Electrospray Ionization Technique Research Articles

Controlled release behaviour of protein-loaded microparticles prepared via coaxial or emulsion electrospray

Biodegradable poly (lactic-co-glycolic acid) (PLGA) microparticles are an effective way to achieve sustained drug release. In this study, we investigated a sustained release model of PLGA microparticles with incorporated protein via either emulsion or coaxial electrospray techniques. PLGA (75:25) was used as the carrier, and bovine serum albumin as a model protein. Coaxial electrospray resulted in a type of core–shell structure with mean diameters of 2.41 ± 0.60 µm and a centralised protein distribution within the core. Emulsion electrospray formed bigger microparticles with mean diameters of 22.75 ± 8.05 µm and a heterogeneous protein distribution throughout the microparticles. The coaxial electrospray microparticles presented a much slighter burst release than the emulsion electrospray microparticles. Loading efficiency was significantly higher (p < 0.05) in the coaxial group than emulsion group. This indicated that both emulsion and coaxial electrospray could produce protein-loaded microparticles with sustained release behaviour, but the former revealed a superior approach for drug delivery.

Simultaneous determination of 24 constituents in Cortex Lycii using high-performance liquid chromatography–triple quadrupole mass spectrometry

A fast high-performance liquid chromatography (HPLC) coupled with electrospray ionization (ESI) tandem mass spectrometry method was developed to determine 24 components including 11 phenolic compounds, 9 phenolic amides, and 4 cyclic peptides in Cortex Lycii. The analytes were quantified by a triple quadrupole instrument in multiple reaction monitoring (MRM) mode. The fragmentation patterns of phenolic amides and cyclic peptides using ESI and collision-induced dissociation (CID) techniques are reported. This assay method was validated with respect to linearity (r2>0.9920), precision, repeatability, and accuracy (recovery rate between 93.0 and 105.9% with RSD<4.4%). The analytical results of 28 batches of Cortex Lycii indicated that cyclic peptides and phenolic amides were not only the abundant constituents, but also the characteristic components for Cortex Lycii to distinguish from the adulterants. Principle component analysis (PCA) was used to discriminate samples from different geographical regions of China, and cyclic peptides were considered to be the chemical markers responsible for the classification. The systematic and integrated assessment of Cortex Lycii provides sufficient evidence for the establishment of the quality standard.

Development of a validated UPLC–qTOF-MS/MS method for determination of bioactive constituent from Glycyrrhiza glabra

An ultra-performance liquid chromatography quadrupole time of flight mass spectrometry (UPLC–qTOF-MS/MS) method was developed and validated for the simultaneous determination of glycyrrhizin and glycyrrhetic acid. These analytes were separated on a reverse phase C18 column using a mobile phase of acetonitrile:2% acetic acid in water (75:25, v/v) with a flow rate of 200μL/min. The qTOF-MS was operated under multiple reaction monitoring (MRM) mode using the electrospray ionization (ESI) technique with positive ion polarity. A comparison of three different extraction techniques i.e. accelerated solvent extraction (ASE), extraction under ultrasonic waves (USW) and the classical extraction by percolation (CE) method was done and quantification of these extracts was also carried out by the proposed method.

C124 マルチジェットモード静電噴霧に関する実験的研究(OS-10:噴霧生成・混相流動)

Recently, micro-combustors which combust hydrocarbon fuels at micro scale are expected to be used as a small energy source. Since fast evaporation of liquid fuel is required, electrospray techniques, which can generate small droplets, have been employed. We focus on the multi-jet-mode electrospray, which produces many jets from a single capillary. We investigate effect of applied voltage to the capillary and fuel flow rate on the jet characteristics by visualization using a laser light sheet and nano flash shadow graph system. The investigation was conducted for some different diameters of capillaries and distance between the capillary grounded mesh.

