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.

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 (C33H35N5O5)2·C4H6O6 in the volume of Injection taken by the formula: 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.