The potential effects of N-Acyl homoserine lactones on aerobic sludge granulation during phenolic wastewater treatment.

The formation of aerobic granular sludge (AGS) is relatively difficult during the treatment of refractory wastewater, which generally shows small granular sizes and poor stability. The formation of AGS is regulated by N-Acyl homoserine lactones (AHLs)-mediated quorum sensing (QS). However, the potential role of AHLs in AGS formation under the toxic stress of refractory pollutants and the heterogeneity in the distribution and function of AHLs across different aggregates are not well understood. This study investigated the potential effects of AHLs on the formation of AGS during phenolic wastewater treatment. The distribution and succession of AHLs across varying granular sizes and development stages of AGS were investigated. Results showed that AGS was successfully formed in 13 days with an average granular size of 335 ± 39 µm and phenol removal efficiency of >99%. The levels of AHLs initially increased and then decreased. C4-HSL and 3-oxo-C10-HSL were enriched in large granules, suggesting they may play a pivotal role in regulating the concentration and composition of extracellular polymeric substances (EPS). The content of EPS constantly increased to 149.4 mg/gVSS, and protein (PN) was enriched in small and large granules. Luteococcus was the dominant genus constituting up to 62% after the granulation process, and exhibited a strong association with C4-HSL. AHLs might also regulate the bacterial community responsible for EPS production, and pollutant removal, and facilitate the proliferation of slow-growing microorganisms, thereby enhancing the formation of AGS. The synthesis and dynamics of AHLs were mainly governed by AHLs-producing bacterial strains of Rhodobacter and Pseudomonas, and AHLs-quenching strains of Flavobacterium and Comamonas. C4-HSL and 3-oxo-C10-HSL might be the major contributors to promoting sludge granulation under phenol stress and play critical roles in large granules. These findings enhance our understanding of the roles that AHLs play in sludge granulation under toxic conditions.

Selective Characterization of Olefins by Paternò-Büchi Reaction with Ultrahigh Resolution Mass Spectrometry.

Petroleum olefins play important roles in various secondary processing procedures and are important feedstocks for the modern organic chemical industry. It is quite challenging to analyze petroleum olefins beyond the gas chromatography (GC)-able range using mass spectrometry (MS) due to the difficulty of soft ionization and the matrix complexity. In this work, a Paternò-Büchi (PB) reaction combined with atmospheric pressure chemical ionization and ultrahigh resolution mass spectrometry (APCI-UHRMS) was developed for selective analysis of olefins. Through the PB reaction, C═C bonds were transformed into four-membered rings of oxetane with improved polarity so that soft ionization of olefins could be achieved. The systematic optimization of PB reaction conditions, as well as MS ionization conditions, ensured a high reaction yield and a satisfied MS response. Furthermore, a sound scheme was set up to discriminate the coexisting unsaturated alkanes in complex petroleum, including linear olefins, nonlinear olefins, cycloalkanes, and aromatics, making use of their different behaviors during the PB reaction and chemical ionization. The developed strategy was successfully applied to the analysis of olefins in fluid catalytic cracking oil slurry, a complex heavy oil sample. This method extended the characterization of petroleum olefins from lower to higher with high efficiency and selectivity to provide a comprehensive molecular library for heavy petroleum samples and process optimization.

Recent advances in mass spectrometry imaging of single cells.

Mass spectrometry imaging (MSI) is a sensitive, specific, label-free imaging analysis technique that can simultaneously obtain the spatial distribution, relative content, and structural information of hundreds of biomolecules in cells and tissues, such as lipids, small drug molecules, peptides, proteins, and other compounds. The study of molecular mapping of single cells can reveal major scientific issues such as the activity pattern of living organisms, disease pathogenesis, drug-targeted therapy, and cellular heterogeneity. Applying MSI technology to the molecular mapping of single cells can provide new insights and ideas for the study of single-cell metabolomics. This review aims to provide an informative resource for those in the MSI community who are interested in single-cell imaging. Particularly, we discuss advances in imaging schemes and sample preparation, instrumentation improvements, data processing and analysis, and 3D MSI over the past few years that have allowed MSI to emerge as a powerful technique in the molecular imaging of single cells. Also, we highlight some of the most cutting-edge studies in single-cell MSI, demonstrating the future potential of single-cell MSI. Visualizing molecular distribution at the single-cell or even sub-cellular level can provide us with richer cell information, which strongly contributes to advancing research fields such as biomedicine, life sciences, pharmacodynamic testing, and metabolomics. At the end of the review, we summarize the current development of single-cell MSI technology and look into the future of this technology.

