The Selisistat cell line mobile phase consisted of 10 mM phosphate buffer (adjusted to pH 4 with triethyl amine) and methanol in the ratio of 50:50 v/v that was set at a flow rate of 1 mL/min. A stock solution was prepared by dissolving accurately weighed 100 mg of acipimox in 100 mL of HPLC grade methanol to yield a final concentration of 1 mg/mL of the drug. The stock solution (1000 μg/mL of acipimox) was diluted suitably and spiked with human blank plasma to get 0.1–30 μg/mL of drug. 500 μL of each

standard solution (drug spiked human plasma) was pipetted into a series of polypropylene tubes and vortexed briefly. 3 mL of tert-butyl methyl ether was added to each tube and caped. All calibration samples were vortexed for approximately for 10 min and centrifuged at 4000 rpm for approximately 5 min at 10 °C. The standard supernatant layer was decanted into each clean polypropylene tube and evaporated to dryness at 40 °C under a stream of nitrogen. Then, the dried extract was reconstituted in 500 μL of mobile phase and a 20 μL aliquot was injected into the chromatographic system using Hamilton syringe. The UV detector was used for the estimation of acipimox at 275 nm to maximize the signal of compounds

and minimize the signal of plasma interferents. Akt inhibitor The compositions of mobile phase were optimized through several trials to achieve good resolution and symmetric peak shape for the drug. Optimization of HPLC conditions performed on chromatographic parameters including retention time, column efficiency (HETP) of the various variations of composition, and velocity of mobile phase. Efficiency values (N) showed the results of ≥4000, this suggested that the sharp peaks produced Histone demethylase enough. Acipimox was eluted at 2.8 min. The typical chromatograms for the blank plasma and sample are given in Fig. 2 and Fig. 3 respectively. The system suitability parameters are given in Table 1. The developed method was evaluated for linearity, selectivity, accuracy and precision, stability during various stress conditions including bench

top stability, freeze thaw stability, stability of stock solutions and dilution integrity and recovery. Blank plasma was tested for endogenous interference. Selectivity was evaluated by extracting different blank plasma samples. The absence of interfering peaks at the same retention time of acipimox was considered as evidence for selectivity of the method. Calibration curve was plotted by taking concentration of analyte in X axis and detector response in Y axis. The developed method was linear in the concentration range of 0.1–30 μg/ml with the correlation coefficient value of 0.998. Slope and intercept of the linearity curve ( Fig. 4) was found to be 50.85 and −1.25 respectively. Recovery of acipimox was evaluated by comparing the detector response of acipimox in three quality control samples (LQC, MQC and HQC) with the response of same in equivalent methanolic solutions (Table 2).