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á561ñ ARTICLES OF BOTANICAL ORIGIN

SAMPLING

To reduce the effect of sampling bias in qualitative and quantitative results, it is necessary to ensure that the composition of
the sample used be representative of the batch of drugs being examined. The following sampling procedures are the minimum
considered applicable to vegetable drugs. Some articles, or some tests, may require more rigorous procedures involving more
containers being sampled or more samples per container.

Gross Sample
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, and then sample
each one as a homogeneous batch. It is recommended to include samples from the first, middle, and last containers where the
No. of Containers in Batch (N) is 11 or more, and each container in the batch is numbered or lettered in order.

No. of Containers in Batch No. of Containers to Be Sampled

(N) (n)

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1–10 All

11–19 11

>19 n = 10 + (N/10)
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(Rounding was calculated as “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 that 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, NLT two cores being taken from
different angles. For materials with component parts >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
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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.
For articles in containers holding <1 kg, mix the contents, and withdraw a quantity sufficient for the tests. For articles in
containers holding 1–5 kg, withdraw equal portions from the upper, middle, and lower parts of the container, each of the
samples being sufficient to carry out the tests. Thoroughly mix the samples, and withdraw an amount sufficient to carry out
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the tests. For containers holding more than 5 kg, withdraw three samples, each weighing NLT 250 g, from the upper, middle,
and lower parts of the container. Thoroughly mix the samples, and withdraw a portion sufficient to carry out the tests.

Laboratory Sample
Prepare the Laboratory Sample by repeated quartering of the gross sample.
[NOTE—Quartering consists of placing the sample, adequately mixed, as an even and square-shaped heap and dividing it
diagonally into four equal parts. The two opposite parts are then taken and carefully mixed. The process is repeated as necessary
until the required quantity is obtained.]
The Laboratory Sample should be of a size sufficient for performing all the necessary tests.

Test Sample
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.

METHODS OF ANALYSIS

Foreign Organic Matter

TEST SAMPLE
Unless otherwise specified in the individual monograph, weigh the following quantities of the Laboratory Sample, taking care
that the withdrawn portion is representative (quartering if necessary).

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Roots, rhizomes, bark, and herbs 500 g

Leaves, flowers, seeds, and fruit 250 g

Cut vegetable drugs (average weight of the pieces is <0.5 g) 50 g

Spread the sample out in a thin layer, and separate the foreign organic matter by hand as completely as possible. Weigh it,
and determine the percentage of foreign organic matter in the weight of drug taken.

Total Ash
Accurately weigh a quantity of the Test Sample, representing 2–4 g of the 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.
[NOTE—For botanicals, a material in conformance with the Loss on Drying test in individual monographs should be used.] 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.

Acid-Insoluble Ash

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Boil the ash obtained as directed in Total Ash with 25 mL of 3 N hydrochloric acid for 5 min, 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.

Water-Soluble Ash
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Boil the ash obtained as directed in Total Ash with 25 mL of water for 5 min. Collect the insoluble matter in a sintered-glass
crucible or on an ashless filter paper. Wash with hot water, and ignite for 15 min at a temperature not exceeding 450°. Subtract
the weight of this residue, in mg, obtained in Total Ash, and calculate the percentage of water-soluble ash with reference to
the weight of sample as determined in Total Ash.
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Alcohol-Soluble Extractives
METHOD 1 (HOT EXTRACTION METHOD)
Transfer about 4 g of air-dried, coarsely powdered material, accurately weighed, to a glass-stoppered conical flask. Add
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100 mL of alcohol, and weigh the flask. Shake, and allow to stand for 1 h. Attach a reflux condenser to the flask, boil gently for
1 h, cool, and weigh. Readjust to the original weight with alcohol. Shake, and filter rapidly through a dry filter. Transfer 25 mL
of the filtrate to a tared flat-bottomed dish, and evaporate on a water bath to dryness. Dry at 105° for 6 h, cool in a desiccator
for 30 min, and weigh without delay. Calculate the content, in mg/g, of alcohol-extractable matter in the test specimen.

METHOD 2 (COLD EXTRACTION METHOD)

Transfer about 4 g of air-dried, coarsely powdered material, accurately weighed, to a glass-stoppered conical flask. Add
100 mL of alcohol, insert a stopper into the flask, and macerate for 24 h, shaking frequently during the first 8 h, and then
allowing to stand. Filter rapidly, taking precautions against loss of alcohol. Evaporate 25 mL of the filtrate to dryness in a tared,
flat-bottomed, shallow dish, and dry at 105° to constant weight. Calculate the content, in mg/g, of alcohol-extractable matter
in the test specimen.

Water-Soluble Extractives
METHOD 1 (HOT EXTRACTION METHOD)
Proceed as directed in Method 1 (Hot Extraction Method) in Alcohol-Soluble Extractives, except use water in place of alcohol.

METHOD 2 (COLD EXTRACTION METHOD)

Proceed as directed in Method 2 (Cold Extraction Method) in Alcohol-Soluble Extractives, except use water in place of alcohol.

Crude Fiber
Exhaust a weighed quantity of the Test Sample, representing about 2 g of the material, 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 min, accurately timed, then pass 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% by titration and free from sodium carbonate. Again reflux the mixture for 30 min,

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accurately timed, then rapidly pass 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.
[NOTE—The boiling with acid and alkali should continue for 30 min, accurately timed, from the time that the liquid (which
is cooled below the boiling point by being added to the cold flask) again boils. After the solution has been brought to boiling,
the heat should be turned low enough just to maintain boiling. During the boiling, the flask should be gently rotated from time
to time to wash down any particles that may adhere to the walls of the flask. A slow current of air introduced into the flask
during the boiling operation aids in preventing excessive frothing.]

