Info

nd

tr

Source: Suzuki et at., 1980. Notes

Capsaicinoids were estimated with GC-MS. NDC — nordihydrocapsaicin; C — capsaicin; DC - dihydrocapsaicin; HC — homocapsaicin; HDC - homodihydrocapsaicin; nd — not detected; cr — trace.

Source: Suzuki et at., 1980. Notes

Capsaicinoids were estimated with GC-MS. NDC — nordihydrocapsaicin; C — capsaicin; DC - dihydrocapsaicin; HC — homocapsaicin; HDC - homodihydrocapsaicin; nd — not detected; cr — trace.

biosynthesis with newly developed methods have led to the statement that, at all stages of growth, the total or individual capsaicinoids based on dry weight is far higher in the placenta than in the pericarp (Table 4.3). This statement allows the conclusion that the main site of capsaicinoids synthesis is the placental tissue of the fruits (Fujiwake et al., 1982a,b; Bernal et al., 1993a,b; Ochoa-Alejo and Gomez-Peralta, 1998).

Bernal et al. (1993a,b) suggested by means of in vitro studies that peroxidases were involved in the degradation of capsaicinoids. Pepper peroxidase, also mainly located in the placenta, was reported to oxidize capsaicin (Bernal et al., 1993a) and dihydrocapsaicin (Bernal et al., 1993b). Therefore, the turnover and degradation of capsaicinoids observed after Capsicum fruits reach maturation could be explained by the activity of peroxidases .

Inventory of analytical methods for capsaicinoids determination

The choice of an analytical method is generally guided by many factors, including the properties of the targeted compounds (polarity, stability, solubility), matrices, time assigned for analysis and additional factors introduced by the laboratory itself. For Capsicum compounds two objectives for determination can be followed: the determination of the heat level of a Capsicum fruit and the determination of its capsaicinoid composition.

Organoleptic and spectrophotometric methods

Procedures including organoleptic (Ramos, 1979) and spectrophotometric methods (Meilgaard et al., 1987) have been developed. Organoleptic methods were preferred by the food industry for the direct heat level measure it offered, while it presented the disadvantage of requiring the extensive training of panelists and the monitoring of their sensitivity for the attainment of consistent results (Bajaj and Kaur, 1979). Spectrophotometric methods use vanadium oxytrichloride or phosphomolybdic acid to produce coloured solutions. Even if it is not specific, the result obtained is proportional to the heat level (Kosuge and Furuta, 1970; Rymal et al., 1979) and therefore can be used as a tool for total capsaicinoid content.

Gas chromatographic methods

A variety of gas chromatographic (GC) methods have been developed for capsaicinoids analysis (Table 4.4) and most of them still present serious limitations. As shown in Table 4.4, sample preparation and extraction are not so different, proceeding by the grinding of the dried Capsicum or the chopping of the fresh fruits into small pieces and then extracting with different solvents. Most of the GC methods need a derivatization step to increase the volatility of the capsaicinoids, and, furthermore, an efficient clean up step is necessary (Todd et al., 1977; Iwai et al., 1979; Krajewska and Powers, 1987; Manirakiza et al., 1999). Two types of derivatization procedures have been reported, trimethylsilylation of capsaicinoids (Lee et al., 1976; Todd et al., 1977; Iwai et al., 1979; Fung et al., 1982)and hydrolysis of capsaicinoids for yielding fatty acids and subsequent esterification (Jurenitsch et al., 1979; Jurenitsch and Leinmuller, 1980). An on-column derivatization has also been reported (Krajewska and Powers, 1987). The use of structurally related codeine as an internal standard was reported (Manirakiza et al., 1999), while tetracosane (Krajewska and Powers, 1987) and squalene (Hawer et al., 1994) have also been used as internal standards. To overcome the problem of tailing peaks and to avoid the use of a derivatization step, Thomas et al. (1998) and Hawer et al. (1994) have recognized the use of a polar capillary analytical column (14% cyanopropylphenyl/86% methyl stationary phase, 30m X 0.25 mm i.d. X 0.25 [Am film thickness) for interaction with polar functional group of the molecules. The resulting chromatogram is shown in Figure 4.4. Furthermore, the use of a thermionic selective detector (TSD) instead of flame ionization detection allows the elimination of sample clean-up (Thomas et al., 1998). Unfortunately, GC/MS was not able to separate or differentiate nonivamide and nordihydrocapsaicin (Kosuge and Furuta, 1970; Saria et al., 1981; Fung et al., 1982; Reilly et al., 2001b), leading to the misidentification of nonivamide as nordihydrocapsaicin. Although GC/MS is believed to offer good results, they still require specific sample preparation and a complex, costly instrument. Limits of quantification were superior to 50ppm.

High Performance Liquid Chromatography methods

As for GC methods, HPLC methods are encumbered by significant difficulties (Table 4.5). For instance, normal phase HPLC was unable to separate all individual capsaicinoids (Dicecco, 1976; Cooper et al., 1991; Constant et al., 1995). Therefore, the results were given as total capsaicinoids even if three major capsaicinoids — capsaicin, dihydrocapsaicin and nordihydrocapsaicin — could be eventually separated. Furthermore, these HPLC methods were applied to relatively capsaicin rich matrices, while poor capsaicinoids matrices required sample clean-ups with a preconcentration step (Peusch et al., 1997).

The reverse phase HPLC was limited by the non-resolution of nonivamide and capsaicin with regards to the similar chromatographic properties in HPLC (Saria et al., 1981; Constant et al., 1995). Constant and coworkers (1995) used complexation chromatography (AgNOj) to separate the coeluting compounds. In most of these HPLC methods, the limit of quantification was superior to 150ppm.

The use of the LC-MS (Reilly et al., 2001a) has been reported to differentiate nonivamide and capsaicin by mass-to-charge ratio. The same authors have reported the use of LC-MS-MS with an electrospray ionization source operating in the selected reaction monitoring

Table 4.4 Gas chromatographic method for capsaicinoid determination in Capsicum products

Matrix

Sample preparation

Extraction

Analysis

Comments

Chilli peppers

Dry at 80°C overnight

With high speed blender/CHCl2,

GC/MS, splitless, DB-5 analytical

Clean chromatogram, but only three

(Capsicum spp.)

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