Negative Electrospray Ionization Mass Spectrometry Research Articles

Aging of α-Pinene Secondary Organic Aerosol by Hydroxyl Radicals in the Aqueous Phase: Kinetics and Products.

The reaction of hydroxyl radicals (OH) with a water-soluble fraction of the α-pinene secondary organic aerosol (SOA) was investigated using liquid chromatography coupled with negative electrospray ionization mass spectrometry. The SOA was generated by the dark ozonolysis of α-pinene, extracted into the water, and subjected to chemical aging by the OH. Bimolecular reaction rate coefficients (kOH) for the oxidation of terpenoic acids by the OH were measured using the relative rate method. The unaged SOA was dominated by the cyclobutyl-ring-retaining compounds, primarily cis-pinonic, cis-pinic, and hydroxy-pinonic acids. Aqueous oxidation by the OH resulted in the removal of early-stage products and dimers, including well-known oligomers with MW = 358 and 368 Da. Furthermore, a 2- to 5-fold increase in the concentration of cyclobutyl-ring-opening products was observed, including terpenylic and diaterpenylic acids and diaterpenylic acid acetate as well as some of the newly identified OH aging markers. At the same time, results obtained from the kinetic box model showed a high degree of SOA fragmentation following the reaction with the OH, which indicates that non-radical reactions occurring during the evaporation of water likely contribute to the high yields of terpenoic aqSOAs reported previously. The estimated atmospheric lifetimes showed that in clouds, terpenoic acids react with the OH exclusively in the aqueous phase. Aqueous OH aging of the α-pinene SOA results in a 10% increase of the average O/C ratio and a 3-fold decrease in the average kOH value, which is likely to affect the cloud condensation nuclei activity of the aqSOA formed after the evaporation of water.

Speciation and molecular characterization of thiophenic and sulfide compounds in petroleum by sulfonation and methylation followed by electrospray mass spectrometry.

Thiophenes and sulfides are the dominant sulfur-containing compounds in petroleum and have been widely ofconcern in the fields of petroleum refining and geochemistry. In this study, a novel approach was developed for selective separation and characterization of petroleum-derived thiophenic and sulfide compounds. Thiophenic compounds were selectively converted to sulfonates in the presence of vitriolic acid and can be characterized by negative ion electrospray mass spectrometry. Thiophenic sulfonates were further separated from the oil by silica chromatography and enabled the molecular characterization of sulfides in the residual oil. Various model sulfur compounds and a vacuum gas oil were used to validate the method; thiophenic and sulfide biomarker compounds in a well-documented crude oil were selectively characterized. The results indicate that the approach is feasible for molecular characterization of thiophenic and sulfide compounds, which is complementary to recently developed methods for separation and/or ionization of sulfur compounds in petroleum.

Unexpected Photocatalytic Degeneration of NAD + for Inducing Apoptosis of Hypoxia Cancer Cells

Unexpected Photocatalytic Degeneration of NAD ⁺ for Inducing Apoptosis of Hypoxia Cancer Cells

HESI-MS/MS Analysis of Phenolic Compounds from Calendula aegyptiaca Fruits Extracts and Evaluation of Their Antioxidant Activities.

Considering medicinal plants as an inexhaustible source of active ingredients that may be easily isolated using simple and inexpensive techniques, phytotherapy is becoming increasingly popular. Various experimental approaches and analytical methods have been used to demonstrate that the genus Calendula (Asteraceae) has a particular richness in active ingredients, especially phenolic compounds, which justifies the growing interest in scientific studies on this genus’ species. From a chemical and biological viewpoint, Calendula aegyptiaca is a little-studied plant. For the first time, high-performance liquid chromatography combined with negative electrospray ionization mass spectrometry (HPLC-HESI-MS) was used to analyze methanolic extracts of Calendula aegyptiaca (C. aegyptiaca) fruits. Thirty-five molecules were identified. Flavonoids (47.87%), phenolic acids (5.18%), and saponins (6.47%) formed the majority of these chemicals. Rutin, caffeic acid hexoside, and Soyasaponin βg’ were the most abundant molecules in the fruit methanolic extract, accounting for 17.49% of total flavonoids, 2.32 % of total phenolic acids, and 0.95% of total saponins, respectively. The antioxidant activity of the fruit extracts of C. aegyptiaca was investigated using FRAP, TAC, and DPPH as well as flavonoids and total phenols content. Because the phenolic components were more extractable using polar solvents, the antioxidant activity of the methanolic extract was found to be higher than that of the dichloromethane and hexane extracts. The IC50 value for DPPH of methanolic extract was found to be 0.041 mg·mL−1. Our findings showed that C. aegyptiaca is an important source of physiologically active compounds.