Understanding of protomers/deprotomers by combining mass spectrometry and computation.

Multifunctional compounds may form different prototropic isomers under different conditions, which are known as protomers/deprotomers. In biological systems, these protomer/deprotomer isomers affect the interaction modes and conformational landscape between compounds and enzymes and thus present different biological activities. Study on protomers/deprotomers is essentially the study on the acidity/basicity of each intramolecular functional group and its effect on molecular structure. In recent years, the combination of mass spectrometry (MS) and computational chemistry has been proven to be a powerful and effective means to study prototropic isomers. MS-based technologies are developed to discriminate and characterize protomers/deprotomers to provide structural information and monitor transformations, showing great superiority than other experimental methods. Computational chemistry is used to predict the thermodynamic stability of protomers/deprotomers, provide the simulated MS/MS spectra, infrared spectra, and calculate collision cross-section values. By comparing the theoretical data with the corresponding experimental results, the researchers can not only determine the protomer/deprotomer structure, but also investigate the structure-activity relationship in a given system. This review covers various MS methods and theoretical calculations and their devotion to isomer discrimination, structure identification, conformational transformation, and phase transition investigation of protomers/deprotomers.

Separation and identification of degradation impurities of esomeprazole sodium.

Drug impurities are important factors that affect drug safety and efficacy. The aim of this study is to separate and confirm the structure of two degradation impurities of esomeprazole sodium, designated X and Y. The impurities X and Y were successfully isolated using preparative HPLC by developing separation methods with the help of ACD/Labs AutoChrom software. There was a steady increase in X and Y impurities in forced esomeprazole sodium degradation. Impurity X was confirmed as 6-methoxy-1h-benzo[d]imidazole-2-yl-4-amino-3,5-dimethylpyridinecarboxylate and impurity Y as 6-methoxy-1h-benzo[d]imidazole-2-yl-4-hydroxy-3,5-dimethylpyridinecarboxylate using nuclear magnetic resonance spectrometry, infrared spectroscopy, and high-resolution mass spectrometry. These findings provide a comprehensive understanding of the impurity profile of esomeprazole sodium because these impurities are reported for the first time. Based on our results, active pharmaceutical ingredient manufacturers can further control process parameters to reduce impurity generation, and drug production manufacturers can optimize the packaging and storage conditions of esomeprazole sodium.

Rapid screening of polybrominated diphenyl ethers in water by solid-phase microextraction coupled with ultrahigh-resolution mass spectrometry.

Polybrominated diphenyl ethers (PBDEs) are considered emerging organic contaminants that attract more attention in the environment. Herein, online coupling of solid-phase microextraction and ultrahigh-resolution mass spectrometry was developed for rapid screening of eight PBDEs in water samples. This procedure was completed in 22 min, about 6 times faster than the routine workflow such as solid-phase extraction coupled with gas chromatography-mass spectrometry. Thermal desorption and solvent-assisted atmospheric pressure chemical ionization were developed for the effective coupling of solid-phase microextraction (SPME) with ultrahigh-resolution mass spectrometry (UHRMS), which contributed to the signal enhancement and made the methodology feasible for environmental screening. The limits of detection and quantification were 0.01-0.50 ng/mL and 0.05-4.00 ng/mL, respectively. The recoveries were 57.2-75.2% for quality control samples at spiking levels of 0.8-10 ng/mL (4-50 ng/mL for BDE209), with relative standard deviation less than 19.0%. Twelve water samples from different river sites near industrial areas were screened using the developed method. The results showed that BDE-209 was the dominant PBDE (1.02-1.28 ng/mL in positive samples), but its amount was lower than the human health ambient water quality criteria. Consequently, the developed method provides a rapid and reliable way of evaluating contamination status and risks of PBDEs in aqueous environment.

Pressure-Induced Hydrogen Transfer in 2-Butyne via a Double CH···π Aromatic Transition State.

