Quantitation of tadalafil in human plasma by liquid chromatography-tandem mass spectrometry with electrospray ionization

A simple, rapid, sensitive and specific liquid chromatography-tandem mass spectrometry method was developed and validated for quantitation of tadalafil (I) in human plasma, a new selective, reversible phosphodiesterase 5 inhibitor. The analyte and internal standard (sildenafil, II) were extracted by liquid-liquid extraction with diethyl ether/dichloromethane (70/30, v/v) using a Glas-Col Multi-Pulse Vortexer. The chromatographic separation was performed on reverse phase Xterra MS C18 column with a mobile phase of 10mM ammonium formate/acetonitrile (10/90, v/v, pH adjusted to 3.0 with formic acid). The protonate of analyte was quantitated in positive ionization by multiple reaction monitoring with a mass spectrometer. The mass transitions m/z 390.4 --> 268.0 and m/z 475.5 --> 58.3 were used to measure I and II, respectively. The assay exhibited a linear dynamic range of 10-1000 ng/mL for tadalafil in human plasma. The lower limit of quantitation was 10 ng/mL with a relative standard deviation of less than 15%. Acceptable precision and accuracy were obtained for concentrations over the standard curve ranges. Run time of 1.2 min for each sample made it possible to analyze a throughput of more than 400 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies.     

Indole-3-piperazinyl derivatives: novel chemical class of 5-HT(6) receptor antagonists

N₁-Arylsulfonyl-3-piperazinyl indole derivatives were designed and identified as a novel class of 5-HT₆ receptors ligands. All the compounds have high affinity and antagonist activity towards 5-HT₆ receptor. The compound 7a (K_i = 3.4 nM, functional assay IC₅₀ = 310 nM) shows enhanced cognitive effect when tested in NORT and Morris water maze models. Synthesis, SAR and PK profile of these novel compounds constitute the subject matter of this Letter.     

High-performance liquid chromatography method for the quantification of pantoprazole in human plasma

A sensitive and selective HPLC method with UV detection (290 nm) was developed and validated for quantitation of pantoprazole, proton-pump inhibitor, in human plasma. Following a single-step liquid-liquid extraction with methyl tert-butyl ether/diethyl ether (70/30, v/v), the analyte and internal standard (zonisamide) were separated using an isocratic mobile phase of 10mM phosphate buffer (pH 6.0)/acetonitrile (61/39, v/v) on reverse phase Waters symmetry C18 column. The lower limit of quantitation was 20 ng/mL, with a relative standard deviation of less than 4%. A linear range of 20-5000 ng/mL was established. This HPLC method was validated with between-batch and within-batch precision of 1.3-3.2% and 0.7-3.3%, respectively. The between-batch and within-batch bias was -0.5 to 8.2 % and -2.5 to 12.1%, respectively. This validated method is sensitive and repeatable enough to be used in pharmacokinetic studies.     

High-performance liquid chromatography method for the quantification of entacapone in human plasma

A simple, sensitive and selective HPLC method with UV detection (315 nm) was developed and validated for quantitation of entacapone in human plasma, the newest addition to the group of antiparkinsonian agents. Following a single-step liquid-liquid extraction (LLE) with ethyl acetate/n-hexane (30/70, v/v), the analyte and internal standard (rofecoxib) were separated using an isocratic mobile phase of 30 mM phosphate buffer (pH 2.75)/acetonitrile (62/38, v/v) on a reverse phase C18 column. The lower limit of quantitation was 25 ng/mL, with a relative standard deviation of less than 8%. A linear range of 25-2500 ng/mL was established. This HPLC method was validated with between-batch and within-batch precision of 2.2-4.2% and 1.7-7.8%, respectively. The between-batch and within-batch accuracy was 98.7-107.5% and 97.5-106.0%, respectively. Frequently coadministered drugs did not interfere with the described methodology. Stability of entacapone in plasma was excellent, with no evidence of degradation during sample processing (autosampler) and 30 days storage in a freezer. This validated method is sensitive, simple and repeatable enough to be used in pharmacokinetic studies.     

Design,synthesis and pharmacological evaluation of 4-(piperazin-1-yl methyl)-N1-arylsulfonyl indole derivatives as 5-HT6 receptor ligands

4-(Piperazin-1-yl methyl)-N₁-arylsulfonyl indole derivatives were designed and synthesized as 5-HT₆ receptor (5-HT₆R) ligands. The lead compound 6a, from this series shows potent in vitro binding affinity, good PK profile, no CYP liabilities and activity in animal models of cognition.     

