Eliminating interference by anthocyanins when determining the porphyrin ratio of red plant leaves

Anthocyanins (Ants) are water-soluble secondary metabolites that are responsible for red colour of plant leaves. To determine photosynthetic pigments, 80% acetone was used to extract Ants from Ant-containing leaves of test plants. However, using the 80% acetone extraction method can lead to interference between chlorophylls (Chls) and Ants. Porphyrins, such as protoporphyrin IX (PPIX), Mg-protoporphyrin IX (MgPP), and protochlorophyllide (Pchlide), are Chl biosynthetic intermediates and demonstrate photospectrometric characteristics similar to those of Chl. Although the ether/water extraction method was able to remove Ants interference when detecting porphyrins, the porphyrins extraction efficiency was lower than that of the 80% acetone extraction method. Low Ants levels interfered with individual porphyrin ratios, and the extent of the effect was correlated with Ants concentrations. We developed the three equations could eliminate interference by Ants when determining the porphyrin molecular percentage (%) and were comprehensively applied to all tested species of Ants-containing leaves.