Rapid determination of rat plasma uridine levels by HPLC-ESI-MS utilizing the Captiva plates for sample preparation

A rapid, accurate and precise HPLC-ESI-MS method for the determination of rat plasma uridine concentrations was developed and is described here. Sample preparation involves methanol precipitation of plasma proteins in a 96-well Captiva protein precipitation filter plate. A clear extract is drawn through the filter plate with vacuum, followed by evaporation of the extract and subsequent reconstitution prior to chromatography on a reversed-phase column with an aqueous mobile phase [0.1% (v/v) glacial acetic acid]. Detection was accomplished by positive-ion electrospray ionization mass spectrometry. A calibration curve ranging in concentration from 0.78 to 25 microM was constructed by best-fit, 1/x weighting linear regression analysis of the calibration standard concentrations vs peak height ratios of analyte with internal standard. The correlation coefficient was >0.995. The overall assay accuracy as shown by the back-calculated concentrations of the calibration curve ranged from 96.6 to 103% with RSD ranging from 4.5 to 20%. While this assay method was developed for the determination of uridine in rat plasma, it could be readily adapted for determination of uridine in plasma from other species, such as human.     

Interaction of dimethyltin(IV) dichloride with 5′‐IMP and 5′‐UMP

The formation constants of the species formed in the systems H⁺ + dimethyltin(IV) + 5′‐IMP and 5′‐UMP, H⁺ + 5′‐IMP and H⁺ + 5′‐UMP have been determined in aqueous solution in the pH range 1.5–9.5 at constant temperature (25 °C) and constant ionic strength (0.1 mol dm⁻³ NaClO₄), using spectrophotometric and potentiometric techniques. ¹H and ³¹P NMR investigations in aqueous solution confirmed the species formation. The precipitated complexes of IMP and UMP by Me₂Sn(IV)²⁺ at low pH values were characterized by elemental analysis and FTIR spectroscopy methods, the bonding sites of the ligands were determined and ruled out purine and pyrimidine moieties (N‐7 and N‐1 in IMP and N‐3 in UMP, respectively) while a bidentated coordination of the phosphate group is concluded in both cases. Finally, the experiments revealed the existence of complexes with trigonal bipyramidal structures that is in agreement with similar systems resulted previously. Copyright © 2008 John Wiley & Sons, Ltd.     

Bioanalytical method validation,biopharmaceutical and pharmacokinetic evaluation of GSK-650394, a serum- and glucocorticoid-regulated kinase 1 inhibitor

GSK-650394 is an inhibitor of serum- and glucocorticoid-regulated kinase 1 that displays potency for treating cancer, hypertension, cardiovascular and neuronal diseases, such as Parkinson’s disease. However, the biopharmaceutical properties and pharmacokinetics of GSK-650394 have not been studied extensively. Also, there are currently no bioanalytical assays available for this new drug candidate. In this study, we developed a simple and sensitive liquid chromatography-tandem mass spectrometry method to quantify GSK-650394 in rat plasma and validated its selectivity, linearity, accuracy and precision, sensitivity, matrix effects, extraction recovery, and stability, following the United States Food and Drug Administration guidelines. In vitro studies showed the biopharmaceutical properties of GSK-650394, including its low solubility in water and simulated gastrointestinal fluids, passive transport in Caco-2 cell monolayers, high plasma protein binding, and primary metabolism by glucuronide conjugation in the small intestine and liver of rats. Following intravenous administration (2 mg/kg) to rats, GSK-650394 exhibited low total clearance (11.18 ± 1.28 mL/min/kg) and volume of distribution at steady-state (346.1 ± 120.6 mL/kg). Following oral administration (2, 5, and 10 mg/kg) to rats, GSK-650394 underwent enterohepatic circulation, with low bioavailability (～9%). The insignificant difference in bioavailability among three oral doses suggests that GSK-650394 may follow linear pharmacokinetics up to an oral dose of 10 mg/kg. In addition, the total form of parent drug and glucuronide conjugate in rat plasma from three oral doses showed a much higher value of area under the plasma concentration versus time curve than the parent drug, indicating that the primary metabolism process of GSK-650394 was glucuronidation. Our findings suggest that the low oral bioavailability of GSK-650394 is associated with its low solubility, instability under acidic gastric conditions, and extensive glucuronidation metabolism.     

Structural Reassignment of Two Polyenol Natural Products

Unusual polyenols that defied chemical principles were reassigned as the nucleosides, adenosine and uridine, using a combination of chemical intuition underpinned by Computer Assisted Structure Elucidation (CASE) and DFT methods.