampled. Samples were analyzed confirming that explosives may be detected in OF; the use of this matrix may be of great interest for investigative purposes as it is less affected by secondary transfer when compared to other commonly sampled matrices. The developed method could be a useful tool for law enforcement authorities for the detection of explosives in forensic potential scenarios, including biological matrices.

Rapid Acid/Base Switching in Flow Injection Analysis and Isocratic Elution Liquid Chromatography with Mass Spectrometric Detection for Improved Sensitivity.

Ion signals in electrospray ionization (ESI) mass spectrometry (MS) are affected by addition of acid or base. Acids or bases are typically added to samples to enhance detection of analytes in positive- or negative-ion mode, respectively. To carry out simultaneous monitoring of analytes with different ionogenic moieties by ESI-MS, a rapid acid/base switching system was developed. The system was further coupled with flow injection analysis (FIA) and liquid chromatography (LC) MS. The two variants enable detection of separated analytes immediately after alternating addition of acid and base. The methods were tested using a set of phospholipids (PLs) as analytes. The rapid acid/base switching enhanced signals of some of the PL analytes in both ion modes of MS. Both FIA-MS and LC-MS with acid/base switching show signal enhancements (∼1.3-23.2 times) of some analyte signals when compared with analysis conducted without acid/base switching. The proposed methods are suitable for simultaneous analysis of cationic and anionic analytes. The FIA-MS and LC-MS methods with acid/base switching were also applied in analysis of lipid extract from real samples (sausage and porcine liver). However, the FIA-MS results were affected by ionization competition and isobaric interference due to the complexity of the sample matrix and diversity of PL species. In contrast, the LC-MS mode provides adequate selectivity to observe signal enhancement for specific analyte ions. Overall, alternating addition of acid and base immediately before the ESI source can improve analytical performance without the need to carry out separate analyses targeting different types of analytes.

Scalable Microfabrication of Multi-Emitter Arrays in Silicon for a Compact Microfluidic Electrospray Propulsion System.

The recent proliferation of SmallSats and their use in increasingly demanding applications require the development of onboard electric propulsion compatible with the power, mass, and volume constraints of these spacecraft. Electrospray propulsion is a promising technology for SmallSats due to its unique high efficiency and scalability across the wide power range of these platforms, for example, from a few watts available in a CubeSat to a few hundred watts in a MiniSat. The implementation of electrospray propulsion requires the use of microfabrication techniques to create compact arrays of thousands of electrospray emitters. This article demonstrates the microfabrication of multi-emitter electrospray sources of a scalable size for electrospray propulsion. In particular, a microfabrication and assembly process is developed and demonstrated by fabricating sources with arrays of 1, 64, and 256 emitters. The electrospray sources are tested in a relevant environment for space propulsion (inside a vacuum chamber), exhibiting excellent propulsive performance (e.g., absence of beam impingement in the extractor electrode, absence of hysteresis in the beam current versus propellant flow rate characteristic, proper operation in the cone-jet electrospraying mode, etc.) and nearly coincident output per emitter. Several design elements contribute to this performance: the even distribution of the propellant among all emitters made possible by the implementation of a network of microfluidic channels in the backside of the emitter array; the small dead volume of the network of microfluidic channels; the accurate alignment between the emitters and extractor orifices; and the use of a pipe-flow configuration to drive the propellant through closed conduits, which protects the propellant.

Developing transmission mode for infrared matrix-assisted laser desorption electrospray ionization mass spectrometry imaging.

RationaleThe development and characterization of the novel NextGen infrared matrix‐assisted laser desorption electrospray ionization (IR‐MALDESI) source catalyzed new advancements in IR‐MALDESI instrumentation, including the development of a new analysis geometry.MethodsA vertically oriented transmission mode (tm)‐IR‐MALDESI setup was developed and optimized on thawed mouse tissue. In addition, glycerol was introduced as an alternative energy‐absorbing matrix for tm‐IR‐MALDESI because the new geometry does not currently allow for the formation of an ice matrix. The tm geom was evaluated against the optimized standard geometry for the NextGen source in reflection mode (rm).ResultsIt was found that tm‐IR‐MALDESI produces comparable results to rm‐IR‐MALDESI after optimization. The attempt to incorporate glycerol as an alternative matrix provided little improvement to tm‐IR‐MALDESI ion abundances.ConclusionsThis work has successfully demonstrated the adaptation of the NextGen IR‐MALDESI source through the feasibility of tm‐IR‐MALDESI mass spectrometry imaging on mammalian tissue, expanding future biological applications of the method.