Characterization of gas phase aggregates of bis(2‐ethylhexyl)sulfosuccinate (AOT) and divalent metal ions. Elimination of radical species in the decomposition pathways of even‐electron [AOTMIICl2]– anions

Structure and properties of even-electron anionic species formed by bis(2-ethylhexyl)sulfosuccinate (AOT) and divalent metal ions (M(II)) with stoichiometry [AOTM(II)Cl(2)](-) have been investigated by using electrospray ionization and different mass spectrometry techniques, such as high resolution, accurate mass measurements, collision-induced dissociation (CID) multiple-stage mass spectrometry. Owing to CID, eliminations of neutrals, mainly consisting in hydrochloric acid, 2-ethyl-1-hexene and 2-ethylhexanol, and an unexpected loss of an alkyl radical have been observed. The radical anions [C(4)HO(6)SM(II)Cl](-•) so produced have been characterized by MS(3) experiments. Density functional theory calculations have been carried out for investigating structure and stability of the ionic species formed in the decomposition pathways.

Simultaneous determination of morinidazole, its N-oxide, sulfate, and diastereoisomeric N+-glucuronides in human plasma by liquid chromatography–tandem mass spectrometry

Morinidazole is a new third-generation 5-nitroimidazole antimicrobial drug. To investigate the pharmacokinetic profiles of morinidazole and its major metabolites in humans, a liquid chromatography–tandem mass spectrometry method was developed and validated for simultaneous determination of morinidazole, its N-oxide metabolite (M4-1), a sulfate conjugate (M7), and two diastereoisomeric N+-glucuronides (M8-1 and M8-2) in human plasma. A simple acetonitrile-induced protein precipitation was employed to extract five analytes and internal standard metronidazole from 50μL human plasma. To avoid the interference from the in-source dissociation of the sulfate and achieve the baseline-separation of diastereoisomeric N+-glucuronides, all the analytes were separated from each other with the mobile phase consisting of 10mM ammonium formate and acetonitrile using gradient elution on a Hydro-RP C18 column (50mm×2mm, 4μm) with a total run time of 5min. The API 4000 triple quadrupole mass spectrometer was operated under the multiple reaction-monitoring mode using the electrospray ionization technique. The developed method was linear in the concentration ranges of 10.0–12,000ng/mL for morinidazole, 1.00–200ng/mL for M4-1, 2.50–500ng/mL for M7, 3.00–600ng/mL for M8-1, and 10.0–3000ng/mL for M8-2. The intra- and inter-day precisions for each analyte met the accepted value. Results of the stability of morinidazole and its metabolites in human plasma were also presented. The method was successfully applied to the clinical pharmacokinetic studies of morinidazole injection in healthy subjects, patients with moderate hepatic insufficiency, and patients with severe renal insufficiency, respectively.

Study on pharmacokinetics of scopolamine hydrobromide gel transdermally administered in rats by microdialysis

Objective: To establish and validate the HPLC-MS/MS method for the determination of scopolamine concentration in dermal microdialysis samples of rats,and study pharmacokinetics of scopolamine hydrobromide gel transdermally administered in rats.Methods: Scopolamine was separated with an Agilent Zorbax Extend C18 column.The mobile phase consisted of methanol-2 mmol/L ammonium formate(25∶75,the pH was adjusted to 3.5 by formic acid) at a flow rate of 0.3 ml/min and the column temperature was 40 ℃.The HPLC-MS/MS system was operated in the mode of multiple reaction monitoring with the electrospray ionization technique in positive mode.Results:Scopolamine was optimized at the transitions m/z 138.1-304.2.Calibration curve of scopolamine was linear within the range of 5.0-1 000 ng/ml.The inter day and intra day precision and accuracy were all less than 15%.Scopolamine could rapidly penetrate the skin and be absorbed.One hundred and fifty min after administration,the concentration of scopolamine reached peak,then dropped and maintained at a relatively high concentration range.The subcutaneous scopolamine concentration was decreased rapidly 9 h later.Conclusion:This method is sensitive and selective,and could be used to determine the concentration of scopolamine in the dermal microdialysis samples of the rats.

