7.3.1. High Performance Liquid (HPLC)

The chromatography principle essentially governs the separation of the components of a mixture; this from a mobile phase fluid (gas, liquid, or supercritical fluid), which drags the sample at constant flow and pressure due to the use of a pump. This pressure flow leads to a column containing the stationary phase, which is either a solid or a liquid bound to a solid [68]. As a result of the interaction between the components of the mixture and the two phases, they allow separation; hence, at the end, it passes through a detector that produces a signal that allows both indicating the moment of appearance of the various components that make up the sample and qualifying it quantitatively and qualitatively [69].

HPLC differs from other forms of chromatography in that it is not limited by the sample's volatility or thermal stability. The main explanation for this is that it is broadly applicable for the study of macromolecules, such as fungal pigments in this case [43,70]. Several authors have reported the use of this method in the molecular characterization of fungal pigments at various stages. Beginning with Lebeau et al. [21], who used UV- VIS and HPLC-DAD (inverse) to identify and elucidate the conformation of two new pigments from *Fusarium oxysporum*, which they called wild-type purple naphthoquinone and bikaverin, respectively [21]. Venkatachalam et al. [22], reported the application of HPLC in some of its variants (PDA-ESI/MS) for the analysis of the pigments produced by *Talaromyces albobiverticillius* 30548 and reported twelve compounds of *Monascus* spp. Further, they characterized and elucidated a new compound called NGABA-PP-V (6-[(Z)- 2-Carboxyvinyl] -N-GABA-PP-V) with a *cis* configuration at the C10-C11 carbon double bond [22]. Finally, Gonçalves et al. [71] contributed by identifying and quantifying the phenolic compounds gallic acid, catechin, chlorogenic acid, caffeic acid, and vanillin found in the ethyl extract of *Penicillium flavigenum* CML2965 using HPLC [71]. Since it is a nonlimiting method, it is obvious that there are millions of works published in its use for the molecular elucidation of fungal pigments, which is why the examples above are just a few of the most recent.
