A: To one part of the filtrate add 10 mL of p-dimethylaminobenzaldehyde TS: a blue color develops (presence of ergotamine).

B: Transfer the remaining part of the filtrate to a small evaporating dish, evaporate on a steam bath to dryness, add 1 mL of hydrochloric acid and 100 mg of potassium chlorate, and evaporate. Invert the dish over a vessel containing ammonium hydroxide: the residue acquires a purple color, which disappears upon the addition of 1 N sodium hydroxide (presence of caffeine).

Mobile phase A— Prepare a degassed mixture of water, acetonitrile, and triethylamine (850:150:0.5), and adjust by the dropwise addition of fluorometric grade sulfuric acid to a pH of 3.1 ± 0.1.

Mobile phase B— Prepare a degassed mixture of water, acetonitrile, and triethylamine (1380:620:1), and adjust by the dropwise addition of fluorometric grade sulfuric acid to a pH of 3.1 ± 0.1. Make any necessary adjustments in pH to meet relative retention times, and make other adjustments if necessary (see System Suitability under Chromatography 621).

Solvent mixture— Mix equal volumes of methanol and tartaric acid solution (1 in 100).

Ergotamine tartrate standard solution— Using an accurately weighed amount of USP Ergotamine Tartrate RS, prepare a solution in Solvent mixture having a known concentration of about 40 µg per mL.

Mixed standard preparation— Into a 10-mL volumetric flask pipet 5 mL of Ergotamine tartrate standard solution, and add about 10 mg of USP Caffeine RS, accurately weighed. Dilute with Solvent mixture to volume, and mix to obtain a solution having known concentrations of about 20 µg of USP Ergotamine Tartrate RS per mL and about 1 mg of USP Caffeine RS per mL.

System suitability preparation— Pipet 5 mL of the Mixed standard preparation and 1 mL of a solution containing about 20 µg of USP Ergotaminine RS per mL into a 10-mL volumetric flask. Dilute with Solvent mixture to volume, and mix.

Assay preparation— Weigh not fewer than 20 Suppositories, and grind to a fine mesh. Transfer a portion of the ground mass, equivalent to about 2 mg of ergotamine tartrate, to a suitable glass-stoppered flask. Add 100.0 mL of Solvent mixture, insert the stopper in the flask, and place it in a water bath maintained at 40. Shake vigorously for 5 minutes, or longer if necessary, until the specimen is completely melted. Sonicate for 30 minutes, and transfer to a freezer for 45 minutes. Pass through a 0.7-µm glass fiber filter, discarding the first 5 to 10 mL of the filtrate.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 4.6-mm × 25-cm column that contains packing L7. The chromatograph is equipped with a 244-nm detector in series with a fluorometric detector operating at an excitation wavelength of 229 nm and an emission wavelength of 435 nm. Equilibrate the system with Mobile phase A. The flow rate is about 2 mL per minute. At 3 minutes after injection of a specimen, or after caffeine has eluted, whichever occurs last, switch to Mobile phase B, and at 23 minutes after the initial injection, return to Mobile phase A. Allow not less than 2 minutes to elapse between injections. Chromatograph the Mixed standard preparation and the System suitability preparation, and record the peak responses as directed for Procedure: the resolution, R, between ergotamine and ergotaminine is not less than 3.0, and the relative standard deviation for replicate injections of the Mixed standard preparation is not more than 2.0%.

Procedure— Separately inject equal volumes (about 10 µL) of the Mixed standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks. The relative retention times are about 4 for ergotamine, 4.5 for ergotaminine, and 1.0 for caffeine. Calculate the quantity, in mg, of caffeine (C8H10N4O2) in the portion of Suppositories taken by the formula: in which C is the concentration, in mg per mL, of USP Caffeine RS in the Mixed standard preparation; and rU and rS are the peak responses obtained from the Assay preparation and the Mixed standard preparation, respectively. Calculate the quantity, in mg, of ergotamine tartrate [(C33H35N5O5)2·C4H6O6] in the portion of Suppositories taken by the formula: in which C is the concentration, in µg per mL, of USP Ergotamine Tartrate RS in the Mixed standard preparation; and IU and IS are the fluorometric responses obtained from the Assay preparation and the Mixed standard preparation, respectively.