Validated LC-MS-MS Method for the Determination of Prodrug of Ginkgolide B in Rat Plasma and Brain: Application to Pharmacokinetic Study

A sensitive, simple and rapid liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous determination of ginkgolide B (GB) and the prodrug of ginkgolide B (PGB) in rat plasma and brain tissue. Detection was performed on a triple quadrupole tandem mass spectrometry in multiple reaction monitoring mode using the electrospray ionization technique in negative ionization mode. The total run time was only 2.0 min. Good linearity was found between 1-200 ng/mL (r > 0.9993) for plasma samples and 0.5-50 ng/mL (r = 0.9995) for the brain tissue samples. The lower limits of quantification of PGB and GB were 1 ng/mL for plasma and 0.5 ng/mL for brain tissue. Intra-day and inter-day precision was less than 11.67%. Intra-day accuracy was in the range of -2.61-10.67%, inter-day accuracy was in the range of -2.36-8.98%. The mean recovery for PGB and GB was between 82.5 and 97.0%. The validated method was successfully applied to a pharmacokinetic study of PGB and GB in rats after intravenous administration.

Electrosprayed polyelectrolyte complexes between mucoadhesive N,N,N,-trimethylchitosan-homocysteine thiolactone and alginate/carrageenan for camptothecin delivery

Novel hydrogel polyelectrolyte complexes (PECs) between the N,N,N,-trimethylchitosan-homocysteine thiolactone (TM-HT-chitosan) and two anionic polymers were investigated. The particles of pure thiolated chitosan and its PECs with alginate and carrageenan were fabricated using the electrospray ionization technique. The hydrogel PEC particles were characterized by scanning electron microscopy, dynamic light scattering, Fourier transform infrared microscopy, thermogravimetric analysis, encapsulation efficiency (EE), mucoadhesive property and in vitro drug release behavior. TM-HT-chitosan/alginate particles could be loaded with camptothecin (CPT), employed as a model anti-cancer drug, at an over 70% EE, and revealed both a reduced burst effect and a prolonged release of CPT over 3 days. The resultant TM-HT-chitosan/alginate PEC particles displayed a 5.60-, 1.86- and 1.55-fold stronger mucoadhesive property compared to that of the unmodified chitosan/alginate PEC at pH 1.2, 4.0 and 6.4, respectively, and this was not affected by the CPT loading level.

Enantioselective LC/ESI‐MS/MS Analysis and Pharmacokinetic and Tissue Distribution Study of (2RS)‐1‐(7‐Methoxy‐1H‐indol‐4‐yloxy)‐3‐(2‐(2‐methoxyphenoxy)ethylamino)‐propan‐2‐ol in Rats

A sensitive and stereospecific liquid chromatography-tandem mass spectrometry method for the quantitative determination of TWo8 enantiomers ((2RS)-1-(7-methoxy-1H-indol-4-yloxy)-3-(2-(2-methoxyphenoxy)ethylamino)-propan-2-ol) was developed and validated in rat serum and some tissues. Racemic TWo8 is a new chemical entity, and it has been shown to possess pharmacological activity in vivo. The assay involved the diastereomeric derivatization of racemic TWo8 with 2,3,4,6-tetra-O-acetyl-beta-glucopyranosyl isothiocyanate. The TWo8 diastereoisomers quantification was performed on a triple quadrupole mass spectrometer employing an electrospray ionization technique. The precursor to the product ion transition for TWo8 derivatives and for the internal standard (carbamazepine) was m/z 776.4 → 387.2 and 237.4 → 194.4, respectively. The assay was validated with a linear range of 10-2000 ng/ml of racemic TWo8. The inter-day precisions for (-)-(S)-TWo8 and (+)-(R)-TWo8 were 2.1% to 14.9% and 1.3% to 14.8%, respectively. The inter-day accuracy for (-)-(S)-TWo8 and (+)-(R)-TWo8 was within 86% to 114% and 91% to 114%, respectively. A pilot pharmacokinetic study of this new β-adrenolytic compound has shown that (-)-(S)-TWo8 is eliminated faster than its antipode. The terminal half-lives of (-)-(S)-TWo8 and (+)-(R)-TWo8 were 3.2 and 3.9 h, respectively. The compound distribution into different organs, evaluated in tissue homogenate samples following TWo8 intravenous administration, showed an enantioselective penetration of TWo8 enantiomers in the liver (p < 0.03), in the kidney (p < 0.001), and in the lungs (p < 0.05). The developed method using liquid chromatography-tandem mass spectrometry method with electrospray ionization could be employed for quantitative determination of compounds with similar structure.

