Eucalyptus Oil

This article is compiled based on the United States Pharmacopeia (USP) – 2025 Edition

Issued and maintained by the United States Pharmacopeial Convention (USP)

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1 DEFINITION

Eucalyptus Oil is obtained by steam distillation and rectification from the fresh leaves or the fresh terminal branchlets of various species of Eucalyptus rich in 1,8-cineole. The species mainly used are Eucalyptus globulus Labill., Eucalyptus polybractea R.T. Baker, and Eucalyptus smithii R.T. Baker. It contains NLT 70.0% and NMT 95.0% of eucalyptol (1,8-cineole, C₁₀H₁₈O).

2 IDENTIFICATION

Change to read:

A. Spectroscopic Identification Tests 〈197〉, Infrared Spectroscopy: 197F (CN 1-May-2020)

B. Identity by Aroma Substance Profile: The retention times of the (+)-α-pinene, β-pinene, (R)-(–)-α-phellandrene, (R)-(+)-limonene, and eucalyptol peaks of the Sample solution correspond to those of the Standard solution, as obtained in the test for Content of Aroma Substances in the Assay.

3 ASSAY

Content of Aroma Substances

Standard solution A: 1.0 μL/mL of (+)-α-pinene, 0.5 μL/mL of β-pinene, 0.5 μL/mL of sabinene, 0.5 μL/mL of (R)-(–)-α-phellandrene, 1.0 μL/mL of (R)-(+)-limonene, 5 μL/mL of USP Eucalyptol RS, and 5 mg/mL of USP Camphor RS in heptane

Standard solution B: 0.01 μL/mL of (R)-(+)-limonene in heptane

Sample solution: 20 μL/mL of Eucalyptus Oil in heptane

3.1 Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: GC

Detector: Flame ionization

Column: 0.25-mm × 60-m fused-silica capillary; 0.25-μm layer of phase G16

Temperatures

Injection port: 220°

Detector: 220°

Column: See Table 1.

Table 1

Carrier gas: Helium

Flow rate: 1.5 mL/min

Injection type: Split ratio, 1:50

Injection volume: 1 μL

3.2 System suitability

Sample: Standard solution A

[Note-The relative retention times are listed in Table 2.]

Table 2

3.3 Suitability requirements

Resolution: NLT 1.5 between (R)-(+)-limonene and eucalyptol

3.4 Analysis

Samples: Standard solution A, Standard solution B, and Sample solution

Identify the peaks in the Sample solution based on those in Standard solution A as well as in Table 3.

Table 3

β-Myrcene elutes before (R)-(–)-α-phellandrene and after sabinene; γ-terpinene and p-cymene elute between eucalyptol and camphor; and terpinen-4-ol and α-terpineol elute after camphor.

Calculate the percentage of (+)-α-pinene [β-pinene, sabinene, (R)-(–)-α-phellandrene, (R)-(+)-limonene, or eucalyptol] in the portion of

Eucalyptus Oil taken:

Result = (r_U/r_S) × (C_S/C_U) × 100

r_U = peak response of (+)-α-pinene [β-pinene, sabinene, (R)-(–)-α-phellandrene, (R)-(+)-limonene, or eucalyptol] from the Sample solution

r_S = peak response of (+)-α-pinene [β-pinene, sabinene, (R)-(–)-α-phellandrene, (R)-(+)-limonene, or eucalyptol] from the Standard solution

C_S = concentration of (+)-α-pinene [β-pinene, sabinene, (R)-(–)-α-phellandrene, (R)-(+)-limonene, or USP Eucalyptol RS] in the Standard solution (μL/mL)

C_U = concentration of Eucalyptus Oil in the Sample solution (μL/mL)

Calculate the percentage of camphor in the portion of Eucalyptus Oil taken:

Result = (r_U /r_S ) × [C_S /(C_U × D)] × 100

r_U = peak response of camphor from the Sample solution

r_S = peak response of camphor from the Standard solution

C_S = concentration of USP Camphor RS in the Standard solution (mg/mL)

C_U = concentration of Eucalyptus Oil in the Sample solution (μL/mL)

D = density of Eucalyptus Oil (mg/μL)

Calculate the percentage of β-myrcene (γ-terpinene, p-cymene, terpinen-4-ol, or α-terpineol) in the portion of Eucalyptus Oil taken:

Result = (r_U /r_T ) × 100

r_U = peak response of β-myrcene (γ-terpinene, p-cymene, terpinen-4-ol, or α-terpineol) from the Sample solution

r_T = sum of the total peak responses, except for the peaks due to solvent, from the Sample solution

Acceptance criteria: See Table 4. Disregard any peak with an area less than the major peak area from Standard solution B, corresponding to 0.05%.

Table 4

Percentage of γ-terpinene: 0.1%–6.0%

Percentage of p-cymene: 0.5%–15.0%

Total percentage of all identified aroma substances [(+)-α-pinene, β-pinene, sabinene, (R)-(–)-α-phellandrene, (R)-(+)-limonene, eucalyptol, camphor, β-myrcene, γ-terpinene, p-cymene, terpinen-4-ol, and α-terpineol]: NLT 98.0%

4 IMPURITIES

Test for Aldehyde

Alcoholic hydroxylamine solution: Dissolve 3.5 g of hydroxylamine hydrochloride in 95 mL of 60% alcohol (v/v) and add 0.5 mL of a 2-mg/mL solution of methyl orange in 60% alcohol (v/v) and sufficient 0.5 M potassium hydroxide in 60% alcohol (v/v) to give a pure yellow color. Dilute with 60% alcohol (v/v) to 100 mL.

Analysis: In a ground-glass-stoppered tube, 25 mm in diameter and 150 mm long, add 10 mL of Eucalyptus Oil. Then add 5 mL of toluene and 4 mL of Alcoholic hydroxylamine solution. Shake vigorously, and titrate immediately with 0.5 M potassium hydroxide in 60% alcohol (v/v) until the red color changes to yellow. Continue the titration with shaking; the endpoint is reached when the pure yellow color of the indicator is permanent in the lower layer after shaking vigorously for 2 min and allowing separation to take place. The reaction is complete in about 15 min.

Repeat the titration using a further 10 mL of Eucalyptus Oil and, as a reference solution for the endpoint, the titrated liquid from the first determination, to which has been added 0.5 mL of 0.5 M potassium hydroxide in 60% alcohol (v/v).

Acceptance criteria: NMT 2.0 mL of 0.5 M potassium hydroxide in 60% alcohol (v/v) is required in the second titration.

5 SPECIFIC TESTS

Specific Gravity 〈841〉: 0.906–0.927, at 20°

Refractive Index 〈831〉: 1.450–1.470, at 20°

Optical Rotation 〈781〉: 0°–10°, at 20°

6 ADDITIONAL REQUIREMENTS

Packaging and Storage: Preserve in tight containers. Do not store above 25°.

USP Reference Standards 〈11〉

USP Camphor RS

USP Eucalyptol RS

USP Eucalyptus Oil RS