Molecular characterization of aromatics in petroleum fractions by combining silica sulfuric acid sulfonation with electrospray ionization high-resolution mass spectrometry

Aromatic hydrocarbons are one of the main components of petroleum and its fractions. However, the characterization of its molecular composition is still a technical challenge for mass spectrometry because of the lack of ionization methods, especially for quantitative analysis. This study presents a novel approach for the molecular characterization of aromatics in petroleum fractions. Aromatic hydrocarbons were converted into their corresponding sulfonates and then analyzed by negative-ion electrospray ionization mass spectrometry. The method showed good selectivity and sensitivity for aromatic hydrocarbons. More importantly, no significant discrimination was found on monoaromatic compouds compared with atmospheric pressure photoionization, which has been considered the most suitable technique for aromatic hydrocarbon analysis. The method was used to characterize two vacuum gas oil samples, and the result was verified by the hydrocarbon group composition obtained by the ASTM standard method. The method enables a semiquantitative anlaysis for molecular characterization of aromatic compounds in heavy petroleum fractions. In addition, this method can be used to distinguish monoaromatic steranes from alkyl naphthalenes which have the same molecular composition.

A Novel Liquid Chromatography-Tandem Mass Spectrometry Method for Simultaneous Quantification of Telmisartan and Chlorthalidone in Rat Plasma and its Application to a Pharmacokinetic Study

A selective, sensitive, rapid, and stable liquid chromatography-tandem mass spectrometric (LC-MS/MS) assay method has been developed for the simultaneous quantification of chlorthalidone (CLT) and telmisartan (TEL) in rat plasma. Sample preparation involved liquid-liquid extraction by ethyl acetate. Analytes and internal standard (IS) were separated on Gemini C18 (50 x 4.6 mm, 5 μm) using a mixture of methanol-2 mM ammonium acetate (80:20 v/v) as the mobile phase at a flow rate of 0.4 mL/min. Hydrochlorothiazide (HCTZ) was used as an internal standard. Detection was carried out using Electrospray positive and negative ionization mass spectrometry using multiple reaction monitoring of the transition at m/z 515.90→276 for TEL and m/z 337.1→190.05 for CLT, m/z 296.1→205.3 for HCTZ respectively. For TEL and CLT, the method was linear (R2 ≥ 0.995) within the range of 6-1200 ng/mL and 3-600 ng/mL respectively. The retention time for TEL, CLT, and IS was found to be 3.65±0.03 min, 1.83±0.02 min, and 1.76±0.01 min. The mean recovery for both analytes and IS was greater than 70 %. The developed method was validated according to the USFDA guideline. The quantitative method was validated for specificity, linearity, accuracy, precision, recovery, dilution integrity, matrix effect, and analyte stability. The method was successfully applied to a pharmacokinetic study in rat plasma following oral administration.

A simplified method for determination of short-, medium-, and long-chain chlorinated paraffins using tetramethyl ammonium chloride as mobile phase modifier

The formation of halide adducts ion is an important pathway to improve the sensitivity of analytes in liquid chromatography (LC) combined with negative electrospray ionization (ESI) mass spectrometry (MS). Although adding modifier in mobile phase is generally the simplest way to form anions adducts, the formation of halide adducts ion requires a complex post-column addition strategy since traditional halide ionization enhancement reagents are incompatible with LC systems. To solve this problem, the volatile organochlorine salt tetramethyl ammonium chloride (TMAC) was therefore investigated as a potentially non-corrosive mobile phase modifier that was confirmed to be compatible with both LC and MS systems in this study. When short-chain, medium-chain, and long-chain chlorinated paraffins (CPs) were determinated simultaneously by ultra-high performance LC combined with ESI high resolution MS (UPLC-ESI-HRMS), all of them tended to ionize by forming [M+Cl]− ions and exhibited excellent sensitivity with the instrumental detection limits of 1-4 pg/μL. Meanwhile, their sensitivities towards CPs were less dependent on their Cl content with the total relative response factors of 0.8-3.5. The method's utility was demonstrated through determination of CPs in surface soil and chicken muscle samples. This was an effective and practical method to enhance the selectivity for [M + Cl]− ions and improve sensitivity towards CPs with various carbon lengths. Importantly, post-column addition was not required, and thus the analytical procedure was simplified. The method has also improved sensitivity towards some other organohalides and may be generally useful in the determination of challenging organic analytes.