Localization and Quantification of Drugs in Animal Tissues by Use of Desorption Electrospray Ionization Mass Spectrometry Imaging

Mass spectrometric imaging (MSI) has emerged as a powerful technique to obtain spatial arrangement of individual molecular ions in animal tissues. Ambient desorption electrospray ionization (DESI) technique is uniquely suited for such imaging experiments, as it can be performed on animal tissues in their native environment without prior treatments. Although MSI has become a rapid growing technique for localization of proteins, lipids, drugs, and endogenous compounds in different tissues, quantification of imaged targets has not been explored extensively. Here we present a novel MSI approach for localization and quantification of drugs in animal thin tissue sections. DESI-MSI using an Orbitrap mass analyzer in full scan mode was performed on 6 μm coronal brain sections from rats that were administered 2.5 mg/kg clozapine. Clozapine was localized and quantified in individual brain sections 45 min postdose. External calibration curves were prepared by micropipetting standards with internal standard (IS) on top of the tissues, and average response factors were calculated for the scans in which both clozapine and IS were detected. All response factors were normalized to area units. Quantifications from DESI-MSI revealed 0.2-1.2 ng of clozapine in individual brain sections, results that were further confirmed by extraction and liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis.

Development and validation of LC‐MS/MS method for the estimation of β‐hydroxy‐β‐methylbutyrate in rat plasma and its application to pharmacokinetic studies

A simple, sensitive and specific high-performance liquid chromatography mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of β-hydroxy-β-methyl butyrate (HMB) in small volumes of rat plasma using warfarin as an internal standard (IS). The API-4000 LC-MS/MS was operated under the multiple reaction-monitoring mode using the electrospray ionization technique. A simple liquid-liquid extraction process was used to extract HMB and IS from rat plasma. The total run time was 3 min and the elution of HMB and IS occurred at 1.48 and 1.75 min respectively; this was achieved with a mobile phase consisting of 0.1% formic acid in a water-acetonitrile mixture (15:85, v/v) at a flow rate of 1.0 mL/min on a Agilent Eclipse XDB C(8) (150 × 4.6, 5 µm) column. The developed method was validated in rat plasma with a lower limit of quantitation of 30.0 ng/mL for HMB. A linear response function was established for the range of concentrations 30-4600 ng/mL (r > 0.998) for HMB. The intra- and inter-day precision values for HMB were acceptable as per Food and Drug Administration guidelines. HMB was stable in the battery of stability studies, viz. bench-top, autosampler freeze-thaw cycles and long-term stability for 30 days in plasma. The developed assay method was applied to a bioavailability study in rats.

The Application and Perspective of Extractive Electrospray Ionization on The Protein Analysis*

The extractive electrospray ionization(EESI) technique is an emerging soft ionization technique and it can be used for the sensitive detection of large molecules and small molecules in solid,liquid,gas and viscous samples without sample pretreatment.Based on the brief introduction of the mechanism of EESI,its applications on the protein analysis were reviewed in detail.Compared to the commercial electrospray ionization(ESI) source,EESI maintains the native structure of protein and thus the biological activity of the protein to the most extent,which shows it can be potentially applied in the fields of preparation of protein microarray,high resolution hydrogen/deuterium exchange mass spectrometry(structure analysis of protein),and metrology for protein.

Development and Validation of a Highly Sensitive and Robust LC-MS/MS with Electrospray Ionization Method for Quantification of Rosuvastatin in Small Volume Human Plasma Samples and its Application to a Clinical Study

