Optimization of MS/MS Conditions

The compound-dependent MS parameters, such as precursor ion, product ion, DP and collision energy (ce), were carefully optimized for each target compound individually in both positive and negative ion modes by injecting the individual standard solution into the mass spectrometer. It was observed that the investigated piperamides were adequately ionized in the positive ion ESI mode, whereas phenolics, flavonoids and terpenoid were best ionized in the negative ion ESI mode.

Quantitative Analysis

MS/MS Spectra and MRM Transitions

The precursor-product ion pairs described here were selected for MRM transitions. Piperine (m/z 286 [M+H]+) and piperlonguminine (m/z 274 [M+H]+) generated the same major fragment ion at m/z, 201 in positive ion mode due to the cleavage of the amide group in their structures. A similar fragmentation in piperlongumine at m/z. 318 [M+H]+ led to the product ion at m/z, 221.1. Protocatechuic acid m/z, 153 |M-H]~ and caffeic acid m/z, 179 [M-H| produced major fragment ions at m/z, 109 and 135, respectively, due to loss of C02. Vanillic acid (m/z, 167 | M-H ]~) produced a major fragment ion at m/z 108 due to losses of methyl radical followed by C02 (Gonthier et al. 2003). Ferulic acid at m/z, 193 |M-H]■ also showed losses of methyl radical and C02resulting in the ion at m/z, 134. Rosmarinic acid at m/z, 379 [M-H]~ generated a major fragment ion at m/z, 161 corresponding to [M-H-18()-H20]“ or [caffeic acid-H- H20]‘(Hossain et al. 2010).

Retro Diels-Alder (RDA) reaction gave rise to the ion at m/z, 151 in quercetin, whereas kaempferol yielded a fragment ion at m/z, 239 corresponding to [M-CO-H20]~ (Zhao et al. 2007). RDA reaction in luteolin at m/z 285 [M-H]-) and apigenin at m/z, 269 [M-H]~ yielded product ions at m/z, 133 and 117, respectively. MRM with parent and product ion was not suitable for ursolic acid quantitation as the parent ion isolated in Quadrupole 1 (Ql) passed through Quadrupole 2(Q2) without fragmentation and monitored in Quadrupole 3 (Q3) as such (Xia et al. 2011). The internal standards palmatine at m/z, 352 [M+H]+ and curcumin at m/z, 367 [M-H]~ yielded product ions at m/z. 336 and 217, respectively (Feng et al. 2010; Yang et al. 2007). The MS2 spectra and fragmentations pattern are shown in Figures 3.1 and 3.2.

MRM parameters, DP and ce, were suitably optimized to achieve the most abundant, specific and stable MRM transition for each compound (Table 3.1). A positive/negative switching time of 100 ms was used for the continuous polarity switching between the positive and negative ionization modesin the LC-MS/MS system. The acquisition method built by the software algorithm aligns both polarities at appropriate MRM transitions. This has resulted in the simultaneous determination of both positively and negatively ionized bioactive compounds in shorter analysis time without additional injections into the LC/MS/MS system.

MS/MS spectra and fragmentation of analytes. (Reproduced from Ref. Chandra et al. 2015 with permission from Elsevier.)

FIGURE 3.1 MS/MS spectra and fragmentation of analytes. (Reproduced from Ref. Chandra et al. 2015 with permission from Elsevier.)

MS/MS spectra and fragmentation of analytes and IS. (Reproduced from Ref. Chandra et al. 2015 with permission from Elsevier.)

FIGURE 3.2 MS/MS spectra and fragmentation of analytes and IS. (Reproduced from Ref. Chandra et al. 2015 with permission from Elsevier.)

 
Source
< Prev   CONTENTS   Source   Next >