The commercially available tablets were purchased from the local market. Stock solution of 1000 μg/mL was prepared by accurately weighing 5.00 mg of MMF, transferred into a 5.0 mL clean and dry volumetric flask, and dissolved in methanol. The primary standard solution of concentration of 10 μg/mL was prepared by taking 10 μL stock solutions and diluted to 1.0 mL with methanol. Further a series of working standard solutions of different concentrations were sequentially diluted to the required selleckchem volume. The LC/MS/MS analysis was carried out on Applied Biosystems API 3200 triple quadrupole mass spectrometer attached to LC 20 Series Shimadzu Corporation (Kyoto, Japan), equipped with pump (Shimadzu
LC-10AT VP), auto sampler (Shimadzu SIL-HTC), degasser (Shimadzu FCV-10AL VP) and system controller (Shimadzu SIL-HTC ver 6.03) in NISHKA Scientific and Research Laboratories, Hyderabad. The chromatographic PD0325901 cost analysis was performed under isocratic conditions using 75% acetonitrile containing 2 mM ammonium acetate at pH 5.0 at a flow rate of 600 μL/min and Chromosil ODS-3, C18, 4.6 × 50 mm, 2.5 μm column. The ionization was carried out
by ESI. The source heater temperature was maintained at 300 °C. The analysis was carried out in multiple reaction monitoring (MRM) mode for the transition m/z 434 → 114 at collision energy 30 V. The mass spectral analysis was carried out by direct infusion of 10 μg/mL solution of MMF in to the ESI source at a flow rate of 10 μL/min along with the mobile phase flow rate of 600 μL/min. The obtained mass spectrum showed m/z 434 as a major ion which can be attributed to the MH+ ion of the analyte. This ion was subjected to collision induced dissociation (CID) using nitrogen as a collision gas. The collision energy was tuned in such a way that the intensity of MH+ ion was reduced to a minimum of 20%. The obtained mass spectrum after CID showed m/z 114 as a major fragment. Hence the transition m/z 434 → 114 was used to monitor the analyte peak in LC/MS/MS analysis. The ESI mass
spectra of MMF obtained before and after fragmentation were presented in Fig. 2 and Fig. 3 Histone demethylase respectively. Intra/inter day precision was calculated at three different concentrations of working standard solution of reference MMF by taking measurements of six replicates at each concentration on different occasions. Mean, standard deviation (SD) and percent of relative standard deviation (%RSD) were calculated at each concentration and found to be within the acceptable limits. The results of intra day and inter day precision were presented in Table 1. In proposed method, accuracy was determined at three different concentrations of working standard sample solution of MMF (Tablet) by taking measurements of three replicates at each concentration. The proposed method was found to be highly accurate. The calculated %RSD of peak area, weight found and percent of weight found were found to be 2.382, 0.133 and 0.153; 1.