(Official January 1, 2007)

Chromatographic tubes— Prepare three similar tubes, each about 260 mm long and consisting of about 200 mm of 25-mm tubing and about 6 cm of 6-mm tubing. In each of the tubes, place a pledget of glass wool at a point where the 6-mm tubing is constricted slightly, about 2 cm from the junction.

Citrate buffer— Mix equal volumes of 0.1 M sodium citrate and 0.1 M citric acid.

Standard preparation— Prepare a solution by dissolving an accurately weighed quantity of USP Morphine Sulfate RS, equivalent to about 40 mg of anhydrous morphine, in 0.5 mL of triethylamine contained in a 100-mL volumetric flask, and add methanol to volume. Pipet 10 mL of this solution into a 50-mL volumetric flask, add 1 mL each of triethylamine and hydrochloric acid, and add water-saturated chloroform to volume.

Assay preparation— Evaporate 10.0 mL of Paregoric (equivalent to about 4 mg of morphine) on a steam bath under a stream of air to about 2 mL, and cool. [NOTE—Avoid reducing the volume to less than 2 mL.] Add 0.5 mL of Citrate buffer.

Chromatographic columns— Fill the three tubes with adsorbent prepared as follows, using chromatographic siliceous earth as the base of the adsorbent, and tamp it firmly in place. Pack Column I in two layers, the lower layer consisting of 3 g of chromatographic siliceous earth mixed with 2 mL of Citrate buffer and the upper layer of 3 g of chromatographic siliceous earth mixed with the Assay preparation. Dry-rinse the beaker in which the components of the two layers have been mixed with 1 g of chromatographic siliceous earth, and add it also to the top of Column I. Pack Column II with 3 g of chromatographic siliceous earth mixed with 2 mL of dibasic potassium phosphate solution (1 in 5.75). Pack Column III with 3 g of chromatographic siliceous earth mixed with 2 mL of sodium hydroxide solution (1 in 50). Place a small pad of glass wool above each column packing.

Procedure— [NOTES—(1) Use water-saturated solvents throughout this procedure; (2) prepare eluants fresh daily; and (3) avoid bringing the solutions into contact with metal.] Wash Column I with 100 mL of ether, followed by 100 mL of chloroform, rinse the tip of the column with chloroform, and discard the solvents. In the following operations, rinse each column tip before discarding the column or changing receivers. Mount the three columns vertically so that the effluent from Column I flows into Column II, and the effluent from the latter flows into Column III. Pass through the three columns 5 mL of a 1 in 5 solution of triethylamine in chloroform, followed by four 10-mL portions of a 1 in 100 solution of triethylamine in chloroform, allowing each portion to pass through completely before subsequent additions. Discard Column I. Pass three 5-mL portions of the 1 in 100 solution of triethylamine in chloroform through the two remaining columns. Discard Column II. Wash Column III successively with 10 mL of the 1 in 100 solution of triethylamine in chloroform, 50 mL of chloroform, 2 mL of a 1 in 10 solution of glacial acetic acid in chloroform, and 50 mL of a 1 in 100 solution of glacial acetic acid in chloroform. Discard all washings. Arrange to collect eluate from Column III in a 50-mL volumetric flask containing 10 mL of methanol and 1 mL of hydrochloric acid. Elute the column with 5 mL of a 1 in 5 solution of triethylamine in chloroform, followed by 33 mL of a 1 in 100 solution of triethylamine in chloroform. Dilute with chloroform to volume, and mix. Concomitantly record the spectra of this solution and the Standard preparation in 1-cm cells, with a suitable spectrophotometer, from 255 nm to 360 nm, using chloroform as the blank, and plot the corresponding wavelength-absorbance curves. Correct the absorbance of each solution, at the wavelength of maximum absorbance at about 285 nm, by extrapolating the portion of the base-line curve between 340 nm and 310 nm to this wavelength. Calculate the weight of anhydrous morphine, in mg per 100 mL of Paregoric, taken by the formula: in which W is the weight, in mg, of anhydrous morphine in the 50 mL of Standard preparation, and AU and AS are the corrected absorbances of the solution from the Assay and the Standard preparation, respectively.