Chromatography-electrospray Ionization-triple Quadrupole Mass Research Articles

Organ-level distribution tandem mass spectrometry analysis of three structural types of brassinosteroids in rapeseed.

Brassinosteroids (BRs) are a class of naturally occurring steroidal phytohormones mediating a wide range of pivotal developmental and physiological functions throughout the plant's life cycle. Therefore, it is of great significance to determine the content and the distribution of BRs in plants.Regretfully, although a large number of quantitative methods for BRs by liquid chromatography-tandem mass spectrometry (LC-MS/MS) have been reported, the in planta distribution of BRs is still unclear because of their lower contents in plant tissues and the lack of effective ionizable groups in their chemical structures. We stablished a novel analytical method of BRs based on C18 cartridge solid-phase extraction (SPE) purification, 4-(dimethylamino)-phenylboronic acid (DMAPBA) derivatization, and online valve-switching system coupled with ultra-high performance liquid chromatography-electro spray ionization-triple quadrupole mass spectrometry (UHPLC-ESI-MS/MS). This method has been used to quantify three structural types of BRs (epibrassinolide, epicastasterone, and 6-deoxo-24-epicastaster one) in different organs of Brassica napus L. (rapeseed). We obtained the contents of three structural types of BRs in various organ tissues of rapeseed. The contents of three BRs in rapeseed flowers were the highest, followed by tender pods. The levels of three BRs all decreased during the maturation of the organs. We outlined the spatial distribution maps of three BRs in rapeseed based on these results, so as to understand the spatial distribution of BRs at the visual level. Our results provided useful information for the precise in situ localization of BRs in plants and the metabolomic research of BRs in future work. The in planta spatial distribution of BRs at the visual level has been studied for the first time.

Effect of oridonin on oxylipins in the livers of mice with acute liver injury induced by D-galactosamine and lipopolysaccharide

Background and PurposeOridonin (Ori) has been shown to protect against acute liver injury (ALI) induced by D-galactosamine (D-GalN) and lipopolysaccharide (LPS). Oxylipins are oxidation products of polyunsaturated fatty acids (PUFAs) and are key proinflammatory mediators. This study aimed to investigate the changes in oxylipins in the livers of mice with D-GalN/LPS-induced ALI and the effects of Ori on these changes. Results54 oxylipins in liver tissues were identified and qualitatively and quantitatively analyzed by ultra-performance liquid chromatography-electrospray ionization triple quadrupole mass spectrometry (UPLC-QTRAP/MS/MS). The levels of 12-HETE, 12-HEPE, 14(S)-HDHA, PGE2, dihomo-γ-linolenic acid and 13-HOTrE in the liver were significantly increased in the D-GalN/LPS-induced ALI group compared with the control group, and the levels of EPA and 7-HDHA were significantly decreased. However, pretreatment with Ori dramatically decreased the levels of 12-HETE, 12-HEPE, 14(S)-HDHA, PGE2 and 13-HOTrE compared with those of the ALI group and induced 7-HDHA and 15-oxoETE. Moreover, Ori reduced the protein levels of COX-1, COX-2, ALOX5, ALOX12 and ALOX15 induced by D-GalN/LPS, indicating that Ori altered oxylipins through the COX and LOX pathways. ConclusionsThese results suggest that the protective effect of Ori on ALI is partly mediated by affecting the oxylipin pathway.

Screening of inhibitors of angiotensin-converting enzyme (ACE) employing high performance liquid chromatography-electrospray ionization triple quadrupole mass spectrometry (HPLC-ESI-QqQ-MS)

Angiotensin-converting enzyme (ACE) plays a key role in regulating blood pressure in the body by converting the angiotensin I (AI) into angiotensin II (AII). Angiotensin II is a potent vaso-active peptide that causes arterioles to constrict, resulting in increased blood pressure. A rapid and sensitive method for the identification of inhibitors of ACE was developed, and optimized employing HPLC-ESI-QqQ-MS. In this assay, angiotensin I substrate was converted into the product angiotensin II with the catalytic action of ACE. A calibration curve for depleting concentration of angiotensin I was developed and linearity of R2=0.999 with a remarkably low concentration of substrate range 20–200nM. The limit of detection and quantification of angiotensin I was found to be 1.93 and 5.84nM, respectively. The enzymatic reaction was optimized for incubation time, concentration, and volume of enzyme and substrate. All reactions were performed at 37°C at pH7.5 with standard incubation time of 20min. Two standard inhibitors, Captopril and Lisinopril, were checked through the newly developed method for their inhibitory potential, and their IC50 values were found to be 3.969 and 0.852μM, respectively. Reproducibility and precision analysis of different experiments showed <9.9% RSD. The developed method can be used for the identification of new ACE inhibitors.

