Authors:Y. Liu, S. Wang, C. Wang, G. Chen, H. Cao, Y. Wang, W. Ma, Y. Hu, and Y. Yan
A comparative proteomic analysis of grain proteins during five grain developmental stages of wheat cultivar Chinese Spring (CS) and its 1Sl/1B substitution line CS-1Sl(1B) was carried out in the current study. A total of 78 differentially expressed protein (DEP) spots with at least 2-fold expression difference were detected by two-dimensional electrophoresis (2-DE). Among these, 73 protein spots representing 55 differentially expressed proteins (DEPs) were successfully identified by matrix-assisted laser desorption/ionization time-offlight tandem mass spectrometry (MALDI-TOF/TOF-MS). Differential protein spots between the two genotypes were analyzed by cluster software, which revealed significant proteome differences. There were 39 common spots (including 33 DEPs) that showed significant difference between the two lines across five grain developmental stages, of which 14 DEP spots (including 11 DEPs) were mainly involved in carbohydrate metabolism that were encoded by the genes on 1B chromosome while 25 DEP spots (including 12 DEPs) were mainly related to stress response and gluten quality that were encoded by 1S1 chromosome. These results indicated that the Sl genome harbors more stress and quality related genes that are potential valuable for improving wheat stress resistance and product quality.
Authors:Peiwu Geng, Xinhua Luo, Xiufa Peng, Zixia Lin, Wenhao Chen, Jin Zhang, Congcong Wen, Lufeng Hu, and Siyi Hu
Eupatilin, mainly derived from Artemisia asiatica (Asteraceae), is an O-methylated flavone with various bioactivities. In the present study, a validated ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method was established for the quantification of eupatilin in rat plasma with the internal standard (IS) of tussilagone and the protein precipitation of plasma samples was performed using acetonitrile–methanol (9:1, v/v). The eupatilin and IS were eluted separately on a UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 μm) with the gradient mobile phase consisted of 0.1% formic acid and acetonitrile. The protonated analytes were quantified by multiple reactions monitoring (MRM) mode with an electrospray ionization (ESI) source operated in positive ion mode. The calibration plots were found to be linear over the range from 2 to 1000 ng/mL for eupatilin in rat plasma. Both of the intra-day and inter-day precision variations (RSDs) were ≤13%. The recoveries of eupatilin in rat plasma were between 83.7% and 94.6%, and the accuracy of the method ranged from 95.8% to 107.6%. In addition, the validated method was applied to pharmacokinetic study of eupatilin after an intravenous dose of 2 mg/kg to rats.
Authors:Su-su Bao, Jian Wen, Teng-hui Liu, Bo-wen Zhang, Chen-chen Wang, and Guo-xin Hu
Olmutinib (Olita™) is an oral third-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) which is used to treat non-small cell lung cancer (NSCLC). A simple, rapid, and sensitive method based on ultra-performance liquid chromatography tandem mass spectrometry (UHPLC–MS/MS) has been developed for the determination of olmutinib. Sample preparation was performed following simple one-step protein precipitation with acetonitrile. Olmutinib and internal standard (dasatinib) were separated on an Eclipse Plus C18 RRHD (2.1 × 50 mm, 1.8 μm) column. The mobile phase consisted of acetonitrile–0.1% formic acid in water with gradient elution. A total run time of 1.7 min was achieved. Detection was performed on a positive-ion electrospray ionization mass spectrometer in multiple reaction monitoring (MRM) mode, using transitions of m/z 487.2 → 402.1 for olmutinib and m/z 488.2 → 401 for dasatinib (IS), respectively. The calibration curve (R2 = 0.999) was linear over the range of 1–500 ng/mL. The recovery of olmutinib ranged from 85.8% to 95.5%. This method can be applied to pharmacokinetic studies of olmutinib.
Chronic hepatitis B virus (HBV) carriers may develop hepatocellular carcinoma (HCC) by a wide range of mechanisms including angiogenesis. We show that HBV replication induces the expression of angiogenic proteins interleukin 6 (IL6) and cyclooxygenase-2 (Cox2). Interestingly, ibuprofen (a Cox2 inhibitor) is found to attenuate the levels of IL6 and Cox 2 which are induced by HBV replication.
