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        Capsicum Oleoresin is an ethanolic extract of Chilli Pepper.  It contains not less than 6.5 per cent of total capsaicinoids [capsaicin (C₁₈H₂₇NO₃), dihydrocapsaicin (C₁₈H₂₉NO₃) and nordihydrocapsaicin (C₁₈H₂₇NO₃)], calculated as capsaicin, (C₁₈H₂₇NO₃) on the anhydrous basis. 

Description  Dark red oily liquid.

Solubility  Soluble in acetone, chloroform, ethanol, ether, and volatile oils.  Soluble with opalescence in fixed oils.

Additional information  Nonivamide content in Capsicum Oleoresin should not exceed 5 per cent w/w of total capsaicinoids to prevent adulteration by the synthetic nonivamide.

Packaging and storage  Capsicum Oleoresin shall be kept in tightly closed containers, preferably in glass containers.

Labelling  The label on the container states that if separation occurs, it should be warmed and mixed before use.

Identification  

     A. The retention time of the capsaicinoid peak in the chromatogram of the Assay preparation corresponds to the capsaicin peak of the Standard preparation, as obtained in the Assay. 

        B. Carry out the test as described in the “Thin-Layer Chromatography” (Appendix 3.1), using silica gel GF254 as the coating substance and a mixture of 60 volumes of toluene and  40 volumes of ethyl acetate as the mobile phase.  Apply separately to the plate as bands of  10 mm, 5 μL each of the following two solutions.  Prepare solution (A) by diluting 50 mg  of the sample with 5 mL of n-hexane.  For solution (B), dissolve 1 mg of capsaicin in 1 mL  of methanol.  After removal of the plate, allow it to dry in air and examine under ultraviolet light (254 nm), marking the quenching bands.  The chromatogram obtained from solution (A) shows a quenching band (hR_f value 29 to 31) corresponding to the capsaicin band from solution (B) and other bands may be observed.  Subsequently spray the plate with anisaldehyde TS and heat at 105° for 3 minutes; the band due to capsaicin is purple.  Other two purple and four violet bands are also observed.

Water  Not more than 8.0 per cent w/w (Karl Fischer Method, Appendix 4.12).

Assay Carry out the determination as described in the “Liquid Chromatography” (Appendix 3.5).

     Mobile phase  Prepare a mixture of 40 volumes of acetonitrile and 60 volumes of a 0.1 per cent w/v solution of phosphoric acid.  Make adjustments if necessary.

     Standard preparation  Prepare a solution of Capsaicin RS in methanol having a known concentration of about 200 μg per mL.  Filter a portion of this solution through a 0.2-μm  porosity filter.

     Assay preparation  Transfer about 1 g of Capsicum Oleoresin, accurately weighed, to  a 100-mL volumetric flask, dissolve in and dilute with methanol to volume, and mix.  Filter  a portion of this solution through a 0.2-μm porosity filter.

     Chromatographic system  The chromatographic procedure may be carried out using  (a) a stainless steel column (30 cm × 3.9 mm) packed with octadecylsilane chemically bonded to porous silica or ceramic microparticles (1 to 10 μm) or monolithic rod, (b) Mobile phase at flow rate of 1 mL per minute, and (c) an ultraviolet photometer set at 280 nm.

      To determine the suitability of the chromatographic system, chromatograph Standard preparation, and record the peak response as directed under Procedure: the symmetry factor for the capsaicin peak is not more than 2.0 and the relative standard deviation for replicate injections is not more than 2.0 per cent.  Chromatograph Assay preparation, and record the peak response as directed under Procedure:  the relative retention times are about 0.90 for nordihydrocapsaicin, 0.95 for nonivamide, 1.00 for capsaicin, and 1.30 for dihydrocapsaicin and the resolution factor, R, between the nonivamide peak and the capsaicin peak is not less than 1.5.  

        Procedure  (Note  Use peak areas where peak responses are indicated.)  Separately inject equal volumes (about 10 μL) of Standard preparation and Assay preparation into the  chromatograph, record the chromatograms, and measure the responses for the three  major peaks.

         Calculation  Calculate the percentage of total capsaicinoids, as capsaicin (C₁₈H₂₇NO₃)  in the Capsicum Oleoresin taken by the formula:

(C_s/C_u)(r_u/r_s) X 100,

in which C_s is the concentration, in mg per mL, of Capsaicin RS in Standard preparation,  C_u is the concentration, in mg per mL, of Capsicum Oleoresin in Assay preparation, r_u is the sum of the peak responses for nordihydrocapsaicin, capsaicin, and dihydrocapsaicin obtained from Assay preparation and r_s is the peak response of capsaicin obtained from  Standard preparation.

        Calculate the percentage of capsaicin in the Capsicum Oleoresin taken by the formula: 

(C_s/C_u)(r_u/r_s) X 100,

in which C_s is the concentration, in mg per mL, of Capsaicin RS in Standard preparation, C_u  is the concentration, in mg per mL, of Capsicum Oleoresin in Assay preparation, r_u is the peak response for capsaicin obtained from Assay preparation and r_s is the peak response of  capsaicin obtained from Standard preparation.