Starch Content
METHOD 1
The following is a general procedure for all reducing sugars and may be used to determine the starch content in botanical
articles.
Malt extract: Use clean new barley malt of known efficacy, and grind just before use. Prepare malt extract just before 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 h. [NOTE—If
an electric mixer is used, stir the mixture for 20 min.] 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

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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 h, 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 NLT 1 h.
Centrifuge until the precipitate is closely packed on the bottom of the bottle, and decant the supernatant. Wash the
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precipitate with successive 50-mL portions of alcohol solution (50 in 100) by centrifuging and decanting through a suitable
filter until the washings are sugar free. [NOTE—To test for the presence of sugar, transfer a few drops of the washings to a test
tube, and add 3 or 4 drops of a 20% solution of 1-naphthol in alcohol prepared by dissolving 200 mg of 1-naphthol in 1 mL
of alcohol and 2 mL of water. Shake the test tube well to allow uniform mixing, allow 2–4 mL of sulfuric acid to flow down the
sides of the test tube, and hold the test tube upright. If sugar is present, the interface of the two liquids is colored faint to deep
violet, and on shaking, the whole solution becomes blue-violet.]
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Transfer the residue from the bottle and hardened filter to a beaker with about 50 mL of water. Immerse the beaker in boiling
water, and stir constantly for 15 min or until all of the starch is gelatinized. Cool the beaker to 55°, add 20 mL of Malt extract,
and hold at this temperature for 1 h. Heat again to boiling for a few min, cool to 55°, add 20 mL of Malt extract, and hold at
this temperature for 1 h or until the residue when treated with iodine TS shows no blue tinge upon microscopic examination.
Cool, dilute with water to 250 mL, and filter.
General procedure: Transfer 200 mL of the Test solution to a flask fitted with a reflux condenser, add 20 mL of hydrochloric acid,
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and heat in a boiling water bath for 2.5 h. 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 tartrate 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 min, and continue boiling for exactly
2 min. Pass 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.

Table 1. Determination of the Optimum Aliquot

Expected Starch Content Aliquot
(%) (mL)
60 25

50 35

40 50

30 50

20 50

For solutions of reducing sugars of comparatively high purity, proceed as directed in Method 1A to determine the amount
of reduced copper obtained by weighing the dried cuprous oxide. For solutions of reducing sugars containing large amounts
of organic impurities, including sucrose, proceed as directed in Method 1B to determine the amount of reduced copper obtained
by titration with sodium thiosulfate.
Method 1A: Dry the precipitate obtained in General procedure for 30 min in an oven at 110 ± 2°, cool to room temperature
in a desiccator, and weigh. Refer to Table 2 to find the quantity of dextrose, in mg, corresponding to the weight of cuprous
oxide found. Determine the percentage of dextrose and then the content of starch by the following formula:

Percentage of dextrose = (wt. of dextrose in mg × 0.1 × 500)/(wt. of sample in g × aliquot in mL)

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Content of starch = % dextrose × 0.9

Table 2. Calculating Dextrose (mg) [applicable when Cuprous Oxide (Cu2O) is weighed directly]
Cuprous Dextrose Cuprous Dextrose Cuprous Dextrose Cuprous Dextrose Cuprous Dextrose Cuprous Dextrose
Oxide (D-Glu- Oxide (D-Glu- Oxide (D-Glu- Oxide (D-Glu- Oxide (D-Glu- Oxide (D-Glu-
(Cu2O) cose) (Cu2O) cose) (Cu2O) cose) (Cu2O) cose) (Cu2O) cose) (Cu2O) cose)
10 4.0 90 38.9 170 75.1 250 112.8 330 152.2 410 193.7

12 4.9 92 39.8 172 76.0 252 113.7 332 153.2 412 194.7

14 5.7 94 40.6 174 76.9 254 114.7 334 154.2 414 195.8

16 6.6 96 41.5 176 77.8 256 115.7 336 155.2 416 196.8

18 7.5 98 42.4 178 78.8 258 116.6 338 156.3 418 197.9

20 8.3 100 43.3 180 79.7 260 117.6 340 157.3 420 199.0

22 9.2 102 44.2 182 80.6 262 118.6 342 158.3 422 200.1

24 10.0 104 45.1 184 81.5 264 119.5 344 159.3 424 201.1
26 10.9 106 46.0 186 82.5 266 120.5 346 160.3 426 202.2

28 11.8 108 46.9 188 83.4 268 121.5 348 161.4 428 203.3

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30 12.6 110 47.8 190 84.3 270 122.5 350 162.4 430 204.4

32 13.5 112 48.7 192 85.3 272 123.4 352 163.4 432 205.5

34 14.3 114 49.6 194

36

38
15.2

16.1
116

118
50.5

51.4
196

198
ci 86.2

87.1

88.1
274

276

278
124.4

125.4

126.4
354

356

358
164.4

165.4

166.5
434

436

438
206.5

207.6

208.7
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40 16.9 120 52.3 200 89.0 280 127.3 360 167.5 440 209.8

42 17.8 122 53.2 202 89.9 282 128.3 362 168.5 442 210.9

44 18.7 124 54.1 204 90.9 284 129.3 364 169.6 444 212.0

46 19.6 126 55.0 206 91.8 286 130.3 366 170.6 446 213.1
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48 20.4 128 55.9 208 92.8 288 131.3 368 171.6 448 214.1

50 21.3 130 56.8 210 93.7 290 132.3 370 172.7 450 215.2

52 22.2 132 57.7 212 94.6 292 133.2 372 173.7 452 216.3

54 23.0 134 58.6 214 95.6 294 134.2 374 174.7 454 217.4

56 23.9 136 59.5 216 96.5 296 135.2 376 175.8 456 218.5

58 24.8 138 60.4 218 97.5 298 136.2 378 176.8 458 219.6

60 25.6 140 61.3 220 98.4 300 137.2 380 177.9 460 220.7

62 26.5 142 62.2 222 99.4 302 138.2 382 178.9 462 221.8

64 27.4 144 63.1 224 100.3 304 139.2 384 180.0 464 222.9

66 28.3 146 64.0 226 101.3 306 140.2 386 181.0 466 224.0

68 29.2 148 65.0 228 102.2 308 141.2 388 182.0 468 225.1

70 30.0 150 65.9 230 103.2 310 142.2 390 183.1 470 226.2

72 30.9 152 66.8 232 104.1 312 143.2 392 184.1 472 227.4

74 31.8 154 67.7 234 105.1 314 144.2 394 185.2 474 228.3

76 32.7 156 68.6 236 106.0 316 145.2 396 186.2 476 229.6

78 33.6 158 69.5 238 107.0 318 146.2 398 187.3 478 230.7

80 34.4 160 70.4 240 108.0 320 147.2 400 188.4 480 231.8

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Table 2. Calculating Dextrose (mg) [applicable when Cuprous Oxide (Cu2O) is weighed directly] (continued)
Cuprous Dextrose Cuprous Dextrose Cuprous Dextrose Cuprous Dextrose Cuprous Dextrose Cuprous Dextrose
Oxide (D-Glu- Oxide (D-Glu- Oxide (D-Glu- Oxide (D-Glu- Oxide (D-Glu- Oxide (D-Glu-
(Cu2O) cose) (Cu2O) cose) (Cu2O) cose) (Cu2O) cose) (Cu2O) cose) (Cu2O) cose)
82 35.3 162 71.4 242 108.9 322 148.2 402 189.4 482 232.9