Selective Removal of Thiophenic Compounds in Petroleum by Vitriolic Acid Sulfonation and Molecular Characterization Using Negative-Ion Electrospray Mass Spectrometry

Selective Removal of Thiophenic Compounds in Petroleum by Vitriolic Acid Sulfonation and Molecular Characterization Using Negative-Ion Electrospray Mass Spectrometry

Bisecting GlcNAc Protein N-Glycosylation Is Characteristic of Human Adipogenesis.

Human adipose tissue contains a major source of adipose-derived stem cells (ADSCs) that have the ability to differentiate into various cell types: in vitro, ADSCs can differentiate into mesenchymal lineages including adipocytes, while in vivo, ADSCs become mature adipocytes. Protein glycosylation has been shown to change in stem cell differentiation, and while ADSCs have been acknowledged for their therapeutic potential, little is known about protein glycosylation during human ADSC adipogenic differentiation. In the present study, the global membrane protein glycosylation of native adipocytes was compared to ADSCs from the same individuals as a model of in vivo adipogenesis. For in vitro adipogenesis, ADSCs were adipogenically differentiated in cell culture using an optimized, large-scale differentiation procedure. The membrane glycome of the differentiated ADSCs (dADSCs) was compared with mature adipocytes and the progenitor ADSCs. A total of 137 glycan structures were characterized across the three cell types using PGC-LC coupled with negative-ion electrospray ionization mass spectrometry (ESI-MS)/MS. Significantly higher levels of bisecting GlcNAc-type N-glycans were detected in mature adipocytes (32.1% of total glycans) and in in vitro dADSC progeny (1.9% of total glycans) compared to ADSCs. This was further correlated by the mRNA expression of the MGAT3 gene responsible for the enzymatic synthesis of this structural type. The bisecting GlcNAc structures were found on the majority of human native adipocyte membrane proteins, suggesting an important role in human adipocyte biology. Core fucosylation was also significantly increased during in vivo adipogenesis but did not correlate with an increase in Fut8 gene transcript. Unexpectedly, low abundance structures carrying rare β-linked Gal-Gal termini were also detected. Overall, the N-glycan profiles of the in vitro differentiated progeny did not reflect native adipocytes, and the results show that bisecting GlcNAc structures are a characteristic feature of human adipocyte membrane protein N-glycosylation. Raw MS files are available on GlycoPOST (ID: GPST000153 https://glycopost.glycosmos.org/).

Temperature Dependence of Aqueous-Phase Decomposition of α-Hydroxyalkyl-Hydroperoxides.

Ozonolysis of unsaturated organic species with water produces α-hydroxyalkyl-hydroperoxides (α-HHs), which are reactive intermediates that lead to the formation of H2O2 and multifunctionalized species in atmospheric condensed phases. Here, we report temperature-dependent rate coefficients (k) for the aqueous-phase decomposition of α-terpineol α-HHs at 283-318 K and terpinen-4-ol α-HHs at 313-328 K. The temporal profiles of α-HH signals, detected as chloride adducts by negative-ion electrospray mass spectrometry, showed single-exponential decay, and the derived first-order k for α-HH decomposition increased as temperature increased, e.g., k(288 K) = (4.7 ± 0.2) × 10-5, k(298 K) = (1.5 ± 0.4) × 10-4, k(308 K) = (3.4 ± 0.9) × 10-4, k(318 K) = (1.0 ± 0.2) × 10-3 s-1 for α-terpineol α-HHs at pH 6.1. Arrhenius plot analysis yielded activation energies of 17.9 ± 0.7 (pH 6.1) and 17.1 ± 0.2 kcal mol-1 (pH 6.2) for the decomposition of α-terpineol and terpinen-4-ol α-HHs, respectively. Activation energies of 18.6 ± 0.2 and 19.2 ± 0.5 kcal mol-1 were also obtained for the decomposition of α-terpineol α-HHs in acidified water at pH 5.3 and 4.5, respectively. Theoretical kinetic and thermodynamic calculations confirmed that both water-catalyzed and proton-catalyzed mechanisms play important roles in the decomposition of these α-HHs. The relatively strong temperature dependence of k suggests that the lifetime of these α-HHs in aqueous phases (e.g., aqueous aerosols, fog, cloud droplets, wet films) is controlled not only by the water content and pH but also by the temperature of these media.