A UPLC- MS/MS Method to Quantify β-Sitosterol and Ferulic Acid of Pygeum Africanum Extract in Bulk and Pharmaceutical Preparation.

This study uses a liquid chromatography-electrospray ionization-tandem mass spectrometry method to determine β-Sitosterol and Ferulic acid in Pygeum africanum extract. Chromatographic separation of the two analytes was performed on an ACQUITY UPLC H-Class system coupled with Xevo TQD mass spectrometer and HSS T3 C18 column (2.1 X 50mm, 1.8μm). Mobile phase A consisted of an aqueous solution of 0.1% formic acid (v/v), and mobile phase B was 0.1% formic acid (v/v) in methanol pumped through a gradient elution mode. Mass spectrometer parameters were optimized using an electrospray ionization source in the positive and negative ionization modes. The quantification of the two analytes was performed using multiple reaction monitoring transitions. The method was fully validated per (FDA) guidelines regarding linearity, accuracy, precision, carryover and selectivity. The proposed method was applied successfully to determine the two investigated compounds in commercially available pharmaceutical products.

Determination of Ethyl Glucuronide and Ethyl Sulfate in Dried Blood Spots by UHPLC-MS-MS: Method Validation and Assessment of Ethanol Exposure in Postmortem Samples from Road Traffic Victims.

The determination of ethyl glucuronide (EtG) and ethyl sulfate (EtS) in blood has been proposed in clinical and forensic applications to identify recent alcohol consumption. Also, there is a growing interest on the use of dried blood spots (DBS) in toxicological analysis, allowing increased stability of the analytes and simplifying sample transportation and storage. This study presents the development and validation of a method for quantifying EtG and EtS in DBS using ultra-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS-MS). The DBS samples were extracted with a mixture of methanol and acetonitrile (80:20 v/v) and analyzed using UHPLC-MS-MS with electrospray source in negative mode, after separation with a fluoro-phenyl stationary phase. Validation was performed according to the Scientific Working Group for Forensic Toxicology (SWGTOX) guidelines, with calibrations ranging from 0.10 to 18 µg/mL for EtG and 0.02 to 6 µg/mL for EtS. The analytes were stable in DBS stored from -20 to 45°C for 21 days. The method was successfully applied to capillary and venous DBS samples from 20 volunteers after ethanol ingestion and to DBS samples from 99 fatal victims of road traffic injuries. Capillary DBS was comparable to venous DBS and fresh whole blood in Passing-Bablok and Bland-Altman analysis, with correlation coefficients >0.91 (P < 0.001) for all comparisons. In postmortem application, the DBS EtG and EtS analysis indicated positive exposure to ethanol in 72.7% of the cases (EtG: 0.10-24.0 µg/mL and EtS: 0.03-4.11 µg/mL). The identification of ethanol consumption from blood alcohol concentrations (BACs) and EtG/EtS in DBS was in agreement in 98.6% of positive and 96.3% of negative cases (kappa 0.877, P < 0.001), indicating a high level of concordance with BAC in assessing alcohol use in postmortem samples.

Fast and Sensitive HPLC-ESI-MS/MS Method for Etoricoxib Quantification in Human Plasma and Application to Bioequivalence Study.

Etoricoxib is a non-steroidal anti-inflammatory drug (NSAID) used to treat pain and inflammation. The objective of the current study was to develop a sensitive, fast and high-throughput HPLC-ESI-MS/MS method to measure etoricoxib levels in human plasma using a one-step methanol protein precipitation technique. A tandem mass spectrometer equipped with an electrospray ionization (ESI) source operated in a positive mode and multiple reaction monitoring (MRM) were used for data collection. The quantitative MRM transition ions were m/z 359.15 > 279.10 and m/z 363.10 > 282.10 for etoricoxib and IS. The linear range was from 10.00 to 4000.39 ng/mL and the validation parameters were within the acceptance limits of the European Medicine Agency (EMA) and Food and Drug Analysis (FDA) guidelines. The present method was sensitive (10.00 ng/mL with S/N > 40), simple, selective (K prime > 2), and fast (short run time of 2 min), with negligible matrix effect and consistent recovery, suitable for high throughput analysis. The method was used to quantitate etoricoxib plasma concentrations in a bioequivalence study of two 120 mg etoricoxib formulations. Incurred sample reanalysis results further supported that the method was robust and reproducible.

