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

B: To the other residue add 1mLof hydrochloric acid and 100mg 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 a solution of fixed alkali (presence of caffeine).

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

Apparatus 2: 75rpm.

Time: 30minutes.

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

Procedure— Measure the amount of caffeine in solution in filtered portions of the Medium,suitably diluted,at the wavelength of maximum absorbance at about 273nm,with a suitable spectrophotometer,in comparison with the Standard preparation.Similarly,measure the amount of ergotamine tartrate in solution in filtered portions of the Medium,suitably diluted,in a suitable fluorometer,using 327nm as the excitation wavelength and 427nm 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 30minutes.

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 pHof 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 pHof 2.7±0.1.Make necessary adjustments in pHwith sodium hydroxide solution (1in 20)or with fluorometric grade sulfuric acid to meet relative retention times,and make other adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Solvent mixture— Transfer 10g of tartaric acid to a 1-liter volumetric flask,add 500mLof water,and mix with shaking.Add 330mLof 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 mixturehaving a known concentration of about 40µg per mL.

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

Mixed standard preparation— Pipet 10mLof Ergotamine tartrate standard solutionand 10mLof Caffeine standard solutioninto a 100-mLvolumetric flask.Dilute with Solvent mixtureto volume,and mix to obtain a solution having known concentrations of 4µg of USP Ergotamine Tartrate RSper mLand 0.4mg of USP Caffeine RSper mL.

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

System suitability preparation— Pipet 20mLof Caffeine standard solution,20mLof Ergotamine tartrate standard solution,and 4mLof a solution containing 20µg of USP Ergotaminine RSper mLof Solvent mixture,into a 200-mLvolumetric flask.Dilute with Solvent mixtureto volume,and mix.

Chromatographic system (see Chromatography á621ñ)—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 325nm and an emission wavelength of 435nm.Equilibrate the system with Mobile phase A.The flow rate is about 2mLper minute.At 3minutes after injection of a specimen,or after caffeine has eluted,whichever occurs last,switch to Mobile phase B,and at 18minutes after initial injection,return to Mobile phase A.Wait not less than 2minutes between injections.Chromatograph the Mixed standard preparationand the System suitability preparation,and record the peak responses as directed under Procedure:the tailing factor for the ergotamine peak is not more than 2.0,the resolution,R,between the ergotamine and ergotaminine peaks is not less than 3.0,and the relative standard deviation for replicate injections of the Mixed standard preparationis not more than 2.0%.

Procedure— Separately inject equal volumes (about 20µL)of the Mixed standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.The relative retention times are about 3.5for ergotamine,4for ergotaminine,and 1.0for caffeine.Calculate the quantity,in mg,of caffeine (C8H10N4O2)in the portion of Tablets taken by the formula: in which Cis the concentration,in mg per mL,of USP Caffeine RSin the Mixed standard preparation,and rUand rSare the 254-nm peak responses obtained from the Assay preparationand 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 Cis the concentration,in µg per mL,of USP Ergotamine Tartrate RSin the Mixed standard preparation,and IUand ISare the fluorometric responses obtained from the Assay preparationand the Mixed standard preparation,respectively.