84 36.2 164 72.3 244 109.9 324 149.2 404 190.5 484 234.1

86 37.1 166 73.2 246 110.8 326 150.2 406 191.5 486 235.2

88 38.0 168 74.1 248 111.8 328 151.2 408 192.6 488 236.3

Method 1B
SODIUM THIOSULFATE SOLUTION: Transfer 3.9 g of sodium thiosulfate, accurately weighed, to a 100-mL volumetric flask,
dissolve in and dilute with water to volume, and mix.
POTASSIUM IODIDE SOLUTION: Dissolve 42 g of potassium iodide in 100 mL of water.
SODIUM ACETATE SOLUTION: Dissolve 5.74 g of sodium acetate in 10 mL of water.
COPPER SOLUTION: Transfer about 0.3 g of pure electrolytic copper, accurately weighed, to a 250-mL flask, add 5 mL of
nitric acid to dissolve the copper, add about 25 mL of water, and boil to expel red fumes. Add about 5 mL of bromine TS, and
boil until the bromine is completely removed. Cool, add 10 mL of Sodium acetate solution followed by 10 mL of Potassium iodide
solution, and titrate with Sodium thiosulfate solution to a light yellow color. Add enough starch TS to produce a marked blue
color, and continue the titration. As the endpoint nears, add 2 g of potassium thiocyanate, and stir until completely dissolved.
Continue titration until the precipitate is completely white. One mL of Sodium thiosulfate solution is equivalent to about 10 mg

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of copper. [NOTE—It is essential that the concentration of Potassium iodide solution be carefully regulated. If the solution contains
less than 320 mg of copper at the completion of titration, add 4.2–5 g of potassium iodide to make a total solution of 100 mL.
If greater amounts of copper are present, add Potassium iodide solution slowly, with constant agitation, from the buret in amounts
proportionately greater.]
PROCEDURE: Wash the precipitated cuprous oxide obtained in General procedure with water, cover this filter with a watch
glass, and dissolve the cuprous oxide with 5 mL of nitric acid directed under the watch glass with a pipette. Collect the filtrate
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in a 250-mL flask, wash the watch glass, and the filter with water. Collect all the washings in the flask. Boil the contents of the
flask to expel red fumes. Add about 5 mL of bromine TS, and boil until the bromine is completely removed. Cool, and proceed
as directed in Copper solution beginning with “add 10 mL of Sodium acetate solution”. From the volume of Sodium thiosulfate
solution consumed, obtain the weight of copper, in mg, and multiply the weight of copper by 1.1259 to obtain the weight, in
mg, of cuprous oxide. From Table 2, find the quantity of dextrose, in mg, corresponding to the weight of cuprous oxide. The
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content of starch is equivalent to the weight, in mg, of dextrose obtained times 0.9. Conduct a blank determination, using
50 mL of alkaline cupric tartrate TS and 50 mL of Malt extract. If the weight of the cuprous oxide so obtained exceeds 0.5 mg,
correct the result of the determination accordingly. [NOTE—The alkaline cupric tartrate TS deteriorates on standing, and the
quantity of cuprous oxide obtained in the blank determination increases.]

METHOD 2
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The following method is specific for dextrose (glucose), and because of its extreme sensitivity it may account for differences
noted between values obtained from the same specimen. Duplicate determinations do not vary more than 2%.
Glucoamylase solution: Prepare a solution of glucoamylase in water containing 30 International Units (IU)/mL. Use
glucoamylase obtained preferably from Rhizopus delemar. The total glucoamylase activity of the test specimen being used should
be NLT 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/mL of USP Dextrose RS. 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/mL of USP Dextrose RS, respectively. [NOTE—Allow
4 h 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 h. [NOTE—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 of 5.0–7.0, if necessary. Boil the suspension for about 3 min, transfer the flask to an autoclave, and heat to 135° for
2 h. 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 h to effect hydrolysis, pass 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 20–60 µg of D-glucose to each of five test tubes.

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[NOTE—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 min to develop the color. At the end of 30 min, 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 in 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/mL, of dextrose, and draw the
straight line best fitting the five plotted points. From the graph, determine the concentration, C, in µg/mL, of dextrose in each
of the Test solutions, and calculate the average concentration, in µg/mL, of the solution under test. The percentage of starch
content in the weight of the test specimen taken is calculated by the formula:

(0.9C/106) × V1 × (250/V0)(100/E)(100/W) = 2.25CV1/V0EW

C = average concentration of dextrose in the Test solutions (µg/mL)

V1 = volume of the Test solution if extra dilution is done (mL) (see Note 2 in Test solutions)
V0 = volume of the aliquot taken from the 250-mL volumetric flask (mL)
E = weight of the test specimen (g)
W = percentage of dry weight of the test specimen

[NOTE—V0 is 1.0 when no extra dilution is done.]

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Volatile Oil Determination
Set up a round-bottom, shortneck, 1-L 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 (see Figure 1)).
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Figure 1. Traps for volatile oil apparatus.

Coarsely comminute a sufficient quantity of the material to yield from 1–3 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 h, or until the volatile oil has been completely separated from the material 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 material 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.

Water Content
For unground or unpowdered materials, 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 in Water Determination
á921ñ, Method III (Gravimetric), Procedure for Articles of Botanical Origin.

