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Cao D.,Guangdong Pharmaceutical University | Cao D.,China Pharmaceutical University | Cao D.,Shanghai Key Laboratory for Pharmaceutical Metabolite Research | Li W.,China Pharmaceutical University | And 13 more authors.
Biomedical Chromatography | Year: 2014

A simple, rapid and high sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of neostigmine in small-volume beagle dog plasma was developed to assess the plasma pharmacokinetics of neostigmine. After protein precipitation in a Sirocco 96-well filtration plate, the filtrate was directly injected into the LC-MS/MS system. The analytes were separated on a Hanbon Hedera CN column (100×4.6 mm, 5 μm) with a mobile phase composed of methanol-water (60:40, v/v) and the water containing 0.01% formic acid at a flow rate of 0.6mL/min, with a split ratio of 1:1 flowing 300 μL into the mass spectrometer. The run time was 3 min. Detection was accomplished by electrospray ionization source in multiple reactions monitoring mode with the precursor-to-product ion transitions m/z 223.0→72.0 and 306.0→140.0 for neostigmine and anisodamine (internal standard), respectively. The method was sensitive with a lower limit of quantitation of 0.1 ng/mL, and good linearity in the range 0.1-100ng/mL for neostigmine (r≥0.998). All the validation data, such as accuracy, intra-run and inter-run precision, were within the required limits. The method was successfully applied to pharmacokinetic study of neostigmine methylsulfate injection in beagle dogs. © 2013 John Wiley & Sons, Ltd.

Li W.,China Pharmaceutical University | Li W.,Fujian University of Traditional Chinese Medicine | Li W.,Shanghai Key Laboratory for Pharmaceutical Metabolite Research | Wen J.,China Pharmaceutical University | And 13 more authors.
Journal of Separation Science | Year: 2013

A simple, rapid, high-throughput, and highly sensitive LC-MS/MS was developed to determine anisodamine in a small volume (50 μL) of beagle dog plasma using atropine sulfate as the internal standard. The analyte and internal standard were isolated from 50 μL plasma samples after a one-step protein precipitation using Sirocco 96-well protein precipitation filtration plates. The separation was accomplished on a Hanbon Hedera CN column (100 × 4.6 mm, 5 μm) and the run time was 4 min. A Micromass Quatro Ultima mass spectrometer equipped with an ESI source was operated in the multiple reaction monitoring mode with the precursor-to-product ion transitions m/z 306.0→140.0 (anisodamine) and 290.0→123.9 (atropine) used for quantitation. The method was sensitive with a low LOQ of 0.05 ng/mL, and good linearity in the range 0.05-50 ng/mL for anisodamine (r2 ≥ 0.995). All the validation data, such as accuracy, intra- and interrun precision, were within the required limits. The method was successfully applied to the pharmacokinetic study of anisodamine hydrochloride injection in beagle dogs. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Li J.,China Pharmaceutical University | Li J.,Shanghai Key Laboratory for Pharmaceutical Metabolite Research | Li J.,Xuhui Central Hospital of Shanghai | Fang J.,China Pharmaceutical University | And 12 more authors.
Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences | Year: 2014

A simple, rapid and specific high performance liquid chromatography-electrospray ionization tandem mass spectrometry method was developed for simultaneous determination of dabigatran etexilate (BIBR 1048 MS), the intermediate metabolite (BIBR 1087 SE) and dabigatran (BIBR 953 ZW). In this method, a stacked protein precipitation with methanol was performed in Sirocco 96-well filtration plates to extract analytes using only 50. μL plasma. The analysis was performed on an Ultimate TM XB-C18 (4.6 × 50. mm, 5. μm) column using gradient elution with a mobile phase composed of methanol containing 0.01% formic acid and pure water at a flow rate of 0.3. mL/min. The gradient was set to 90% methanol containing 0.01% formic acid for the first 1.0. min, after which it dropped to 10%, and then was kept at 10% for the next 5. min followed by an additional 1.0. min at the initial composition of 90% methanol containing 0.01% formic acid for equilibration. Detection was performed on a triple-quadrupole mass spectrometer electrospray ionization interface in positive ion mode. Linear calibration curves were obtained over the concentration ranges of 1-500. ng/mL for all analytes. The validated LC-MS/MS method for its selectivity, sensitivity, linearity, precision, accuracy, recovery, matrix effect and stability had been successfully applied to a pharmacokinetic study of analytes in rat plasma following a single oral administration of 15. mg/kg dabigatran etexilate. © 2014 Elsevier B.V.

