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Category  Pharmaceutic aid.

Cottonseed Oil is the refined fixed oil obtained from the seeds of cultivated plants of various varieties of Gossypium hirsutum L. or of other species of Gossypium (Family Malvaceae).

Origin of plant  Cottonseed oil-yielding plant is native to the Pacific region, from Mexico to Ecuador and Northeastern Brazil.

Constituents  Cottonseed Oil contains mainly ∝-tocopherol and fatty acids predominantly linoleic, oleic, palmitic, and stearic acids. It also contains sterols such as campesterol, sitosterol, stigmasterol, and avenasterol.

Description  Clear, pale yellow liquid when heated.

Solubility  Practically insoluble in water, freely soluble in methylene chloride and in toluene, very slightly soluble in ethanol.

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

Identification of fixed oils by thin-layer chromatography  Carry out the test as described in Appendix =.

      Results  The chromatogram obtained is similar to the corresponding chromatogram shown in the figure.

Specific gravity  0.915 to 0.921 (Appendix 4.9).

Acid value  Not more than 0.5 (Appendix 5.4); dissolve 10.0 g in 50 mL of a hot mixture of equal volumes of ethanol and toluene, previously neutralized with 0.1 M potassium hydroxide using 0.5 mL ofphenolphthalein TS as indicator. Titrate the solution immediately while still hot.

Peroxide value  Not more than 5.0 (Appendix 5.12).

Unsaponifiable matter  Not more than 1.0 per cent w/w (Appendix 5.8); use 5.0 g.

Composition of fatty acids  Carry out the test as described in the “Gas Chromatography” (Appendix 3.4).

Standard solution  Prepare an ester mixture of known composition containing the esters required in the Cottonseed oil. (Nu-Chek mixture 17A^® or equivalent is suitable.)

0.5 M Methanolic sodium hydroxide solution  Dissolve 2 g of sodium hydroxide in 100 mL of methanol.

Test solution  (Note  If fatty acids containing more than 2 double bonds are present in the sample, remove air from the flask by purging it with nitrogen for a few minutes.) Transfer about 100 mg of the sample, accurately weighed, to a 50-mL conical flask fitted with a suitable water-cooled reflux condenser and a magnetic stir bar. Add 4 mL of 0.5 M Methanolic sodium hydroxide solution, and reflux until fat globules disappear (usually 5 to 10 minutes). Add 5.0 mL of a solution prepared by dissolving 14 g of boron trifluoride in methanol to make 100 mL, swirl to mix, and reflux for 2 minutes. Add 4 mL of n-heptane for chromatography through the condenser, and reflux for 1 minute. Cool, remove the condenser, add about 15 mL of a saturated solution of sodium chloride, shake, and allow the layers to separate. Pass the n-heptane layer through 100 mg of anhydrous sodium sulfate (previously washed with n-heptane for chromatography) into a suitable flask. Transfer 1.0 mL of this solution to a 10-mL volumetric flask, dilute with n-heptane for chromatography to volume, and mix.

System suitability solution  Transfer about 20 mg each of stearic acid, palmitic acid, and oleic acid to a 25-mL conical flask fitted with a suitable water-cooled reflux condenser and a magnetic stir bar, and prepare as directed for Test solution beginning with “Add 5.0 mL of a solution prepared by dissolving . . . .”

Chromatographic system

Detector  Flame ionization.

            Column  A fused-silica capillary column (30 m × 0.53 mm) bonded with a 1.0-µm layer of polyethylene glycol compound (Polyethylene Glycol Compound 20M^®, also known as Carbowax 20M^®, or equivalent.)

Temperature

                Column  The temperature programme is as follows:

                  Injection port  20.

                  Detector  260.

Carrier gas  Helium.

            Flow rate  About 50 cm per second (6.6 mL per minute).

(Note  The relative retention times are about 0.87, 0.99, and 1.0 for methyl palmitate, methyl stearate, and methyl oleate, respectively.)

System suitability

            Sample  System suitability solution.

Suitability requirements

Resolution  Not less than 1.5 between methyl stearate and methyl oleate peaks.

            Relative standard deviation  The relative standard deviation of the peak area responses for the palmitate and stearate peaks for replicate injections is not more than 6.0 per cent. The relative standard deviation of the peak area response ratio of the palmitate to stearate peaks from these replicate injections is not more than 1.0 per cent.

Procedure  Separately inject equal volumes (about 1 µL) of Standard solution and Test solution into the chromatograph, and record the chromatograms. Identify the peak due to the fatty acid ester peaks in the chromatogram of the Test solution by comparing the retention times of these peaks with those in the chromatogram of the Standard solution, and measure the peak areas for all of the fatty acid ester peaks in the chromatogram from the Test solution.

Calculation  Calculate the percentage content of the components of Cottonseed Oil taken by normalization procedure. 

Limits  Cottonseed Oil exhibits the composition profiles of fatty acids in Table 1 below.

Table 1