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Category  Carminative; pharmaceutic aid.

        Peppermint Oil is obtained by steam distillation from the aerial parts of the flowering plant of Mentha × piperita L. (Family Lamiaceae). 

Constituents  Peppermint Oil contains menthol and menthone as the major compounds.  Other monoterpenes include cineole, menthyl acetate and limonene.

Description  Colourless, pale yellow or pale greenish yellow liquid; odour, characteristic; taste, characteristic followed by a sensation of cold.

Solubility  Miscible with ethanol and with dichloromethane.

Other relevant information  It darkens in colour and becomes viscous on keeping.  On cooling to a low temperature, separation of menthol occurs, especially when a few crystals of this substance are added to start crystallization.

Packaging and storage  Peppermint Oil shall be kept in well-filled, tightly closed containers, protected from light, and at a temperature not exceeding 25º.

Identification Carry out the test as described in the “Thin-Layer Chromatography” (Appendix 3.1).
Standard solution Dissolve 50 mg of menthol, 20 µL of cineole, 10 mg of thymol, and 10 µL of menthyl acetate in toluene and dilute to 10 mL with the same solvent.
Test solution  Mix 100 mg of the sample with toluene and dilute to 10 mL with the same solvent.
Adsorbent  Silica gel F254 (TLC or HPTLC plate).
Mobile phase  Ethyl acetate and toluene (5:95).
Application  Apply 10 µL (or 1 µL) of Standard solution and 20 µL (or 2 µL) of Test solution as 10-mm (or 8-mm) bands.
Development and drying  Allow the solvent front to ascend 15 cm (or 6 cm) above the line of application.  Dry the developed plate in air.
Detection A  Examine the plate under ultraviolet light (254 nm).
Results A  When examined under ultraviolet light (254 nm), the test solution may show a quenching band of carvone or pulegone in the middle of the chromatogram.  A quenching band of thymol obtained from the standard solution appears in the middle of the chromatogram.
Detection B  Spray the plate with anisaldehyde TS and heat at 105° for 5 to 10 minutes.  Examine immediately in daylight.
Results B  When examined under visible light, the test solution shows a violet-blue band due to menthyl acetate, a violet-blue or brown band due to cineole in the middle of the chromatogram, and an intense blue or violet band due to menthol in the lower third of the chromatogram, corresponding in colour and R_f to the bands shown by the standard solution.  An intense violet-red band of hydrocarbons, a brownish yellow band of menthofuran in the upper third of the chromatogram, a greenish blue band of menthone in the middle of the chromatogram are present; a light pink or greyish blue or greyish green band of carvone, pulegone, isomenthone in the middle of the chromatogram may be present.
The standard solution shows a pink band of thymol in the middle of the chromatogram.
The retention time of the peaks due to limonene, cineole, menthone, menthofuran, isomenthone, menthyl acetate, and menthol in the chromatogram of the Peppermint Oil correspond to those in the chromatogram of the Standard solution, as obtained in the Chromatographic profile.  Isopulegol, pulegone and carvone may be present in the chromatogram.

Relative density  0.900 to 0.916 (Appendix 4.9).

Optical rotation  −30° to −10° (Appendix 4.8).

Refractive index  1.457 to 1.467 (Appendix 4.7).

Acid value  Not more than 1.4; use 5.0 g (Appendix 5.4).

Chromatographic profile  Carry out the test as described in the “Gas Chromatography” (Appendix 3.4).
Standard solution (a) Dissolve 10 µL of limonene, 20 µL of cineole, 40 µL of menthone, 10 µL of menthofuran, 10 µL of (+)-isomenthone, 40 µL of menthyl acetate, 20 µL of isopulegol, 60 mg of menthol, 20 µL of pulegone, 10 µL of piperitone, and 10 µL of carvone in heptane and dilute to 10.0 mL with the same solvent.
Standard solution (b) Dissolve 5 µL of isopulegol in heptane and dilute to 10.0 mL with the same solvent.  Dilute 0.1 mL of the solution to 5.0 mL with heptane.
Test solution  Mix 0.20 mL of Peppermint Oil with heptane and dilute to 10.0 mL with the same solvent.
Chromatographic system
DETECTOR  Flame ionization
COLUMN  A fused-silica capillary column (60 m × 0.25 mm) packed with macrogol 20000 (0.25 µm).
TEMPERATURE
     Column The temperature programmed is as  follows:

                    Injection port 200°
                    Detector 220°
              CARRIER GAS  Helium
              SPLIT RATIO  1:50
              FLOW RATE  1.5 mL per minute
        System suitability
               SAMPLE  Standard solution (a)

        Suitability requirements
               Resolution  Not less than 1.5 between limonene and cineole peaks and not less than 1.5 between piperitone and carvone peaks.
Procedure  Inject a portion (about 1 mL) of Standard solution (a), Standard solution (b) and Test solution into the chromatograph.  Identify the peaks due to the components in Peppermint Oil, using the retention time determined from the chromatogram obtained from the standard solution (a).  Measure the areas of the principal peaks in the chromatogram obtained from the test solution.
Calculation  Calculate the percentage content of the components of Peppermint Oil taken by normalization procedure. 
Limits
— limonene:  1.0 per cent to 3.5 per cent,
— 1,8-cineole:  3.5 per cent to 8.0 per cent,
— menthone:  14.0 per cent to 32.0 per cent,
— menthofuran:  1.0 per cent to 8.0 per cent,
— isomenthone:  1.5 per cent to 10.0 per cent,
— menthyl acetate:  2.8 per cent to 10.0 per cent,
— isopulegol:  not more than 0.2 per cent,
— menthol:  30.0 per cent to 55.0 per cent,
— pulegone:  not more than 3.0 per cent,
— carvone:  not more than 1.0 per cent,
disregard limit:  the area of principal peak in the chromatogram obtained from the standard solution (b) (0.05 per cent).
The ratio of cineole content to limonene content is a minimum of 2.