A: To one part of the filtrate add 10mLof 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 1mLof hydrochloric acid and 100mg 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 a solution of fixed alkali (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 pHof 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 pHof 3.1±0.1.Make any necessary adjustments in pHto meet relative retention times,and make other adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

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

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.

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

System suitability preparation— Pipet 5mLof the Mixed standard preparationand 1mLof a solution containing about 20µg of USP Ergotaminine RSper mLinto a 10-mLvolumetric flask.Dilute with Solvent mixtureto volume,and mix.

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

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 244-nm detector in series with a fluorometric detector operating at an excitation wavelength of 229nm 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 23minutes after the initial injection,return to Mobile phase A.Allow not less than 2minutes to elapse between injections.Chromatograph the Mixed standard preparationand the System suitability preparation,and record the peak responses as directed under Procedure: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 10µL)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 4for ergotamine,4.5for ergotaminine,and 1.0for caffeine.Calculate the quantity,in mg,of caffeine (C8H10N4O2)in the portion of Suppositories 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 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 Suppositories 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.