Identification—
Transfer a volume of Oral Solution, equivalent to about 100 mg of diphenoxylate hydrochloride, to a separator, add 1 mL of 3 N hydrochloric acid and sufficient water to make about 100 mL, and extract with five 25-mL portions of a mixture of chloroform and isopropyl alcohol (9:1). After each extraction transfer the bottom chloroform layer to a second separator, passing each portion through a sintered-glass filter. Wash the combined chloroform solutions with two 25-mL portions of water, discard the washings, and evaporate the chloroform on a steam bath to dryness. To the residue add 10 mL of ether previously saturated with hydrochloric acid, carefully evaporate to dryness, then add a second portion of the acid-saturated ether, and again evaporate to dryness. Make a slurry of the residue with
n-hexane, allow the solids to settle, and carefully decant the supernatant. Dry the solids at 105
for 1 hour: the diphenoxylate hydrochloride so obtained meets the requirements for
Identification tests
A and
B under
Diphenoxylate Hydrochloride.
Assay for diphenoxylate hydrochloride—
Transfer an accurately measured volume of Oral Solution, equivalent to about 100 mg of diphenoxylate hydrochloride, to a separator, add 4 mL of 3 N hydrochloric acid, and extract with six 30-mL portions of chloroform. Wash the combined chloroform extracts with 25 mL of water, and discard the washing. Transfer the chloroform to a beaker, and evaporate nearly to dryness. Add 100 mL of glacial acetic acid and 4 mL of
mercuric acetate TS to the beaker, and titrate with 0.05 N perchloric acid in dioxane VS, determining the endpoint potentiometrically. Each mL of 0.05 N perchloric acid is equivalent to 24.45 mg of diphenoxylate hydrochloride (C
30H
32N
2O
2·HCl).
Assay for atropine sulfate—
pH 2.8 Buffer—
Dissolve 1.9 g of aminoacetic acid and 1.5 g of sodium chloride in 250 mL of water. Adjust by the gradual addition of about 85 mL of 0.1 N hydrochloric acid to a pH of 2.8.
Internal standard solution—
Transfer about 20 mg of homatropine hydrobromide to a 200-mL volumetric flask, dissolve in and dilute with water to volume, and mix.
Standard preparation—
Transfer about 25 mg of
USP Atropine Sulfate RS, accurately weighed, to a 200-mL volumetric flask. Dissolve in and dilute with water to volume, and mix. Pipet 2 mL of the resulting solution into a 125-mL separator containing about 50 mL of water. Add 2.0 mL of
Internal standard solution, 10.0 mL of
pH 2.8 Buffer, and 25 mL of water-saturated methylene chloride. Insert the stopper, shake for 2 minutes, and allow the layers to separate. Discard the lower, organic layer. Repeat the extraction four times, using 25-mL portions of water-saturated methylene chloride each time, allowing the layers to separate and discarding the organic layer each time. To the remaining aqueous layer add 3 mL of 0.1 N sodium hydroxide, and shake briefly. Using a pH meter, adjust the solution to a pH of 9.0 ± 0.3. Immediately add 10 mL of water-saturated methylene chloride, insert the stopper, and shake. Transfer the lower, organic layer to a 50-mL container. Repeat the extraction twice more with 10-mL portions of water-saturated methylene chloride, combining the organic extracts. Under a stream of nitrogen, evaporate the organic extracts to dryness. Dissolve the residue in 0.1 mL of methylene chloride.
Assay preparation—
Transfer an accurately measured volume of Oral Solution, equivalent to about 250 µg of atropine sulfate, to a 125-mL separator. Proceed as directed for Standard preparation, beginning with “Add 2.0 mL of Internal standard solution.”
Chromatographic system (see Chromatography 
621
)—
The gas chromatograph is equipped with a flame-ionization detector and a 4-mm × 1.2-m glass column that contains 3% phase G3 on support S1. The column is maintained at a temperature of 230
, and the injection port and detector temperatures are maintained at 250
. The carrier gas is helium, flowing at a rate of 40 mL per minute. Chromatograph the
Standard preparation, and record the peak responses as directed for
Procedure: the resolution,
R, between atropine and the internal standard is not less than 2.0; and the relative standard deviation for replicate injections is not more than 2.5%.
Procedure—
Separately inject equal volumes (about 2 µL) of the
Standard preparation and the
Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the quantity, in µg, of atropine sulfate [(C
17H
23NO
3)
2·H
2SO
4·H
2O] in each mL of the Oral Solution taken by the formula:
2(694.85/676.83)(C/V)(RU / RS),
in which 694.85 and 676.83 are the molecular weights of atropine sulfate monohydrate and anhydrous atropine sulfate, respectively;
C is the concentration, in µg per mL, of
USP Atropine Sulfate RS (corrected to the monohydrate) in the
Standard preparation; V is the volume, in mL, of Oral Solution taken; and
RU and
RS are the peak response ratios of atropine to the internal standard obtained from the
Assay preparation and the
Standard preparation, respectively.
