A: Mix one of the residues with 5 mL of tartaric acid solution (1 in 100), and add 10 mL of p-dimethylaminobenzaldehyde TS: a blue color develops (presence of ergotamine).

B: To the other residue add 1 mL of hydrochloric acid and 100 mg of potassium chlorate, and evaporate on a steam bath to dryness. 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).

Medium: tartaric acid solution (1 in 100); 900 mL.

Apparatus 2: 75 rpm.

Time: 30 minutes.

Standard preparation— Using suitable quantities of USP Ergotamine Tartrate RS and USP Caffeine RS, accurately weighed, prepare a solution, using the Medium, having known concentrations of about 1 µg of ergotamine tartrate and 100 µg of caffeine in each mL.

Procedure— Measure the amount of caffeine in solution on filtered portions of the Medium, suitably diluted, at the wavelength of maximum absorbance at about 273 nm, with a suitable spectrophotometer, in comparison with the Standard preparation. Similarly, measure the amount of ergotamine tartrate in solution on filtered portions of the Medium, suitably diluted, in a suitable fluorometer, using 327 nm as the excitation wavelength and 427 nm as the emission wavelength, in comparison with the Standard preparation.

Tolerances— Not less than 70% (Q) of the labeled amount of ergotamine tartrate ((C33H35N5O5)2·C4H6O6) and not less than 75% (Q) of the labeled amount of caffeine (C8H10N4O2) is dissolved in 30 minutes.

(Official January 1, 2007)

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 2.7 ± 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 2.7 ± 0.1. Make necessary adjustments in pH with sodium hydroxide solution (1 in 20) or with fluorometric grade sulfuric acid to meet relative retention times, and make other adjustments if necessary (see System Suitability under Chromatography 621).

Solvent mixture— Transfer 10 g of tartaric acid to a 1-L volumetric flask, add 500 mL of water, and mix with shaking. Add 330 mL of alcohol, and mix. Dilute with water to volume, and mix. Use a freshly prepared mixture.

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.

Caffeine standard solution— Using an accurately weighed amount of USP Caffeine RS, prepare a solution in Solvent mixture having a known concentration of about 4 mg per mL.

Mixed standard preparation— Pipet 10 mL of Ergotamine tartrate standard solution and 10 mL of Caffeine standard solution into a 100-mL volumetric flask. Dilute with Solvent mixture to volume, and mix to obtain a solution having known concentrations of 4 µg of USP Ergotamine Tartrate RS per mL and 0.4 mg of USP Caffeine RS per mL.

Assay preparation— Weigh and finely powder not fewer than 20 Tablets. Transfer an accurately weighed portion of the powder, equivalent to about 10 mg of ergotamine tartrate, to a 250-mL volumetric flask. Add 150 mL of Solvent mixture and 20 drops of benzalkonium chloride solution (1 in 2). Shake by mechanical means for 45 minutes. [NOTE—Two to 3 mL of methanol may be added, if necessary, to break up bubbles that form during shaking.] Dilute with Solvent mixture to volume, and mix. Filter through a 0.5-µm membrane, discarding the first 20 mL of the filtrate. Pipet 5 mL of the subsequent filtrate into a 50-mL volumetric flask, dilute with Solvent mixture to volume, and mix.

System suitability preparation— Pipet 20 mL of Caffeine standard solution, 20 mL of Ergotamine tartrate standard solution, and 4 mL of a solution containing 20 µg of USP Ergotaminine RS per mL of Solvent mixture, into a 200-mL volumetric flask. Dilute with Solvent mixture to volume, and mix.

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 254-nm detector in series with a fluorometric detector operating at an excitation wavelength of 325 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 18 minutes after initial injection, return to Mobile phase A. Wait not less than 2 minutes between injections. Chromatograph the Mixed standard preparation and the System suitability preparation, and record the peak responses as directed for Procedure: the tailing factor for the ergotamine peak is not more than 2.0; 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 20 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 3.5 for ergotamine, 4 for ergotaminine, and 1.0 for caffeine. Calculate the quantity, in mg, of caffeine (C8H10N4O2) in the portion of Tablets 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 254-nm 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 Tablets 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.