**4.3 Identification**

After the extraction of bioactive compounds, the separation, identification and quantitation are necessary to sudied. In the past few decades, there are a huge number of published reports on HPLC analysis of extracted bioactive compounds from rice grains describe as the most widely used analytical method. Recently, Prabhakaran et al. (2019) reported the analyzed method of selected phenolic compounds in rice grains and its by-products using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) [33].

In addition, our group developed an identification and quantification techniques for phenolic acids and anthocyanins in pigmented rice by using UPLC-ESI-QqQ-MS/MS analysis [13]. The analysis was performed using a UPLC coupled with a mass spectrometer. The separation was carried out by UPLC HSS T3 column 1.8 μm, 2.1 × 100 mm. Column temperature was maintained at 35°C. The mobile phase consisted of 0.1% formic acid (solvent A) and 0.1% formic acid in acetonitrile (solvent B) and the flow rate was set at 0.4 mL/min. The injection volume was 2.0 μL. A stepwise gradients B (%) including an initial isocratic at 2.0% for 1 min, then linear gradient to 98% in 5 min, and by return to the initial condition of 2% B in 7 min. Therefore, the total operation time was 12 min. The solvents and extracts were previously filtered through a 0.45 μm filter membrane. Mass spectral data were obtained in positive or negative mode with a mass range between *m/z* 0 to *m/z* 500. The Multiple Reaction Monitoring (MRM) transitions and compound parameters for the target phenolic compounds were developed. Identification was confirmed by comparing *m/z* values, retention times and fragmentation patterns with those of references standards. In addition, the concentration of phenolic compounds was quantified using external standard method. Our study showed that eight target phenolic compounds were detected and identified in both the unbound and polyphenol-rich bound fractions of pigmented rice [13]. The identification of compounds was carried out by applying one quantification transition (quantifier ion) and/or one or two confirmation transitions (qualifier ions) to assess the detection

and quantification specific to each compound (**Table 1**). Positive ionization mode was selected for caffeic, ferulic acids, (+)-catechin and anthocyanins while negative ionization mode was applied for *p*-coumaric acid and quercetin, due to the chemical structures of the analyses and their ionization behavior observed in ion mode.