The mechanism of attenuation of angiogenic proteins by ibuprofen was further investigated. Our results show that HBx is involved in the increase of the expression of Cox2 through the NFκB pathway. However, the expression of Cox2 is decreased when the HBx-expressing cells are incubated with ibuprofen. The contrasting effect of HBx on Cox2 is found to be determined by differential dimer formation among the members of the NFκB family of proteins, including NFκB, RelA, and C-rel. Specifically, HBx alone results in dimer formation between NFκB and RelA, while the combined presence of HBx and ibuprofen leads to the formation of NFκB and C-rel. Additional information on the interaction network involving HBx, ibuprofen, and NFκB pathways is revealed by two-dimensional liquid chromatography-tandem mass spectrometry proteomics analysis. Taken together, our findings provide new insights on the angiogenesis induced by HBV replication.
Authors:B. Sas, G. Domány, Ilse Gyimóthy, Katalin Gaál Kovácsné, and M. Süth
The effect of free-range versus cage management system on corticosterone transfer into the eggs was studied in laying hens. Hungarian Yellow laying hens (age: 21 weeks, body weight: 2.0 ± 0.5 kg) were divided into two groups in the spring: Group I, free-range keeping (n = 15 layers, density: ≯ 0.5 bird/m2) in outdoor runs, with continuous access to a commercial layer feed; Group II, hens kept in battery cages (n = 17 layers, density: 2 birds/m2, natural light, continuous access to feed and water). Eggs were collected after a one-week adaptation period on days 2, 7 and 16. Corticosterone (CST) was extracted from homogenised egg samples using an ASE-200 Accelerated Solvent Extractor and then assayed by liquid chromatography linked with tandem mass spectrometry (LC-MS/MS) [Thermo Quest Surveyor high-performance liquid chromatography (HPLC) interfaced via Atmospheric Pressure Chemical Ionisation (APCI) ion source to Finnigan/Thermo Quest LCQ Deca MS/MS] using dexamethasone as internal standard with positive APCI ionisation. CST concentrations of whole eggs laid by free-range hens on days 2, 7 and 16 were 0.370 ± 0.218, 0.259 ± 0.066 and 0.915 ± 0.745 ng·g-1, respectively, while those of eggs laid by caged hens were 0.206 ± 0.157, 0.223 ± 0.165 and 0.184 ± 0.110 ng·g-1 at the above sampling times. It is concluded that in free-range laying hens the sharp changes of environmental weather conditions significantly increased the corticosterone content of eggs, while the environmentally controlled and closed battery cage management technology resulted in relatively uniform corticosterone concentrations in the whole eggs.
Authors:M. Crevar-Sakač, Z. Vujić, Z. Vujčić, B. Marković, and D. Vasiljević
A simple and sensitive liquid chromatography—tandem mass spectrometry method was developed for the quantification of atorvastatin, ortho-hydroxyatorvastatin, para-hydroxyatorvastatin, and atorvastatin lactone in rat plasma. Solid-phase extraction was used for preparation of samples. Rosuvastatin was chosen as an internal standard. Chromatographic separation was achieved on ZORBAX Eclipse C18 Analytical, 4.6 × 100 mm (3.5 μm) column with a gradient mobile phase composed of acetonitrile and 0.1% acetic acid, at a flow rate of 400 μL min−1. The column was kept at constant temperature (25 °C), and autosampler tray temperature was set at 4 °C. The following selected reaction monitoring (SRM) transitions were selected: (m/z, Q1 → Q3, collision energy) atorvastatin (559.47 → 440.03, 22 eV), atorvastatin lactone (541.36 → 448.02, 19 eV), ortho-ohydroxyatorvastatin (575.20 → 440.18, 20 eV), para-hydroxyatorvastatin (575.54 → 440.18, 20 eV), and rosuvastatin (482.25 with selected combination of two fragments 257.77, 31 eV, and 299.81, 35 eV) in positive ion mode. The method was validated over a concentration range of 0.5–20 ng mL−1 for ortho-hydroxyatorvastatin and para-hydroxyatorvastatin and 0.1–20 ng mL−1 for atorvastatin and atorvastatin lactone with excellent linearity (r2 ≥ 0.99). This method demonstrated acceptable precision and accuracy at four quality control concentration levels. The detection limits were 0.1 and 0.13 ng mL−1 for orth-ohydroxyatorvastatin and para-hydroxyatorvastatin, respectively, and 0.05 ng mL−1 for atorvastatin and atorvastatin lactone. All analytes were found to be stable at examined conditions. Validated method was applied for determination of atorvastatin and its metabolites in plasma of experimental animals.
