The distribution of different virulence grass carp reovirus strains in some neglected tissues.

Grass carp reovirus (GCRV) is the causative agent of hemorrhagic disease in infected grass carp. During an outbreak, a mortality rate of up to 85% can be experienced, thus leading to substantial economic losses. The current understanding of disease pathogenesis is limited, with the distribution and dynamics of replication amongst different GCRV strains in vivo largely unknown. We determined distribution of different GCRV strains in infected grass carp, especially in some neglected tissues, such as the gill, brain, blood and so on. The results showed elevated viral RNA copy numbers in the blood, with some tissues such as the kidney, heart, brain, and bladder exhibiting even higher viral loads following infection with the virulent GCRV-CL strain. Even more interesting is that the brain exhibited the highest viral load, with a copy number of 800,000 following GCRV-CL infection. Overall, this study provides further insight into GCRV viral load distributions following infection and potentially identified some new viral tropism sites to provide a foundation for further studies aimed at characterizing GCRV viral pathogenesis.

Quantitative determination of endogenous sorbitol and fructose in human erythrocytes by atmospheric-pressure chemical ionization LC tandem mass spectrometry.

Evaluation of different extraction methods for quantification of endogenous sorbitol and fructose in human red blood cells (RBCs) and matrix effects in ESI and APCI showed that protein-precipitation followed by mixed-mode solid-phase extraction was more effective extraction method and APCI more effective ionization method. Then the LC/APCI-MS/MS method was fully validated and successfully applied to analysis of clinical RBC samples. The concentrations of endogenous sorbitol and fructose were determined using calibration curves employing sorbitiol-13C6 and fructose-13C6 as surrogate analytes. The method has provided excellent intra- and inter-assay precision and accuracy with a linear range of 50.0-10,000 ng/mL (correlation coefficient >0.999) for sorbitol-13C6 and 250-50000 ng/mL (correlation coefficient >0.999) for fructose-13C6 in human RBCs.

Quantitative determination of endogenous sorbitol and fructose in human nerve tissues by atmospheric-pressure chemical ionization liquid chromatography/tandem mass spectrometry.

Attachment of anions to sorbitol and fructose has been shown to enhance sensitivity in both electrospray ionization (ESI) and atmospheric-pressure chemical ionization (APCI) mass spectrometry. The post-column addition of CHCl3 produced Cl-adducts of sorbitol and fructose but their signals were suppressed due to the elevated background. Different chlorinated compounds and different additive methods were systematically investigated to form more abundant Cl-adduct precursor ions and deprotonated product ions. The major causes of the high background were explored and effective methods were developed to improve the signal-to-noise ratios and reproducibility. The compositions of mobile phase, percentages of organic modifiers (MeCN, MeOH and water), columns, oven temperature, flow rates and different gradients were investigated to separate sorbitol from fructose along with their isomers including glucose, galactose, mannose, sorbose, mannitol, and dulcitol. The optimized separation was achieved on a Luna 5 mu NH2 100A column (150 x 4.6 mm) using a mobile phase containing MeCN with 0.1% of CH2Cl2 and 50% MeOH in water at a flow rate of 800 microL/min and an oven temperature of 40 degrees C using a gradient liquid chromatography (LC) system. Human nerve tissue samples were extracted by protein precipitation followed by mixed-mode solid-phase extraction. The LC/ESI-MS/MS method produced higher peak intensities than LC/APCI-MS/MS. However, there were matrix effects from extracted tissues in LC/ESI-MS/MS but not in LC/APCI-MS/MS. Consequently, APCI proved to be the more effective method of ionization. Then the LC/APCI-MS/MS method was fully validated and successfully applied to analysis of clinical samples. The concentrations of endogenous sorbitol and fructose were determined using calibration curves employing sorbitol-13C6 and fructose-13C6 as surrogate analytes. The method has provided excellent intra- and inter-assay precision and accuracy with linear ranges of 0.2-80 ng/mg for sorbitol and 1-400 ng/mg for fructose in human nerve tissues.

Method development and validation for quantitative determination of methadone enantiomers in human plasma by liquid chromatography/tandem mass spectrometry.

