**2.1. General experimental procedures**

1H and 13C NMR spectra were recorded on a Brucker AM-200 and 500MHz) chemical shifts are given in d values referred to internal tetramethlysilane (TMS), EIMS (MS Agilent 5973; 70eV) and Infrared (IV) spectra were recorded on a Nicolet spectrophotometer with Fourier transform Model Magna–IR 760 wavelengths are expressed in reciprocal centimeter (cm-1).

HPLC analysis were performed at room temperature using the following system: second pump system (Shimadzu LC10AD model, Japan) a photodiode array detector (Shimadzu, SPD10ADVP model), an auto injector (Shimadzu SIL10ADVP model) an oven (Shimadzu, CTO 8-A model) and under the following conditions: Shimpack C-8 (1cm x 4.6mm i.d.) guard column and C-18 column (25cmx4.6mm i.d.; 5µm particle size) was from Zorba Zx provided by Agilent Technologies (USA). The system was controlled by Class Vp (Shimadzu, Japan) software 5.16. The gradient mobile phase was carried acetonitrile (Tedia, Brazil) and water HPLC grade. The mobile phase was degassed with helium and the flow rate adjusted to 1mL. min-1. The water was acidified with TFA (0.05% v/v). All samples were injected automatically (10µ L) in triplicate.

In GC/FID experiments, 1mg each extracts [August 2009 to July 2010] and standard (Carl Roth – Karlsruhe, Germany) were transferred into glass vial and submitted methlylation with CH2N2 with 100% yield. Those samples were dissolved in CH2Cl2 in concentrations 1.4mg/mL and injected for GC-FID and GC-MS analysis. The identification of methylated betulinic acid in extracts was done with use Wiley and NBS peak matching library search system. Authentic standard of the betulinic acid and data reported in the literature were also used for further identification as described.

was constructed using the average values of the detector response. The detector response was

= 0.9994, Figure 2b).

http://dx.doi.org/10.5772/55868

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Analysis of the Presence of the Betulinic Acid in the Leaves of *Eugenia florida* by…

linear to the concentration internal of 0.1 to 0,5µg.mL-1 (r2

**Figure 2.** Calibration curve of Betulinic acid, A: GC-FID and B: HPLC-UV