Fat Storage-inducing Transmembrane Protein 2 Is Required for Normal Fat Storage in Adipose Tissue

Triglycerides within the cytosol of cells are stored in a phylogenetically conserved organelle called the lipid droplet (LD). LDs can be formed at the endoplasmic reticulum, but mechanisms that regulate the formation of LDs are incompletely understood. Adipose tissue has a high capacity to form lipid droplets and store triglycerides. Fat storage-inducing transmembrane protein 2 (FITM2/FIT2) is highly expressed in adipocytes, and data indicate that FIT2 has an important role in the formation of LDs in cells, but whether FIT2 has a physiological role in triglyceride storage in adipose tissue remains unproven. Here we show that adipose-specific deficiency of FIT2 (AF2KO) in mice results in progressive lipodystrophy of white adipose depots and metabolic dysfunction. In contrast, interscapular brown adipose tissue of AF2KO mice accumulated few but large LDs without changes in cellular triglyceride levels. High fat feeding of AF2KO mice or AF2KO mice on the genetically obese ob/ob background accelerated the onset of lipodystrophy. At the cellular level, primary adipocyte precursors of white and brown adipose tissue differentiated in vitro produced fewer but larger LDs without changes in total cellular triglyceride or triglyceride biosynthesis. These data support the conclusion that FIT2 plays an essential, physiological role in fat storage in vivo.     

High-performance liquid chromatography method for the quantification of rabeprazole in human plasma using solid-phase extraction

A simple, sensitive and selective HPLC method with UV detection (284 nm) was developed and validated for quantitation of rabeprazole in human plasma, the newest addition to the group of proton-pump inhibitors. Following solid-phase extraction using Waters Oasistrade mark SPE cartridges, the analyte and internal standard (Pantoprazole) were separated using an isocratic mobile phase of 5 mM ammonium acetate buffer (pH adjusted to 7.4 with sodium hydroxide solution)/acetonitrile/methanol (45/20/35, v/v) on reverse phase Waters symmetry C(18) column. The lower limit of quantitation was 20 ng/mL, with a relative standard deviation of less than 8%. A linear range of 20-1000 ng/mL was established. This HPLC method was validated with between- and within-batch precision of 2.4-7.2% and 2.2-7.3%, respectively. The between- and within-batch bias was -1.7 to 2.6% and -2.6 to 2.1%, respectively. Frequently coadministered drugs did not interfere with the described methodology. Stability of rabeprazole in plasma was excellent, with no evidence of degradation during sample processing (autosampler) and 3 months storage in a freezer. This validated method is sensitive, simple and repeatable enough to be used in pharmacokinetic studies.     

Proliferating cell nuclear antigen (PCNA) as a proliferative marker during embryonic and adult zebrafish hematopoiesis

We investigated the expression of proliferative cell nuclear antigen (PCNA) in zebrafish to delineate the proliferative hematopoietic component during adult and embryonic hematopoiesis. Immunostaining for PCNA and enhanced green fluorescence protein (eGFP) was performed in wild-type and fli1-eGFP (endothelial marker) and gata1-eGFP (erythroid cell marker) transgenic fish. Expression of PCNA mRNA was examined in wild-type and chordin morphant embryos. In adult zebrafish kidney, the renal tubules are surrounded by endothelial cells and it is separated into hematopoietic and excretory compartments. PCNA was expressed in hematopoietic progenitor cells but not in mature neutrophils, eosinophils or erythroid cells. Some PCNA+ cells are scattered in the hematopoietic compartment of the kidney while others are closely associated with renal tubular cells. PCNA was also expressed in spermatogonial stem cells and intestine crypts, consistent with its role in cell proliferation and DNA synthesis. In embryos, PCNA is expressed in the brain, spinal cord and intermediate cell mass (ICM) at 24 h-post fertilization. In chordin morphants, PCNA is significantly upregulated in the expanded ICM. Therefore, PCNA can be used to mark cell proliferation in zebrafish hematopoietic tissues and to identify a population of progenitor cells whose significance would have to be further investigated.     

Impact of temperature on Na2Sr(PO4)F:Eu3+ phosphor

In this article, we report the synthesis of Na₂Sr_1‐x(PO₄)F:Eu_x phosphor via a combustion method. The influence of different annealing temperatures on the photoluminescence properties was investigated. The phosphor was excited at both 254 and 393 nm. Na₂Sr_1‐x(PO₄)F:Eu_x³⁺ phosphors emit strong orange and red color at 593 and 612 nm, respectively, under both excitation wavelengths. Na₂Sr_1‐x(PO₄)F:Eu_x³⁺ phosphors annealed at 1050°C showed stronger emission intensity compared with 600, 900 and 1200°C. Moreover, Na₂Sr_1‐x(PO₄)F:Eu_x³⁺ phosphor was found to be more intense when compared with commercial Y₂O₃:Eu³⁺ phosphor. Copyright © 2013 John Wiley & Sons, Ltd.     

Selection of suitable housekeeping genes for expression analysis in glioblastoma using quantitative RT-PCR

Background  

Considering the broad variation in the expression of housekeeping genes among tissues and experimental situations, studies using quantitative RT-PCR require strict definition of adequate endogenous controls. For glioblastoma, the most common type of tumor in the central nervous system, there was no previous report regarding this issue.