Simultaneous determination and pharmacokinetic study of six components in beagle dog plasma by UPLC–MS/MS after oral administration of Astragalus Membranaceus aqueous extract

Astragalus Membranaceus (AM) is widely applied in Chinese herbal compound formulas for treating various kinds of diseases. However, relative pharmacokinetics data on AM in nonrodents is still lacking. Here, an UPLC-MS/MS method for determining the six main compounds of AM was developed. The chromatographic separation was carried out by a Waters Acquity UPLC HSS T3 column (100 × 2.1 mm, 1.8μm) with gradient elution of water-formic acid (99.98:0.02, v/v) and acetonitrile-formic acid (99.98:0.02, v/v) at a flow rate of 0.3ml/min within 11 min. Analyses of all compounds were conducted in multiple reaction monitoring mode with a positive/negative ion-switching mode of an electrospray ionization source in a single run. The analytical method was validated in terms of specificity, linearity, accuracy, precision, stability, etc. The method showed excellent linearity (r > 0.999) over certain concentration ranges. The intra-day and inter-day precisions were evaluated, and the RSD values were <12.4%. Furthermore, the validated method was successfully applied to determine the six components in plasma after oral administration of AM aqueous extract to beagle dogs and the pharmacokinetic parameters were obtained. Together, this study provides a reference for medication in the clinical practice of AM.

Simultaneous determination of three naphthoquinones from Arnebia euchroma (Royle) Johnst in beagle plasma by UPLC-MS/MS and application for pharmacokinetics study

Abstract A rapid, simple and efficient ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS) method was established to simultaneous determination of shikonin, isobutyryl shikonin, β, βʹ-dimethylacryl alkanin in beagle plasma and evaluated by using esculetin as internal standard. The electrospray ionization (ESI) source was operated in negative ionization mode. Multi-reaction monitoring (MRM) was used to quantitatively analyzed shikonin m/z 287.0 → 217.9, isobutyryl shikonin m/z 357.0 → 268.9, β, βʹ-dimethylacryl alkanin m/z 370.0 → 270.1 and esculetin m/z 177.0 → 89.0, respectively. The method was validated for selectivity, linearity, lower limit of quantification, precision, accuracy, recovery, matrix effect and stability. All validation parameters met the acceptance criteria according to regulatory guidelines. This method was successfully applied for the pharmacokinetic study of shikonin, isobutyryl shikonin, β, βʹ-dimethylacryl alkanin in beagle dogs plasma after oral administration of A. euchroma extract.

A Sensitive Liquid Chromatography-Mass Spectrometry Method for Determination of 14-Deoxy-12(R)-Sulfo Andrographolide Concentration in Rat Plasma and its Application to a Pharmacokinetic Study.

Andrographolide is a promising natural substance with numerous pharmacotherapy uses. 14-deoxy-12(R)-sulfo andrographolide (SAP) is the main metabolite of andrographolide in the intestine. To investigate the pharmacokinetic properties of SAP, a precise and sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the determination of SAP concentration in rat plasma was developed and validated in this study. Chromatographic separation was achieved on an Acpuity UPLC BEH C18 column with gradient elution that consisted of methanol and water at a flow rate of 0.3 mL/min. MS/MS detection was carried out by the multiple reaction monitoring (MRM) mode with negative electrospray ionization (ESI-) source, with the transitions of m/z 413.2→m/z 287.2 for SAP and m/z 269→m/z 133 for genistein [which was used as an internal standard (IS)]. The calibration curve of SAP was linear over the concentration range of 5-120 ng/mL. The selectivity, precision, accuracy, extraction recovery, matrix effect, and stability of the method were within acceptable ranges. This SAP quantification method was then successfully applied to a pharmacokinetic study of SAP. The area under the curve (AUC) of SAP in rats treated with SAP at 60 mg/kg by intravenous administration was 7498.53 ± 2405.02 mg/L·min. The AUC of SAP in rats treated with SAP at 60 mg/kg by oral administration was 97.74 ± 39.56 mg/L·min. Thus, the absolute oral bioavailability of SAP was determined to be 1.40%.

