A: Reflux 1 g of sample with 15 mL of 0.5 N potassium hydroxide solution in dehydrated alcohol for 1 hour. Add 15 mL of water, acidify with diluted hydrochloric acid (about 6 mL). Oil drops or a white to yellowish-white solid is produced that is soluble in 5 mL of hexane. Remove the hexane layer, extract again with 5 mL of hexane and again remove the hexane layer. Keep the resulting aqueous layer for Identification tests B and C.

B: Evaporate 1 mL of the aqueous layer resulting from Identification test A in a porcelain dish. The residue meets the requirements of the test for Citrate 191.

C: Transfer 5 mL of the aqueous layer resulting from Identification test A into a test tube. Add excess calcium hydroxide as a powder, place in boiling water for 5 minutes, shaking several times, cool, and filter. Transfer one drop of the filtrate into a test tube, and add about 50 mg of potassium hydrogen sulfate. On top of the test tube, place a filter paper moistened with a reagent for acrolein consisting of a mixture of a 5% nitroprusside solution in water and a 20% piperidine solution in water (1:1). Heat the test tube. If the filter paper turns blue, the presence of glycerin is indicated. The color changes to light red after addition of sodium hydroxide TS.

Standard preparation— Dissolve an accurately weighed quantity of USP Citric Acid RS in water, and dilute quantitatively, and stepwise if necessary, with water to obtain a solution having a known concentration of about 230 µg per mL.

Test preparation— Transfer about 150 mg of Monoglyceride Citrate, accurately weighed, into a saponification flask, add 50 mL of 4% potassium hydroxide solution in dehydrated alcohol, and reflux for 1 hour. Acidify the reaction mixture with hydrochloric acid to a pH of 2.8 to 3.2, transfer into a 400-mL beaker, and evaporate to dryness on a steam bath. Quantitatively transfer the contents of the beaker into a separator, using no more than 50 mL of water, and extract with three 50-mL portions of petroleum ether, discarding the extracts. Transfer the water layer to a 100-mL volumetric flask, dilute with water to volume, and mix.

Procedure— Pipet 2.0 mL each of the Standard preparation and of the Test preparation into separate 40-mL graduated centrifuge tubes. Add 2 mL of a 1 in 2 sulfuric acid solution and 11 mL of water to each tube. Boil for 3 minutes, cool, and add 5 mL of bromine TS to each tube. Dilute to 20 mL, allow to stand for 10 minutes, and centrifuge. Transfer 4.0 mL of each solution into separate 19- × 110-mm test tubes, add 1 mL of water, 0.5 mL of a 1 in 2 sulfuric acid solution, and 0.3 mL of 1 M potassium bromide, and shake. Add 0.3 mL of 1.5 N potassium permanganate, shake, and allow to stand for 2 minutes. Add 1 mL of a saturated solution of ferrous sulfate, shake, allow to stand for 2 minutes, and then dilute to 10 mL with water. Add 10.0 mL of n-hexane (previously washed with sulfuric acid, followed by a water wash, and then dried over anhydrous sodium sulfate), shake vigorously for 2 minutes, and centrifuge at low speed for 1 minute. Transfer 5.0 mL of the hexane extract into a 20- × 145-mm tube containing 10.0 mL of 4% sodium sulfide solution, and briefly shake vigorously (three oscillations only). Centrifuge the mixture at low speed for 1 minute. Immediately determine the absorbance of each aqueous layer in a 1-cm cell at 450 nm with a suitable spectrophotometer, using a reagent blank in the reference cell. Calculate the quantity, in mg, of citric acid in the Monoglyceride Citrate taken by the formula: in which C is the concentration, in µg per mL, of USP Citric Acid RS in the Standard preparation; and AU and AS are the absorbances obtained from the Test preparation and the Standard preparation, respectively.