Where external examination of containers, markings, and labels indicates that the batch can be considered to be homogeneous, take individual samples from the number of randomly selected containers indicated below. Where the batch cannot be considered to be homogeneous, divide it into sub-batches that are as homogeneous as possible, then sample each one as a homogeneous batch.

(Round calculated “n” to next highest whole number.)

Samples are taken from the upper, middle, and lower sections of each container. If the crude material consists of component parts which are 1 cm or less in any dimension, and in the case of all powdered or ground materials, withdraw the sample by means of a sampling device that removes a core from the top to the bottom of the container, not less than two cores being taken in opposite directions. For materials with component parts over 1 cm in any dimension, withdraw samples by hand. In the case of large bales or packs, samples should be taken from a depth of 10 cm because the moisture content of the surface layer may be different from that of the inner layers.

Prepare the gross sample by combining and mixing the individual samples taken from each opened container, taking care not to increase the degree of fragmentation or significantly affect the moisture content.

Prepare the laboratory sample by repeated quartering of the gross sample.

The laboratory sample should be of a size sufficient for performing all the necessary tests.

Unless otherwise directed in the individual monograph or test procedure below, prepare the test sample as follows:

Decrease the size of the laboratory sample by quartering, taking care that each withdrawn portion remains representative. In the case of unground or unpowdered drugs, grind the withdrawn sample so that it will pass through a No. 20 standard-mesh sieve, and mix the resulting powder well. If the material cannot be ground, reduce it to as fine a state as possible, mix by rolling it on paper or sampling cloth, spread it out in a thin layer and withdraw the portion for analysis.

Roots, rhizomes, bark, and herbs	500 g
Leaves, flowers, seeds, and fruit	250 g
Cut vegetable drugs (average weight of the pieces is less than 0.5 g)	50 g

Accurately weigh a quantity of the Test Sample, representing 2 to 4 g of the air-dried material, in a tared crucible, and incinerate, gently at first, and gradually increase the temperature to 675 ± 25, until free from carbon, and determine the weight of the ash. If a carbon-free ash cannot be obtained in this way, extract the charred mass with hot water, collect the insoluble residue on an ashless filter paper, incinerate the residue and filter paper until the ash is white or nearly so, then add the filtrate, evaporate it to dryness, and heat the whole to a temperature of 675 ± 25. If a carbon-free ash cannot be obtained in this way, cool the crucible, add 15 mL of alcohol, break up the ash with a glass rod, burn off the alcohol, and again heat the whole to a temperature of 675 ± 25. Cool in a desiccator, weigh the ash, and calculate the percentage of total ash from the weight of the drug taken.

Boil the ash obtained as directed under Total Ash, above, with 25 mL of 3 N hydrochloric acid for 5 minutes, collect the insoluble matter on a tared filtering crucible or ashless filter, wash with hot water, ignite, and weigh. Determine the percentage of acid-insoluble ash calculated from the weight of drug taken.

Boil the ash obtained as directed for Total Ash with 25 mL of water for 5 minutes. Collect the insoluble matter in a sintered-glass crucible or on an ashless filter paper. Wash with hot water, and ignite for 15 minutes at a temperature not exceeding 450. Subtract the weight of this residue, in mg, obtained under Total Ash, and calculate the percentage of water-soluble ash with reference to the weight of sample as determined under Total Ash.

Exhaust a weighed quantity of the Test Sample, representing about 2 g of the drug, with ether. Add 200 mL of boiling dilute sulfuric acid (1 in 78) to the ether-exhausted marc, in a 500-mL flask, and connect the flask to a reflux condenser. Reflux the mixture for 30 minutes, accurately timed, then filter through a linen or hardened-paper filter, and wash the residue on the filter with boiling water until the effluent washing is no longer acid. Rinse the residue back into the flask with 200 mL of boiling sodium hydroxide solution, adjusted to 1.25 percent by titration and free from sodium carbonate. Again reflux the mixture for 30 minutes, accurately timed, then rapidly filter through a tared filter, wash the residue with boiling water until the last washing is neutral, and dry it at 110 to constant weight. Incinerate the dried residue, ignite to constant weight, cool in a desiccator, and weigh the ash: the difference between the weight obtained by drying at 110 and that of the ash represents the weight of the crude fiber.