Hydrogen transfer (H-transfer) is an important elementary reaction in chemistry and bioscience. It is often facilitated by the hydrogen bonds between the H-donor and acceptor. Here, at room temperature and high pressure, we found that solid 2-butyne experienced a concerted two-in-two-out intermolecular CH···π H-transfer, which initiated the subsequent polymerization. Such double H-transfer goes through an aromatic Hückel six-membered ring intermediate state via intermolecular CH···π interactions enhanced by external pressure. Our work shows that H-transfer can occur via the CH···π route in appropriate conformations under high pressure, which gives important insights into the H-transfer in solid-state hydrocarbons.

Ultra-fast screening of free fatty acids in human plasma using ion mobility mass spectrometry.

Free fatty acids are involved in many metabolic regulations in the human body. In this work, an ultra-fast screening method was developed for the analysis of free fatty acids using trapped ion mobility spectrometry coupled with mass spectrometry. Thirty-three free fatty acids possessing different unsaturation degrees and different carbon chain lengths were baseline separated and characterized within milliseconds. Saturated, monounsaturated, and polyunsaturated free fatty acids showed different linearities between collision cross-section values and m/z. The establishment of correlations between structures and collision cross-section values provided additional qualitative information and made it possible to determine free fatty acids which were out of the standards pool but possessed the confirmed linearity. The gas-phase separation made the quantitative analysis reliable and repeatable at a much lower time cost than chromatographic methods. The sensitivity was comparable to and even better than the reported results. The method was validated and applied to profiling free fatty acids in human plasma. Saturated free fatty acids abundance in the fasting state was found to be lower than that in the postprandial state, while unsaturated species abundance was found higher. The method was fast and robust with minimum sample pretreatment, so it was promising in the high-throughput screening of free fatty acids.

Tracing the migration and transformation of metabolites in xylem during wood growth by mass spectrometry imaging.

Metabolites in the xylem experience several migration and transformation processes during tree growth. Their composition and distributions can reflect the environment that the wood lived through. Herein, a matrix-assisted laser desorption/ionization mass spectrometry imaging method was developed to investigate the migration and transformation of metabolites in the xylem during heartwood formation and after mechanical injury. The thickness of the wood slice, the type of matrix and its manner of deposition were optimized to improve ionization response and spatial resolution. The mass difference correlation (MDC) data processing method was proposed to improve the efficiency of compound identification, in which the compounds were classified by their molecular weight. The compound species was identified by results calculated using MDC and the experimental results from MS/MS. The directly identified metabolites, whose type and number were found to be quite different between sapwood and heartwood, demonstrated the transformation and migration of metabolites from sapwood to heartwood. Additionally, two kinds of resins produced from different positions were identified by MSI simultaneously, even though their heterogeneous distribution was not visible in optical images. The origin and type of the two resins were deduced from the identified compounds and their molecular distribution. This work provides a method to directly reveal metabolite migration and transformation mechanisms in xylem during wood growth.

Expansion of the composition library for chemodiversity of hardwood extractives at molecular level by ultrahigh-resolution mass spectrometry.

To enhance the characterization of wood extractives at molecular level, a detailed ultrahigh-resolution mass spectrometry (UHRMS)-based analytical methodology was developed in this work. The analytical strategies, including selection of compatible solvent for extraction, evaluation of ionization solvent for effective electrospray ionization, and multi-dimensional data analysis, were established to ensure a comprehensive characterization of complex compositions in wood extractives. Extraction capability of seven solvents with varied polarities was examined by a standard reference material of hardwood biomass and evaluated based on thousands of compounds which were much more than those discovered before. With a variety of data-processing approaches, including compound type distribution, double bond equivalent versus carbon number plot, and van Krevelen diagram, the chemodiversity of the extractives was fully explored from different perspectives. This work greatly expanded the compound library of wood extractives and could also provide guidance for the integrated composition analysis of other biomass materials.

Antitumor and off-target effects of cholesterol-conjugated let-7a mimics in an orthotopic hepatocellular carcinoma xenograft nude mouse model.

