Assay—
pH 9.5 Phosphate buffer—
Dissolve 34.8 g of dibasic potassium phosphate in 900 mL of water, and adjust to a pH of 9.5, determined electrometrically, by the addition of 3 N hydrochloric acid or sodium hydroxide, with mixing.
Internal standard solution—
Dissolve about 40 mg of
USP Homatropine Hydrobromide RS, accurately weighed, in about 25 mL of dilute sulfuric acid (1 in 350) in a 50-mL volumetric flask, add the same dilute acid to volume, and mix. Prepare fresh on the day of use.
Standard preparation—
Dissolve about 10 mg of
USP Scopolamine Hydrobromide RS, accurately weighed, in about 5 mL of dilute sulfuric acid (1 in 350) in a 10-mL volumetric flask, add the same dilute acid to volume, and mix (
Solution A). Dissolve about 20 mg of
USP Atropine Sulfate RS, accurately weighed, in about 25 mL of dilute sulfuric acid (1 in 350) in a 50-mL volumetric flask, add 2.0 mL of
Solution A, and mix. Add dilute sulfuric acid (1 in 350) to volume, and mix. Prepare fresh on the day of use.
Extraction blank—
Place about 10 mL of dilute sulfuric acid (1 in 350) in a 60-mL separator. Proceed as directed under
Assay preparation, beginning with “then add 15 mL of chloroform.” The blank chromatogram contains no significant interferences at the locus of atropine, scopolamine, or homatropine.
Assay preparation—
Weigh accurately about 0.5 g of Extract, transfer to a 125-mL conical flask, and add 40 mL of dilute sulfuric acid (1 in 350). Heat to a temperature not above 45
, and stir to hasten solution. Filter the solution through filter paper into a 100-mL volumetric flask. Wash the flask and the filter with two 20-mL portions of warmed dilute sulfuric acid (1 in 350), and collect the washings in the 100-mL volumetric flask. Add dilute sulfuric acid (1 in 350) to volume, and mix.
Pipet 10 mL of this solution into a 60-mL separator. To the separator add 1.0 mL of
Internal standard solution, then add 15 mL of chloroform, shake vigorously, allow the layers to separate, and discard the chloroform layer. (If emulsions are formed, a
mixed solvent consisting of chloroform and isopropyl alcohol (10:3) may be substituted for chloroform throughout the extraction procedure.) Add another 15 mL of chloroform, and extract again, discarding the chloroform phase. Add 15 mL of
pH 9.5 Phosphate buffer and sufficient 1 N sodium hydroxide to yield a final pH between 9.0 and 9.5. Add 15 mL of chloroform, shake vigorously, and allow the layers to separate. Filter the organic phase through 10 g of anhydrous sodium sulfate (see
Suitability for alkaloid assays under
Sodium Sulfate,
Anhydrous, in the section
Reagents,
Indicators,
and Solutions), previously washed with chloroform and supported in a funnel with a small pledget of glass wool, into a suitable container. Extract again with two 15-mL portions of chloroform, again collecting the clarified organic phase. Wash the sodium sulfate and the tip of the funnel with 5 mL of chloroform. Evaporate the combined organic phases under reduced pressure, at a temperature below 45
, add 1 mL of chloroform, and mix to dissolve the alkaloids, taking care to wet the sides of the container.
Standard curve—
Prepare three
Standard solutions as follows. Pipet into three separate 60-mL separators 1.0-, 2.0-, and 3.0-mL portions, respectively, of
Standard preparation, and add 9.0, 8.0, and 7.0 mL, respectively, of dilute sulfuric acid (1 in 350). Proceed as directed under
Assay preparation, beginning with “add 1.0 mL of
Internal standard solution.”
Chromatographic system—
Under typical condition, the instrument contains a 1.2-m × 4-mm glass column packed with 3% G3 on S1AB. The column may be cured and conditioned as specified under
Gas Chromatography (see
Chromatography 
621
). The column is maintained at a temperature of about 215
, and the injection port and detector block at about 240
, and dry helium is used as a carrier gas at a flow rate of about 65 mL per minute.
System suitability—
Chromatograph six to ten injections of the solution, and record peak areas as directed for
Procedure. The analytical system is suitable for conducting this assay if the relative standard deviation for the ratio,
RA, calculated by the formula:
100 × (standard deviation / mean ratio),
does not exceed 2.0%; the resolution,
R, between
aH and
aA is not less than 3; and the tailing factor (the sum of the distances from peak center to the leading edge and to the tailing edge divided by twice the distance from peak center to the leading edge), measured at 5% of the peak height of
aA, does not exceed 2.0.
Procedure—
Inject a portion (about 5 µL) of each
Standard solution into a suitable gas chromatograph equipped with a flame-ionization detector. Measure the areas,
aA,
aH, and
aS, of the atropine, homatropine, and scopolamine peaks, respectively, in each chromatogram, and calculate the ratios
AA and
AS by the formulas:
aA / aH and aS / aH.
Plot the
Standard curves of the values of
RA and
RS against the amounts, in mg, of atropine and scopolamine in the solutions. (The ratio of the molecular weight of atropine to that of anhydrous atropine sulfate is 0.8551, and the ratio of the molecular weight of scopolamine to that of anhydrous scopolamine hydrobromide is 0.7894.) Inject a portion of the
Assay preparation into the chromatograph, obtain the chromatogram area ratios, measure the peak areas, and calculate the area ratios, as with the
Standard solutions. Record from the
Standard curve the quantities, in mg, of atropine and scopolamine in the volume of specimen taken. Add the quantity, in mg, of atropine and scopolamine, and multiply by 10 to obtain the weight, in mg, of alkaloids in the portion of Extract taken.