Malt Extract— Use clean new barley malt of known efficacy, and grind just before use. Prepare malt extract just prior to use. For every 80 mL of malt extract needed, digest 5 g of ground malt with 100 mL of water at room temperature for 2 hours. [note—If an electric mixer is used, stir the mixture for 20 minutes.] Filter to obtain a clear extract, filtering again, if necessary, and mix the infusion well.

Test Solution— Extract about 5 g of the finely ground test specimen with five 10-mL portions of ether, using a filter that will completely retain the smallest starch granule. Allow the ether to evaporate from the residue, and wash with 250 mL of aqueous alcohol solution (10 in 100). Carefully wash the residue from the paper into a 500-mL beaker with about 100 mL of water. Heat to about 60 (avoiding, if possible, gelatinizing starch), and allow to stand for about 1 hour, stirring frequently to effect complete solution of sugars. Transfer to a wide-mouth bottle, rinse the beaker with a little warm water, and cool. Add an equal volume of alcohol, mix, and allow to stand for not less than 1 hour.

General Procedure— Transfer 200 mL of the Test Solution to a flask fitted with a reflux condenser, add 20 mL of hydrochloric acid, and heat in a boiling water bath for 2½ hours. Cool, nearly neutralize with sodium hydroxide TS, complete neutralization with sodium carbonate TS, dilute with water to 500 mL, mix, and filter. The volume of aliquot taken depends on the starch content of the specimen under test (see Table 1). The aliquot should contain between 100 and 200 mg of dextrose. Transfer 50 mL of the filtrate to a 400-mL alkali-resistant glass beaker, add 50 mL of alkaline cupric tartarate TS, cover the beaker with a water glass, and heat. Adjust the flame in the burner so that the contents of the flask begin to boil in 4 minutes and continue boiling for exactly 2 minutes. Filter the hot solution at once through a sintered-glass filter. Wash the precipitate of cuprous oxide thoroughly with water at about 60, then with 10 mL of alcohol, and finally with 10 mL of ether.

method 1b—

Glucoamylase Solution— Prepare a solution of glucoamylase in water containing 30 International Units (IU) per mL. Use glucoamylase obtained preferably from Rhizopus delemar. The total glucoamylase activity of the test specimen being used should be not less than 150 IU.

Acetate Buffer Solution— Dissolve 16.4 g of sodium acetate in 100 mL of water, add 12.0 mL of glacial acetic acid, and mix. The pH of this solution is 4.8.

Phosphate Buffer— Dissolve 3.63 g of tris (hydroxymethyl) aminomethane and 5.0 g of monobasic sodium phosphate in 50.0 mL of water. At 37, adjust with phosphoric acid to a pH of 7.0, dilute with water to 100.0 mL, and mix. [note—The pH of the buffer medium is sensitive to temperature and should be adjusted to the desired pH at the temperature to be used during incubation.]

Enzyme Solution— Dissolve 30 mg of glucose oxidase (Type II from Aspergillus niger), 3 mg of peroxidase (Type I from horseradish), and 10 mg of potassium ferrocyanide in 100 mL of Phosphate Buffer. [note—This mixture can be stored in a refrigerator for up to 10 days.]

18 N Sulfuric Acid— Add slowly, while stirring, 54 mL of sulfuric acid to 102 mL of water, allow to cool to 25, and mix.

Standard Solutions— Dissolve an accurately weighed quantity of USP Dextrose RS in water to obtain a solution containing 1.0 mg of USP Dextrose RS per mL. Quantitatively dilute a known volume of this solution with water to obtain Standard Solutions A, B, C, D, and E, having known concentrations of 10, 20, 25, 40, and 50 µg per mL of USP Dextrose RS, respectively. [note—Allow 4 hours for complete mutarotation before use.]