To explore the antitumor and potential off-target effects of systemically delivered cholesterol-conjugated let-7a mimics (Chol-let-7a) and control mimics (Chol-miRCtrl) on hepatocellular carcinoma in vivo. Methods: The antitumor effects of two intravenous dosing regimens of Chol-let-7a on heptocellular carcinoma growth were compared using an orthotopic xenograft mouse model. Off-targets were analyzed with histopathological and ultrapathological features of heparenal tissue and cells in the Chol-let-7a-, Chol-miRCtrl-, and saline-treated (blank) xenograft mice and normal control mice. Then, let-7a abundance in orthotopic tumors, corresponding paracancerous hepatic tissue, and normal liver tissue from healthy nude mice was examined by reverse transcription-polymerase chain reaction. The distribution of Chol-let-7a and Chol-miRCtrl in vivo was examined by whole-animal imaging and frozen-sections observation. The experiments were approved by the Institutional Research Board of Peking Union Medical College Hospital. Results: Continuous treatment with Chol-let-7a resulted in tumors that were 35.86% and 40.02% the size of those in the Chol-miRCtrl and blank xenograft group (P < 0.01 and P < 0.01, respectively), while intermittent dosing with Chol-let-7a resulted in tumors that were 65.42% and 56.66% the size of those in the Chol-miRCtrl and the blank control group, respectively (P < 0.05 and P < 0.05). In addition, some histopathological and ultrapathological features were only observed after treatment with the two cholesterol-conjugated molecules, however mild with intermittent dosing Chol-let-7a treatment, such as diffuse sinusoidal dilation and edema, primarily around the centrolobular vein in heptic tissues; mild hypercellularity with dilated capillary lumens in the renal tissue; and some organelle abnormalities found in heptic and renal cells. Furthermore, whole-animal imaging showed that Chol-let-7a and Chol-miRCtrl were predominantly distributed in the liver, kidney, and bladder regions after injection, and that the concentration of Chol-let-7a and Chol-miRCtrl in the kidney and the bladder decreased much slowly in the xenograft animals, especially in the Chol-miRCtrl group. Finally, RT-PCR analysis showed that let-7a levels were significantly increased in Chol-let-7a-treated xenografts compared with Chol-miRCtrl group (P=0.003) and blank xenograft group (P=0.001); however, the level was only equivalent to 50.6% and 40.7% of that in paracancerous hepatic tissue and hepatic tissue in normal mice, respectively. Conclusions: Chol-let-7a, administered either continuously or intermittently, showed effective antitumor efficacy. Chol-let-7a had some off-target effects, such as mild acute hepatitis-like inflammation and non-specific drug-induced kidney injury. The intermittent dosing regimen resulted in less damage than the continuous regimen, while maintaining relatively satisfactory antitumor efficacy, which could be useful for the investigation and possible clinical use of miRNA treatment regimens in the future.

Mass spectrometry imaging for direct visualization of components in plants tissues.

Mass spectrometry is considered the most informative technique for components identification and has been widely adopted in plant sciences. However, the spatial distribution of compounds in the plant, which is vital for the exploration of plant physiological mechanisms, is missed in MS analysis. In recent years, mass spectrometry imaging has brought a great breakthrough in plant analysis because it can determine both the molecular compositions and spatial distributions, which is conducive to understand functions and regulation pathways of specific components in plants. Mass spectrometry imaging analysis of plant tissue is toward high sensitivity, high spatial resolution, and even single-cell analysis. Despite many challenges and technical barriers, such as difficulties of sample pretreatment caused by morphological diversity of plant tissues, obstacles for high spatial resolution imaging, and so on, lots of researches have contributed to remarkable progress, including improvement in tissue preparation, matrix innovation, and ionization mode development. This review focuses on the advances of mass spectrometry imaging analysis of plants in the last 5 years, including commonly used ionization techniques, technical advances, and recent applications of mass spectrometry imaging in plants.

Diacylglycerols ions as novel marker indicators for the classification of edible oils using ultrahigh resolution mass spectrometry.

Diacylglycerols (DAGs) ions, instead of triacylglycerols (TAGs) ions, were established as marker indicators for an improved classification of edible oils using ultrahigh resolution mass spectrometry (UHRMS). DAGs ions can be used not only to identify triacylglycerols (TAGs) and their embedded fatty acids (FAs), but also to distinguish positional isomers of TAGs. In this work, DAGs ions were determined in edible oils by direct infusion atmospheric pressure chemical ionization-ultrahigh resolution mass spectrometry (APCI-UHRMS), where the ultrahigh resolving power up to 500,000 FWHM (full width at half maximum) can provide accurate molecular compositions and detailed fingerprints MS spectra in a minute. A total of 146 samples belonging to 22 species of plant oils and animal fats, were characterized. Chemometric analyses were performed using principal component analysis, partial least square-discriminant analysis and orthogonal partial least squares-discriminant analysis. DAGs ions were proved to be better than TAGs ions as marker indicators in the chemometric analyses. An overall correct rate of 93.40% was achieved for the classification of tested samples. In addition, blend oils and gutter oils were also characterized by this developed method.