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TEST FOR AFLATOXINS

[CAUTION—Aflatoxins are highly dangerous, and extreme care should be exercised in handling aflatoxin materials.]
Where the individual monograph calls for compliance with the limits for aflatoxins, the limits are NMT 5 ppb for
aflatoxin B1 (AFB1) and NMT 20 ppb for the sum of aflatoxins B1 (AFB1), B2 (AFB2), G1 (AFG1), and G2 (AFG2). The extent of
testing may be determined using a risk-based approach that considers the likelihood of contamination. The presence of
unexpected contamination with aflatoxins is to be considered in determining compliance. The following analytical procedures
are provided for determining compliance. Unless otherwise specified in the individual monograph, use Method I. If system
suitability fails, use either Method II or Method III.

Method I
This TLC test is provided to detect the possible presence of AFB1, AFB2, AFG1, and AFG2 in any material of plant origin.

ZINC ACETATE–ALUMINUM CHLORIDE REAGENT

Dissolve 20 g of zinc acetate and 5 g of aluminum chloride in sufficient water to make 100 mL.

SODIUM CHLORIDE SOLUTION

Dissolve 5 g of sodium chloride in 50 mL of water.

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TEST SOLUTION 1
Grind about 200 g of plant material to a fine powder. Transfer about 50 g of the powdered material, accurately weighed,
to a glass-stoppered flask. Add 200 mL of a mixture of methanol and water (17:3). Shake vigorously by mechanical means for
NLT 30 min, and filter. [NOTE—If the solution has interfering plant pigments, proceed as directed for Test Solution 2.] Discard
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the first 50 mL of the filtrate, and collect the next 40-mL portion. Transfer the filtrate to a separatory funnel. Add 40 mL of
Sodium Chloride Solution and 25 mL of solvent hexane, and shake for 1 min. Allow the layers to separate, and transfer the lower
aqueous layer to a second separatory funnel. Extract the aqueous layer in the separatory funnel twice, each time with 25 mL
of methylene chloride, by shaking for 1 min. Allow the layers to separate each time, separate the lower organic layer, and collect
the combined organic layers in a 125-mL conical flask. Evaporate the organic solvent on a water bath. Transfer the remaining
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extract to an appropriate sample tube, and evaporate to dryness on a water bath. Cool the residue. If interferences exist in the
residue, proceed as directed for Cleanup procedure in Test Solution 2. Otherwise, dissolve the residue obtained above in 0.2 mL
of a mixture of chloroform and acetonitrile (9.8: 0.2), and shake by mechanical means if necessary.

TEST SOLUTION 2
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Collect 100 mL of the filtrate from the start of the flow, and transfer to a 250-mL beaker. Add 20 mL of Zinc Acetate–Aluminum
Chloride Reagent and 80 mL of water. Stir, and allow to stand for 5 min. Add 5 g of a suitable filtering aid, such as diatomaceous
earth, mix, and filter. Discard the first 50 mL of the filtrate, and collect the next 80-mL portion. Proceed as directed for Test
Solution 1, beginning with “Transfer the filtrate to a separatory funnel”.
Cleanup procedure: Place a medium-porosity sintered-glass disk or a glass wool plug at the bottom of a 10-mm × 300-mm
chromatographic tube. Prepare a slurry of 2 g of silica gel with a mixture of ethyl ether and solvent hexane (3:1), pour the slurry
into the column, and wash with 5 mL of the same solvent mixture. Allow the absorbent to settle, and add to the top of the
column a layer of 1.5 g of anhydrous sodium sulfate. Dissolve the residue obtained above in 3 mL of methylene chloride, and
transfer it to the column. Rinse the flask twice with 1-mL portions of methylene chloride, transfer the rinses to the column, and
elute at a rate NMT 1 mL/min. Add successively to the column 3 mL of solvent hexane, 3 mL of ethyl ether, and 3 mL of
methylene chloride; elute at a rate NMT 3 mL/min; and discard the eluates. Add to the column 6 mL of a mixture of methylene
chloride and acetone (9:1), and elute at a rate NMT 1 mL/min, preferably without the aid of vacuum. Collect this eluate in a
small vial, add a boiling chip if necessary, and evaporate to dryness on a water bath. Dissolve the residue in 0.2 mL of a mixture
of chloroform and acetonitrile (9.8: 0.2), and shake by mechanical means if necessary.

TEST SOLUTION 3
If interferences still exist in the residue, proceed as directed for Cleanup procedure with IAC in Test Solution in Method II.

AFLATOXIN STANDARD SOLUTION

[CAUTION—Aflatoxins are highly toxic. Handle with care.]
Prepare an Aflatoxin Standard Solution containing AFB1, AFB2, AFG1, and AFG2 at 2.0, 0.50, 2.0, and 0.50 µg/mL, respectively,
in acetonitrile as follows. First prepare individual stock solutions of aflatoxins AFB1, AFB2, AFG1, and AFG2 in acetonitrile, each
having a nominal concentration of 10 µg/mL.1 Determine the concentration of each aflatoxin stock solution by measuring the
absorbance (A) at a wavelength of maximum absorption close to 360 nm and using the equation:

1 Suitable aflatoxin individual standards are available from Romer Labs, catalog #001012, 001013, 001014, 001015; and Sigma-Aldrich, catalog #A6636,
A9887, A0138, A0263.

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Aflatoxin (µg/mL) = (A × Mr × 1000)/ε

A = absorbance
Mr = molecular weight
ε = molar absorptivity (see Table 3)

Table 3. Molecular Weights and Molar Absorptivities for Aflatoxins

Molecular Weight Molar Absorptivity
Aflatoxin (Mr) Solvent (ε)
AFB1 312 Acetonitrile 20700

AFB2 314 Acetonitrile 22500

AFG1 328 Acetonitrile 17600

AFG2 330 Acetonitrile 18900

Finally, to obtain the Aflatoxin Standard Solution, transfer an accurate volume of each aflatoxin stock solution to the same
volumetric flask, and dilute with acetonitrile to volume. Calculate the concentration of each aflatoxin in the Aflatoxin Standard
Solution:

CAF = C′AF × VTS/VAF

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CAF = concentration of the relevant aflatoxin in the Aflatoxin Standard Solution
C′AF = concentration of the relevant aflatoxin in the corresponding stock solution, as determined above
VTS = volume of the volumetric flask used to prepare the final Aflatoxin Standard Solution
VAF = volume of each individual aflatoxin stock solution transferred to the volumetric flask used to prepare the Aflatoxin
Standard Solution
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Store in a refrigerator. Equilibrate to room temperature before use.