Development and Validation of a High-Throughput Ultrahigh-Performance Liquid Chromatography–Mass Spectrometry Approach for Screening of Oxylipins and Their Precursors

Lipid mediators, highly bioactive compounds synthesized from polyunsaturated fatty acids (PUFAs), have a fundamental role in the initiation and signaling of the inflammatory response. Although extensively studied in isolation, only a limited number of analytical methods offer a comprehensive coverage of the oxylipin synthetic cascade applicable to a wide range of human biofluids. We report the development of an ultrahigh-performance liquid chromatography-electrospray ionization triple quadrupole mass spectrometry (UHPLC-MS) assay to quantify oxylipins and their PUFA precursors in 100 μL of human serum, plasma, urine, and cell culture supernatant. A single 15 min UHPLC run enables the quantification of 43 oxylipins and 5 PUFAs, covering pro and anti-inflammatory lipid mediators synthesized across the cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450 (CYP450) pathways. The method was validated in multiple biofluid matrixes (serum, plasma, urine, and cell supernatant) and suppliers, ensuring its suitability for large scale metabonomic studies. The approach is accurate, precise, and reproducible (RSD < 15%) over multiple days and concentrations. Very high sensitivity is achieved with limits of quantification inferior to picograms for the majority of analytes (0.05-125 pg) and linear range spanning up to 5 orders of magnitude. This enabled the quantification of the great majority of these analytes at their low endogenous level in human biofluids. We successfully applied the procedure to individuals undergoing a fasting intervention; oxylipin profiles highlighted significantly altered PUFA and inflammatory profiles in accordance with previously published studies as well as offered new insight on the modulation of the biosynthetic cascade responsible for the regulation of oxylipins.

Quantification of steroidal alkaloids in Buxus papillosa using electrospray ionization liquid chromatography–triple quadrupole mass spectrometry

Buxus papillosa is one of the most extensively studied species of the genus Buxus known to possess steroidal alkaloids which can be used for assessing the various pharmacological activities of this plant. This paper describes the liquid chromatography–electrospray ionization triple quadrupole mass spectrometry (LC–ESI-QQQ-MS) method for the quantification of six steroidal alkaloids as chemical markers in the extracts of leaves, roots and stems of B. papillosa. Quantitative MS/MS analysis was carried out by optimization of the most sensitive transition for each analyte. This has yielded detection and quantification limits of 0.486–8.08ng/mL and 1.473–24.268ng/mL, respectively for all analytes. Linearity of response was also achieved and the regression coefficient found to be >0.99 for all analyzed compounds. The newly developed MRM (Multiple Reaction Monitoring) method showed excellent sensitivity for the quantification of steroidal alkaloids within 15min run time. This paper describes the application of LC–QQQ-MS technique for steroidal alkaloids analysis in plant samples.

Comprehensive ultra-performance liquid chromatographic separation and mass spectrometric analysis of eicosanoid metabolites in human samples

Over the past decade, the number of known eicosanoids has expanded immensely and we have now developed an ultra-performance liquid chromatography–electrospray ionization triple quadrupole mass spectrometric (UPLC-QTRAP/MS/MS) method to monitor and quantify numerous eicosanoids. The UPLC-QTRAP/MS/MS approach utilizes scheduled multiple reaction monitoring (MRM) to optimize sensitivity, number of metabolites that can be analyzed and the time requirement of the analysis. A total of 184 eicosanoids including 26 deuterated internal standards can be separated and monitored in a single 5min UPLC run. To demonstrate a practical application, human plasma samples were analyzed following solid-phase extraction (SPE) and the recovery rate and matrix effects were determined for the 26 deuterated internal standards added to the plasma. The method was validated and shown to be sensitive with the limit of quantitation at pg levels for most compounds, accurate with recovery rates of 70–120%, and precise with a CV<30 for all compounds. Also, the method showed a linear response over a range spanning several orders of magnitude. In a QC human plasma sample, we identified and rigorously quantified over 120 eicosanoids.

Phosphorylcholine-containing lipid molecular species profiling in biological tissue using a fast HPLC/QqQ-MS method

A fast reversed-phase liquid chromatography-electrospray ionization triple quadrupole mass spectrometry method was developed for the molecular species profiling of glycerophosphocholine (GPC) and sphingomyelin (SM) in total lipid extracts. A two-stage mass spectrometry strategy was adopted to analyze in detail the composition of lipid molecular species. Precursor ion analysis was first conducted to obtain the preliminary composition profile of the phosphorylcholine-containing lipid. The product ion spectra were sequentially acquired for each recorded signal to determine the molecular structure of the lipid. A total of 150 GPCs and 12 SMs were identified in the fetal mouse lung with relative amounts ranging from 13.7% to less than 0.002% (normalizing by the total signal response). A column packed with core-shell particles was used to obtain excellent chromatographic separation with a shorter time demand in a conventional high-performance liquid chromatography system. Considering the compromise between the chromatographic efficiency and the electrospray signal response, the optimization of the mobile phase improves the chromatographic plate number to approximately 40,000 and the detection limits to less than 0.001 mg/L. The applicability of the method was validated through a study of chemically induced early lung maturation. The metabolic alteration in the fetal mouse lung was clearly reflected in the GPC and SM composition with several characteristics of the molecular structure that related to the character of the phospholipid layer upon the epithelial lining of alveoli and the relevant cell function. The results indicated that this analytical strategy is reliable for comprehensive molecular species profiling of GPC and SM and might be extended to the analysis of other phospholipids.

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 (R2) about 0.9999. The limits of detection (S/N=3) for three BRs were found to be 2.00–8.00ng/L while the limits of quantification (S/N=10) were 6.00–23.0ng/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 0.055ng/g fresh weight (FW) 24-epiBR and 0.070ng/g (FW) 28-epihomoBR were successfully detected from only 2g A. thaliana plants.