Assay—
Chloroform—
Use chloroform that recently has been saturated with water.
Standard preparation—
Dissolve about 10mg of
USP Ergotamine Tartrate RS,accurately weighed,with warming if necessary,in 50mLof diluted alcohol and sufficient water to give a concentration of 50.0µg per mL.
Ergotamine preparation
—Pipet a volume of Injection,equivalent to about 5mg of ergotamine tartrate,into a beaker.Add 5mLof
Chloroformand 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 1g for each mLof the Injection taken plus 3g in addition).Pack the mixture in a chromatographic tube about 2.5cm in diameter and about 30cm in length.Rinse the sides of the beaker with 2mLof
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 9g of the siliceous earth with 7mLof citric acid solution (1in 4).Place a mixture of 2g of the siliceous earth and 2mLof 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 90mLof Chloroformto the upper tube,and receive the eluate from the lower tube in a 200-mLvolumetric flask.Rinse the tip of the upper tube with Chloroform.Pass sufficient Chloroformthrough 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-mLbeaker containing 10g of sodium bicarbonate,and mix.Cautiously add 50mLof water,with continuous stirring.Wash the mixture with water into a 250-mLseparator,and extract the ergotamine with four 15-mLportions of Chloroform.Pass the extracts through a glass wool filter,combining them in a 100-mLvolumetric flask,wash the filter,and dilute with Chloroformto volume.This is the Ergotamine preparation.
Pipet 10mLof the Ergotamine preparationand 20mLof the Ergotamine preparationinto separate,small conical flasks,and evaporate with the aid of a current of air to dryness.
Total alkaloid preparation—
Pipet a volume of Injection,equivalent to about 2.5mg of ergotamine tartrate,into a 50-mLvolumetric flask,add 25mLof alcohol,then add tartaric acid solution (1in 100)to volume.
Procedure—
Pipet 5mLeach of the Standard preparationand the Total alkaloid preparationinto separate,small conical flasks.To the dried residues of Ergotamine preparationand Ergotamine preparationadd 5.0mLof a freshly prepared solution of equal volumes of alcohol and tartaric acid solution (1in 100).In turn,place each flask in an ice bath,and swirl continuously while adding,dropwise,10.0mLof p-dimethylaminobenzaldehyde TS.Allow to stand in subdued light at room temperature for not less than 90minutes and not more than 2hours.Concomitantly determine the absorbances of the four solutions at the wavelength of maximum absorbance at about 545nm,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
6in the volume of Injection taken by the formula:
0.05C(AU/AS),
in which
Cis the concentration,in µg per mL,of
USP Ergotamine Tartrate RSin the
Standard preparation,and
AUand
ASare the absorbances of the solutions from the
Total alkaloid preparationand 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 preparationand AUis as defined in the preceding paragraph.Calculate the percentage of ergotaminine tartrate taken by the formula:
(A¢¢/AU)50,
in which A¢¢represents the absorbance of the solution from the Ergotamine preparation.