AFLATOXIN SOLUTION
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Dilute the Aflatoxin Standard Solution with acetonitrile to obtain a solution having a concentration of 0.4 µg/mL each
of AFB1 and AFG1, and 0.1 µg/mL each of AFB2 and AFG2.

PROCEDURE
Separately apply 2.5, 5, 7.5, and 10 µL of the Aflatoxin Solution and three 10-µL applications of either Test Solution 1, Test
O

Solution 2, or Test Solution 3 to a suitable TLC plate (see Chromatography á621ñ) coated with a 0.25-mm layer of chromatographic
silica gel mixture. Superimpose 5 µL of the Aflatoxin Solution on one of the three 10-µL applications of the Test Solution. Allow
the spots to dry, and develop the chromatogram in an unsaturated chamber containing a solvent system consisting of a
mixture of chloroform, acetone, and isopropyl alcohol (85:10:5) until the solvent front has moved NLT 15 cm from the origin.
Remove the plate from the developing chamber, mark the solvent front, and allow the plate to air-dry. Locate the spots on the
plate by examination under UV light at 365 nm.

SYSTEM SUITABILITY
The four applications of the Aflatoxin Solution appear as four clearly separated blue fluorescent spots. Observe any spot
obtained from the Test Solution that coincides in hue and position with those of the Aflatoxin Solution. Any spot obtained from
the Test Solution with the superimposed Aflatoxin Solution is not less intense than that of the corresponding Aflatoxin Solution.

ACCEPTANCE CRITERIA
No spot from any of the other applications of the Test Solution corresponds to any of the spots obtained from the applications
of the Aflatoxin Solution. If any spot of aflatoxins is obtained in the Test Solution, match the position of each fluorescent spot of
the Test Solution with those of the Aflatoxin Solution to identify the type of aflatoxin present. The intensity of the aflatoxin spot,
if present in the Test Solution, when compared with that of the corresponding aflatoxin in the Aflatoxin Solution will give an
approximate concentration of aflatoxin in the Test Solution. Where the individual monograph calls for compliance with the
limits for aflatoxins, the limits are NMT 5 ppb for AFB1 and NMT 20 ppb for the sum of AFB1, AFB2, AFG1, and AFG2, except
when otherwise indicated.

Method II
SODIUM CHLORIDE SOLUTION
See Method I.

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PHOSPHATE BUFFERED SALINE SOLUTION

Prepare 10 mM phosphate buffer solution containing 0.138 M sodium chloride and 0.0027 M potassium chloride in water,
and adjust with 2 M sodium hydroxide to a pH of 7.4.2

IMMUNOAFFINITY COLUMN
Before conditioning, adjust the immunoaffinity column (IAC) to room temperature. For conditioning, apply 10 mL of
Phosphate Buffered Saline Solution onto the column and let it flow through the column by gravity force at a rate of 2–3 mL/min.
Leave 0.5 mL of the Phosphate Buffered Saline Solution on top of the column until the Test Solution is applied.

TEST SOLUTION
Sample extraction: Transfer about 5 g of a representative powdered sample, accurately weighed, to a glass-stoppered flask.
Add 20 mL of a mixture of methanol and water (17:3). Shake vigorously by mechanical means for NLT 30 min, and filter. Discard
the first 5 mL of the filtrate, and collect the next 4-mL portion. Transfer the filtrate to a separatory funnel. Add 4 mL of Sodium
Chloride Solution and 2.5 mL of hexane, and shake for 1 min. Allow the layers to separate, and transfer the lower aqueous layer
to a second separatory funnel. Extract the aqueous layer in the separatory funnel twice, each time with 2.5 mL of methylene
chloride, by shaking for 1 min. Allow the layers to separate each time, separate the lower organic layer, and collect the combined
organic layers in a 50-mL conical flask. Evaporate the organic solvent on a water bath. Transfer the remaining extract to an
appropriate sample tube, and evaporate to dryness on a water bath. Cool the residue. If interferences exist in the residue,
proceed as directed for Cleanup procedure with IAC. Otherwise, dissolve the residue obtained above in 200 µL of acetonitrile,

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and shake by mechanical means if necessary.
Cleanup procedure with IAC: The residue is dissolved in 5 mL of a mixture of methanol and water (6:4) and then diluted with
5 mL of water. This extract is applied onto a conditioned IAC. The IAC is rinsed twice with 10 mL of Phosphate Buffered Saline
Solution, and the elution is performed slowly with 2 mL of methanol. Evaporate the eluate with nitrogen, and dissolve the residue
in 200 µL of acetonitrile. ci
AFLATOXIN SOLUTION
[CAUTION—Aflatoxins are highly toxic. Handle with care.]
Dilute the Aflatoxin Standard Solution with acetonitrile to obtain a solution containing 0.04 µg/mL each of AFB1 and AFG1,
and 0.01 µg/mL each of AFB2 and AFG2.
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ANALYSIS
Separately apply 5, 7.5, and 10 µL of Aflatoxin Solution and three 10-µL applications of the Test Solution to a suitable HPTLC
plate (see Chromatography á621ñ) coated with a 200-µm layer of chromatographic silica gel mixture. Superimpose 5 µL of
Aflatoxin Solution on one of the three 10-µL applications of the Test Solution. Allow the spots to dry, and develop the
O

chromatogram in a saturated chamber containing a solvent system consisting of a mixture of chloroform, acetone, and water
(140: 20: 0.3) until the solvent front has moved NLT 72 mm from the origin (80 mm from the lower edge of the plate). Remove
the plate from the developing chamber, mark the solvent front, and allow the plate to air-dry for 5 min. Locate the spots on
the plate by scanning fluorescence density (>400 nm) under UV light at 366 nm. Match the position of each fluorescent spot
of the Test Solution with those of Aflatoxin Solution to identify the type of aflatoxin present. The concentration of aflatoxins in
the Test Solution can be calculated from the calibration curve obtained from the scan data with Aflatoxin Solution.