Authors:Narendra A. Gajbhiye, Jayanti Makasana, Tushar Dhanani, and Raju Saravanan
Aegle marmelos Correa (Bael tree) is a medicinal fruit tree, widely used for healing purposes in various systems of medicines. Coumarins and alkaloids present in various parts of bael tree including roots and fruit pulp are the primary active constituents implicated for its biological activities. An efficient liquid chromatography–electrospray ionization—tandem mass spectrometry (LC—ESI—MS/MS) method was developed for identification and simultaneous determination of four coumarin derivatives, namely, umbelliferone, psoralene, marmin, and imperatorin, and an alkaloid, skimmianine, in root and stem bark of A. marmelos. The chromatographic separation of analytes was performed on Altima C18 (50 × 4.6 mm, 3 μm) column using methanol and 0.1% acetic acid in water (54:46, v/v) as the mobile phase under isocratic conditions. The LC–MS/MS parameters were optimized in the positive ionization mode using electrospray ionization source. The quantification of the analytes was performed using multiple reaction monitoring (MRM) transitions, umbelliferone (m/z 163.1 → 107.1), psoralene (m/z 187.2 → 131.1), marmin (m/z 333.5 → 163.2), imperatorin (m/z 271.1 → 203.1), and skimmianine (m/z 260.1 → 227.0). The extraction method was standardized for optimum yield of coumarin derivatives and the alkaloid in different extraction solvents. Higher yield of the analytes was found in methanolic extracts in comparisons to petroleum ether, chloroform, ethyl acetate, ethanol, and water. The method was validated for linear range, intra- and inter-batch precision and accuracy. The distribution of coumarin derivatives and an alkaloid was found to vary significantly in different plant samples, and their concentration was much higher in roots as compared to stem bark.
Authors:Biljana K. Tubić, Bojan D. Marković, Sandra S. Vladimirov, Slavica M. Ristić, Branka M. Ivković, Miroslav M. Savić, Jelena M. Poljarević, and Tibor J. Sabo
A series of new (S,S)-ethylenediamine-N,N′-di-2-(3-cyclohexyl)propanoate esters has shown cytotoxic activity towards human leukemic cell lines. The aim of this study was to develop and validate a bioanalytical method for quantification of (S,S)-O,O-diethyl-ethylenediamine-N,N′-di-2-(3-cyclohexyl)propanoate dihydrochlorides (DE-EDCP) and its metabolite, substituted propanoic acid (EDCP), in mouse serum by ultra high-performance liquid chromatography—tandem mass spectrometry (UHPLC—MS/MS). Structural analog, derivative of 1,3-propanediamine, was used as an internal standard (IS). Sample preparation employed protein precipitation by acetonitrile and subsequent centrifugation. Optimal UHPLC separation conditions were set to achieve simultaneous determination of both compounds in a short run time of 6 min. Additionally, the selected reaction monitoring (SRM) mode developed in this method allowed a highly sensitive, accurate, and precise identification of compounds of interest. The lower limit of quantitation (LOQ) was 1.3 ng mL−1 for DE-EDCP and 0.3 μg mL−1 for EDCP. The calibration curves were linear over the concentration range of 1.3–26.7 ng mL−1 and 0.3–6.7 μg mL−1 for DE-EDCP and EDCP, respectively. Precision (%CV) and accuracy (%RE) for DE-EDCP and EDCP ranged from 3.5% to 16.0% and from 1.8% to 14.4%, respectively.