Test Solutions— Extract about 5 g of finely ground test specimen with five 25-mL portions of 80% alcohol, and filter. Remove all the alcohol from the residue by drying in an air oven at 105 for about 8 hours. [Note 1—Any traces of alcohol remaining in the residue will inhibit glucoamylase.] Cool, and transfer the flask containing the dried test specimen to a desiccator. Transfer about 1 g, accurately weighed, of the test specimen to a previously tared flask, add 25 mL of water, and adjust with phosphoric acid to a pH between 5.0 and 7.0, if necessary. Boil the suspension for about 3 minutes, transfer the flask to an autoclave, and heat to 135 for 2 hours. Remove the flask from the autoclave, maintain the temperature near 55, and add 2.5 mL of Acetate Buffer Solution and sufficient water to adjust the total weight of the solution to 45 ± 1 g. Immerse the flask in a water bath maintained at 55 ± 1, and add 5 mL of Glucoamylase Solution. Continuously swirl the flask for 2 hours to effect hydrolysis, filter through filter paper into a 250-mL volumetric flask, wash quantitatively with water, and collect all the washings in the flask. Dilute the contents of the flask with water to volume, and mix. Transfer 1 mL of an aliquot containing 20 to 60 µg of d-glucose to each of five test tubes. [Note 2—In order to obtain the range of concentration of glucose in the hydrolysate, quantitatively dilute, if necessary, with water to volume.] Add 2 mL of Enzyme Solution to each of the five test tubes, and place the test tubes in the dark at 37 ± 1 for exactly 30 minutes to develop the color. At the end of 30 minutes, add 2 mL of 18 N Sulfuric Acid to each of the test tubes to stop the reaction, and mix.

Control Solution— Transfer an accurately weighed quantity of about 0.4 g of starch to a previously tared flask and proceed as directed under Test Solutions beginning with “add 25 mL of water and, adjust the pH with phosphoric acid.”

Procedure— Concomitantly determine the absorbances of the Standard Solutions and the Test Solutions at the wavelength of maximum absorbance at about 540 nm, with a suitable spectrophotometer, using the Control Solution as the blank to set the instrument. Plot the absorbance values of the Standard Solutions versus concentration, in µg per mL, of dextrose, and draw the straight line best fitting the five plotted points. From the graph so obtained, determine the concentration, C, in µg per mL, of dextrose in each of the Test Solutions, calculate the average concentration, in µg per mL, of the solution under test. The percentage of starch content in the weight of the test specimen taken by the equation is calculated by the formula: in which E is the weight, in g, of the test specimen taken; V0 is the volume, in mL, of the aliquot taken from the 250-mL volumetric flask; W is the percentage of dry weight of the test specimen; and V1 is the volume, in mL, if extra dilution is done (see Note 2 under Test Solutions). [note—V0 is 1.0 when no extra dilution is done.]

Set up a round-bottom, shortneck, 1-liter flask in a heating mantle set over a magnetic stirrer. Insert an egg-shaped stirring bar magnet in the flask, and attach a cold-finger condenser and an appropriate volatile oil trap of the type illustrated.

Coarsely comminute a sufficient quantity of the drug to yield from 1 to 3 mL of volatile oil. Small seeds, fruits, or broken leaves of herbs ordinarily do not need comminution. Very fine powders are to be avoided. If this is not possible, it may be necessary to mix them with purified sawdust or purified sand. Place a suitable quantity of the drug, accurately weighed, in the flask, and fill it one-half with water. Attach the condenser and the proper separator. Boil the contents of the flask, using a suitable amount of heat to maintain gentle boiling for 2 hours, or until the volatile oil has been completely separated from the drug and no longer collects in the graduated tube of the separator.

