**6. Experimental**

The quantification of capsaicin in pepper spray can be made by GC-MS/FID [14]. This experimental result presented here aims to exemplify the quantitative and qualitative analysis of the samples of the commercial Brazilian pepper spray. About 500 μl of solution sample was weighed on an analytical balance and the contents were solubilized with HPLC grade acetone and transferred quantitatively to a 1 ml volumetric flask. The solution was swollen and an aliquot was conducted for analysis by gas chromatography.

A model GC-2010 equipped with a mass spectrometer GCMS-QP2010 Ultra and an automatic sampler AOC-5000 (Shimadzu) was used. The chromatographic separation was performed using RTx-5MS capillary column (Restek) with a stationary phase containing 5% phenylmethyl and 95% polydimethylsiloxane (30 m × 0.25 mm × 0.25 μm of film). The temperatures of the injector and the transfer line of the mass spectrometer were 300°C. The samples were injected with a split ratio of 1:50. The oven temperature program followed the following conditions: 50°C (2 min), heating rate from 10°C/min to 300°C (2 min). Helium was used as the carrier gas in a flow rate of 1.2 ml/min. The mass spectrometer was operated in electron impact ionization mode, with acquisition in the scan mode with m/z between 50 and 500.

To identify the compounds, a comparison of the spectra with the NIST library was carried out. Only substances whose similarity was greater than 80% were considered, compared with data from the library. **Figure 1** shows the chromatogram result with the solvent around 7.2 min and capsaicin and dihydrocapsaicin peak around 24 min.

With the purpose of quantifying, the capsaicin and dihydrocapsaicin are integrated together and the calibration curve was made by this result against

**Figure 1.** *GC-FID chromatogram of capsaicin and dihydrocapsaicin peaks with retention time around 24 min.*

standard solutions made with Sigma-Aldrich Capsaicin (purity capsaicin: 61.1%, dihydrocapsaicin: 31.2% LOT#: LRAA9221 09 September 2015). The regression coefficient R<sup>2</sup> was 0.999 and uncertainty was 0.9%. The subsequent dilution was satisfactory by the statistical linear fit results. In this way, it is possible to quantify the capsaicinoids, that is, capsaicin and dihydrocapsaicin together, on the pepper spray solution.