Zhou Y.,Fudan University | Pu H.-H.,Xuhui Central Hospital of Shanghai | Xu X.-Y.,Fudan University | Li B.,Fudan University | And 2 more authors.
Fudan University Journal of Medical Sciences | Year: 2010

Objective: To study the changes of renal vitamin D metabolism and skeleton phenotypes in ovariectomized rats and effects of estrogen treatment. Methods: Female SD rats (n = 30) were randomly divided into 3 groups (n = 10 for each). Bilateral ovariectomy for ovariectomy (OVX) and OVX-estrogen (OVX-E) groups, and sham surgery for SHAM group were performed at 6 month of age respectively. Meanwhile, estrogen (1 mg/kg Nilestriol in 2 mL drink water) in OVX-E group was administered twice a week by gavages from 6 to 18 weeks after operation. Same volume of drink water was used as the control for OVX and SHAM groups. The serum vitamin D levels, calcium and phosphate, and skeleton phenotypes were investigated at the middle or end of experiments. Results: The serum 1,25(OH)2D level in OVX rats was 63.1 pmol/L, increased by 64% (P<0. 001) compared with 38. 5 pmol/L in SHAM. The high 1,25(OH)2D level was back down to 24. 7 pmol/L by estrogen treatment in OVX-E (- 60. 8% versus OVX, P<0. 001), which was lower than that in SHAM (P<0. 01). The osteoporotic phenotypes in OVX rats were showed by the decrease (- 9. 5% in femur, P<0. 001 and-9.8% in lumbar, P<0. 001) of volume bone mineral density (vBMD), and the decrease of bone volume as determined by histomorphometry analysis. Bone resorption was activated after ovariectomy as showed by the increased urine PyD excretion (58. 7%, P<0. 05). Meanwhile, the calcium levels in serum and urine of OVX rats were lower than that in SHAM. The skeleton phenotypes were improved by Nilestriol treatment showed by increased vBMD and bone volume, increased serum calcium and decreased urine PyD excretion, while the urine Ca excretion still kept in lower level (- 60%, versus SHAM, P<0. 01). Conclusions: The serum 1,25(OH)2D level was increased in ovariectomized osteoporotic rats, which was fell down by estrogen treatment along with improved bone quality and structure.

Wang S.,China Pharmaceutical University | Wang S.,Shanghai Key Laboratory for Pharmaceutical Metabolite Research | Wang S.,Shanghai Research Center for Drug Metabolism | Wang S.,Xuhui Central Hospital of Shanghai | And 14 more authors.
Analytica Chimica Acta | Year: 2012

A comprehensive two-dimensional (2D) separation is one that employs two separation dimensions (columns) and draws on all of the available resolving power from each of the dimensions of separate the components in a sample. In this study, a comprehensive 2D chromatography approach was developed for the separation and identification of membrane permeable compounds in a famous traditional Chinese medicine of Schisandra chinensis. The first dimensional column was the immobilized liposome chromatography (ILC) column, which mimics the biological membranes and can be used to study drug-membrane interactions in liquid chromatography. Using an automatic ten-port switching valve equipped with two sample loops, the section of the first-dimension was introduced in the second-dimension consist of a silica monolithic column. More than 40 components in Schisandra chinensis were resolved by using the developed separation system and among them 14 compounds were identified interacting with the ILC column based on their retention action, UV and mass data. With this comprehensive 2D-HPLC system, the three-dimensional chromatographic fingerprints of Schisandra chinensis were preliminarily established and processed by using principal component analysis and hierarchical clustering analysis. The obtained information can distinguish the unacceptable samples of the quality control. The result demonstrated that the 2D biochromatography system has been demonstrated to have more advantages of finding strong binding bioactive components, providing an enhanced peak capacity, good sensitivity and powerful resolution biological fingerprinting analysis of complex TCMs, which was a useful means to control the quality of and to clarify the membrane permeability of the compounds in Schisandra chinensis. © 2011 Elsevier B.V.

Pu H.-H.,Fudan University | Pu H.-H.,Xuhui Central Hospital of Shanghai | Duan J.,Fudan University | Wang Y.,Fudan University | And 3 more authors.
Placenta | Year: 2012

Our previous study has demonstrated that thymic stromal lymphopoietin (TSLP) stimulates trophoblast proliferation and invasion, suggesting TSLP plays an important role in the placentation in early human pregnancy, but the intracellular molecular mechanism is currently unknown. The present study is undertaken to investigate whether the STAT3-c-Myc signaling pathway is involved in TSLP-mediated trophoblast proliferation. Primary human first-trimester trophoblasts were treated with TSLP only, or TSLP combined with different signaling inhibitors (STAT3, STAT5, AKT, and ERK). The levels of STAT3 tyrosine phosphorylation and c-Myc expression were determined by using Western blot analysis, and the proliferation of trophoblasts was analyzed by BrdU cell proliferation assay. JEG-3 cells were transfected with siRNA targeting to c-Myc, and the proliferation was determined in JEG-3 cells treated with TSLP only, or TSLP combined with c-Myc silencing. It was revealed that treatment with TSLP significantly enhanced STAT3 phosphorylation and c-Myc expression in human trophoblasts. The effect of TSLP upregulation on trophoblast proliferation was abrogated completely by either STAT3 inhibitor or c-Myc siRNA silence. We further found that the upregulation of TSLP on c-Myc expression was abrogated completely by the STAT3 inhibitor, which suggests that the intracellular STAT3 phosphorylation is an upstream signal of c-Myc expression in the TSLP-stimulated trophoblast proliferation. These results suggest that TSLP may upregulate c-Myc expression through activation of STAT3 pathway, thereby inducing trophoblast proliferation. © 2012 Elsevier Ltd. All rights reserved.

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