SYSTEM SUITABILITY
The four applications of Aflatoxin Solution appear as four clearly separated blue fluorescent spots. Observe any spot obtained
from the Test Solution that coincides in hue and position with those of Aflatoxin Solution. Any spot obtained from the Test
Solution with the superimposed Aflatoxin Solution is not less intense than that of the corresponding Aflatoxin Solution. The mean
recovery of spiked AFB1 and AFG1 is NLT 70%.

ACCEPTANCE CRITERIA
Where the individual monograph calls for compliance with the limits for aflatoxins, the limits are NMT 5 ppb for AFB1 and
NMT 20 ppb for the sum of AFB1, AFB2, AFG1, and AFG2, except when otherwise indicated.

Method III
This test method is provided as an example for the detection of the possible presence of AFB1 and total aflatoxins (AF: sum
of AFB1, AFB2, AFG1, and AFG2). It has been shown to be suitable for powdered ginseng and ginger. Its suitability to other articles
of botanical origin must be demonstrated.

2A suitable powder mixture is available from Sigma as PBS P-3813.

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0.1 M PHOSPHATE BUFFER SOLUTION

Dissolve 8.69 g of anhydrous disodium phosphate and 4.66 g of anhydrous monosodium phosphate or 5.36 g of
monosodium phosphate monohydrate in 800 mL of water, adjust with 2 M sodium hydroxide to a pH of 7.4, add 10 mL of
polysorbate 20, and dilute with water to 1 L.

PHOSPHATE BUFFERED SALINE SOLUTION

Prepare as directed in Method II.

WORKING AFLATOXIN STANDARD SOLUTIONS

Prepare six solutions by diluting suitable volumes of Aflatoxin Standard Solution with methanol and water (1:1). The final
aflatoxin concentrations in each Working Aflatoxin Standard Solution are shown in Table 4. Store in a refrigerator, and equilibrate
to room temperature before use. Prepare the solutions daily.

Table 4. Preparation of Working Aflatoxin Standard Solutions

Working Final Aflatoxin Concentration of Working Aflatoxin Standard Solution (ng/mL)
Aflatoxin Aflatoxin
Standard Standard
Solutions Solution (µL) AFB1 AFB2 AFG1 AFG2 ΣAF
1 0 0 0 0 0 0

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2 12.5 0.25 0.0625 0.25 0.0625 0.625

3 25 0.5 0.125 0.5 0.125 1.25

4 50 1 0.25 1 0.25 2.5

5 100 2 0.5 2 0.5 5

6 200 4
ci 1 4 1 10

IMMUNOAFFINITY COLUMN3
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Use an IAC that contains monoclonal antibodies cross reactive toward AFB1, AFB2, AFG1, and AFG2. The immunoaffinity
columns have a minimum capacity of NLT 100 ng of total aflatoxin and give a recovery of NLT 80% for AFB1, AFB2, AFG1,
and AFG2 when 5 ng of each AFB1, AFB2, AFG1, and AFG2 is applied in 10 mL of 10% methanol in Phosphate Buffered Saline
Solution (v/v).

TEST SOLUTION
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Extraction: Weigh 5 g of a representative test sample in a 50-mL centrifuge tube. Add 1 g of sodium chloride and 25 mL of a
mixture of methanol and 0.5% sodium bicarbonate (7:3). Mix on a vortex mixer until sample particles and extract solvent are
well mixed. Shake at 400 rpm for 10 min. Centrifuge for 10 min at 7000 rpm (g value = 5323 mm/s2) or at a speed that can
result in a firm pellet of residues. Immediately pipette 7 mL into a 50-mL centrifuge tube, add 28 mL of 0.1 M Phosphate Buffer
Solution, mix, and filter through glass microfiber paper. Collect 25 mL of filtrate (equivalent to 1 g of test sample) into a 25-mL
graduated cylinder, and proceed immediately with IAC chromatography.
IAC cleanup: [NOTE—For IAC cleanup, columns must be kept at room temperature for at least 15 min before use.] Remove
the top cap from the column, and connect it with the reservoir. Remove the end cap from the column, and attach it to the
column manifold (the fit must be tight). Let the liquid in the column pass through until the liquid is about 2–3 mm above the
column bed. Pass 25 mL of filtrate into the reservoir. Let the filtrate flow through the column by gravity force. Let the column
run dry. To start the flow again easily, remove the column from the manifold, add about 2 mL of Phosphate Buffered Saline
Solution into the column, reattach the column to the reservoir, and wash the column with an additional 3 mL of Phosphate
Buffered Saline Solution and then with 5 mL of water (the 5 mL of Phosphate Buffered Saline Solution can be added directly to
the column reservoir if other techniques are used to dislodge the air bubble at the end of the column and to start flow again
easily). Let the column run dry, then force 3 mL of air through the column with a syringe. Elute with 1 mL of methanol, and
collect the analytes in a 3-mL volumetric flask, letting the eluate drip freely. Let the column run dry. Let stand for 1 min, then
elute with an additional 1 mL of methanol, and collect in the same volumetric flask. Let the column run dry, and force 10 mL
of air through the column. Dilute the eluate with water to volume. Use this as the Test Solution, and perform the analysis of
aflatoxins immediately.

SYSTEM SUITABILITY SOLUTION

Prepare a spiked sample by adding 5 mL of Working Aflatoxin Standard Solution 5 to a 5-g sample and repeating the procedure
for the Test Solution, using 20 mL instead of 25 mL of the mixture of methanol and 0.5% sodium bicarbonate (700:300).

3 AflaOchraTest column (G1017; Vicam, Watertown, MA, USA) or equivalent. Aflatoxin/OTA immunoaffinity columns are suitable.