Analysis of Flavonoids in Dalbergia odorifera by Ultra-Performance Liquid Chromatography with Tandem Mass Spectrometry.

Dalbergia odorifera, a traditional Chinese medicine, has been used to treat cardio- and cerebrovascular diseases in China for thousands of years. Flavonoids are major active compounds in D. odorifera. In this paper, a rapid and sensitive ultra-high performance liquid chromatography-triple quadrupole mass spectrometry method was developed and validated for simultaneous determination of 17 flavonoids in D. odorifera. Quantification was performed by multiple reaction monitoring using electrospray ionization in negative ion mode. Under the optimum conditions, calibration curves for the 17 analytes displayed good linearity (r2 > 0.9980). The intra- and inter-day precisions (relative standard deviations) were lower than 5.0%. The limit of quantitation ranged from 0.256 to 18.840 ng/mL. The mean recovery range at three spiked concentrations was 94.18–101.97%. The validated approach was successfully applied to 18 samples of D. odorifera. Large variation was observed for the contents of the 17 analytes. Sativanone and 3′-O-methylviolanone were the dominant compounds. The fragmentation behaviors of six flavonoids were investigated using UPLC with quadrupole time-of-flight tandem mass spectrometry. In negative ion electrospray ionization mass spectrometry, all the flavonoids yielded prominent [M − H]− ions. Fragments for losses of CH3, CO, and CO2 were observed in the mass spectra. Formononetin, liquiritigenin, isoliquiritigenin, sativanone, and alpinetin underwent retro-Diels–Alder fragmentations. The proposed method will be helpful for quality control of D. odorifera.

Cis/Trans Isomerization of Unsaturated Fatty Acids in Polysorbate 80 During Light Exposure of a Monoclonal Antibody-Containing Formulation.

Light exposure of a monoclonal antibody formulation containing polysorbate 80 (PS80) leads to cis/trans isomerization of monounsaturated and polyunsaturated fatty acids. This cis/trans isomerization was monitored by positive electrospray ionization mass spectrometry of intact PS80 components as well as by negative ion electrospray ionization mass spectrometry analysis of free fatty acids generated via esterase-catalyzed hydrolysis. The light-induced cis/trans isomerization of unsaturated fatty acids in PS80 required the presence of the monoclonal antibody, or, at a minimum (for mechanistic studies), a combination of N-acetyltryptophan amide and glutathione disulfide, suggesting the involvement of thiyl radicals generated by photoinduced electron transfer from Trp to the disulfide. Product analysis confirmed the conversion of PS80-bound oleic acid to elaidic acid; furthermore, together with linoleic acid, we detected conjugated linoleic acids in PS80, which underwent light-induced cis/trans isomerization.

Rapid screening of very long-chain fatty acids from microorganisms

The analysis of triacylglycerols and phospholipids - phosphatidylcholines allowed the use of shotgun lipidomics to identify very long-chain fatty acids and very long-chain polyunsaturated fatty acids in microalgae. These fatty acids were determined in triacylglycerols by positive electrospray ionization of neutral loss scans of different fatty acids, e.g. 24:0, 24:1ω9, 24:6ω3, 26:0, 26:1ω9, 28:0, 28:1ω9, 28:2ω6, and 28:8ω3. Likewise, very long-chain fatty acids in phosphatidylcholines were identified by negative electrospray ionization mass spectrometry in the selected ion-monitoring of the two most important ions (R1COO− and R2COO−). The limit of detection was determined at 10 nmol/L (∼11 pg/μL) in triacylglycerols and 8.6 nmoles/L (∼8 pg/μL) in phosphatidylcholines. The use of liquid chromatography–mass spectrometry is suitable for very long-chain polyunsaturated fatty acids with up to 8 double bonds due to the time of analysis as well as for reasons of lower thermal stability of polyunsaturated fatty acids towards saturated fatty acids, but gas chromatography–mass spectrometry is better suited for the analysis of saturated very long-chain fatty acids.

Application of Molecularly Imprinted Polymers (MIP) and Magnetic Molecularly Imprinted Polymers (mag-MIP) to Selective Analysis of Quercetin in Flowing Atmospheric-Pressure Afterglow Mass Spectrometry (FAPA-MS) and in Electrospray Ionization Mass Spectrometry (ESI-MS).