Rapid characterization and pharmacokinetic study of aristolochic acid analogues using ion mobility mass spectrometry.

Aristolochic acid analogues (AAAs), naturally existing in herbal Aristolochia and Asarum genera, were once widely used in traditional pharmacopeias because of their anti-inflammatory properties, but lately they were identified as potential nephrotoxins and mutagens. A method for rapid characterization of AAAs in serum was developed using ion mobility spectrometry coupled with mass spectrometry (IMS-MS). Five AAAs, containing four aristolochic acids and one aristolactam, were separated and identified within milliseconds. AAAs were separated in gas phase based on the difference of their ion mobility (K0), and then identified based on their K0 values, m/z, and product ions from MS/MS. Quantitative analysis of AAAs was performed using an internal standard with a satisfactory sensitivity. Limits of detection (signal-to-noise = 3) and quantification (signal-to-noise = 10) were 1-5 ng/mL and 3-8 ng/mL, respectively. The method was validated and successfully applied to the pharmacokinetics study of AAAs in rats, offering a promising way for fast screening and evaluation of AAAs in biological samples.

Comprehensive Composition, Structure, and Size Characterization for Thiophene Compounds in Petroleum Using Ultrahigh-Resolution Mass Spectrometry and Trapped Ion Mobility Spectrometry.

Thiophene compounds are the main concern of petroleum desulfurization, and their chemical composition and molecular configuration have critical impacts on thermodynamic and kinetic processes. In this work, atmospheric pressure chemical ionization (APCI) was employed for effective ionization of thiophene compounds in petroleum with complex matrix, in which carbon disulfide was used for generating predominant [M]+• ions without the need of derivatization as for electrospray ionization. APCI coupled with ultrahigh-resolution mass spectrometry (UHRMS) was successfully applied to the composition characterization of thiophene compounds in both a low boiling petroleum fraction and a whole crude oil. APCI coupled with trapped ion mobility spectrometry (TIMS) was developed to determine the shape and size of thiophene compounds, providing configuration information that affects the steric hindrance and diffusion behavior of reactants in the desulfurization reaction, which has not been previously reported. Moreover, the comprehensive experimental structural data, expressed as the collision cross section (CCS) of the ions as surrogates of molecules, provided clues to the factors affecting the desulfurization reactivity of thiophene compounds. Further exploration showed that not only qualitative analysis of thiophene compounds can be achieved from the correlation between m/z and CCS, but also molecular size was found to be correlated with CCS that can be used as structural analysis. Overall, the molecular composition and dimension analysis together can provide substantial information for the desulfurization activity of thiophene compounds, facilitating the desulfurization process studies and catalyst design.

Spatial Distribution of Endogenous Molecules in Coffee Beans by Atmospheric Pressure Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging.

Mass spectrometry imaging (MSI) is a promising chemical imaging method. Among various endogenous molecules, mapping the concentration and the spatial distribution of specific compounds in the coffee bean tissue is of tremendous significance in its function research, as these compounds are critical to grading coffee beans at the molecular level, determining the geographical origin, and optimizing storage conditions of coffee beans. In this paper, we established an atmospheric pressure (AP) matrix-assisted laser desorption/ionization (MALDI) MSI method for the microscopic distribution analysis of endogenous molecules, for example, sucrose, caffeine, and caffeoylquinic acid, in the coffee bean endosperm. Experiments were done on the differences between coffee beans from eight countries. Principal component analysis (PCA) was performed using IMAGEREVEAL software. The results showed that the chemical composition and relative content of coffee beans from different origins are different. Our work provides a detection method that may be used for coffee bean quality identification, efficient use, product traceability, and product counterfeiting.

Analytical strategies for chemical characterization of bio-oil.

Bio-oils, produced by biomass pyrolysis, have become promising candidates for feedstocks of high value-added chemicals and alternative sources for transportation fuels. Bio-oil is such a complicated mixture that contains nonpolar hydrocarbons and polar components which cover almost all kinds of organic oxygenated compounds such as carboxylic acids, alcohols, aldehydes, ketones, esters, furfurals, phenolic compounds, sugar-like material, and lignin-derived compounds. Comprehensive characterization of bio-oil and its subfractions could provide insight into the conversion process of biomass processing, as well as its further utilization as transportation fuels or chemical raw materials. This review focuses on advanced analytical strategies on in-depth characterization of bio-oil, which is concerned with gas chromatography, high-resolution mass spectrometry, FTIR spectroscopy and NMR spectroscopy, offering complementary information for previous reviews.