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CHROMATOGRAPHIC SYSTEM
Detector: Fluorescence, excitation wavelength (Ex) 362 nm and emission wavelength (Em) 440 nm
Column: 4.6-mm × 15-cm; 3-µm packing L1
Flow rate: 0.8 mL/min
Mobile phase: Isocratic
For post-column derivatization with PHRED cell:4 Water, methanol, and acetonitrile (60:25:15)
For post-column derivatization with Kobra cell:5 A solution prepared by mixing 1 L of a mixture of water, methanol, and
acetonitrile (60:25:15); 350 µL of 4 M nitric acid; and 120 mg of potassium bromide
Post-column derivatization (PCD) systems
PHRED cell: Post-column photochemical derivatization cell
Kobra cell: Electrochemical cell, post-column bromination derivatization cell

ANALYSIS
Post-column derivatization for aflatoxins: Use a UV or Kobra cell. Inject 50 µL of reagent blank (Working Aflatoxin Standard
Solution 1), Working Aflatoxin Standard Solutions 2–6, or the Test Solution into the LC column. Identify the aflatoxin peaks in the
Test Solution by comparing the retention times with those of the working standards. The aflatoxins elute in the
order AFG2, AFG1, AFB2, and AFB1. After passing through the PHRED or Kobra cell, the AFG1 and AFB1 have been derivatized to
form AFG2a (derivative of AFG1) and AFB2a (derivative of AFB1). [NOTE—The chemical structures of the derivatives resulting from
electrochemical bromination and photolysis are not the same. The structures of AFB1 and AFG1 photolysis products have not
been established.] The retention times of AFG2, AFG2a, AFB2, and AFB2a are between about 14 and 27 min using the PHRED cell;

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retention times are shorter using the Kobra cell. The peaks should be baseline resolved. Construct standard curves for each
aflatoxin. Determine the concentration of each aflatoxin in the Test Solution from the calibration curve.
Aflatoxins calibration curves: Calibration curves are prepared for each of the aflatoxins using the Working Aflatoxin Standard
Solutions containing the four aflatoxins described. These solutions cover the range of 0.25–4 ng/mL for AFB1 and AFG1, and the
range of 0.0625–1 ng/mL for AFB2 and AFG2. Make the calibration curves before analysis according to Table 4, and check the
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plot for linearity. If the test portion area response is outside (higher) the calibration range, then the Test Solution should be
diluted with a mixture of methanol and water (1:1, v/v) and reinjected into the LC column.
Quantitation of aflatoxins: Quantitation of aflatoxins is performed by measuring peak areas at each aflatoxin retention time
and comparing them with the corresponding calibration curve.
ffi
SYSTEM SUITABILITY
The mean recovery of spiked AFB1 (2 µg/kg) and the total of aflatoxins (5 µg/kg) is NLT 68% and 70%, respectively. The
relative standard deviation (RSD) is NMT 10% for AFB1 and for the total of aflatoxins.

CALCULATIONS
O

Plot the peak area (response, y-axis) of each of the toxin standards against the concentration (ng/mL, x-axis) and determine
the slope (S) and y-intercept (a). Calculate the level of toxin in the sample by the following formula:

Toxin (µg/kg) = {[(R − a)/S] × V/W} × F

R = peak area of the Test Solution
a = y-intercept of the calibration curve
S = slope of the calibration curve
V = final volume of the injected Test Solution (mL)
W = 1 g of test sample passed through the immunoaffinity column
F = dilution factor, 1 when V = 3 mL

The total of aflatoxins is the sum of AFG2, AFG1, AFB2, and AFB1.

ACCEPTANCE CRITERIA
Where the individual monograph calls for compliance with the limits for aflatoxins, the limits are NMT 5 ppb for AFB1 and
NMT 20 ppb for the sum of AFB1, AFB2, AFG1, and AFG2, except when otherwise indicated.

4 PHRED™ Photochemical Reactor (AURA Industries, New York, NY, USA) or equivalent. Avoid looking at the UV lamp.
5 KobraCell™ (R-Biopharm Inc., Marshall, MI, USA) or equivalent. Set at 100 mA. Do not turn on the current until the LC pump is operating to avoid
overheating the cell membrane.

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Change to read:
PESTICIDE RESIDUE ANALYSIS

Definition
Where used in this Pharmacopeia, the designation “pesticide” applies to any substance or mixture of substances intended
to prevent, destroy, or control any pest, unwanted species of plants, fungus, or animals causing harm during or otherwise
interfering with the production, processing, storage, transport, or marketing of pure articles. The designation includes
substances intended for use as growth regulators, defoliants, or desiccants, and any substance applied to crops before or after
harvest to protect the product from deterioration during storage and transport.

Limits
Within the United States, many botanicals are treated as dietary supplements and are subject to the statutory provisions that
govern foods but not drugs in the Federal Food, Drug, and Cosmetic Act. Limits for pesticides in foods are determined by the
Environmental Protection Agency (EPA) as indicated in the Code of Federal Regulations (40 CFR §180) or the Federal Register
(FR). In addition, the FDA establishes action levels for unavoidable pesticide residues (21 CFR §109 and 21 CFR §509). For
pesticide chemicals without EPA-established tolerance levels or FDA action levels, the residues should be below the detection
limit of the specified method. Results less than the EPA detection limits are considered zero values. The limits contained herein,
therefore, are not applicable in the United States when articles of botanical origin are labeled for food purposes. The limits,
however, may be applicable in other countries. Unless otherwise indicated in the monograph, the article to be examined

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complies with the limits given in Table 5. The limits for suspected pesticides that are not listed in Table 5 must comply with the
regulations of the EPA. For instances in which a pesticide is not listed in Table 5 or in EPA regulations, calculate the limit by the
formula:

Limit (mg/kg) = AM/100B

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where A is the acceptable daily intake (ADI), as published by the Food and Agriculture Organization of the United Nations (FAO)
and the World Health Organization (WHO), in mg/kg of body weight; M is body weight, in kilograms (60 kg); and B is the daily
dose of the article, in kilograms.
If the article is intended for the preparation of extracts, tinctures, or other pharmaceutical forms of which the preparation
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method modifies the content of pesticides in the finished product, calculate the limits by the formula:

Limit (mg/kg) = AME/100B

where E is the extraction factor of pesticide in the preparation method, determined experimentally as the ratio between the
original pesticide content in the plant material and the final pesticide content in the preparation; B is the daily dose of the
preparation in kilograms; and A and M are as defined above.
O

A total or partial exemption from the test may be granted when the complete history (nature and quantity of the pesticides
used, date of each treatment during cultivation and after harvest) of the treatment of the batch is known and can be checked
precisely according to good agricultural and collection practices (GACP).