Development of validated UHPLC–PDA with ESI–MS-MS method for concurrent estimation of magnoflorine, berbamine, columbamine, jatrorrhizine, palmatine and berberine in Berberis aristata

Abstract A validated UHPLC-PDA with an ESI-MS/MS method has been developed for simultaneous estimation of six bioactive alkaloids (magnoflorine, berbamine, columbamine, jatrorrhizine, palmatine and berberine) in the different extracts of the roots of Berberis aristata DC (Family:Berberdiaceae). It is an important medicinal herb native to Northern Himalaya and commonly known as ‘daruharidra’, ‘daruhaldi’, ‘Indian barberry’ or ‘tree turmeric’. An insight into the research literature uncovered reports on isoquinoline alkaloids like magnoflorine, berbamine, columbamine, jatrorrhizine, palmatine, and berberine as major bioactives in B. aristata roots, possessing different pharmacological and therapeutic effects. In the present study, these aforementioned alkaloids were separated on Phenomenex Luna®, 5 µm-C8 analytical column. The HPLC-MS analysis was performed at a flow rate of 0.90 mL min−1. Each alkaloid that is resolved was characterized by precursor ions and fragment ions with electrospray ionization (ESI) source in both positive and negative ionization using scan mode. The limit of detections (LODs) were 0.087, 0.727, 0.035, 0.124, 0.782 and 0.794 μg mL−1 for magnoflorine, berbamine, columbamine, jatrorrhizine, palmatine and berberine, respectively. The proposed UHPLC-PDA method was fully validated according to international (ICH) guidelines and was found to be selective, sensitive and highly accurate for the concomitant estimation of the aforementioned symbolic bio-markers of B. aristata roots.

Antioxidant Activity, Total Phenolic and Flavonoid Content and LC–MS Profiling of Leaves Extracts of Alstonia angustiloba

Plants have a wide range of active compounds crucial in treating various diseases. Most people consume plants and herbals as an alternative medicine to improve their health and abilities. A. angustiloba extract showed antinematodal activity against Bursaphelenchus xylophilus, antitrypanosomal action against Trypanosoma brucei and anti-plasmodial activity against the chloroquine-resistant Plasmodium falciparum K1 strain. Moreover, it has demonstrated growth inhibitory properties towards several human cancer cell lines, such as MDA-MB-231, SKOV-3, HeLa, KB cells and A431. DPPH and ABTS assays were carried out to determine the antioxidant activity of the aqueous and 60% methanolic extract of A. angustiloba leaves. Moreover, total phenolic and flavonoid contents were quantified. The presence of potential active compounds was then screened using liquid chromatography coupled with a Q-TOF mass spectrometer (LC–MS) equipped with a dual electrospray ionisation (ESI) source. The EC50 values measured by DPPH for the 60% methanolic and aqueous extracts of A. angustiloba leaves were 80.38 and 94.11 µg/mL, respectively, and for the ABTS assays were 85.80 and 115.43 µg/mL, respectively. The 60% methanolic extract exhibited the highest value of total phenolic and total flavonoid (382.53 ± 15.00 mg GAE/g and 23.45 ± 1.04 mg QE/g), while the aqueous extract had the least value (301.17 ± 3.49 mg GAE/g and 9.73 ± 1.76 mg QE/g). The LC–MS analysis revealed the presence of 103 and 140 compounds in the aqueous and 60% methanolic extract, respectively. It consists of phenolic acids, flavonoids, alkaloids, amino acids, glycosides, alkaloids, etc. It can be concluded that the therapeutic action of this plant is derived from the presence of various active compounds; however, further research is necessary to determine its efficacy in treating diseases.

Study on metabolites in vivo of Dangefentong Capsules based on UHPLC-Q/Orbitrap-MS/MS

Dangefentong Capsules is a new traditional Chinese medicine preparation for the treatment of diabetic peripheral neuropathy. It is based on the Salviae Miltiorrhizae Radix et Rhizoma-Puerariae Lobatae Radix herb pair with salvianolic acids, tanshinones and pueraria flavonoids as main components. Studying the chemical composition in vivo of Dangefentong Capsules and its metabolites is of great significance for making clear its pharmacodynamic material basis and the action mechanism. The UHPLC-Q/Orbitrap-MS/MS was applied to rapidly analyze the metabolites and metabolic pathways of Dangefentong Capsules in Beagle dogs after gavage. Eclipse plus C_(18) column(2.1 mm×50 mm, 1.8 μm) was used, and gradient elution was performed with 0.1% formic acid aqueous solution(A)-formic acid acetonitrile solution(B). A heated electrospray ion source(HESI) was employed. The scanning mode was set as the positive and negative ion mode, and the mass scanning range was m/z 100-1 000. The plasma, urine and feces samples were collected after male Beagle dogs were administered with Dangefentong Capsules. The prototype components and metabolites were identified by UHPLC-Q/Orbitrap-MS/MS analysis combined with reference substances and references. The results showed that 107 chemical components were identified, including 58 prototype components and 49 metabolites. The identified prototype components included 42 components from Salviae Miltiorrhizae Radix et Rhizoma and 16 components from Puerariae Lobatae Radix. The metabolites consist of 21 and 28 metabolites of Salviae Miltiorrhizae Radix et Rhizoma and Puerariae Lobatae Radix, respectively. They are mainly derived from the methylation, hydroxylation, sulfation and glucuronidation of salvianolic acids, tanshinones and pueraria flavonoids. This research rapi-dly analyzes the chemical components in vivo of Beagle dogs administered with Dangefentong Capsules, laying a basis for illustrating the pharmacodynamic material basis and mechanism of Dangefentong Capsules.