If a proper quantity of the volatile oil has been obtained in the graduated tube of the separator, it can be read to tenths of 1 mL, and the volume of volatile oil from each 100 g of drug can be calculated from the weight of the drug taken. The graduations on the separator “for oils heavier than water” are so placed that oil remains below the aqueous condensate that automatically flows back into the flask.

For unground or unpowdered drugs, prepare about 10 g of the Laboratory Sample by cutting, granulating, or shredding, so that the parts are about 3 mm in thickness. Seeds or fruits smaller than 3 mm should be cracked. Avoid the use of high-speed mills in preparing the sample, and exercise care that no appreciable amount of moisture is lost during the preparation and that the portion taken is representative of the Laboratory Sample. Determine the water content as directed for Procedure for Articles of Botanical Origin in the Method III (Gravimetric) under Water Determination 921.

Table 3

If the article is intended for the preparation of extracts, tinctures, or other pharmaceutical forms whose preparation method modifies the content of pesticides in the finished product, calculate the limits by the formula:

in which E is the extraction factor of the preparation method, determined experimentally; and A, M, and B are as defined above.

note—Conduct tests without delay to avoid possible degradation of the residues. If this is not possible, store the samples in hermetic containers suitable for food contact, at a temperature below 0, and protected from light.

Table 4

Quantitative Analysis of Organophosphorus Insecticides—

Test Solution— Concentrate Solution B almost to dryness, with the aid of a stream of helium, dilute with toluene to 100 µL, and mix.

Standard Solution— Prepare at least three solutions in toluene containing each of the pesticides of interest and carbophenothion at concentrations suitable for plotting a calibration curve.

Chromatographic System— The gas chromatograph is equipped with an alkali flame-ionization detector or a flame-photometric detector and a 0.32-mm × 30-m fused silica column coated with a 0.25-µm layer of phase G1. Hydrogen is used as the carrier gas. Other gases, such as helium or nitrogen, may also be used. The injection port temperature is maintained at 250, and the detector is maintained at 275. The column temperature is maintained at 80 for 1 minute, then increased to 150 at a rate of 30 per minute, maintained at 150 for 3 minutes, then increased to 280 at a rate of 4 per minute, and maintained at this temperature for 1 minute. Use carbophenothion as the internal standard. [Note—If necessary, use a second internal standard to identify any possible interference with the peak corresponding to carbophenothion.] Inject the chosen volume of each solution, record the chromatograms, and measure the peak responses: the relative retention times are approximately those listed in Table 5. Calculate the content of each pesticide from the peak areas and the concentrations of the solution.

Test Solution— Concentrate Solution C almost to dryness, with the aid of a stream of helium or oxygen-free nitrogen, dilute with toluene to 500 µL, and mix.

Standard Solution— Prepare at least three solutions in toluene containing each of the pesticides of interest and carbophenothion at concentrations suitable for plotting a calibration curve.

Chromatographic System— The gas chromatograph is equipped with an electron-capture detector, a device allowing direct on-column cold injection, and a 0.32-mm × 30-m fused silica column coated with a 0.25-µm layer of phase G1. Hydrogen is used as the carrier gas. Other gases, such as helium or nitrogen, may also be used. The injection port temperature is maintained at 275, and the detector is maintained at 300. The column temperature is maintained at 80 for 1 minute, then increased to 150 at a rate of 30 per minute, maintained at 150 for 3 minutes, then increased to 280 at a rate of 4 per minute, and maintained at this temperature for 1 minute. Use carbophenothion as the internal standard. [note—If necessary, use a second internal standard to identify any possible interference with the peak corresponding to carbophenothion.] Inject the chosen volume of each solution, record the chromatograms, and measure the peak responses: the relative retention times are approximately those listed in Table 6. Calculate the content of each pesticide from the peak areas and the concentrations of the solutions.