Supercritical fluid extraction with carbon nanotubes as a solid collection trap for the analysis of polycyclic aromatic hydrocarbons and their derivatives.

A supercritical fluid extraction (SFE) method with an online solid collection trap has been developed for the quantitative analysis of 16 polycyclic aromatic hydrocarbons (PAHs) and 15 typical PAH derivatives in solid matrix. Compared with liquid trapping and C18 solid-phase trapping, multi-walled carbon nanotubes (CNTs) were proved to be the most efficient trapping sorbent for the collection of PAHs and their nitro-, oxy- and alkyl-derivatives. The proposed extraction-collection procedure was systematically optimized in terms of pressure, temperature, extraction time, trapping materials, supercritical fluid flow rate, co-solvent type, and co-solvent percentage, taking into account the interaction between these variables. The whole extraction process could be completed in 15min followed by GC-MS analysis. Quantitative recoveries of PAHs and their derivatives from spiked soil samples (50ngg(-1)) were obtained in the range of 62.9-111.8% with the precisions (RSD, intra-day) ranged from 1.9% to 13.7%. The developed SFE method with online CNTs trapping followed by GC-MS analysis has been demonstrated to be an efficient way for quantitative analysis of trace-level PAHs and their nitro-, oxy-, and alkyl-derivatives in soil samples.

Normal phase LC coupled with direct analysis in real time MS for the chiral analysis of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol and jasmonic acid.

Normal phase chiral LC (NPLC) has been proved to be powerful and efficient for chiral separation. However, the combination of NPLC with ESI or atmospheric pressure chemical ionization MS is restricted by the poor ionization efficiency and thermal fragmentations of analytes to some extent. Direct analysis in real time MS (DART-MS) is an ambient ionization technique that shows high ionization efficiency of the analytes in the normal phase mobile phase. In this work, we coupled chiral NPLC to DART-MS for the chiral qualitative and quantitative analysis of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol and jasmonic acid enantiomers. Satisfactory results for the enantiomers of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol operating in the positive mode were obtained in terms of linearity (2.5-250 µg/mL, R(2) , 0.999-1.000) and repeatability (25 µg/mL, RSDs, 4.7-5.6%). Moreover, chiral NPLC-DART-MS resulted in the simultaneous chiral separation and detection of jasmonic acid enantiomers, which are very difficult to be analyzed by NPLC-ESI-MS and NPLC-APCI-MS. Compared with the coupled techniques of NPLC-ESI-MS and NPLC-APCI-MS, NPLC-DART-MS showed advantages in increasing the ionization efficiency and reducing the in-source thermal fragmentation of analytes.

A new derivatization approach for the rapid and sensitive analysis of brassinosteroids by using ultra high performance liquid chromatography-electrospray ionization triple quadrupole mass spectrometry.

In this work, a new labeling reagent, 2-bromopyridine-5-boronic acid (BPBA), was introduced to derivatize brassinosteroids (BRs). The BPBA not only provided a very simple and rapid labeling procedure, but also remarkably increased the detection sensitivity of BRs. Based on this new labeling reaction, a rapid and sensitive method for BRs' analysis in Arabidopsis thaliana was established by using the ultra high performance liquid chromatography-electrospray ionization triple quadrupole mass spectrometry (UHPLC-ESI-QqQ-MS). The extraction and purification procedure of the plant sample was also simplified and improved in this work. Good linearities were obtained for three BRs with the determination coefficients (R(2)) about 0.9999. The limits of detection (S/N=3) for three BRs were found to be 2.00-8.00 ng/L while the limits of quantification (S/N=10) were 6.00-23.0 ng/L. The RSD % for all three samples are lower than 8.67% (n=5). The recoveries of three BRs spiked in A. thaliana samples were from 76.9% to 86.1%. Using this method, the endogenous 0.055 ng/g fresh weight (FW) 24-epiBR and 0.070 ng/g (FW) 28-epihomoBR were successfully detected from only 2g A. thaliana plants.