Glyceryl Behenate
(glis' er il be hen' ate).
DEFINITION
Glyceryl Behenate is a mixture of glycerides of fatty acids, mainly behenic acid.
IDENTIFICATION
•  A. Thin-Layer Chromatographic Identfication Test
Standard solution:  60 mg/mL of USP Glyceryl Behenate RS in chloroform
Sample solution:  60 mg/mL of Glyceryl Behenate in chloroform
Chromatographic plates:  Use suitable thin-layer chromatographic plates (see Chromatography 621) coated with a 0.25-mm layer of chromatographic silica gel. Predevelop the plates by placing in a chromatographic chamber saturated with ether. Remove the plates from the chamber, allow the ether to evaporate, and immerse the plates in a 2.5% solution of boric acid in alcohol. After about 1 min, withdraw the plates, allow them to dry at ambient temperature, and activate them at 110 for 30 min. Keep the plates in a desiccator.
Chromatographic system  
Mode:  TLC
Application volume:  10 µL
Spray reagent:  0.02% solution of dichlorofluorescein in alcohol
Developing solvent system:  Chloroform and acetone (96:4)
Analysis 
Samples:  Standard solution and Sample solution
Develop in Developing solvent system until the solvent front has moved about 12 cm. Remove the plate from the chamber, and allow the solvent to evaporate. Spray the chromatogram with Spray reagent. Examine the spots under short-wavelength UV light.
Acceptance criteria:  The RF values of the spots from the Sample solution correspond to those from the Standard solution.
•  B.
Standard solution:  Dissolve 22 mg of USP Glyceryl Behenate RS in 1 mL of toluene in a screw-capped vial having a polytef-lined septum. Add 0.4 mL of 0.2 N methanolic (m-trifluoromethylphenyl) trimethylammonium hydroxide. Attach the cap, and mix. Allow the vial to stand at room temperature for NLT 30 min.
Sample solution:  Dissolve 22 mg of Glyceryl Behenate in 1 mL of toluene in a screw-capped vial having a polytef-lined septum. Add 0.4 mL of 0.2 N methanolic (m-trifluoromethylphenyl) trimethylammonium hydroxide. Attach the cap, and mix. Allow the vial to stand at room temperature for NLT 30 min.
Chromatographic system 
Mode:  GC
Detector:  Flame ionization
Column:  4-mm × 1.8-m, packed with 10% liquid phase G7 on support S1A
Column temperature:  225
Analysis 
Samples:  Standard solution and Sample solution
Introduce a suitable volume into the gas chromatograph, and record the resulting chromatogram.
Acceptance criteria:  The retention time of the main peak of the Sample solution corresponds to that of the main peak of the Standard solution. The ratio of the response of the main peak to the sum of all the responses is NLT 0.83.
IMPURITIES
•  Residue on Ignition 281: NMT 0.1%
•  Heavy Metals, Method II 231: NMT 10 ppm
•  Content of 1-Monoglycerides
Periodic acid solution:  Dissolve 5.4 g of periodic acid in 100 mL of water, and add 1900 mL of glacial acetic acid. Store in a light-resistant, glass-stoppered bottle or in a clear glass-stoppered bottle protected from light.
Chloroform:  Use chloroform that meets the following test. To each of three 500-mL flasks add 50.0 mL of Periodic acid solution. Add 50 mL of chloroform and 10 mL of water to two of the flasks, and add 50 mL of water to the third flask. To each flask add 20 mL of potassium iodide TS, mix gently, and proceed as directed in the Analysis below, beginning with “allow to stand for NLT 1 min”. The difference between the volumes of 0.1 N sodium thiosulfate required in the titrations with and without the chloroform is NMT 0.5 mL.
Sample:  Melt a quantity of Glyceryl Behenate at a temperature not higher than 80.