Table 5
Limit
Substance (mg/kg)
Acephate 0.1

Alachlor 0.05

Aldrin and dieldrin (sum of) 0.05

Azinphos-ethyl 0.1

Azinphos-methyl 1

Bromide, inorganic (calculated as bromide ion) 125

Bromophos-ethyl 0.05

Bromophos-methyl 0.05

Bromopropylate 3

Chlordane (sum of cis-, trans-, and oxychlordane) 0.05

Chlorfenvinphos 0.5

Chlorpyriphos-ethyl 0.2

Chlorpyriphos-methyl 0.1

Chlorthal-dimethyl 0.01

Cyfluthrin (sum of) 0.1

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Table 5 (continued)
Limit
Substance (mg/kg)
λ-Cyhalothrin 1

Cypermethrin and isomers (sum of) 1

DDT (sum of o,p′-DDE, p,p′-DDE, o,p′-DDT, p,p′-DDT, o,p′-TDE, and p,p′-TDE) 1

Deltamethrin 0.5

Diazinon 0.5

Dichlofluanid 0.1

Dichlorvos 1

Dicofol 0.5

Dimethoate and omethoate (sum of) 0.1

Dithiocarbamates (expressed as CS2) 2

Endosulfan (sum of isomers and endosulfan sulphate) 3

Endrin 0.05

Ethion 2

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Etrimphos 0.05

Fenchlorophos (sum of fenchlorophos and fenchlorophos-oxon) 0.1

Fenitrothion 0.5

Fenpropathrin
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Fensulfothion (sum of fensulfothion, fensulfothion-oxon, fensulfothion-oxon sulfone, and
fensulfothion sulfone)
0.03

0.05

Fenthion (sum of fenthion, fenthion-oxon, fenthion-oxon sulfone, fenthion-oxon sulfoxide,

fenthion sulfone, and fenthion-sulfoxide) 0.05
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Fenvalerate 1.5

Flucythrinate 0.05

τ-Fluvalinate 0.05

Fonophos 0.05
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Heptachlor (sum of heptachlor, cis-heptachlorepoxide, and trans-heptachlorepoxide) 0.05

Hexachlorbenzene 0.1

Hexachlorocyclohexane (sum of isomers α-, β-, δ-, and ε-) 0.3

Lindan (γ-hexachlorocyclohexane) 0.6

Malathion and malaoxon (sum of) 1

Mecarbam 0.05

Methacriphos 0.05

Methamidophos 0.05

Methidathion 0.2

Methoxychlor 0.05

Mirex 0.01
Monocrotophos 0.1

Parathion-ethyl and paraoxon-ethyl (sum of) 0.5

Parathion-methyl and paraoxon-methyl (sum of) 0.2

Pendimethalin 0.1

Pentachloranisole 0.01

Permethrin and isomers (sum of) 1

Phosalone 0.1

Phosmet 0.05

Piperonyl butoxide 3

Pirimiphos-ethyl 0.05

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Table 5 (continued)
Limit
Substance (mg/kg)
Pirimiphos-methyl (sum of pirimiphos-methyl and N-desethyl-pirimiphos-methyl) 4

Procymidone 0.1

Profenophos 0.1

Prothiophos 0.05

Pyrethrum (sum of cinerin I, cinerin II, jasmolin I, jasmolin II, pyrethrin I, and pyrethrin II) 3

Quinalphos 0.05

Quintozene (sum of quintozene, pentachloraniline, and methyl pentachlorphenyl sulfide) 1

S-421 0.02

Tecnazene 0.05

Tetradifon 0.3

Vinclozolin 0.4

Qualitative and Quantitative Analysis of Pesticide Residues Use analytical procedures validated e.g., in
accordance with the latest version of the EU guideline on analytical quality control and validation procedures for pesticide

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residue analysis [NOTE—Current version Document No. ▲SANTE/12682/2019, https://ec.europa.eu/food/system/files/2020-01/
pesticides_mrl_guidelines_wrkdoc_2019-12682.pdf▲ (ERR 1-Nov-2021)] or the EPA method validation principles (OPPTS 860.1340)
that satisfy the following criteria. The method, especially with respect to its purification steps, is suitable for the combination
of pesticide residue and substance under test, and is not susceptible to interference from co-extractives; the limit of
quantification for each pesticide matrix combination to be analyzed is NMT the corresponding tolerance limit: the method is
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shown to recover between 70% and 120% of each pesticide with a repeatability NLT 20% RSD [NOTE—lower recoveries may
be acceptable in certain cases as discussed in SANTE/11813/2017] ; and the concentrations of test and reference solutions and
the setting of the apparatus are such that a linear response is obtained from the analytical detector.

LIMITS OF ELEMENTAL IMPURITIES

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The levels of elemental impurities should be restricted as shown in Table 6 unless otherwise stated in the individual
monograph. Specific monographs may provide different limits for articles that are typically used in large quantities.

Table 6. Limits of Elemental Impurities

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Limit
Element (µg/g)

Arsenic (inorganic)a 2

Cadmium 0.5

Lead 5

Mercury (total) 1
b
Methylmercury (as Hg) 0.2

a Arsenic may be measured using a nonspeciation procedure under the assumption that all arsenic contained in the supplement is in the inorganic form. Where
the limit is exceeded using a nonspeciation procedure, compliance with the limit for inorganic arsenic shall be demonstrated on the basis of a speciation procedure.
b Methylmercury determination is not necessary when the content for total mercury is less than the limit for methylmercury.

Articles are tested according to the procedures set forth in Elemental Impurities—Procedures á233ñ. Where speciation is
required, the procedures given in Elemental Contaminants in Dietary Supplements á2232ñ are used for testing.

USP REFERENCE STANDARDS á11ñ

USP Dextrose RS

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