Molecularly imprinted polymer (MIP) and magnetic molecularly imprinted polymer (mag-MIP) for solid extraction and pre-concentration of quercetin have been successfully prepared by thermal polymerization method using quercetin (Q) as a template, acrylamide (AA) as a functional monomer, and ethylene glycol dimethacrylate (EGDMA) as a cross-linking agent. The MIP and mag-MIP were successfully applied in analysis of quercetin by mass spectrometry (MS) methods. To perform ambient plasma ionization experiments, a setup consisting of the heated crucible, a flowing atmospheric-pressure afterglow (FAPA) plasma ion source, and amaZon SL ion trap (Bruker, Bremen, Germany) was used. The heated crucible with programmable temperature allowed desorption of the analytes from MIPs structure which resulted in their direct introduction into the ion stream. The results of Q-MIP/Q-mag-MIP and FAPA-MS measurements were compared with those of the analysis of quercetin by the ESI-MS method without extractions and pre-concentration of analytes on polymers. Limits of detection (LOD) for quercetin solutions in both positive and negative ESI-MS were established at 10−8 M and 10−7 M, respectively. The linearity (R2 = 0.9999) of the proposed analytical procedure for quercetin determination in positive ions was provided in the range between 10−4 M and 10−7 M. Moreover, the same parameters were established for FAPA-MS in positive ions, reaching LOD at 0.005 mg/gMIP and the linearity of the method in the range of 0.015–0.075 mg/gMIP with the correlation coefficient value R2 = 0.9850.

Arsenolipids in the green alga Coccomyxa (Trebouxiophyceae, Chlorophyta)

Lipid-like compounds containing a dimethylarsinoyl group, i.e. Me2As(O)-, have been identified by liquid chromatography/inductively coupled plasma mass spectrometry (LC/ICP-MS) and non-aqueous reversed-phase high-performance liquid chromatography (positive and/or negative high-resolution tandem electrospray ionization mass spectrometry (NARP-HPLC/HR-ESI+(−)-MS/MS) from three strains of green algae of the genus Coccomyxa (Trebouxiophyceae, Chlorophyta). The algae were cultivated in a medium containing 10 g arsenic/L, i.e. 133.5 mmol/L of Na2HAsO4.7H2O. After extraction by methyl-tert-butyl ether (MTBE), total lipids were analyzed by ICP-MS or ESI-MS without any further separation or fractionation. A total of 39 molecular species of arsenic triacylglycerols (AsTAG), 15 arsenic phosphatidylcholines (AsPC), 8 arsenic phosphatidylethanolamines (AsPE), 6 arsenic phosphatidylinositols (AsPI), 2 arsenic phosphatidylglycerols (AsPG) and 5 unknown lipids (probably ceramides) were identified. The structures of all molecular species were confirmed by tandem MS. Dry matter of the individual strains contained different amounts of total arsenolipids, i.e. C. elongata CCALA 427 (0.32 mg/g), C. onubensis (1.48 mg/g), C. elongata S3 (2.13 mg/g). On the other hand, there were only slight differences between strains in the relative abundances of individual molecular species. Possible biosynthesis of long-chain lipids with the end group Me2As(O) has also been suggested.

A Biosensor-Based Quantitative Analysis System of Major Active Ingredients in Lonicera japonica Thunb. Using UPLC-QDa and Chemometric Analysis.

In the study, a surface plasmon resonance-based (SPR-based) competitive assay was performed to analyze different compounds’ inhibitory activity to TNF-α, an important pro-inflammatory cytokine in the pathogenesis of chronic inflammatory diseases. Moreover, the single mass spectrometry (MS) detection method was coupled with an ultra-high-performance liquid chromatography (UPLC) system for the routine quality control (QC) of a traditional Chinese medicine (TCM). The above quality control strategy was evaluated with Lonicera japonica Thunb. Analytes were firstly separated on a Waters ACQUITYTM UPLC HSS T3 column (2.1 × 50 mm; particle size = 1.8 μm) using a 0.1% formic acid gradient elution, then detected by negative ESI mass spectrometry. The limits of quantification (LOQ) for analytes reached 0.005–0.56 μg/mL. The LOD of the QDa detector was lower than that of the PDA detector, indicating its wider detection range. The QDa detector was also more suitable for the analysis of the complex matrix of TCM. The method showed excellent linearity, with regression coefficients higher than 0.9991. The average recoveries of the investigated analytes were in the range of 98.78–105.13%, with an RSD below 3.91%. The inter-day precision range (n = 3 days) was 2.51–4.54%. Compared to other detectors, this strategy could be widely applied in the quantitative analysis of TCM. In addition, the chemically latent data could be revealed using chemometric analysis. Importantly, this study provides an efficient screening method for small-molecule inhibitors targeting the TNF-α pathway.

