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                        Zingiber Montanum Extract is the liquid extract obtained by hydrodistillation from the fresh rhizome of Zingiber montanum (J. König) Link ex A. Dietr. (Z. cassumunar Roxb.) (Family Zingiberaceae). It contains not less than 90.0 per cent and not more than 110.0 per cent of the labelled amounts of terpinen-4-ol (C₁₀H₁₈O) and sabinene (C₁₀H₁₆); the labelled amounts of terpinen-4-ol and of sabinene are not less than 1.5 per cent.

Description  Pale yellow to brownish yellow liquid; odour, strong and characteristic.

Packaging and storage  Zingiber Montanum Extract shall be kept in well-filled, tightly closed containers, preferably of metal or glass, protected from light, and stored at a temperature not exceeding 25°.

Labelling  The label on the container states (1) the amounts of terpinen-4-ol and sabinene; (2) the expiration date.

Identification

A. The chromatogram of the Assay preparation shows several peaks, two of which correspond to those of the Standard preparations, as obtained in the Assay (Fig. 1).

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 98 volumes of toluene and 2 volumes of ethyl acetate as the mobile phase and allowing the solvent front to ascend 8 cm above the line of application. Apply separately to the plate, 5 mL each of solutions (A), (B), and (C). Prepare solution (A) by diluting 1 mL of the sample with 9 mL of ethanol. For solution (B), dilute 1 mL of terpinen-4-ol with 9 mL of ethanol. For solution (C), dilute 1 mL of (E)-1-(3,4-dimethoxyphenyl) butadiene (DMPBD) with 9 mL of ethanol. After removal of the plate, allow it to dry in air and examine under ultraviolet light (254 nm), marking the quenching spots. The chromatogram obtained from solution (A) shows a quenching spot (hRfvalue 46 to 50), corresponding to the DMPBD spot from solutions (C). Subsequently spray the plate with anisaldehyde TS and heat at 105° for 10 minute. The chromatogram obtained from solution (A) shows a purple spot (hRf value 28 to 32) due to the terpinen-4-ol spot from solution (B) and a blue spot due to the DMPBD spot from solution (C). Three purple spots are also observed (Fig. 2).

Relative density  0.8756 to 0.9836 (Appendix 4.9).

Refractive index  1.4807 to 1.5049, at 20° (Appendix 4.7).

Optical rotation  –34.1° to –26.1°, at 20° (Appendix 4.8).

Assay  Carry out the determination as described in the “Gas Chromatography” (Appendix 3.4).

                Standard preparation A  Dissolve an accurately weighed quantity of Terpinen-4-ol RS in sufficient ethanol. Dilute quantitatively and stepwise with ethanol to obtain five solutions, each having a known concentration of 0.5, 1, 2, 3, and 4 mg per mL of terpinen-4-ol.       

                        Standard preparation B   Dissolve an accurately weighed quantity of Sabinene RS in sufficient ethanol. Dilute quantitatively and stepwise with ethanol to obtain five solutions, each having a known concentration of 0.5, 1.5, 2.5, 3.5, and 4.5 mg per mL of sabinene.       

                        Assay preparation  Dissolve  an accurately weighed quantity of Zingiber Montanum Extract in sufficient ethanol and dilute quantitatively with ethanol to obtain a final concentration of about 5 mg per mL.

                        Chromatographic system  The chromatographic procedure may be carried out using (a) a fused silica column (30 m × 0.32 mm) packed with 5 per cent phenyl-95 per cent methylpolysiloxane (0.25 µm) on silanized diatomaceous support, (b) the injection port and the detector block maintained at 150°, (c) split ratio 1:100, (d) helium for chromatography as the carrier gas at a flow rate of about 1.8 mL per minute, and (e) a flame ionization detector at 300º. The step gradient of temperature is as follows: 

                To determine the suitability of the chromatographic system, chromatograph each of Standard preparation A and Standard preparation B and record the peak response as directed under Procedure and Calculation: the relative standard deviation for replicate injections is not more than 2.0 per cent.

                Procedure  Separately inject about 1 μL each of Standard preparation A and Standard preparation B into the chromatograph, record the chromatograms and measure the responses for the terpinen-4-ol peak and sabinene peak. Plot the readings and draw the standard curve of best fit:  the curves show the correlation coefficient of not less than 0.995. Inject about 1 μL of Assay preparation into the chromatograph, record the chromatogram, and measure the responses for terpinen-4-ol peak and sabinene peak.

                Calculation  By reference to the standard curves, calculate the contents of terpinen-4-ol (C₁₀H₁₈O) and sabinene (C₁₀H₁₆) in the portion of the Extract taken.