Standard solution— Prepare an ester mixture containing methyl linoleate, methyl oleate, methyl palmitate, methyl stearate, and methyl linolenate (50:35:7:5:3). [NOTE—Ester mixtures are available commercially from Nu-Chek-Prep, Inc., P.O. Box 295, Elysian, MN 56028. Typical Nu-Chek-Prep ester mixtures useful in this test include Nu-Chek 15A. This mixture may contain other components.]

Test solution— Transfer about 100 mg of the test specimen 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 N methanolic sodium hydroxide solution, and reflux until fat globules disappear (usually 5 to 10 minutes). Add 5 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 chromatographic n-heptane through the condenser, and reflux for 1 minute. Cool, remove the condenser, add about 15 mL of saturated sodium chloride solution, shake, and allow the layers to separate. Pass the n-heptane layer through 0.1 g of anhydrous sodium sulfate (previously washed with chromatographic n-heptane) into a suitable flask. Transfer 1.0 mL of this solution to a 10-mL volumetric flask, dilute with chromatographic n-heptane to volume, and mix.

Chromatographic system (see Chromatography 621)— The gas chromatograph is equipped with a flame-ionization detector maintained at a temperature of about 250, a splitless injection system, and a 0.25-mm × 30-m fused-silica capillary column bonded with a 0.25-µm layer of phase G5. The column temperature is maintained at 120 for about 2 minutes after injection, and the temperature is then increased at the rate of 4 per minute to 240 and maintained at 240 for 5 minutes. The injection port temperature is maintained at about 220. The carrier gas is hydrogen, with a flow rate of about 1 mL per minute. Chromatograph 1 µL of the Standard solution, and record the peak responses as directed for Procedure: the relative retention times are about 0.87 for methyl palmitate, 0.99 for methyl stearate, and 1.0 for methyl oleate; the resolution, R, between methyl stearate and methyl oleate is not less than 1.5; and the relative standard deviation of the peak area responses for the palmitate and stearate peaks for replicate injections is not more than 6.0%. 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%.

Procedure— Inject a volume (about 1 µL) of the Test solution into the chromatograph, record the chromatogram, and measure the areas of the five major peaks for the methyl esters of the fatty acids, which elute in the following order: palmitate, stearate, oleate, linoleate, and linolenate. Calculate the relative amounts of palmitate, stearate, oleate, linoleate, and linolenate, expressed as percentages of the total area of the five major peaks: for generic Oil, between 3% and 10% of methyl palmitate is found, between 2% and 8% of methyl stearate is found, between 14% and 24% of methyl oleate is found, between 63% and 73% of methyl linoleate is found, and between 0% and 3% of methyl linolenate is found; for mid-oleic Oil, between 2% and 9% of methyl palmitate is found, between 2% and 8% of methyl stearate is found, between 40% and 70% of methyl oleate is found, between 15% and 40% of methyl linoleate is found, and between 0% and 3% of methyl linolenate is found; and for high-oleic Oil, between 2% and 9% of methyl palmitate is found, between 2% and 8% of methyl stearate is found, between 70% and 90% of methyl oleate is found, between 5% and 15% of methyl linoleate is found, and between 0% and 3% of methyl linolenate is found.

Solvent— Mix 60 mL of glacial acetic acid and 40 mL of chloroform.

Potassium iodide solution— Prepare a saturated solution of potassium iodide in freshly boiled and cooled water, and store it protected from light. [NOTE—Discard the solution if it becomes colored upon addition of Solvent and iodine-free starch TS.]

Procedure— Transfer about 10 g of Oil, accurately weighed, to a conical flask; add 30 mL of Solvent; and swirl to dissolve. Add 0.5 mL of Potassium iodide solution; swirl for 1.0 minute; add 30 mL of water; and titrate with 0.01 N sodium thiosulfate VS, with vigorous agitation, to a light yellow color. Add 2.0 mL of iodine-free starch TS, and continue the titration until the blue color has disappeared. Perform a blank determination, and make any necessary correction. Calculate the amount of peroxide, in mEq per kg, in the portion of Oil taken by the formula: in which V is the volume, in mL, of sodium thiosulfate used in the titration; N is the actual normality of sodium thiosulfate VS; and W is the weight, in g, of Oil taken: not more than 10.0 mEq per kg is found.

(Official January 1, 2007)NF24