A: Transfer, with the aid of water, a portion of the Injectable Suspension, freshly mixed and free from air bubbles, equivalent to about 400,000 Penicillin G Units, to a separator, add 50 mL of chloroform, and shake by mechanical means for 15 minutes. Allow to separate, and filter the lower chloroform layer through about 4 g of anhydrous sodium sulfate supported on a pledget of glass wool, collecting the filtrate in a 100-mL volumetric flask. Repeat the extraction with two 25-mL portions of chloroform, combining the filtrates in the 100-mL volumetric flask. Dilute with chloroform to volume, and mix. [NOTE—Retain the aqueous phase for Identification test B.] Prepare a Standard solution of USP Penicillin G Procaine RS in chloroform containing about 4.5 mg per mL. Apply separately 10 µL of each solution to a thin-layer chromatographic plate (see Chromatography 621) coated with a 0.25-mm layer of chromatographic silica gel mixture. Allow the spots to dry, and develop the chromatogram in a solvent system consisting of a mixture of butanol, isopropyl alcohol, acetone, and water (4:4:2:2) until the solvent front has moved about three-fourths of the length of the plate. Remove the plate from the developing chamber, mark the solvent front, and allow the solvent to evaporate. Expose the plate to iodine vapors in a closed chamber for about 15 minutes, and locate the spots: the RF values and colors of the two principal spots obtained from the test solution correspond to those obtained from the Standard solution.

B: Dilute the aqueous phase retained from Identification test A with water to obtain a test solution containing about 5 mg of dihydrostreptomycin per mL. Prepare a Standard solution of USP Dihydrostreptomycin Sulfate RS in water containing 6.5 mg per mL. Apply separately 30 µL of each solution to a thin-layer chromatographic plate (see Chromatography 621) coated with a 0.25-mm layer of chromatographic silica gel mixture. Allow the spots to dry, and develop the chromatogram in a solvent system consisting of a mixture of n-propyl alcohol, water, pyridine, and glacial acetic acid (15:12:10:2) until the solvent front has moved about three-fourths of the length of the plate. Remove the plate from the developing chamber, mark the solvent front, and allow the solvent to evaporate. Spray the plate with a reagent prepared by dissolving 2 g of ninhydrin in 100 mL of alcohol and adding 20 mL of glacial acetic acid, heat the plate at 110 for 10 minutes, and examine the chromatograms: the RF value and color of the principal spot obtained from the test solution correspond to those obtained from the Standard solution.

C: The chromatogram of the Assay preparation obtained as directed in the Assay for chlorpheniramine maleate exhibits a major peak for chlorpheniramine, the retention time of which corresponds to that exhibited in the chromatogram of the Standard preparation similarly determined, both relative to the internal standard.

D: The chromatogram of the Assay preparation obtained as directed in the Assay for dexamethasone exhibits a major peak for dexamethasone, the retention time of which corresponds to that exhibited in the chromatogram of the Standard preparation similarly determined, both relative to the internal standard.

(Official January 1, 2007)

Standard preparation— Using USP Penicillin G Potassium RS, prepare as directed for Standard preparation under Iodometric Assay—Antibiotics 425.

Assay preparation— Dilute an accurately measured volume of Injectable Suspension, freshly mixed and free from air bubbles, quantitatively with Buffer No. 1 to yield a solution containing about 2000 Penicillin G Units per mL. Pipet 2 mL of this solution into each of two glass-stoppered, 125-mL conical flasks.

Procedure— Proceed as directed for Procedure under Iodometric Assay—Antibiotics 425, except in the Blank Determination to add 0.1 mL of 1.2 N hydrochloric acid immediately before the 10.0 mL of 0.01 N iodine VS. Calculate the quantity, in Penicillin G Units, in the portion of Injectable Suspension taken by the formula: in which L is the labeled quantity, in Penicillin G Units, in the volume of Injectable Suspension taken, and D is the concentration, in Penicillin G Units per mL, of the Assay preparation, on the basis of the labeled quantity in the portion of Injectable Suspension taken and the extent of dilution, and the other terms are as defined therein.

Internal standard solution— Prepare a solution of brompheniramine maleate in water having a concentration of about 7 mg per mL.

