2 11 In Vitro HPLC Analysis of DE The samples of DE in vitro exp

2.11. In Vitro HPLC Analysis of DE The samples of DE in vitro experiments were analyzed using an HPLC system consisting of a system controller (SCL-10 ATVP; Shimadzu, Japan), a binary pump (LC-10 ATVP, Shimadzu), a UV-VIS detector (SPD-10 AVP, Shimadzu), a column oven, and an autoinjector (SIL-10A, Shimadzu). The separation method was under the www.selleckchem.com/products/Paclitaxel(Taxol).html following conditions: C18 reversed phase analytical Inhibitors,research,lifescience,medical column (4.6 × 150mm2, 5μm, Shim-pack VP-ODS). The mobile phase was 60:40 (v/v) methanol-ammonium acetate buffer (0.05M, pH 4.0), column temperature of 40°C, UV detective wavelength of 257nm, flow rate of 1.0mL/min, and injection volume of 10μL. The data were acquired

and analyzed by Shimadzu Inhibitors,research,lifescience,medical Class-VP chromatography software. There was no interference from skin and a well-separated peak was detected at the retention time of 9.1 ± 0.1min with the sensitivity of 0.02μg/mL. The peak area correlated linearly with DE concentration in the range from 1 to 500μg/mL. 2.12. In Vivo UPLC-MS/MS Analysis of DE The analyte was recovered from plasma samples by liquid-liquid extraction (LLE) after thawed thoroughly at room temperature [19]. A 100μL aliquot of plasma, 10μL ibuprofen (1μg/mL) as internal standard (IS), and 10μL 0.1 HCl (1mol/L) were pipetted into 1.5mL centrifuge tubes. Samples were extracted using 1mL ethyl acetate and the tubes were vortexed for 2min prior Inhibitors,research,lifescience,medical to centrifugation

at 17,800×g for 3min. Then 800μL supernatant from each centrifuge tube was pipetted into sample insert and evaporated to dryness completely at 40°C with a vacuum centrifugal concentrator (miVac Inhibitors,research,lifescience,medical DUO, Genevac). Samples were then reconstituted with 200μL 50:50 (v/v) methanol-water, the sample vials were vortexed for a further 1min and centrifuged

at 17,800×g for 3min, and then the supernatants were used for analysis. Analysis of DE and plasma was performed with UPLC-MS/MS system equipped with a system controller (SCL-10 ATVP; Shimadzu), a binary pump (LC-10 ATVP; Shimadzu), a UV-VIS detector (SPD-10 AVP, Shimadzu), a column oven, and an Inhibitors,research,lifescience,medical auto injector (SIL-10A; Shimadzu) with an electrospray ionization (ESI) interface. The UPLC separation method was under the following conditions: C18 reversed phase analytical column (Shim-pack XR-ODS) (2.0 I.D. × 75mm2, 1.6μm), mobile phase of methanol and 10mM ammonium acetate buffer, column temperature of 40°C, detective wavelength Journal of Clinical Oncology of 257nm, flow rate of 0.3mL/min, and injection volume of 5μL (see Table 5). A gradient elution was carried out using a mobile phase consisted of a mixture of A (10mM ammonium acetate buffer) and B (methanol) at a flow rate of 0.3mL/min according to the following multistep gradients shown in Tables ​Tables22 and ​and33. Table 2 Composition of the formulation for optimization (a). Table 3 Composition of the formulation for optimization (b). Table 5 Gradient conditions for UPLC.

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