Analysis:  Transfer 1 g of the Sample to a 100-mL beaker, and dissolve in 25 mL of Chloroform. Transfer the solution, with the aid of an additional 25 mL of Chloroform, to a separator, wash the beaker with 25 mL of water, and add the washing to the separator. Tightly place the stopper in the separator, shake vigorously for 30–60 s, and allow the layers to separate. Add 1–2 mL of glacial acetic acid to break any emulsion formed. Collect the aqueous layer in a glass-stoppered 500-mL conical flask, and extract the nonaqueous layer again, using two 25-mL portions of water. [Note—Retain the combined aqueous extracts for the test for Limit of Free Glycerin. ]
Transfer the nonaqueous layer to a glass-stoppered 500-mL conical flask, and add 50.0 mL of Periodic acid solution to this flask and to a blank flask containing 50 mL of Chloroform and 10 mL of water, swirling the flasks during the addition. Allow to stand for 30–90 min. To each flask add 20 mL of potassium iodide TS, and allow to stand for 1–5 min before titrating. Add 100 mL of water, and titrate with 0.1 N sodium thiosulfate VS, using a magnetic stirrer to keep the solution mixed, to the disappearance of the brown iodine color. Then add 2 mL of starch TS, and continue the titration to the disappearance of the blue color. [Note—If the Glyceryl Behenate titration is less than 0.8 of the blank titration, discard, and repeat using a smaller weight of Glyceryl Behenate. ]
Calculate the percentage of 1-monoglycerides as glyceryl monobehenate:
Result = [(B S) × F × N × 100]/W
B== volume of 0.1 N sodium thiosulfate consumed by the blank (mL)
S== volume of 0.1 N sodium thiosulfate consumed by the Glyceryl Behenate (mL)
F== equivalency factor of glyceryl monobehenate, 207.3 mg/mEq
N== normality of the sodium thiosulfate
W== weight of Glyceryl Behenate taken (g)
Acceptance criteria:  12.0%–18.0%
•  Limit of Free Glycerin
Periodic acid solution and Chloroform:  Prepare as directed in the test for Content of 1-Monoglycerides.
Analysis:  To the combined aqueous extracts obtained as directed in the test for Content of 1-Monoglycerides add 50.0 mL of Periodic acid solution. Prepare a blank by adding 50.0 mL of Periodic acid solution to a glass-stoppered conical flask containing 75 mL of water. Allow to stand for NLT 30 min but NMT 90 min. To each flask add 20 mL of potassium iodide TS, and allow to stand for NLT 1 min but NMT 5 min before titrating. Add 100 mL of water, and titrate with 0.1 N sodium thiosulfate VS, using a magnetic stirrer to keep the solution mixed, to the disappearance of the brown iodine color. Then add 2 mL of starch TS, and continue the titration to the disappearance of the blue color.
Calculate the percentage of free glycerin:
Result = [(B S) × F × N × 100]/W
B== volume of 0.1 N sodium thiosulfate consumed by the blank (mL)
S== volume of 0.1 N sodium thiosulfate consumed by the Glyceryl Behenate (mL)
F== equivalency factor of glycerin, 23.0 mg/mEq
N== normality of the sodium thiosulfate
W== weight of Glyceryl Behenate taken (g)
Acceptance criteria:  NMT 1.0%
SPECIFIC TESTS
•  Fats and Fixed Oils, Acid Value, Method I 401
Sample:  10 g
Analysis:  Suspend the Sample in 50 mL of a mixture of alcohol and ether (1:1), which has been neutralized to phenolphthalein with 0.1 N sodium hydroxide, contained in a flask. Connect the flask with a suitable condenser, and warm, with frequent shaking, for 10 min. Continue as directed under Fats and Fixed Oils 401, Acid Value, beginning with “Add 1 mL of phenolphthalein TS”.
Acceptance criteria:  NMT 4
ADDITIONAL REQUIREMENTS
•  Packaging and Storage: Preserve in tight containers at a temperature not higher than 35.
•  USP Reference Standards 11
USP Glyceryl Behenate RS Click to View Structure
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