A high-throughput, simple, highly sensitive and specific LC-MS/MS method (liquid chromatography coupled with tandem mass spectrometry) has been developed for the estimation of rosuvastatin (CAS 287714-41-4, RST) with 100 microl human plasma using atorvastatin (CAS 134523-00-5) as an internal standard (IS). The API-4000 LC-MS/MS was operated under the multiple reaction-monitoring mode (MRM) using the electro spray ionization technique. The assay procedure involved direct precipitation of RST and IS from plasma with acetonitrile. Sample preparation with this method yielded clean extracts and consistent recoveries: 91.39% for RST and 99.28% for IS. The total chromatographic run time was 3.5 min and the elution of RST and IS occurred at 2.5 and 3.1 min, respectively; this was achieved with a mobile phase consisting of 0.05 mol/L formic acid: acetonitrile (20:80, v/v) at a flow rate of 0.50 ml/min on an Inertsil ODS-3 column (4.6 x 100 mm, 3.0 microm). The developed method was validated in human plasma with a limit of quantitation of 0.05 ng/ml. A linear response function was established for the range of concentrations of 0.05 to 50.0 ng/ml with a correlation coefficient (r) of 0.999. The inter- and intra-day precision in the measurement of RST quality control (QC) samples at 0.05, 0.15, 25 and 40 ng/ml were in the range of 6.55 to 11.40% relative standard deviation (RSD) and 1.76 to 11.17% RSD, respectively. Accuracy in the measurement of QC samples for RST was in the range of 95.02 to 101.37% of the nominal values. RST was stable in the battery of stability studies viz., bench-top, auto-sampler and freeze-thaw cycles. The stability of RST was established for 1 month at -80 degrees C. The application of the assay to a clinical study confirmed the utility of the assay to derive human pharmacokinetic parameters.

New Rapid HPLC Method for Separation and Determination of Benzo[A]Pyrene Hydroxyderivatives

A new rapid, one-step semi-quantitative HPLC-MS method applicable for the separation and characterization of the group of total 10 BaP hydroxyderivatives (1-hydroxy benzo[a]pyrene, 2-hydroxybenzo[a]pyrene, 3-hydroxybenzo[a]pyrene, 4-hydroxybenzo [a]pyrene, 7-hydroxybenzo[a]pyrene, 8-hydroxybenzo[a]pyrene, 9-hydroxybenzo[a]py rene, 10-hydroxybenzo[a]pyrene, 12-hydroxybenzo[a]pyrene and benzo[a]pyrene-9,10-dihydrodiol) was developed, involving electrospray ionization technique (ESI) in positive mode. Its qualitative and quantitative characteristics were selected by the analysis of the model OH-BaP mixture consisted of compounds mentioned above. The model system offered the linear response in the whole concentration range used (50–1000 ng.mL−1) as well as the resolution sufficient for semi-quantitative purposes (RS > 0.5). Subsequently, the suitability of the developed method was tested on BaP decomposition products to be formed during photooxidation at λ = 365 nm. These analyses confirmed the applicability of the proposed method, succeeding in the identification of 5 BaP hydroxyderivatives formed upon the BaP photooxidation experiment.

Electrospray Ionization Using Wooden Tips

Electrospray ionization (ESI) is a mass spectrometric technique widely used in various fields including chemistry, biology, medicine, pharmaceutical industry, clinical assessment, and forensic science. In this study, we report a simple and economical ESI-mass spectrometry (MS) technique, which makes use of disposable wooden tips (wooden toothpicks) for loading and ionization of samples. Samples could be loaded by normal pipetting onto the tip or simply dipping the tip into sample solutions. The hydrophilic and porous nature of wood allows effective adhesion of the sample solution for durable ion signals. The tip can be directly connected to nano-ESI ion sources of various mass spectrometers. Upon application of high voltage to the tip, desirable mass spectra could be obtained. We demostrated that this new technique is applicable for analysis of various samples, including organic compounds, organometallic compounds, peptides, proteins, and samples that cannot be directly analyzed by conventional ESI techniques, e.g., slurry samples and powder samples. The slim and hard properties of the wooden tip enable sampling from specific locations such as corners and small openings, indicating potential applications of the new technique in forensic investigations. The observation of electrospray ionization from wooden materials also allows us to get new insights into the materials that can be directly ionized for mass spectrometric analysis.

