Unground Digitalis— This occurs as more or less crumpled or broken leaves.The leaf blades are ovate,oblong-ovate to ovate-lanceolate,mostly 10to 35cm in length and 4cm to 11cm in width and contracted into a winged petiole.The apex is obtuse;the margin irregularly crenate or serrate;the lower surface densely pubescent,the upper surface wrinkled and finely hairy.The venation is conspicuously reticulate,the mid-rib and principal veins broad and flat,and the lower veins are continued into the wings of the petiole.The color of the upper surface is dark green,of the lower surface grayish from the dense pubescence,the larger veins often purplish.The odor is slight when dry,peculiar and characteristic when moistened.

Histology— Digitalis shows an upper epidermis whose cells possess slightly wavy anticlinal walls,numerous hairs,and no stomata;a lower epidermis with wavy anticlinal walls,numerous oval stomata,and many hairs,and frequently not attached over irregular areas to the cell layer within,especially near the veins;a broad chlorenchyma of a single layer of short palisade cells and several layers of spongy parenchyma;and numerous vascular bundles in the larger veins and petioles,separated by vascular rays one cell in width.On the apex of each marginal tooth one or two water stomata occur.

Ground Digitalis— This is dark green in color.Present are chiefly numerous irregular fragments of epidermis and chlorenchyma;nonglandular hairs that are frequently curved or crooked,up to 500µm in length,uniseriate,two-to eight-celled,some of the cells collapsed so that the planes of adjoining cells may be at right angles,the terminal cell pointed or rounded;few,small glandular hairs,usually with a one-or two-celled stalk,and a one-or two-celled head;fragments of veins and petioles with annular,reticulate,spiral and simple pitted vessels and tracheids.Calcium oxalate is absent.

Standard preparation— Weigh the contents of 1container of USP Digitalis RSto the nearest mg,either in the original container or in a weighing bottle,and transfer to a dry,hard-glass,glass-stoppered container or centrifuge tube of at least 50-mLcapacity.Complete the weighing within 5minutes after opening the ampul.Add a menstruum consisting of 4volumes of alcohol and 1volume of water so that the total volume of menstruum added corresponds to 10mLfor each g of powder.Insert the stopper,the upper third of which is greased lightly with petrolatum.Shake the mixture for 24±2hours at 25±5by mechanical means,which continuously brings the solid material into fresh contact with the liquid phase.Immediately thereafter transfer,if necessary,to a centrifuge tube,centrifuge,and decant the supernatant tincture into a dry,hard-glass bottle having a tight closure.Preserve under refrigeration,and use within 30days.

Assay preparation— Transfer about 5g of Digitalis,reduced to a fine powder and accurately weighed,to a hard-glass,glass-stoppered container or centrifuge tube of at least 50-mLcapacity.Proceed as directed under Standard preparation,beginning with “Add a menstruum.”Preserve under refrigeration,and use within 30days.

Pigeons— Employ adult pigeons free from gross evidence of disease or emaciation,and of such weight that the heaviest weighs less than twice the weight of the lightest.Divide the pigeons into groups as nearly alike as practicable with respect to breed and weight so that the average weight of the group assigned by random choice to the Standard preparationshall not differ by more than 30%from the average weight of the group assigned to the preparation to be assayed.Withhold food but not water during the period 16to 28hours prior to use.Preparatory to injection,lightly anesthetize the pigeon with ether,and immobilize it;expose an alar vein,and cannulate with a suitable cannula.Maintain the anesthesia during cannulation and throughout the subsequent injection period at such a level that pain is absent,the pupillary and corneal reflexes are present,and the voluntary musculature is not relaxed beyond permitting the pigeon to make some voluntary movement occasionally.

Preparation of test dilutions— On the day of the assay,dilute portions of the Standard preparationand of the preparation to be assayed (Assay preparation)with isotonic sodium chloride solution in such a way that the estimated fatal dose of each dilution will be 15mLper kg of body weight.

Injection of test dilutions— Arrange to inject the appropriate test dilution by suitable means such as a small-bore buret calibrated to 0.05mL.Start the injection after ensuring the absence of air bubbles from the injection apparatus,by infusing,within a few seconds,a volume of the test dilution equivalent to 1mLper kg of body weight.Repeat this dose at 5-minute intervals thereafter until the pigeon dies of cardiac arrest.

Calculation of potency— Tabulate and average the number of doses of the Standard preparation,designating the average bar(z)S,and likewise obtain the corresponding average,bar(z)U,for the Assay preparation.Compute the potency in USP Digitalis Units per mL(i.e.,per 100mg)of the Assay preparationas: where Requals vS/vU,in which vSis the number of USP Digitalis Units per mLof Standard preparationdilution,and vUis the volume,in mL,of Assay preparationper mLof dilution.Compute the log confidence interval,L(see Equation (31)under Confidence Intervals for Individual Assaysin Design and Analysis of Biological Assays á111ñ).If Lexceeds 0.30,repeat the assay or inject more pigeons with one or both preparations until the confidence interval is 0.30or less.