Determination of Mg-Proto IX via HPLC

Mg-Proto IX was analyzed by high performance liquid chromatography method according to Strand et al. [37 (link)]. Leaf material (0.2 g) was homogenized in 1 mL acetone (0.1 M NH₄OH, 9:1 [v/v]) and homogenized and centrifuged with ice-cooling. The residue was resuspended again, and the same procedures described above were repeated. The collected supernatants were mixed and centrifuged prior to high performance liquid chromatography (Waters, Milford, USA). The eluate (0–10 min) was passed through an ACQUITY Ultra Performance Liquid Chromatography (UPLC) Fluorescence (FLR) Detector (Waters, Milford, USA), with initial excitation wavelength of 417 nm and an emission wavelength 595 nm, and secondary excitation and emission at 402 nm and 633 nm, respectively [68 (link)]. Mg-Proto IX was identified and quantified using authentic standards.