Differentiation of isomeric 2‐aryldimethyltetrahydro‐5‐quinolinones by electron ionization and electrospray ionization mass spectrometry

A series of isomeric 2-aryl-6,6-dimethyltetrahydro-5-quinolinones (set I) and 2-aryl-7,7-dimethyltetrahydro-5-quinolinones (set II) were studied under positive ion electron ionization (EI) and electrospray ionization (ESI) techniques. Under EI conditions, the molecular ions were found to be less stable in set I isomers, and they resulted in abundant fragment ions, i.e., [M-CH(3)](+), [M-CO](+.), [M-HCO](+), [M-(CH(3),CO)](+), and [M-(CH(3),CH(2)O)](+), when compared with set II isomers. In addition, the set I isomers showed specific fragment ions corresponding to [M-OH](+) and [M-OCH(3)](+). The retro-Diels-Alder (RDA) product ion was always higher in set II isomers. The ESI mass spectra produced [M + H](+) ions, and their decomposition showed favorable loss of CH(3) radical, CH(4) and C(2)H(6) molecules in set I isomers. The set II isomers, however, showed predominant RDA product ions, and specific loss of H(2)O. The selectivity in EI and ESI was attributed to the instability of set I isomers by the presence of a gem-dimethyl group at the α-position, and it was supported by the data from model compounds without a gem-dimethyl group. Density functional theory (DFT) calculations successfully corroborated the fragmentation pathways for diagnostic ions. This study revealed the effect of a gem-dimethyl group located at the α-position to the carbonyl having aromatic/unsaturated carbon on the other side of the carbonyl group.

Real-time measurement of UV-inactivated Escherichia coli bacterial particles by electrospray-assisted UVAPS spectrometry

The ultraviolet aerodynamic particle sizer (UVAPS) is a novel commercially available aerosol spectrometer for real-time continuous monitoring of viable bioaerosols, based on fluorescence from living microorganisms. In a previous study, we developed an electrospray-assisted UVAPS using biological electrospray techniques, which have the advantage of generating non-agglomerated single particles by the repulsive electrical forces. With this electrospraying of suspensions containing microorganisms, the analytical system can supply more accurate and quantitative information about living microorganisms than with conventional aerosolization. Using electrospray-assisted UVAPS, we investigated the characteristics of bacterial particles with various viabilities in real-time. Escherichia coli was used as the test microorganism, and its initial viability was controlled by the degree of exposure to UV irradiation. In the stable cone-jet domain, the particle size distributions of test bacterial particles remained almost uniform regardless of the degree of UV inactivation. However, the fluorescence spectra of the bacterial particles changed with the degree of UV inactivation. The fluorescence characteristics of UV-inactivated bacterial particles tended to show a similar decline with viability, determined by the sampling and culture method, although the percentage showing fluorescence was higher than that showing viability.

A rapid LC‐MS/MS method for quantitation of eszopiclone in human plasma: application to a human pharmacokinetic study

A highly reproducible, specific and cost-effective LC-MS/MS method was developed for simultaneous estimation of eszopiclone (ESZ) with 50 μL of human plasma using paroxetine as an internal standard (IS). The API-4000 LC-MS/MS was operated under the multiple reaction-monitoring mode using the electrospray ionization technique. A simple liquid-liquid extraction process was used to extract ESZ and IS from human plasma. The total run time was 1.5 min and the elution of ESZ and IS occurred at 0.90 min; this was achieved with a mobile phase consisting of 0.1% formic acid-methanol (15:85, v/v) at a flow rate of 0.50 mL/min on a Discover C(18) (50 × 4.6 mm, 5 µm) column. The developed method was validated in human plasma with a lower limit of quantitation of 0.1 ng/mL for ESZ. A linear response function was established for the range of concentrations 0.10-120 ng/mL (r > 0.998) for ESZ. The intra- and inter-day precision values for ESZ were acceptable as per FDA guidelines. Eszopiclone was stable in the battery of stability studies, viz. bench-top, autosampler and freeze-thaw cycles. The developed assay method was applied to an oral bioequivalence study in humans.

The Future of Biological Mass Spectrometry

Biological applications of mass spectrometry have grown exponentially since the discovery of MALDI and electrospray ionization techniques. This growth has been further fueled by the massive volume of DNA sequence information that is now available. An ambitious goal of some of this research is to monitor the level and modification of all proteins and metabolites in a biological sample such as plasma. A major research effort in mass spectrometry and related disciplines has been expended over the past several years toward reaching this and other less ambitious goals, and considerable progress has been made; but the presently available tools are clearly not sufficient for these very difficult tasks. In this "critical insight" discussion we suggest that recent advances in time-of-flight (TOF) technology with MALDI ionization may provide some important new tools for achieving the goals of biological research.