A high-throughput method for quantitative determination of methadone enantiomers in human plasma was developed and validated by liquid chromatography/tandem mass spectrometry. The effects of pH and of types and concentrations of mobile-phase modifiers on the enantioselectivity of (R)- and (S)-methadone were investigated on a Chiral-AGP column. A baseline separation of the enantiomers was achieved with a retention time of less than 5 min. Ionization suppression and other matrix effects were evaluated. Morphine, cocaine, 6-monoacetylmorphine, benzoylecgonine and ecgonine methyl ester did not interfere with the performance of the assay. The specificity, linearity, intra- and inter-assay precision and accuracy, and extraction recovery were fully evaluated. The method showed excellent reproducibility (overall coefficient of variance < 8%) and accuracy (overall bias < 2.7%) with a broad linear range. The enantiomers were stable in human plasma after five freeze-thaw cycles, under bench-top storage at room temperature (RT) for 6h, in the extract reconstitution solution at RT for 17 h, and in processed-extracts stored at RT for 142 h. This validated LC/MS/MS assay offers high-throughput and improved specificity, sensitivity, linear range and ruggedness over previously published methods and has been successfully applied to the analysis of clinical samples.

Ionization enhancement in atmospheric pressure chemical ionization and suppression in electrospray ionization between target drugs and stable-isotope-labeled internal standards in quantitative liquid chromatography/tandem mass spectrometry.

The phenomena of ionization suppression in electrospray ionization (ESI) and enhancement in atmospheric pressure chemical ionization (APCI) were investigated in selected-ion monitoring and selected-reaction monitoring modes for nine drugs and their corresponding stable-isotope-labeled internal standards (IS). The results showed that all investigated target drugs and their co-eluting isotope-labeled IS suppress each other's ionization responses in ESI. The factors affecting the extent of suppression in ESI were investigated, including structures and concentrations of drugs, matrix effects, and flow rate. In contrast to the ESI results, APCI caused seven of the nine investigated target drugs and their co-eluting isotope-labeled IS to enhance each other's ionization responses. The mutual ionization suppression or enhancement between drugs and their isotope-labeled IS could possibly influence assay sensitivity, reproducibility, accuracy and linearity in quantitative liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS). However, calibration curves were linear if an appropriate IS concentration was selected for a desired calibration range to keep the response factors constant.

Isolation and immunomodulatory effect of flavonol glycosides from Epimedium hunanense.

The ethyl acetate and n-butanol fractions from aerial parts of E. hunanense were initially screened to find active fractions with immunomodulatory activity. Nine compounds, tricin (1), luteolin (2), thalictoside (3), icariin I (4), baohuoside I (5), quercitrin (6), icariin (7), epimedin C (8), and B (9) were isolated from this species for the first time, and 3 was isolated for the first time from flavonoid extracts of the genus. Their structures were established by chemical and spectroscopic methods. The immunomodulatory effects of the n-butanol fraction and epimedin C isolated from the fraction were investigated. Hydrocortisone acetate (HCA) was used as an immunosuppressant to inhibit the immune response of mice. The n-butanol fraction and epimedin C significantly enhanced the response of spleen antibody-forming cells (SAFC) to near normal in the mice treated with HCA. They also significantly enhanced lymphocyte proliferation and caused a significant recovery of interleukin-2 (IL-2) production in the mice inhibited with HCA. In conclusion, they are active principles with immunoenhancing effects.

Characterization of flavonoids in extracts from four species of Epimedium by micellar electrokinetic capillary chromatography with diode-array detection.

A micellar electrokinetic capillary chromatographic (MEKC) method with diode-array detection is developed for the characterization of pharmacologically active flavonoids in extracts prepared from Epimedium brevicornum, E. humanense, E. coactum, and E. truncatum. The pK(a) values of icarin, epimedin B, and epimedin C are determined by spectrophotometry. Optimal separation of icarin, epimedin B and C, and eight other compounds is achieved by determining pK(a) values and by systematically optimizing electrolytic and instrumental parameters. The repeatability of analyses and the reliability of identification are evaluated by the marker technique. Calculated for relative migration times of flavonoids in the extracts, the repeatability of the analyses varies from 0.7 to 6.4% (nine replicates). For migration indices calculated with two markers, however, the repeatability almost falls below 0.5%. The distribution of the flavonoids is found to differ both qualitatively and quantitatively among the four species. The MEKC technique appears to provide a powerful tool for the identification and quality control of plant drugs and for phytotaxonomic investigations.

[Studies on the chemical constituents of Epimedium acuminatum Franch].

Five flavonoids were isolated from the aerial parts of Epimedium acuminatum. Their structures were determined chemically and spectroscopically to be baohuoside VI, tricin, icaritin, icariside I and baohuoside I.