# Mass Spectrometry: A Rosetta Stone to Learn How Fungi Interact and Talk
## 3 Common Mass Spectrometry-Based Strategies Used in Mycology Studies
### Identification of Microbial Chemical Compounds
#### 3.2.2 Identification of Non-Volatile Compounds of Fungal Origin  
Due to the chemical diversity of non-volatile compounds that are secreted/produced by fungi, it is practically impossible to extract quantitatively all specialized metabolites using a single sample preparation method. Consequently, sample preparation is an important aspect of specialized metabolite profiling and will, independently of the method, lead to a bias toward certain types of compounds. For example, for the extraction of non-polar components, such as lipids, polyketides and terpenoids, one may use water-immiscible solvents such as ethyl acetate or dichloromethane. Other water-immiscible solvents such as ethers, chloroform and carbon tetrachloride are less common due to their toxicity and negative environmental effects [134; 179]. Additionally, pH is crucial for the extraction, due to ionizable moieties in the chemical structure of the metabolites being extracted into the organic phase to a much higher degree in their neutral form than in a charged state. In most of the cases is a low pH extraction necessary, but it can be replaced in some cases with a neutral extraction (e.g., for stability reasons) [118; 165]. To extract polar components, one may use acetone, methanol and ethanol [143; 150; 144]. The most common solvent used to extract polar intracellular secreted compounds is methanol (see Figure 2). Nevertheless, it has been reported that methanol and ethanol extraction can yield in the extraction of non-polar waves, sterols, and triglycerides, as well. Thus, it is necessary to perform a second extraction step, such as extraction with ethyl acetate [118; 165]. A major obstacle in any sample preparation is the possibility of unwanted chemical reactions taking place, such as precipitation, complex formation and degradation, among others. To reduce this risk, fast extraction procedures are preferred. This means extraction procedures of 10 or fewer minutes [118; 165; 166]. One of the most common methods used to achieve the best separation of secreted specialized metabolites produced by fungi is reverse phase (C-18) chromatography since it is more than ideal to interact with polar and some non-polar metabolites [180; 181; 182; 150; 180; 181; 182]. In some cases, before injecting samples on LC, samples are needed to be purified on small solid phase extraction (SPE) columns to remove chromatography-impairing lipids and phospholipids [147; 148; 183]. Interestingly, performing liquid separations at low pH (by addition of formic or acetic acid) is statistically better since sharper peaks can be obtained on the chromatogram. Formic acid and acetic acid are acid additives that are compatible with mass spectrometry detection, meanwhile, at the same time, they keep acidic moieties in specialized metabolites protonated. Nevertheless, some metabolites can be affected by the addition of acidifiers or if the mass spectrometry measurement is done in the negative mode [149; 184; 185]. Other chromatographic techniques use two consecutive separation columns (2-dimensional liquid chromatography) or columns of different sorbent chemistry, such as ionic exchangers(see Table 1) [14; 113; 129; 134; 143]. The most common ionization techniques used in combination with 2D liquid chromatography are the ambient pressure ionization techniques, such as electrospray ionization (ESI) [134; 135; 137; 138; 140; 141; 142; 143; 144; 145; 150] and atmospheric pressure chemical ionization (APCI) [139; 146]. These are all soft ionization techniques (i.e., they produce limited chemical fragmentation). Hence, mass spectrometry signals are extremely clean and easily interpretable. The possibility of adducts formations as well as multiple charge metabolites can make researchers disagree with the \"easily interpretable\" statement [131; 140; 146].