The validation process was performed in accordance with the regulatory guidance/guideline, and all of the obtained results met the established acceptance criteria. The newly developed and validated UHPLC—MS/MS method is rapid, sensitive, and selective, and it can be successfully applied to drug monitoring in nonclinical studies.
Galangin (GAL), the major bioactive flavonol extracted from Alpinia officinarum Hance (Zingiberaceae), has attracted much attention due to its multiple biological activities. To develop a fast, reliable, and sensitive ultrahigh-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method for the quantification of GAL in rat plasma and mouse tissues. UHPLC–MS/MS using electrospray ionization operating in negative-ion mode was used to determinate GAL in 18 rats receiving three doses of GAL (2 and 9 mg/kg by intravenous injection, 5 mg/kg by oral administration), with six rats for each dose. Blood samples were collected at 0.0333, 0.25, 0.5, 1, 2, 4, 6 and 8 h. A total of 25 mice received 18 mg/kg GAL by intraperitoneal injection. Liver, heart, lung, spleen, brain, and kidney tissue samples were collected at 0.25, 0.5, 2, 4, and 6 h. The precision of the method was better than 12.1%, while the accuracy ranged from −4.8% to 8.1%. The results of pharmacokinetics demonstrated rapid GAL absorption (tmax of 0.25 h), fast elimination (t1/2 <1.1 h) after three different dosages, and an absolute bioavailability of ~7.6%. Tissue distribution analysis revealed abundant GAL in liver, kidney, spleen, and lung and smaller amounts in brain. The developed method proved fast (3 min), efficient, and reliable, with high selectivity for the quantitative analysis of GAL in biological samples. This is the first study to identify the target tissues of GAL, and the results may help to elucidate the mechanisms underlying its therapeutic effects in vivo.
Schizonepeta tenuifolia Briq. (ST) has been used as an aromatic exterior-releasing medicine in clinical practice for thousands of years in China. Previous researches have revealed both volatile oil (STVO) and aqueous extract (STAE) from ST showed significant pharmacological activities, such as anti-virus, anti-inflammation, anti-oxidation, and immunoregulation. However, the influence between each other was still unknown. The purpose of this study was to compare the pharmacokinetic profiles of three main flavonoids (luteoloside, apigetrin, and hesperidin) in STAE to illustrate the influence of STVO. A liquid chromatography-tandem mass spectrometry (HPLC-MS) method was established to quantitatively analyze the three absorbed ingredients in the plasma of healthy rats. Biological samples were analyzed on an Agilent Eclipse Plus C18 column (3.0 mm × 150 mm, 3.5 μm) with gradient mobile phase (containing 0.2% formic acid and acetonitrile) at a flow rate of 0.8 mL/min. All the analytes and quercitrin (IS) were investigated with an electrospray ionization source (ESI) using multiple-reaction monitoring (MRM) in negative ionization mode. In addition, this quantitative method showed good linearities (r ≥ 0.9995) and the lower limits of quantification were 0.590–1.19 ng/mL. The intra- and inter-day precisions ranged 3.47–10.45% and 4.29–11.28% for the three analytes. The mean extraction recoveries were in the range of 77.41–109.79% and the average matrix effects were within 83.41–112.67%. The validated method has been fully applied to compare the pharmacokinetic parameters of the three flavonoid glycosides in rat plasma after oral administration of STAE and STAE+STVO. In comparison of luteoloside, apigetrin, and hesperidin in STAE group, it was found that different degree of increasing existed for the time to reach the maximum concentration (Tmax), elimination half-life time (T1/2), the area under the concentration curves (AUC0→t and AUC0→∞) and the maximum concentrations (Cmax) in STAE+STVO group. As can be seen from above results, STVO could improve the absorption and bioavailability of the three analytes. These findings would provide some active and strong basis of safe clinical application for ST and further exploitation for STVO from the perspective of drug–drug interaction.