8 μm particle sizes on Agilent 1200 Series UPLC interfaced to an Agilent 6520 Accurate-Mass QTOFMS. A volume of 20 μl of each sample was injected by auto-sampler to the column. Mobile phase comprised solvent A (water containing 0.1% formic acid) and solvent B (acetonitrile containing 0.1% formic acid) was used in gradient mode. The following gradient elution was carried out: eluent B 5–20%from 8 selleck to 15 min; eluent B 45–65% from 22 to 30 min; eluent B 65–90% from 35 to 40 min (to wash the column); eluent B 5% for 40–45 min (for column equilibration). The flow rate of
the solvent was maintained 0.2 ml/min. The mass spectrometer was operated in positive mode in the m/z range 100–1100 at acquisition rate of 2 MS/MS and 3 MS spectra/s with following parameters: gas temperature MK0683 mouse 350 °C, nebulizer 45 psi, drying gas flow 11 L/min, capillary 3.5 V, skimmer voltage 65 V and fragmentor voltage 175 V. Instrument
was calibrated and tuned as per instruction of manufacturer. To assure mass accuracy of recorded ions, continuous calibrations with internal and infused standards with samples (lidocaine, D-camphor, 5, 7-isoflavone) were performed during analysis. MassHunter Workstation software (MassHunter version 3.1) was used for UPLC–QTOFMS data processing which includes of peak detection, chromatographic alignment, background removal, normalization and mass filtering. The raw data set acquired were initially analyzed by Molecular Features (MFs) extraction software for the detection of the compounds. The list of chemically qualified MFs was generated by eliminating interferences and reducing data complexity. Molecular formulae were estimated Chlormezanone on the basis of fragment patterns of ions. Different intensity threshold from 1000 to 10,000 cpu was used for molecular feature extraction in the full retention time range. Background subtracted data of compound exchange (.cef) files was exported into the Mass Profiler Professional (MPP) software package
(Agilent Technologies, version B 02.02). MPP was used for statistical evaluation of technical reproducibility and comparison of samples. In MPP, the retention time and m/z alignment across the sample sets was performed using a tolerance window of 0.2 min and 20 mDa. Molecular Features were reduced stepwise based on frequency of occurrence, abundance of respective MFs in classes and one-way analysis of variance (ANOVA). A probability level of p < 0.05 was applied to reduce nonsignificant molecular features. Compounds that satisfied fold change cut-off 2.0 in at least one condition pair were selected for further analysis and differentiation. Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were performed using MPP. The MS/MS were performed in positive ion mode with optimized parameters. As juice of T.