*2.4. Oosporein Characterization by HPLC Tandem Mass Spectrometry*

The filtered total aqueous extract at the final point of fermentation was analyzed by reversed-phase high-performance liquid chromatography (HPLC) equipped with an autosampler (Varian ProStar 410, Walnut Creek, CA, USA), ternary pump (Varian ProStar 230I, USA), and PDA detector (Varian ProStar 330, USA). A sample (5 μL) was injected into a Denali C-18 column (150 mm × 2.1 mm, 3.1 μm, Grace, Deerfield, IL, USA). The oven temperature was 30 ◦C. The elution gradient was formic acid (0.2 % *v*/*v*, solvent A)

and acetonitrile (solvent B) with initial gradient course of 3% B, 5–15 min; 16% B linear, 15–45 min; and 50% B linear. The flow rate was 0.2 mL/min and the elution was monitored at 287 nm. Liquid chromatography–ion trap mass spectrometry (Varian 500-MS IT Mass Spectrometer, USA) equipped with an electrospray ion source was used. The MS analysis was performed in the negative mode [M-H]−<sup>1</sup> using nitrogen as the nebulizing gas and helium as the damping gas. The parameters of the ion source were as follows: spray voltage of 5.0 kV, capillary voltage of 90.0 V, and temperature of 350 ◦C. Full scan spectra were acquired in the m/z range 100–2000, and, subsequently, the MS/MS analyses were performed on a series of selected ions. The data were collected and processed using MS Workstation software (V 6.9).

**Figure 2.** Evolution of CO2 production during fermentation of *Beauveria bassiana* PQ2 for the production of aerial conidia and oosporein. The different colors of open circles are the repetitions.
