Porphyrin identification in chloroform–methanol extracts was carried out by LC–MS on an Agilent 1290 Infinity chromatograph (USA) with an Agilent 6460 TQ mass spectrometer (USA).
An Agilent Eclipse Plus C18 1.8 um 2.1 × 50 mm column was used with mobile phase: A—0.1% formic acid in water, B—0.1% formic acid in acetonitrile; Gradient A–B; column temperature—40 °C, flow rate—0.4 ml/min, detection—UV/VIS 400 nm, MS ESI(+) full scan. The mass spectra were acquired with positive electrospray ionization (ESI) with the ion spray voltage set at 5.5 kV. We used 1 μg of the porphyrin standards for the initial tests. Coproporphyrin III, uroporphyrin III and coproporphyrin III tetramethyl ester (BenchChem, USA) were used as standards. Due to the use of 0.1% formic acid in this type of LC–MS analysis, separate data on Zn–porphyrins were lost and included in the total amount as free porphyrins.
An Agilent Eclipse Plus C18 1.8 um 2.1 × 50 mm column was used with mobile phase: A—0.1% formic acid in water, B—0.1% formic acid in acetonitrile; Gradient A–B; column temperature—40 °C, flow rate—0.4 ml/min, detection—UV/VIS 400 nm, MS ESI(+) full scan. The mass spectra were acquired with positive electrospray ionization (ESI) with the ion spray voltage set at 5.5 kV. We used 1 μg of the porphyrin standards for the initial tests. Coproporphyrin III, uroporphyrin III and coproporphyrin III tetramethyl ester (BenchChem, USA) were used as standards. Due to the use of 0.1% formic acid in this type of LC–MS analysis, separate data on Zn–porphyrins were lost and included in the total amount as free porphyrins.