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stance, vinculin, is worth exploring. Rhein, kaempferol, aloe emodin, emodin, and chrysophanol standards were purchased from Sigma Aldrich , while the physcion standard was purchased from Micro Source Discovery System . Ammonium acetate buffer was purchased from Afatinib Fluka . HPLC grade acetonitrile, ethanol, methanol, water, and Whatman no. 1 filter paper were obtained from Fisher Scientific . Doubly distilled deionized water, used throughout the study, was obtained by use of an Elga Genetic Ultra Pure water polishing system from US Filter . Maxi clean RP solid phase extraction C18 cartridges and 0.45 m filters were purchased from Altech . C. alata root samples were collected by the Center of Agricultural Research, Suriname, and identified at the National Herbarium of the University of Suriname, Paramaribo, Suriname .
2.2. Preparation of standard solutions Standard stock solutions of compounds 2 6 were prepared in ethanol at a concentration of 0.5 mg mL, while rhein was prepared at 0.25 mg mL. Standard mixture solutions were prepared in ethanol at various concentration levels Afatinib in the range of 5 250 ppm. All solutions were filtered prior to analysis through a 0.45 m syringe filter and injected four times into the HPLC. The calibration curve for each compound was constructed by plotting the peak area as a function of the standard analyte concentration. 2.3. Sample preparation The C. alata root samples were oven dried at 40 C for five days. Lenalidomide The dried roots were ground by use of a Wiley Mill grinder to particle sizes of 6 mm or smaller.
Ten grams of ground roots were extracted with 100 mL ethanol on an orbital shaker for 12 hours at room temperature. The extraction procedure was repeated two times, after which the two extracts were combined and filtered using Whatman no. 1 filter paper. The extraction solvent was removed by use of rotary evaporation and the residue PARP was reconstituted in 10 mL ethanol and diluted with water . Solid phase extraction was used to remove unwanted interfering phytochemicals from the root extract. The SPE procedure was performed Lenalidomide on an Altech extraction manifold system. SPE C18 cartridges were first conditioned with 4 mL methanol, followed with 4 mL water. Following the conditioning step, 4 mL of the diluted root extract was loaded onto the cartridge. After sample loading, the interfering compounds were removed with 2 mL of 10 aqueous ethanol.
Finally, the fraction containing compounds 1 6 were eluted with 2 mL of hot ethanol . The vacuum pressure was kept at 10 mm Hg during the pre conditioning step and was held constant at 2 mm Hg during the loading and eluting steps. Four replicate SPE extracts were collected. Each eluate was diluted to 5 mL with ethanol. The diluted SPE root Afatinib extract, the eluate, was then filtered through a 0.45 m syringe filter and injected into the HPLC. Each diluted SPE root extract was injected into the HPLC five times, and the average peak area was reported and used for analyte quantification. Separation and quantitative analyses of compounds 1 6 were performed on a Shimadzu HPLC system consisting of an SCL 10A system controller, two LC 10AD pumps, a DGU 14A degasser, an SIL 10AD auto injector and an SPD 10AV UV VIS detector .
Separation of the analytes was performed at 40 C on a Phenomenex Luna C18 column, 100 pore size, 5 m particle size, 250 4.6 mm ID column containing a guard column . The analytes were eluted isocratically at a flow rate of 0.4 mL min using an acetonitrile methanol buffer , where the Lenalidomide buffer is 10 mM ammonium acetate at pH 6.8. The injection volume was 10 L. 2.5. LC APCI MS analysis Analyte identification was performed by use of a Shimadzu LCMS 2010 system . Operating conditions for the HPLC were as described in the previous section. The mass spectrometer used for the identification of the analytes consists of a Q array octapolequadrupole mass analyzer with an APCI interface used in the negative ionization mode and coupled to the Phenomenex Luna C18 column described above.
The APCI probe was operated at a temperature of 400 C, while the CDL and block temperatures were operated at 200 C. The detector voltage was 1.5 kV and the probe was operated in the negative ionization mode with a voltage Lenalidomide of ?4.0 kV. The nebulizing gas was nitrogen at a flow rate of 2.5 L min. The optimum operating conditions for the LC APCI MS were determined for the separation and identification of compounds 1 6 in the scan mode with minimum fragmentation of the analytes. The scan rate of the mass analyzer was at 1s scan within the mass range of m z 100 1000. 2.6. Method validation Precision of the method was obtained by calculating the relative standard deviation from repeated injections of the standard mixture solutions at 15, 45, and 75 ppm for all analytes, except for kaempferol that was determined at 30, 90, and 150 ppm. The intra day precision was determined by six replicate injections, while the inter day precision was determined by six injections for six days, for both