Advancing Cyclic Ion Mobility Mass Spectrometry Methods for Studying Biomolecules: Toward the Conformational Dynamics of Mega Dalton Protein Aggregates.

Native mass spectrometry is a powerful tool for the analysis of noncovalent complexes. When coupled with high-resolution ion mobility, this technique can be used to investigate the conformational changes induced in said complexes by different solution or gas-phase conditions. In this study, we describe how a new-generation high-resolution ion mobility instrument equipped with a cyclic ion mobility cell can be utilized for the analysis of large biomolecular systems, including temperature-induced protein aggregates of masses greater than 1.5 MDa, as well as a 63 kDa oligonucleotide complex. The effects of and the interplay between the voltages applied to the different components of the cyclic ion mobility spectrometry system on ion transmission and arrival time distribution were demonstrated using biomolecules covering the m/z range 2000-10,000. These data were used to establish a theoretical framework for achieving the best separation in the cyclic ion mobility system. Finally, the cyclic ion mobility mass spectrometer was coupled with a temperature-controlled electrospray ionization source to investigate high-mass protein aggregation. This analysis showed that it was possible to continuously monitor the change in abundance for several conformations of MDa aggregates with increasing temperature. This work significantly increases the range of biomolecules that can be analyzed by both cyclic ion mobility and temperature-controlled electrospray ionization mass spectrometry, providing new possibilities for high-resolution ion mobility analysis.

Simultaneous Determination and Pharmacokinetics Study of Three Triterpenes from Sanguisorba officinalis L. in Rats by UHPLC–MS/MS

A selective and rapid ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method was established and validated for the determination of ziyuglycoside I, 3β,19α-dihydroxyurs-12-en-28-oic-acid 28-β-d-glucopyranosyl ester, and pomolic acid in rats after the oral administration of ziyuglycoside I, 3β,19α-dihydroxyurs-12-en-28-oic-acid 28-β-d-glucopyranosyl ester, pomolic acid, and Sanguisorba officinalis L. extract. The separation was carried out on an ACQUITY UPLC®HSS T3 column (2.1 mm × 100 mm, 1.8 μm), using methanol and 5 mmol/L ammonium acetate water as the mobile phase. The three compounds were quantified using the multiple reaction monitoring mode with the electrospray ion source in both the positive and negative mode. Liquid-liquid extraction was applied to the plasma sample preparation. Bifendate was selected as the internal standard. The intra-day and inter-day precision and the accuracy of the method were all within receivable ranges. The lower limit of quantification of ziyuglycoside I, 3β,19α-dihydroxyurs-12-en-28-oic-acid 28-β-d-glucopyranosyl ester, and pomolic acid were 6.50, 5.75, and 2.63 ng/mL, respectively. The extraction recoveries of analytes in rat plasma ranged from 83 to 94%. The three components could be rapidly absorbed into the blood (Tmax, 1.4–1.6 h) both in the single-administration group or S. officinalis extract group, but the first peak of PA occurred at 0.5 h and the second peak at 4–5 h in the S. officinalis extract. Three compounds were eliminated relatively slowly (t1/2, 7.3–11 h). The research was to establish a rapid, sensible, and sensitive UHPLC–MS/MS method using the multi-ion mode for multi-channel simultaneous mensuration pharmacokinetics parameters of three compounds in rats after oral administration of S. officinalis extract. This study found, for the first time, differences in the pharmacokinetic parameters of the three compounds in the monomer compounds and S. officinalis extract administration, which preliminarily revealed the transformation and metabolism of the three compounds in vivo.