Analysis of oligonucleotides by ion-pair reversed-phase liquid chromatography coupled with positive mode electrospray ionization mass spectrometry.

Oligonucleotides are usually analyzed by ion-pair reversed-phase liquid chromatography (IP-RPLC) coupled with negative mode electrospray ionization mass spectrometry (ESI-MS) due to their highly negative charged phosphodiester backbones. Herein, the signal suppression effect of triethylamine (TEA) adducts caused the ion-pair reagent TEA/hexafluoroisopropanol (HFIP) is greatly alleviated after improving the in-source energy in positive mode ESI-MS. This strategy is applied for different RNA sequencing through analyzing their formic acid hydrolysates via IP-RPLC MS. Comparing with negative ion mode, we demonstrate that IP-RPLC MS analysis in positive ion mode is more suitable for RNA sequencing with fewer contaminant interferences. Finally, simultaneous online separation and detection of oligonucleotides and protein digests are achieved in positive ion mode IP-RPLC MS analysis with little interference to each other.

A strategy for validating concentrations of oxylipin standards for external calibration

Quantitative analysis of oxylipins by means of chromatography/mass spectrometry is based on (external) calibration with standard compounds. Therefore, the quality of analytical standards is of fundamental importance for accurate results. Recently launched certified standards with an assured concentration within a narrow range are useful tools to verify analytical standards. However, such standards are only available for a few compounds.Based on the exemplary comparison of certified with none certified standards we suggest a tiered approach to validate and control the concentrations when preparing an external calibration based on non-certified oxylipin standards. Concentrations are evaluated by means of liquid chromatography negative electrospray ionization mass spectrometry (LC-ESI(-)-MS) in selected ion monitoring mode and UV spectroscopy.Based on the suggested approach, more than 50% of the standards in our calibration mix could be validated. Though most of the non-certified standards are of good quality, several oxylipin concentrations differ considerably demonstrating that a quality control strategy as suggested here is a mandatory prerequisite for quantitative oxylipin metabolomics.

Characterization of oil sands naphthenic acids by negative-ion electrospray ionization mass spectrometry: Influence of acidic versus basic transfer solvent

Considerable effort and progress has been made over the past decade with respect to development of analytical tools for the determination of naphthenic acids and related components in environmental samples. However, experimental variables that influence the analytical results have not been fully explored. The relative contributions of Ox classes are of particular interest in data obtained using negative-ion electrospray ionization mass spectrometry. Using two types of ultrahigh resolution mass spectrometers (Orbitrap and FT-ICR), the apparent pH of the transfer solvent was observed to have a significant impact upon compound class distributions. A basic transfer solvent favored the detection of Ox species of lower oxygen content, while acidic pH favored the preferential observation of organic compounds with higher oxygen contents. These observed trends were independent of the instrument type. In addition, when using an acidic transfer solvent, the overall observed response was reduced by a factor of ∼20. Thus, the apparent pH of the transfer solvent has critical influence upon detection and upon the profile of different components observed within a complex mixture. In turn, this significantly impacts oil sands environmental monitoring for toxicity, forensic interpretation, and quantitation; when comparing data sets from different laboratories, these findings should therefore be taken into account.

Pharmacokinetics and tissue distribution of oroxin B in rats using a validated LC–MS/MS assay

A highly sensitive LC-MS/MS method was developed to measure oroxin B in rat plasma and tissue homogenates. The analyte and IS were isolated from biological matrices by a simple protein precipitation followed by centrifugation. Detection was conducted by electrospray negative-ionization mass spectrometry in selected-reaction monitoring mode. The assay was linear in the concentration range 4.52-904 ng/mL with intra- and inter-day precision of <14.41%. It was successfully applied to the pharmacokinetics and tissue distribution studies of oroxin B after an intravenous dose of 1.0 mg/kg in rats. The results would be useful for further development of oroxin B.