Standard preparation— Dissolve an accurately weighed quantity of USP Chlorpheniramine Maleate RS in water to obtain a stock solution having a known concentration of about 6 mg per mL. Transfer 5.0 mL of this solution to a 50-mL centrifuge tube. Add 5.0 mL of Internal standard solution, and adjust with sodium hydroxide solution (1 in 2) to a pH of about 10. Add 25.0 mL of hexanes, place the cap on the tube, shake for about 2 minutes, and centrifuge. Use the upper hexanes layer as the Standard preparation.

Assay preparation— Transfer an accurately measured volume of Injectable Suspension, freshly mixed and free from air bubbles, equivalent to about 30 mg of chlorpheniramine maleate, to a 50-mL centrifuge tube. Proceed as directed under Standard preparation, beginning with “Add 5.0 mL of Internal standard solution.” Use the upper hexanes layer as the Assay preparation.

Chromatographic system (see Chromatography 621)— The gas chromatograph is equipped with a flame-ionization detector, and contains a 4-mm × 1.8-m glass column packed with 1.2% liquid phase G16 and 0.5% potassium hydroxide on 100- to 120-mesh support S1A. The column is maintained isothermally at about 180, and the injection port and the detector block are maintained at about 200. Dry nitrogen is used as the carrier gas at a flow rate of about 50 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the resolution, R, between the analyte and internal standard peaks is not less than 2.0, and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 1.5 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks. The relative retention times are about 0.75 for chlorpheniramine and 1.0 for brompheniramine. Calculate the quantity, in mg, of chlorpheniramine maleate (C16H19ClN2·C4H4O4) in each mL of the Injectable Suspension taken by the formula: in which C is the concentration of USP Chlorpheniramine Maleate RS in the stock solution used to prepare the Standard preparation, V is the volume, in mL, of Injectable Suspension taken, and RU and RS are the peak response ratios of the chlorpheniramine maleate peak to the internal standard peak obtained from the Assay preparation and the Standard preparation, respectively.

Mobile phase— Prepare a suitable filtered mixture of water and acetonitrile (2:1). Make adjustments if necessary (see System Suitability under Chromatography 621).

Internal standard solution— Dissolve about 30 mg of beclomethasone in 2 mL of methanol in a 50-mL volumetric flask, dilute with methylene chloride to volume, and mix.

Standard preparation— Transfer about 25 mg of USP Dexamethasone RS, accurately weighed, to a 50-mL volumetric flask. Add about 1 mL of methanol, swirl to dissolve, dilute with methylene chloride to volume, and mix. Transfer 5.0 mL of this solution to a suitable flask, and add 5.0 mL of Internal standard solution. Heat the flask on a steam bath, and evaporate under a stream of nitrogen just to dryness. Add 10.0 mL of methanol to the flask, and swirl to dissolve the residue. This Standard preparation contains about 0.25 mg of USP Dexamethasone RS and 0.3 mg of beclomethasone per mL.

Assay preparation— Transfer an accurately measured volume of Injectable Suspension, freshly mixed and free from air bubbles, equivalent to about 2.5 mg of dexamethasone, to a separator containing 50 mL of 0.1 N hydrochloric acid, add 5.0 mL of Internal standard solution, and extract with four 25-mL portions of methylene chloride, combining the extracts in a second separator. Wash the combined extracts with 50 mL of sodium bicarbonate solution (1 in 20), filtering the lower methylene chloride layer through about 4 g of anhydrous sodium sulfate supported on a cotton pledget previously washed with methylene chloride, and collecting the filtrate in a suitable flask. Wash the aqueous layer with 25 mL of methylene chloride, and filter the lower methylene chloride layer through the same filter, collecting the filtrate in the same flask. Heat the flask on a steam bath, and evaporate under a stream of nitrogen just to dryness. Add 10.0 mL of methanol, and swirl to dissolve the residue.

Chromatographic system (see Chromatography 621)—The liquid chromatograph is equipped with a 254-nm detector and a 4-mm × 30-cm column that contains packing L1. The flow rate is about 1.2 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the resolution, R, of the analyte and the internal standard peaks is not less than 2.0, and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 10 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks. The relative retention times are about 0.8 for dexamethasone and 1.0 for beclomethasone. Calculate the quantity, in mg, of dexamethasone (C22H29FO5) in each mL of the Injectable Suspension taken by the formula: in which C is the concentration, in mg per mL, of USP Dexamethasone RS in the Standard preparation, V is the volume, in mL, of Injectable Suspension taken, and RU and RS are the peak response ratios of the dexamethasone peak to the internal standard peak obtained from the Assay preparation and the Standard preparation, respectively.