Assay—
Chloroform—
Use chloroform that recently has been saturated with water.
Standard preparation—
Dissolve about 10 mg of
USP Ergotamine Tartrate RS, accurately weighed, with warming if necessary, in 50 mL of diluted alcohol and sufficient water to give a concentration of 50.0 µg per mL.
Ergotamine preparation
—Pipet a volume of Injection, equivalent to about 5 mg of ergotamine tartrate, into a beaker. Add 5 mL of
Chloroform and a portion of sodium bicarbonate approximately equivalent in weight to one-tenth that of the portion of Injection taken. Mix, and add sufficient chromatographic siliceous earth to make a fluffy mixture (about 1 g for each mL of the Injection taken plus 3 g in addition). Pack the mixture in a chromatographic tube about 2.5 cm in diameter and about 30 cm in length. Rinse the sides of the beaker with 2 mL of
Chloroform. Add sufficient chromatographic siliceous earth to make a fluffy mixture, and transfer it to the column.
Prepare a second column using a mixture of 9 g of the siliceous earth with 7 mL of citric acid solution (1 in 4). Place a mixture of 2 g of the siliceous earth and 2 mL of water on top of the second column. Insert a pledget of glass wool, and mount the tube containing the specimen so that the eluate from it will drain into the tube containing the citric acid solution. Add a total of 90 mL of
Chloroform to the upper tube, and receive the eluate from the lower tube in a 200-mL volumetric flask. Rinse the tip of the upper tube with
Chloroform. Pass sufficient
Chloroform through the lower tube to dilute the eluate to volume. This eluate is the
Ergotamine preparation.
Extrude the adsorbent from the second column by means of slight air pressure into a 600-mL beaker containing 10 g of sodium bicarbonate, and mix. Cautiously add 50 mL of water, with continuous stirring. Wash the mixture with water into a 250-mL separator, and extract the ergotamine with four 15-mL portions of
Chloroform. Pass the extracts through a glass wool filter, combining them in a 100-mL volumetric flask, wash the filter, and dilute with
Chloroform to volume. This is the
Ergotamine preparation.
Total alkaloid preparation—
Pipet a volume of Injection, equivalent to about 2.5 mg of ergotamine tartrate, into a 50-mL volumetric flask, add 25 mL of alcohol, then add tartaric acid solution (1 in 100) to volume.
Procedure—
Pipet 5 mL each of the
Standard preparation and the
Total alkaloid preparation into separate, small conical flasks. To the dried residues of
Ergotamine preparation and
Ergotamine preparation add 5.0 mL of a freshly prepared solution of equal volumes of alcohol and tartaric acid solution (1 in 100). In turn, place each flask in an ice bath, and swirl continuously while adding, dropwise, 10.0 mL of
p-dimethylaminobenzaldehyde TS. Allow to stand in subdued light at room temperature for not less than 90 minutes and not more than 2 hours. Concomitantly determine the absorbances of the four solutions at the wavelength of maximum absorbance at about 545 nm, with a suitable spectrophotometer, against a reagent blank.
Calculation—
Calculate the quantity of total alkaloids in terms of mg of (C
33H
35N
5O
5)
2·C
4H
6O
6 in the volume of Injection taken by the formula:
0.05C(AU / AS),
in which
C is the concentration, in µg per mL, of
USP Ergotamine Tartrate RS in the
Standard preparation, and
AU and
AS are the absorbances of the solutions from the
Total alkaloid preparation and the
Standard preparation, respectively.
Calculate the percentage of ergotamine tartrate taken by the formula:
(A¢ / AU)50,
in which
A¢ represents the absorbance of the solution from the
Ergotamine preparation and
AU is as defined in the preceding paragraph. Calculate the percentage of ergotaminine tartrate taken by the formula:
(A¢¢ / AU)50,
85
—
It contains not more than 357.0 USP Endotoxin Units per mg of ergotamine tartrate.